Molecular Characterization of Prostate Hyperplasia in Prolactin Transgenic Mice Karin Dillner

Transcription

Molecular Characterization of Prostate
Hyperplasia in Prolactin Transgenic Mice
Karin Dillner
Department of Physiology and Pharmacology
Sahlgrenska Academy, Göteborgs University
Sweden 2003
All previously published papers were reproduced with permission from the
publishers.
Printed by Svenska Tryckpoolen AB
© Karin Dillner, 2003
ISBN 91-628-5652-9
Molecular Characterization of Prostate Hyperplasia in Prolactin Transgenic Mice
ABSTRACT
Benign prostatic hyperplasia (BPH) and prostate cancer are age-related diseases, affecting a
majority of elderly men in the western world, and are known to be influenced by several
different hormones, including sex hormones. Although the hormone prolactin (PRL) is
well known to exert trophic effects on prostate cells, its involvement in the pathophysiology
is still poorly characterized. In order to evaluate the potential role of PRL in promoting
prostate growth, we used PRL-transgenic mouse models that develop prostate phenotypes.
The Mt-PRL transgenic mouse model, ubiquitously overexpressing the rat PRL
transgene, develops a dramatic prostate hyperplasia with concurrent chronic
hyperprolactinemia and elevated serum androgen levels. In a castration and androgenresubstitution study, we demonstrated that supraphysiological serum androgen levels are
not required for the progress of prostate hyperplasia in adult Mt-PRL transgenic mice.
Furthermore, androgen treatment does not induce prostate hyperplasia in wildtype mice. To
address the role of local PRL action in the prostate, a new transgenic mouse model (PbPRL) was generated using the prostate-specific probasin minimal promoter to drive
expression of the rat PRL gene. The androgen-dependency of the probasin promoter
resulted in onset of the PRL transgene expression at puberty. The Pb-PRL transgenic mice
also develop a significant prostate hyperplasia, evident from 10 weeks of age and the
hyperplasia increases with age. In contrast to the Mt-PRL transgenic mice, the Pb-PRL
transgenic mice display normophysiological serum androgens levels throughout animal life
span. The prostates of both the Mt- and Pb-PRL transgenic mice display a prominent
stromal hyperplasia with mild epithelial dysplastic features, leading to an increased
stromal/epithelial ratio. Accumulation of secretory material is also a major characteristic.
Immunohistochemistry analysis of both the PRL transgenic models’ prostates showed an
increased androgen receptor distribution in both the epithelial and stromal cells.
Microdissections demonstrated an increased ductal morphogenesis in the Mt-PRL prostate
compared to Pb-PRL and controls, indicating that PRL stimulates, directly or indirectly via
increased androgen action, prostate ductal morphogenesis in the developing prostate gland.
The use of differential gene expression technologies enabled characterization of the
molecular mechanisms involved in the prostate hyperplasia. Of particular interest is the
potential significance of reduced apoptosis for the development/progression of the prostate
phenotype. This finding was further confirmed by immunohistochemical analysis using two
different apoptosis markers. Moreover, in line with the prominent expansion of the stromal
compartment, were the identified changes in gene expression seen in the PRL transgenic
prostate, suggesting that activation of the stroma is important for the development of the
prostate hyperplasia.
Altogether, there are histological and molecular similarities between the prostate
hyperplasia of PRL-transgenic mice and human prostate pathology, including both BPH
and prostate cancer.
Key words: Prolactin-transgenic, mouse, prolactin, prostate hyperplasia, gene expression
analysis
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Karin Dillner
TABLE OF CONTENTS
ABSTRACT....................................................................................................... 1
TABLE OF CONTENTS ................................................................................. 2
ORIGINAL PAPERS ....................................................................................... 4
LIST OF ABBREVIATIONS.......................................................................... 5
INTRODUCTION ............................................................................................ 6
The Prostate Gland...........................................................................................6
Prostate development ....................................................................................6
Prostate anatomy and structure in human and rodents.................................7
Prostate disorders .............................................................................................8
Benign Prostatic Hyperplasia........................................................................8
Possible theories of BPH etiology .................................................................9
Premalignant lesions of the prostate...........................................................11
Prostate carcinoma ......................................................................................11
Possible theories of prostate cancer etiology .............................................12
Prolactin...........................................................................................................13
Gene, structure, and variants.......................................................................13
Control of prolactin synthesis, secretion and regulation............................15
The prolactin receptor .................................................................................15
Prolactin signal transduction.......................................................................16
Action of prolactin in the prostate gland .....................................................16
Proliferation.................................................................................................17
Apoptosis.....................................................................................................17
Citrate production........................................................................................18
Prolactin in prostate pathophysiology .........................................................18
Prolactin in human prostate cancer and BPH.............................................18
Experimental animal data ...........................................................................19
Rodent models of prostate disease................................................................20
Transgenic prostate hyperplasia models.....................................................21
Rodent models of prostate cancer...............................................................21
Other genetically engineered mouse models with prostate phenotype .....22
Mouse models genetically engineered in the prolactin signaling pathway22
Mouse models genetically engineered in other hormones..........................23
Hormone/growth factor regulation of the prostate ....................................24
Action of androgens in the prostate............................................................24
Interactions between prolactin and androgens in the prostate gland .........25
Action of estrogens in the prostate .............................................................26
Interactions between prolactin and estrogens in the prostate gland ..........27
Action of other peptide hormones and growth factors in the prostate ......27
Functional genomics in the study of the prostate gland ............................28
AIMS OF THE THESIS ................................................................................ 29
METHODOLOGICAL CONSIDERATIONS ........................................... 30
Transgenic animals.........................................................................................30
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Approaches to gene expression analysis ......................................................32
cDNA representational difference analysis (RDA) ...................................32
Sequence analysis ........................................................................................ 34
cDNA microarray analysis .........................................................................35
Array design and Printing ........................................................................... 36
Target preparation....................................................................................... 37
Hybridization ............................................................................................... 37
Image analysis and Normalization.............................................................. 38
Data Analysis and Statistical Evaluation.................................................... 39
Experimental design .................................................................................... 40
Microarray databases.................................................................................. 41
Comparisons between cDNA RDA and cDNA microarray analyis .........42
Verification strategies .................................................................................43
cDNA microarray analysis.......................................................................... 43
Real-time RT-PCR ....................................................................................... 43
Assessment of apoptotic activity ...................................................................46
RESULTS AND TECHNICAL COMMENTS ........................................... 47
Paper I..............................................................................................................47
Paper II ............................................................................................................49
Paper III...........................................................................................................53
Paper IV...........................................................................................................55
DISCUSSION .................................................................................................. 58
CONCLUSIONS ............................................................................................. 67
ACKNOWLEDGEMENTS........................................................................... 68
REFERENCES................................................................................................ 70
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ORIGINAL PAPERS
This thesis is based on the following papers, which are referred to in the text
by their Roman numbers (I-IV);
I. Kindblom J, Dillner K, Ling C, Törnell J and Wennbo H
Progressive Prostate Hyperplasia in Adult Prolactin Transgenic Mice is
Not Dependent on Elevated Androgen Serum Levels.
Prostate 2002 Sep 15;53(1):24-33.
II. Dillner K, Kindblom J, Flores-Morales A, Pang ST, Törnell J,
Wennbo H and Norstedt G
Molecular Characterization of Prostate Hyperplasia in ProlactinTransgenic Mice Using cDNA Representational Difference Analysis.
Prostate 2002 Jul 1;52(2):139-49.
III. Kindblom J, Dillner K, Sahlin L, Robertson F, Ormandy CJ,
Törnell J and Wennbo H
Prostate Hyperplasia in a Transgenic Mouse with Prostate-Specific
Expression of Prolactin
Endocrinology, 2003, in press.
IV. Dillner K, Kindblom J, Flores-Morales A, Shao R, Törnell T,
Norstedt G and Wennbo H
Gene Expression Analysis of Prostate Hyperplasia In Mice
Overexpressing the Prolactin Gene Specifically in the Prostate.
Submitted for publication.
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LIST OF ABBREVIATIONS
-/aa
AP
AR
BPH
cDNA
Cy
DLP
DP
DP1, 2, 3
ECM
ER
EST
FDR
GH
hPRL
LP
MMP
mRNA
Mt-1
Mt-PRL
Pb
Pb-PRL
PIF
PIN
PL
PRL
PRLR
PSA
RDA
rPRL
RT-PCR
SAM
ssDNA
Stat
SMA
TUNEL
TURP
UGM
UGS
UTR
VP
homozygous gene-deficiency
amino acids
anterior prostate
androgen receptors
benign prostatic hyperplasia
complementary deoxyribonucleic acid
cyanine
dorsolateral prostate
dorsal prostate
difference products 1, 2, 3
extracellular matrix
estrogen receptor
expressed sequence tags
false discovery rate
growth hormone
human PRL
lateral prostate
matrix metalloproteinase
messenger ribonucleic acid
metallothionein-1 gene
The metallothionein-1 promoter - rat prolactin gene
probasin gene
The minimal probasin promoter - rat prolactin gene
prolactin inhibiting factors
Prostatic intra-epithelial neoplasia
placental lactogen
prolactin
prolactin receptor
prostate specific antigen
representational difference analysis
rat prolactin
reverse transcription polymerase chain reaction
Significance Analysis of Microarrays
single stranded DNA
signal transducers and activators of transcription
Statistics of Microarrays Analysis
terminal deoxynucleotidyl transferase dUTP nick end labeling
transurethral resection of the prostate
urogenital mesenchyme
urogenital sinus
untranslated region
ventral prostate
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INTRODUCTION
THE PROSTATE GLAND
The prostate gland is an exocrine gland that is found only in mammals. The
main function of the gland is to produce a major fraction of the seminal fluid,
including enzymes, amines, lipids and metal ions. One unique function of the
prostate gland is the capacity to produce, accumulate and secrete high levels
of citrate [1]. The prostate varies in its anatomy, biochemistry and pathology
between different species. The mature mammalian prostate is a glandular
organ consisting of epithelial and stromal cell types that are hormonally
regulated. The epithelium consists of a single layer of polarized columnar
epithelial cells together with basal and neuroendocrine cells. The epithelial
cells supply secretions that empty through ducts into the urethra to form the
major component of the seminal plasma of the ejaculate. The surrounding
stromal compartment comprises of fibroblasts, smooth muscle cells and
loose collagenous extracellular matrix (ECM), in addition to neuronal,
lymphatic and vascular components.
Interest in understanding the biology of the prostate has largely been driven
by the high incidence of prostate diseases, including benign prostatic
hyperplasia (BPH) and prostate cancer.
PROSTATE DEVELOPMENT
The development of the male reproductive tract is dependent upon androgens
and mesenchymal-epithelial interactions [2]. The initial event in prostatic
morphogenesis is the outgrowth of solid cords of epithelial cells, so-called
prostatic buds, from the urogenital sinus epithelium into the surrounding
urogenital sinus mesenchyme. In rodents, this occurs in a precise spatial
pattern that establishes the lobar subdivisions of the prostate [2, 3]. In
rodents, the critical time period for ductal budding and the consequent
process of ductal growth and branching initiate around day 15 of gestation
and conclude approximately 4-5 weeks postpartum [4-6]. The branching
morphogenesis is almost entirely complete by 2 weeks after birth in the
mouse [4]. At this time, serum testosterone levels are still low and the
increase in prostatic wet weight is modest. As shown by neonatal castration
studies, the neonatal prostatic ductal morphogenesis is sensitive to, but does
not require, chronic androgen stimulation [7]. The prepubetal growth of the
prostatic ductal network is considered non-uniform, where the growth is
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highest in the distal region, at the ductal tips, and much lower in the
proximal region closest to the urethra [8, 9]. At puberty, the testosterone
levels raise significantly, and the rodent prostatic wet weight and DNA
content increase more rapidly [7]. In contrast, the human prostate
morphogenesis occurs entirely during the fetal period, with ductal
development primarily occurring in the first half of gestation [10].
PROSTATE ANATOMY AND STRUCTURE IN HUMAN AND RODENTS
There are fundamental differences between the prostate anatomy in human,
dog and other primates and non-primates, e.g. rodents. The human prostate is
associated with the urethra contiguously below the urinary bladder and
prostatic ducts emanate from the urethra radiating towards the periphery
completely surrounding the urethra. The adult human gland can be divided
into four zones based on morphology; the anterior fibromuscular stroma, the
central zone, the peripheral zone and the transition zone. The two latter are
of more clinical interest because prostatic carcinoma arises nearly
exclusively from the peripheral zone and BPH from the transition zone [11].
In contrast, the process of branching morphogenesis in rodents ultimately
gives rise to three distinct bilaterally symmetrical prostatic lobes: the anterior
prostate (AP; also known as the coagulating gland), the dorsolateral prostate
(DLP), and the ventral prostate (VP). The DLP is sometimes further divided
into the dorsal prostate (DP) and the lateral prostate (LP). Individual lobes
are located in specific positions around the urethra, but not completely
circumscribing it [2]. This explains why rodents, in contrast to most humans,
do not suffer from urinary tract symptoms following prostate enlargement.
The ducts of each of the rodent prostatic lobes have a characteristic
branching pattern [4]. The VP and LP lobes are attached to the urethra by
two or three main ducts that show extensive so-called “oak tree” branching,
whereas the DP lobe demonstrates multiple main urethral ducts with less
extensive so-called “palm tree” branching morphology [4]. Furthermore, the
ductal system also shows regional variation in morphology and functional
activity [12] and therefore ductal system of each lobe can be further
subdivided into regional segments, defined as proximal, intermediate and
distal with respect to the urethra [13]. The VP has no clear homologous
counterpart in the prostate of higher animals, whereas the DLP are
considered the most homologous to the human prostate [14, 15].
The prostate tissue can be divided into epithelial and stromal parts and the
proportion between epithelial and stromal compartments differs between
species. In the adult rat the stromal:epithelial ratio is 1:5, whereas in humans,
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the stromal and epithelial cells are present in approximately the same number
in the normal prostate [16, 17].
Another important species difference between rodent and human prostate is
the presence of the androgen-regulated serine protease, prostate specific
antigen (PSA) in human. PSA is produced by both prostate epithelial cells
and prostate cancer cells and is the most commonly used serum marker for
prostate cancer as well as to monitor responses to therapy. Genes related to
human PSA have been detected in several non-human primate species, but
not in other mammalian species, including rodents [18].
PROSTATE DISORDERS
Prostate gland disorders are age-related diseases affecting a majority of
elderly men in the western world. Among mammals with a prostate gland,
humans and dogs are the only species known to suffer from BPH and
prostate carcinoma [19].
BENIGN PROSTATIC HYPERPLASIA
BPH is characterized as a slow, progressive enlargement of the prostate gland,
which eventually causes obstruction and subsequent problems with urination.
However, BPH is believed to be neither a premalignant lesion nor a precursor
of prostate cancer. The incidence of BPH is increasing dramatically with age
from about 50% at 50 year of age to 90 % by the ninth decade of life [20]. The
BPH progression is characterized by hyperplasia of both the stromal and
epithelial compartments. When calculating the stromal:epithelial ratio, clinical
reports have firmly established a dominance of the stromal compartment in
BPH tissues, which is in contrast to the balanced epithelial and stromal
distribution in normal prostate tissue [21-24]. Furthermore, in symptomatic
BPH patients the stromal:epithelial ratio has been reported to be significantly
higher than in asymptomatic patients [22].
Testosterone is the principal circulating androgen. In men, it is secreted
primarily by the testis, with the adrenal glands providing a minor
contribution. To be maximally active in the prostate, testosterone must first
be converted to dihydroxy testosterone (DHT) by the enzyme 5-alpha
reductase. DHT is about five times more potent as an androgen within cells
than testosterone, and it binds readily to the androgen receptors (AR) in the
nucleus. Androgens are clearly required for development of BPH and
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reduction of androgenic effects through 5-alpha reductase inhibitors is utilized
in the pharmacotherapy of BPH. Treatment with 5-alpha reductase inhibitors
rapidly reduces DHT serum levels and over time results in an average
decrease in prostate volume [25]. In addition, alpha-1 adrenoreceptor
antagonists are increasingly used, either given in combination with a 5-alpha
reductase inhibitor or separately [26]. The mechanism of action of the alpha1 adrenoreceptor antagonists is primarily to reduce the contractility of the
smooth muscle cells in the bladder neck and prostatic urethra which result in
an improved urinary flow. The traditional surgical techniques such as
transurethral resection of the prostate are still appropriate for some patients,
although with improved medical treatments now available, the number of
men undergoing surgery is most likely declining [27].
Possible theories of BPH etiology
Despite of BPH’s obvious importance as a major health problem, little is
known in terms of the biological processes that contribute to the
pathogenesis of BPH. However, a number of theories have been proposed
over recent years to explain the etiology of the pathological phase of BPH
and the most typical will be described briefly below. Although they may
show some degree of contradictions, they most likely contribute together to
the pathogenesis of BPH.
One of the theories, the dihydroxy testosterone theory, was originally based
on the failure of BPH to develop in men castrated prior to puberty. Although
controversy still exists, a decreased testosterone/DHT ratio, due to both
decrease in plasma testosterone levels and possibly an increase in DHT
levels, in elderly men with BPH, may be involved in the etiology of BPH
[28, 29]. DHT levels in BPH may be higher than in normal prostate tissue.
The local levels of DHT may be increased by age, testicular endocrine
function declines steadily with age and at 75 years of age, mean plasma
testosterone levels are reported to be around 65% of levels in young males
[30] and the decrease in bio-active (non sex hormone-binding globulin
(SHBG)-bound) testosterone levels is even more pronounced [31]. This is
likely due in part to the recognized increase in SHBG binding capacity
associated with ageing. [32-34]. The DHT theory proposes that there is a
shift in prostatic androgen metabolism that occurs with aging, which leads to
an abnormal accumulation of the more potent DHT in the prostate, thus
producing the enlarged prostate. Although the level of DHT in BPH tissue
might not be elevated compared to normal tissue, it is very likely that the 5alpha-reductase activity and AR levels are greater in BPH tissue than in
controls. It is the binding of DHT to the AR which is important in
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stimulating cell proliferation, and prostatic cells may therefore gradually
become more and more sensitive to androgens with ageing [35]. Moreover,
the reduction in prostate size upon suppression of androgen-mediated action,
either by blocking secretion of circulating testosterone and adrenal androgen,
inhibiting 5 alpha-reductase to prevent DHT formation, or blocking DHT
binding to AR, have further proved the DHT theory [25, 36].
Another theory, the embryonic reawakening theory, was originally based on
BPH histopathological features from which McNeal concluded that the
prostate stroma undergoes an “embryonic reawakening”, resulting in
inductive effects of the local stroma, which in turn induces hyperplastic
changes in the epithelium through stromal-epithelial interactions. Somehow
a shift of stromal-epithelial interactions occurs with aging, which leads to the
inductive effect on prostatic growth.
A further theory is the stem cell theory [37]. The stem cell is a proliferative
cell but the number of these cells within the prostate is unknown but believed
to be very low. The normal behavior of stem cells include: (i) relatively
undifferentiated; (ii) their numbers are preserved; (iii) unlimited proliferating
potential; (iv) easily adapt to the environment; and (v) finally, but maybe the
most important, they are pluripotent, which means that they can give rise to a
number of different cell types. According to this theory, BPH could occur as
a result from changed properties of the stem cells giving rise to a clonal
expansion of cell populations [38].
One more theory, the estrogen-androgen imbalance theory, suggests that an
age-associated imbalance between circulating estrogens and testosterone plays
a role in the pathogenesis of BPH [39]. In humans, the serum testosterone and
free-testosterone levels decrease with age, but the serum estradiol level is
constant throughout life. Therefore, with age, creating an estrogen-dominant
status compared to that at younger ages. These endocrine changes at mid-life
have been extensively investigated through the past 30 years, and are
commonly referred to as the “andropause” [40]. This results in a gradual, but
significant, increase in the ratio of estradiol/testosterone in the serum [41].
Estrogen plays an important role in prostate pathophysiology (for more
information, see section “ACTION OF ESTROGENS IN THE PROSTATE”).
An additional theory, the reduced apoptosis theory, suggests that a reduced
rate of apoptosis is involved in the etiology of BPH [42], based on the
observations of reduced apoptotic activity in BPH tissue compared to control
[43, 44]. A homeostasis appears to exist after the prostate has reached its
adult size, whereby the rates of prostatic cell growth and prostatic apoptosis
are in equilibrium. This ensures that neither involution nor overgrowth takes
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place, so that prostate size is constant. The reduced apoptosis theory suggests
that the increased prostate volume in BPH is a function of a decrease in the
rate of cell death perhaps in parallel with an increase in cell proliferation.
PREMALIGNANT LESIONS OF THE PROSTATE
Prostatic intra-epithelial neoplasia (PIN) is associated with various alterations
in prostatic cellular architecture such as dysplastic foci present in the prostatic
ducts and acini [45]. Other histological or biological changes that have been
reported include: decreased secretory differentiation, nuclear and nucleolar
abnormalities, neovascularity, increased proliferative potential and genetic
instability with variation of DNA content. Based on the morphological
features, PIN can be divided into low and high grades. PIN is most
commonly found in the peripheral zone of the human prostate. Genetic events
in PIN have been linked to the development of prostatic carcinoma.
However, detailed analysis of the genetic alterations in PIN and matched
cancer samples has been limited by the small size of foci of PIN, as well as
by the marked morphologic heterogeneity and multi-focality of both lesions
[46, 47]. Although, it seems like high-grade PIN is a precursor lesion to
prostate carcinoma, the lack of adjacent high-grade PIN in many early
cancers indicates the contradictory.
PROSTATE CARCINOMA
Prostate carcinoma remains one of the most common malignant diseases and
is a leading cause of cancer-related deaths among men in the industrialized
world. However, the vast majority of men harboring pathologic evidence of
prostate cancer are not clinically diagnosed with this disease and it is far
more common to die with prostate cancer than as a direct result of the
disease. The development of new capillary blood vessels (angiogenesis)
might well be one of the first steps in cancer progression. This may be
induced by the abnormal tumor expression of growth factors, such as
vascular endothelial growth factor (VEGF) and basic fibroblast growth factor
(FGF) [48]. Further tumor progression and eventual metastasis may result
from the fact that malignant cells are less adhesive to one another than
normal cells. Cadherins are cell surface glycoproteins that are required for
cell adhesion. Changes in the gene which controls cadherins could well be
involved in progression and metastasis [49]. Extension of the tumor into the
ECM is probably a complex alteration involving mediators between the
malignant cells and the adhesive proteins of the ECM, e.g. integrins and
fibronectin [50].
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Karin Dillner
Possible theories of prostate cancer etiology
The exact cause of prostate cancer is unknown, and part of the problem is the
variability and heterogeneity of the tumor within the prostate gland.
However, the most established risk factors for development of prostate
cancer include ageing, race, diet and a family history of prostate cancer. In
addition, a number of theories for its pathogenesis have been suggested over
recent years and together these theories most likely contribute to
development of the disease.
One theory suggests an imbalance in growth regulation in the prostate. As in
BPH, stromal-epithelial interactions and growth factors may also play a role
in the pathogenesis of malignant disease of the prostate. These important
local regulatory factors are involved in a balance which controls, not only
cell growth, but also apoptosis. Inappropriate regulation of growth factors,
which are produced not only by the target cells themselves, but also by
neighboring cells, could develop a significant imbalance which, if prolonged,
would be an important step in the genesis of the cascade of events which
ultimately leads to prostate cancer.
Another possible theory proposes that the stroma undergoes an activation
process, resulting in a formation of a so-called reactive stroma. There are
considerable evidence that neoplastic stroma is different from the stroma of
normal tissue. In an effort to maintain tissue homeostasis, the stromal
compartment reacts to tumorigenic epithelium in a process similar to the
generation of granulation tissue in wound repair stroma [51]. This activation
of the stroma, resulting in a so-called “reactive stroma” and includes
phenotypical changes of the stroma cells to a more myofibroblast-like
phenotype (transient form between fibroblasts and smooth-muscle cells). The
formation of reactive stroma is known to occur in many human cancers,
including prostate, and is likely to promote tumorigenesis [52]. Furthermore,
it is characterized by ECM remodeling, elevated protease activity, increased
angiogenesis and an influx of inflammatory cells.
An additional theory involves the possibility of genetic instability in the
growing tumor. This genetic instability refers to accumulation of several
genetic defects that can occur either at the nucleotide level (e.g., insertion,
deletion, or base substitution) or at the chromosomal level (such as, loss or
gain of an entire chromosome or small portions) [53]. The genetic instability
may result in the stimulation of proto-oncogene and/or inactivation of tumor
suppressor genes. Carcinogenesis may develop when the genetic restraint and
control in the growth of the cell is lost. Abnormal intracellular behavior can be
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induced by oncogene activation or by a change in activity or character of the
tumor suppressor genes. Proto-oncogenes are normal cellular genes involved
in the regulation of growth and cellular differentiation, for example c-ras and
c-myc. The simultaneous activation of these oncogenes could override the
inhibitory restraints of neighboring cells and allow tumor proliferation. In
parallel with oncogenes, normal cell also contain genes which protect against
cancer, so-called tumor suppressor genes, for example the p53 and
retinoblastoma (Rb) genes. The normal role of these genes include control of
cell division, cell cycle check points and DNA repair, all to reduce and control
the proliferation activity of the cells. It is known that loss of these genes may
result in cancer, and it seems probable that the prostate tumors that occur in
younger men, which appear to have a familial basis, may also be the result of
specific gene deletions [53]. Furthermore, it is suggested that increased
genetic instability is associated with decreased androgen-responsiveness and
progressive behavior of human prostate tumors. Changes may take place
which allow the development of androgen-insensitive cells and the death of
androgen-sensitive cells. This would provide a further movement away from
the modulating influence of androgens on the growth factors associated with
normal cell regulation. However, it remains unclear whether this genomic
instability is causing the progression of cancer or is the consequence of cancer
[53].
PROLACTIN
Prolactin (PRL) has classically been considered as a pituitary-derived
peptide hormone but over the last decade expression of the PRL gene has
also been demonstrated in several extrapituitary tissues [54]. More than 70
years ago, PRL was found to be a pituitary factor that stimulates mammary
gland development and lactation in rabbits, but since then PRL has been
demonstrated to regulate more than 300 different biological functions,
including reproduction, lactation growth, development, metabolism,
immunomodulation, osmoregulation and behaviour [55].
GENE, STRUCTURE, AND VARIANTS
PRL is a member of the PRL/PL/GH hormone family, to which among
others growth hormone (GH) and placental lactogen (PL) also belong to.
They all share genomical, structural, biological and immunological features
[56, 57]. More recently, this family has been linked to a still more extended
family of proteins, referred to as hematopoietic cytokines [58].
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Karin Dillner
The gene encoding PRL is unique and is found in all vertebrates [55]. The rat
PRL (rPRL) gene is located on chromosome 17, is approximately 10 kb long
and composed of five exons and four introns. The human PRL (hPRL) gene
is also approximately 10 kb, but located on chromosome 6 and contains an
additional exon at the 5'-end [59]. This extra exon is only transcribed in
extrapituitary sites, generating a 134 bp longer transcript differing in the 5´untranslated region, compared to the pituitary transcripts [54]. The mature
form of the protein contains 199 residues (23 kDa) and is folded into an allα-helix protein. Although the tertiary structure has not been determined,
PRL is predicted to adopt the four-helix bundle folding described for the
GHs [55, 60].
Extrapituitary PRL protein is identical to pituitary PRL, but different
promoters drive the expression of PRL in pituitary and extrapituitary sites in
humans [61]. Pituitary PRL is controlled by a proximal promoter, which
requires the Pit-1 transcription factor for trans-activation. In human, the
promoter is divided into a proximal region and a distal enhancer, both of
which are necessary for optimal pituitary-specific expression. The pituitarytype promoter and its regulation by dopamine, estrogens, neuropeptides and
some growth factors have been well characterized [58]. In contrast, the
synthesis of extrapituitary PRL is driven by a superdistal promoter, located
5.8 kb upstream of the pituitary start site. This promoter is silenced in the
pituitary, does not bind Pit-1 and is not affected by dopamine or estrogens
[60]. The superdistal promoter contains binding sites for several transcription
factors but its regulation is poorly understood [62].
The PRL isoform 16K, was discovered more than 20 years ago as the Nterminal 16-kDa fragment resulting from the proteolysis of rat PRL by
acidified mammary extracts [63]. The protease responsible for the cleavage
of rat PRL into 16K PRL was identified as cathepsin D, whose implication in
tumor progression is relevant [64]. 16K PRL was shown to have lost PRLR
binding ability but otherwise to have acquired the ability to specifically bind
another membrane receptor [65] through which it exerts anti-angiogenic
activity [66]. Although this receptor is still not identified, some of its
downstream signaling targets have been elucidated [67-70].
Moreover, a PRL-related hormone called proliferin (also known as mitogenregulated protein (MRP)) [71] has been identified as a growth factorinducible gene in immortalized mouse fibroblasts [72, 73], but in vivo it is
produced primarily by the trophoblast giant cells [74]. Interestingly,
reactivation of the proliferin gene expression has been associated with
increased angiogenesis, as shown in a cell culture model of fibrosarcoma
tumor progression [75].
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CONTROL OF PROLACTIN SYNTHESIS, SECRETION AND REGULATION
In contrast to what is seen with all the other pituitary hormones, the
hypothalamus tonically suppresses PRL secretion from the pituitary. If the
pituitary stalk is cut, PRL secretion increases, while secretion of all the other
pituitary hormones falls dramatically due to loss of hypothalamic releasing
hormones. Dopamine serves as the major inhibiting factor on PRL secretion.
Dopamine is secreted into portal blood by hypothalamic neurons, binds to
receptors on lactotrophs, and inhibits both the synthesis and secretion of PRL.
Agents and drugs that interfere with dopamine secretion or receptor binding
lead to changes in secretion of PRL. In addition to tonic inhibition by
dopamine, PRL secretion is positively regulated by several hormones,
including thyroid-releasing hormone (TRH), oxytocin, gonadotropin-releasing
hormone (GrRH) and vasoactive intestinal polypeptide (VIP) [76, 77].
Moreover, estrogens provide a well-studied positive regulation of PRL
synthesis and secretion [78, 79].
THE PROLACTIN RECEPTOR
The PRL receptor (PRLR) belongs to the class 1 cytokine receptor
superfamily and they all share a homology in their extracellular regions,
characterized by the conserved cysteine residues and the tryptophan-serinex-tryptophan-serine motif [55]. The cytoplasmic domain of the PRLR lacks
any intrinsic enzymatic activity; however, it includes a proline-rich motif
(‘box 1’) that couples to protein kinase signaling molecules which in turn
activate downstream effectors.
A single PRLR gene exists from which several PRLR isoforms derive. The
PRLR isoforms differ in the length and composition and are referred to as
long, intermediate or short PRLR with respect to their size. In human, one
long, one intermediate and two short isoforms have been identified
(reviewed in [55]). In rat, all three isoforms are present, whereas, in mice,
one long and three short isoforms have been identified [80, 81]. Regardless
of post-transcriptional splicing events, the extracellular ligand-binding
domain is identical in all isoforms.
The PRLR binds to at least three types of ligands: PRL, PL, and primate
GHs [57]. Activation of the cell surface receptor involves dimerization of
two PRLR molecules [57], which is mediated by a single molecule of ligand
[82]. The ligand binds in a two-step process in which site 1 on the PRL
ligand molecule binds to one receptor molecule, after which a second
receptor molecule binds to site 2 on the hormone, forming a homodimer
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consisting of one molecule of PRL and two receptor molecules. [57]. Once
bound to one of its ligand, PRLR triggers intracellular signaling cascades.
Like all cytokine receptors, PRLR lacks intrinsic enzymatic activity and
therefore transduces its signal inside the cell via a wide number of associated
kinases.
PRLR is virtually expressed in all tissues [55]. However, because of the
extremely broad distribution of PRLR, it is currently difficult to propose a
general overview of its regulation of expression [55].
PROLACTIN SIGNAL TRANSDUCTION
The main and best-known cascades involve the Jak/Stat pathway, the RasRaf-MAPK pathway, and the Src tyrosine kinases (e.g. Fyn), but other
transducing proteins are also involved [55, 83]. Site-directed mutational
studies have identified specific tyrosine residues within the PRLR
cytoplasmic domain that can be phosphorylated and participate in recruiting
Stats, insulin receptor substrates (IRS), and adaptor proteins to the receptor
complex [55]. Depending on the presence or absence of these features, the
various PRLR isoforms are expected to exhibit different signaling properties.
For example, the short PRLR is not tyrosine-phosphorylated, which prevents
this isoform from interacting directly with SH2-containing proteins, such as
Stat factors. However, these interactions may also be mediated by certain
adaptor proteins [84]. The PRLR signaling pathways can be negatively
regulated by protein tyrosine phosphatases, although their mechanism of
action is still poorly understood [84, 85]. Recently, the SOCS (suppressor of
cytokine signaling) gene family was identified and they function by
negatively regulating the Jak/Stat pathway at the level of activation [86].
Finally, another emerging field in PRLR signaling is the occurrence of cross
talk with members of other receptor families, such as tyrosine kinases [87,
88] or nuclear receptors [89].
ACTION OF PROLACTIN IN THE PROSTATE GLAND
PRL-mediated effects in the prostate are well described and supported by
both in vivo and in vitro studies in rodent and human tissues. The presence of
PRLR in both human and rodent prostate are well known [90-93]. Moreover,
the PRL ligand has been demonstrated to be locally expressed both in human
and rat prostate epithelium [93, 94]. The expression of PRL ligand in the rat
DP and LP was found to be androgen dependent in vivo as well as in organ
cultures [94]. These results could indicate a role for PRL as an
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autocrine/paracrine growth factor, regulated by androgen, as well as
mediating androgenic downstream effects in the rat prostate. Most of the
described PRL prostatic effects have been studied in intact animals.
However, several reports indicate that PRL exert many androgenindependent effects [95, 96].
PROLIFERATION
In human BPH organ cultures, human primary prostate epithelium and in the
androgen refractory human prostate cancer cell lines PC-3 and DU145, PRL
has been shown to stimulate growth and significantly increase the cell
proliferation rate [93, 97-99]. In one of these studies, DHT, estrogen and
progesterone were assessed in parallel with PRL, but they all were found to
exert weaker proliferating effects than PRL [99]. Moreover, PRL has been
found to up-regulate ornithine decarboxylase (ODC) in the LP of rat. ODC is
a rate-limiting enzyme in polyamine biosynthesis, and polyamines have been
classified as growth mediators due to their effects on DNA- and RNAsynthesis in somatic cells [100, 101].
Several in vivo studies in rodents, have demonstrated the growth-promoting
effects of PRL on the prostate [102-104]. To add to these studies are our own
group’s generated PRL-transgenic mice, which develop a dramatic prostate
enlargement [105, 106] (see the section RODENT MODELS OF PROSTATE
DISEASE).
APOPTOSIS
The concept of PRL regulation of target tissue size by controlling not only
proliferative activity, but also apoptosis, is relatively new. PRL has been
shown to significantly inhibit apoptosis in vitro in androgen deprived DP and
LP prostate cultures, as assessed by nuclear morphology and in situ DNA
fragmentation analysis [107]. This indicates a possible physiological role for
PRL as a survival factor for prostate epithelium. In earlier in vivo work, a
significant delay of castration-induced regression of the rat LP was noted in
pituitary graft bearing animals [95, 108, 109]. In addition, these studies also
indicated that AR did not mediate PRL actions on the prostate gland, as
evidenced by the failure of flutamide, an AR antagonist, to inhibit the delay
in prostatic regression. These results also reveal a lobe-specific response to
PRL in the androgen-deprived prostate. Taken together, these observations
suggest that in addition to known trophic actions in target tissues, PRL may
regulate cell number by prolonging survival through anti-apoptotic
mechanisms.
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CITRATE PRODUCTION
The major function of the prostate gland is to accumulate and secrete
extraordinarily high levels of citrate. In addition to citrate, the normal and
BPH prostate also accumulate the highest levels of zinc in the body. In
prostate cancer the capability for citrate production has been found to be lost
and the ability for high zinc accumulation diminished [110, 111].
In several different species and model systems, PRL has been shown to
androgen-independently stimulate citrate production, by direct regulation on
enzymes involved in the citrate production, including mitochondrial
aspartate aminotransferase, m-AAT, [112, 113], pyruvate dehydrogenase,
PDH E1α [114, 115], m-aconitase [116] and aspartate transporter [14]. In
addition, studies have revealed that the accumulation of zinc in the prostate
also is regulated by PRL, independently of androgens. PRL increases both
cellular and mitochondrial zinc levels of citrate-producing prostate cells
[117]. Moreover, the regulation of the ZIP-type plasma membrane zinc
uptake transporter has been reported to be regulated by PRL [118]. It is
suggested that this ZIP-type zinc transporter is responsible for the ability to
accumulate and transport high amounts of zinc in prostate cells.
PROLACTIN IN PROSTATE PATHOPHYSIOLOGY
Although PRL is well known to exert trophic effects on prostate cells, its
role in the development and regulation of the age-dependent disorders, BPH
and prostate cancer, is still poorly characterized. In order study the
participation of PRL in the regulation of proliferative prostatic disorders
several different experimental animal models have been used.
PROLACTIN IN HUMAN PROSTATE CANCER AND BPH
The role of PRL in human prostate biology and pathophysiology is not well
known. The altered endocrine status of aging men is likely to be of importance
for development of prostate pathophysiology. Testosterone and GH levels
decrease while estrogen levels increase with age. Conflicting data exists
whether the circulating PRL levels increase or not with increasing age in the
human male [32, 119-122]. Moreover, in a subset of aged men, an increase of
TRH-stimulated PRL secretion together with an increase in circulating PRL
level have been demonstrated [123, 124].
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There is no clear correlation between serum PRL levels and risk of BPH or
prostate cancer. More than 20 years ago, a beneficial effect of
hypophysectomy in combination with castration compared to castration alone
was observed in patients with disseminated prostate cancer [125, 126]. This
indicated a role for one or more pituitary hormones, such as PRL, in advanced
prostate cancer. Furthermore, significantly higher PRL serum levels have been
reported in patients with prostate cancer [127, 128] and patients with BPH
[129]. However, other studies report no differences in serum PRL levels
between prostatic carcinoma patients and age-matched controls [121, 130].
Moreover, there is evidence that elevated PRL serum levels correlate with
poorer prognosis in patients with advanced prostate [131, 132]. Although
there are conflicting data, some clinical trials of advanced prostate cancer
treatment have indicated a significant improvement in clinical response when
combining conventional treatment with PRL suppression [128, 133-136].
There are clinical studies that have indicated increased prostatic tissue levels
of PRL in patients with BPH [137] and prostate cancer [138]. Interestingly,
PRL serum levels have been reported to transiently decrease following
prostatectomy or transurethral resection of the prostate, TURP, [129, 139,
140], indicating loss of local PRL production or a prostatic influence on
pituitary PRL secretion. Similar results have been presented in rodents [141].
Using immunohistochemistry, Nevalainen et al. reported local production of
PRL in human prostate tissue [93]. Moreover, this study showed the
presence of PRLR in the human prostate. The staining of the receptor was
localized mainly to the secretory epithelium, but faint staining was also
noted in the prostatic stroma. Collectively, these data provide significant
support for the existence of an autocrine/paracrine loop of PRL in the human
prostate. Furthermore, using in situ hybridization and immunohistochemistry
Leav et al [91] demonstrated an increased PRLR expression levels in
dysplastic lesions, whereas in lower grade carcinomas the receptor expression
levels approximated those found in normal prostatic epithelium. Results from
this study suggest that PRL plays a role in the development and maintenance
of the human prostate and may participate in early neoplastic transformation
of the gland.
EXPERIMENTAL ANIMAL DATA
Enhanced growth of rodent prostate lobes after pituitary grafting under the
renal capsule [102], or local grafting to a specific lobe [103, 104] has been
reported. In rat, anterior pituitary grafting to the LP results in significant
growth specifically in the LP compared to controls [103]. These results
indicated a local direct effect of PRL on the LP. In mice, implantation of a
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single anterior pituitary into the VP of intact mice results in a significant
increase in VP weight and the area occupied by the glands of VP associated
with the elevation of circulating PRL. Furthermore, hyperplastic lesions were
noted in the grafted prostate lobes of these animals [104].
In another work, hyperprolactinemia has been reported to induce prostatic
dysplasia in vivo. Noble rats, treated with testosterone and estradiol-17β2 for
a prolonged time period, develop DLP dysplasia, a pre-neoplastic lesion. In
these rats, the dysplasia was mediated via estradiol-induced
hyperprolactinemia, as evidenced by effective inhibition of dysplastic
evolution through the co-treatment of bromocriptin (a dopamine antagonist)
[142].
Furthermore, animals which are exposed to a transient increase in PRL
secretion prior to puberty have been shown to develop LP inflammation
(prostatitis) as adults [143]. A recent study reports that early lactational
exposure to atrazine, a toxic agent that suppresses suckling-induced PRL
release, leads to altered PRL regulation and subsequent prostatitis in the
male offspring. The mechanistic explanation is that without early lactational
exposure to PRL (postnatal day 1-9), tuberoinfundibular neuronal growth is
impaired and as a consequence prepubertal PRL levels become elevated.
This results in higher incidence and severity of LP inflammation in the
offspring, evident at 120 days of age [144].
In addition to the abovementioned short PRL-treatment studies, also
prolonged treatment of PRL has been shown to induce dramatic enlargement
of the prostate as shown in our PRL transgenic mice which ubiquitously
express the rat PRL transgene (Mt-PRL) [105] (see the following section).
RODENT MODELS OF PROSTATE DISEASE
Because the rodent prostate does not spontaneously develop prostate
carcinoma and benign hypertrophy or hyperplasia, the usefulness of studying
the mouse prostate as a model of human disease is frequently addressed.
However, the known heterogeneity of pathological prostate changes in the
human prostate gland and the multifaceted nature of prostate disease have
prompted the development of less complex, complementary model systems
to study the etiology of prostate disease. Both prostate cancer and benign
hypertrophy or hyperplasia can be induced in the rodent prostate through
genetic modulation or chemical induction and several such models have been
established. The advent of transgenic techniques in mice have put increasing
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focus on the mouse as a model organism for in vivo studies aiming at
understanding gene function and by this gain insights into human
pathophysiological conditions. Moreover, the mouse genome project will
soon be completed which will enable a direct comparison between the mouse
and human genes.
TRANSGENIC PROSTATE HYPERPLASIA MODELS
Male mice overexpressing the rat PRL gene, Mt-PRL transgenic mice,
develop a dramatic enlargement of the prostate gland, which shows
similarities prostatic hyperplasia in humans. These animals were generated
using a construct consisting of the rat PRL gene under the control of the
ubiquitous metallothionein (Mt) promoter, which gives the transgene a general
transcription in virtually all cell types. Expression of transgene was detected in
all parts of the prostate (DP, VP, LP, AP). The prostate enlargement is mainly
characterized by an expansion of the stromal compartment and areas of
glandular hyperplasia with accumulation of secretory material [105]. Although
dysplastic epithelial features were detected in individual prostates from older
PRL-transgenic animals, no development of prostate carcinoma has been
observed. The PRL-transgenic animals display, in addition to high serum
levels of PRL, approximately a three-fold increase in serum androgen levels
compared to wildtype littermates. The degree of prostate enlargement showed
no correlation to circulating levels of PRL or testosterone.
RODENT MODELS OF PROSTATE CANCER
There are several rodent models for human prostate cancer. One of the most
well known is the Dunning-3327 rat prostatic adenocarcinoma model [145].
There are several recently established transgenic mouse models for use in
prostate cancer studies [146]. The purpose of utilizing these animal models is
to identify specific molecular changes in early malignant disease. As the
mouse does not spontaneously develop prostate malignancy, different
transgenic strategies for in vivo tumor induction have been developed
including the use of the the SV40 early genes, such as the tumorigenic T
antigen (Tag). Transgenes are usually under the control of a prostate-specific
promoter region such as probasin or C3, directing expression to prostate
epithelial cells.
The transgenic models of prostate cancer can be divided into two main types.
The first consists of models resulting from enforced expression of SV40
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early genes. Two frequently used models are the TRAMP (transgenic mouse
model for prostate carcinoma) model and the C3(1)-Tag transgenic model,
which utilizes the minimal rat probasin promoter to drive the expression of
the Tag gene. In addition, a number of transgenic lines use the long probasin
promoter to express large SV40 early genes. These models are well
characterized and widely distributed, displaying progressive disease ranging
from epithelial hyperplasia or PIN to adenocarcinoma and development of
metastases [147].
The second type of transgenic mice utilizes the promoters mentioned above
to express various “natural” molecules that have previously been suggested
to play a role in development of prostate cancer. The list is extensive but
includes c-myc, Bcl-2 and dominant negative transforming growth factor
beta (TGFß). Interestingly, the majority of these models only display a
relatively mild phenotype, primarily epithelial hyperplasia or PIN. Moreover,
these phenotypes usually not arise until the mice are of advanced age.
OTHER GENETICALLY ENGINEERED MOUSE MODELS WITH PROSTATE
PHENOTYPE
Mouse models genetically engineered in the prolactin signaling pathway
Null mutated mice have been generated both for the PRL ligand, PRL-/-[148],
and the PRLR-/- [149]. PRL-/- males are reported fertile [148], whereas
studies of male PRLR-/- mice have demonstrated both a subset of completely
infertile males and a general latency to first successful mating [150].
Moreover, the studies of the prostate gland in PRLR-/- males did reveal only
subtle histological alterations and the PRL-/- prostate has not been very well
characterized. Taken together, the data from these two knockout mouse
models indicate that PRL action is not of essential importance for male
fertility and normal anatomical development of the prostate gland. However,
studies of more functional aspects of the gland need to be carried out in these
animals.
PRL can activate several of the Stat proteins, including Stat 1, 3, 5a, and 5b,
but the two latter acts as the major mediator [55]. Stat5a-/- and Stat5b-/knockout mice have confirmed these molecules as the major transducers of
PRL signaling in both prostate and mammary gland [151], and also shown
similar phenotype to those of the PRL-/- and PRLR-/- knockout mouse
models, mainly emphasizing the irreplaceable role of PRL in reproduction
and mammary gland development. PRL signaling in rat prostate tissue is
primarily transduced via Stat5a and Stat5b, likely supporting the viability of
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prostate epithelial cells during long-term androgen deprivation [152]. In the
prostate, studies in Stat5a-/- knockout mice have provided evidence for a
direct role of Stat5a in the maintenance of normal tissue architecture and
function of the mouse prostate [153]. Lack of Stat5a function results in a
distinct prostatic phenotype characterized by an increased occurrence of cyst
formation with disorganization and detachment of prostate epithelial cells. In
addition to PRL, other polypeptide factors, such as GH, insulin-like growth
factor I (IGF-I), epidermal growth factor (EGF) and interleukin-6 (IL-6) are
known to activate Stat5.
Mouse models genetically engineered in other hormones
The AR transgenic mice overexpress the AR specifically in prostate
secretory epithelium [154]. The earliest alteration observed in the AR
transgenic mouse prostates was an extensive 5-fold increase in the
proliferation of secretory epithelial cells, as evidenced by immunostaining of
the proliferating marker Ki-67, in the absence of histological abnormalities.
Proliferation in these glands was associated with increased apoptosis,
possibly accounting for the absence of hyperplasia. Older AR transgenic
mice developed focal areas of intraepithelial neoplasia, resembling human
high-grade PIN, but no further malignancy has been observed. A certain
resistance to malignant transformation in the mouse prostate compared to
humans has been suggested. No reports of any tumorigenic effects of
exogenously added androgens in these models are available.
The recent generation and characterization of the various estrogen modulated
mouse models (αERKO, βERKO, αβERKO and ArKO) have provided new
insights regarding the role of estrogens in prostate growth and development
[155]. A specific direct response to estrogens is the induction of changes in
the prostatic epithelium, termed squamous metaplasia [156-159]. Tissue
recombinant studies using epithelium and stroma from wildtype and
transgenic mice lacking a functional ERα (αERKO) or ERβ (βERKO) have
demonstrated that the development of squamous metaplasia is mediated
through stromal ERα [160, 161]. Furthermore, a distinct phenotype of focal
epithelial hyperplasia in the VP has been reported in aging mice lacking
functional ERβ (βERKO) [162, 163], while no apparent prostate pathology
or enlargement has yet been reported in αERKO or the double knockout
αβERKO [155]. Altogether, these findings indicate an anti-proliferative role
for epithelial ERβ and also suggest that an unbalanced stromal ERα in action
could contribute to the phenotype observed.
The ArKO (aromatase knockout) mouse lacks endogenous estrogen
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Karin Dillner
production due to a non-functional aromatase enzyme. In the ArKO mouse,
the combined effects of estrogen absence and elevated androgen and PRL
levels result in a moderate prostate enlargement with hyperplasia evident in
all lobes and tissue compartments [161]. Moreover, an associated upregulation of epithelial AR was demonstrated in the ArKO mouse and has
been suggested to contribute to the observed phenotype. In the absence of
endogenous estrogen (ArKO) or ERs (αERKO and βERKO), prostate
development occurs normally, suggesting that intact estrogen signaling is not
essential for the initiation of neonatal prostate growth. The histological
appearance of the prostate hyperplasia in ArKO male mice is strikingly
similar to that of the Mt-PRL-transgenic mice.
In contrast, the AROM+ mice, which overexpress the aromatase gene,
resulting in elevated estrogens levels, combined with significantly reduced
testosterone and FSH levels, and elevated levels of PRL and corticosterone
[164]. AROM+ males present a multitude of severe structural and functional
alterations in the reproductive organs. Furthermore, squamous metaplasia
has been seen in the prostatic collecting ducts, consistent with high levels of
endogenous estrogens. Some of the abnormalities, such as non-descended
testes and undeveloped prostate, resemble those observed in animals exposed
perinatally to high levels of exogenous estrogen, indicating that the elevated
aromatase activity results in excessive estrogen exposure during early phases
of development.
HORMONE/GROWTH FACTOR REGULATION OF THE
PROSTATE
All lobes are responsive to both estrogens and to androgens, but to varying
degrees; the VP is more sensitive to androgens and the AP more sensitive to
estrogens [159, 165]. In rat prostate, both testosterone and estrogen have been
shown to regulate the level of the long PRL receptor mRNAs in a tissuespecific manner [92]. In addition to steroid hormones, several different growth
factors and other pituitary hormones have been shown to regulate cellular
growth, differentiation and apoptosis.
ACTION OF ANDROGENS IN THE PROSTATE
Androgen is a critical factor for the survival of prostatic epithelial cells.
Underdeveloped prostate gland is seen in eunuchs who lack androgen
stimulation since childhood [166]. Castration-induced androgen-withdrawal
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regress the the number of epithelial cells in the prostate gland via an active
process of apoptosis [167, 168]. Apoptosis can be observed within one day
after castration and nearly 2/3 of epithelial cells are lost in the VP by seven
days of castration [169]. In contrast, testosterone replacement to castrated rats
stimulates the re-growth of the gland to its normal size via proliferation of
new epithelial cells from basal cells [170].
INTERACTIONS BETWEEN PROLACTIN AND ANDROGENS IN THE
PROSTATE GLAND
PRL has been shown to potentiate the action of androgens in the support and
stimulation of prostatic growth and metabolism [171-173]. This has been
hypothezised to be accomplished through increasing prostate receptivity to
androgens, mainly by affecting AR levels and 5-alpha reductase activity.
Results suggest that PRL is involved in regulating AR synthesis, at least
partially by direct action on the prostate gland. In immature,
hypophysectomized male rats, PRL treatment can significantly increase AR
mRNA levels [174]. Findings in adult, castrated and pituitary grafted rats
suggest that PRL promotes LP growth via an increase in nuclear AR levels,
and thus optimizes tissue response to circulating testosterone [175].
Furthermore, pituitary grafting in immature rats can produce a significant
increase in the weight of the seminal vesicles and the VP and AP [176]. In
the VP, nuclear AR content increased, whereas the cytosolic AR content
decreased, suggesting increased translocation of the AR to the nucleus. In a
study on human BPH patients, cytosolic and nuclear levels of AR were
shown to be proportional to plasma PRL levels [177]. These findings
indicate plasma PRL involvement in the regulation of AR content also in the
benign human prostate.
Recently the existence of crosstalk between the signal transduction systems
of steroid hormones and peptide hormones/growth factors were recognized
[178-180] which provides a mechanism for locally produced growth factor
influence on AR activation. In the progression of prostate cancer to an
androgen-independent state, local growth factors, such as PRL, may prove
instrumental in regulation of cell growth.
In rat, hyperprolactinemia by pituitary grafting can lead to increased 5-alpha
reductase activity in the testis [181] but indications of a PRL-induced increase
in 5-alpha reductase activity in the prostate is limited [182]. PRL mediation of
steroid uptake through alterations of the plasma membrane permeability in
human BPH tissue has also been reported [183].
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To interpret findings in rodent versus human studies, one needs to be aware of
the important differences in influence of PRL on circulating androgen levels.
In man, PRL is known to decrease circulating androgen levels through
depression of gonadotrophine release from the pituitary gland [184], whereas
in rodents, PRL can elevate circulating androgen levels by increasing the
response to luteinizing hormone in the testis [185].
ACTION OF ESTROGENS IN THE PROSTATE
A hierarchy of estrogen responsiveness in the three prostatic lobes has been
revealed in male mice, with the AP being the most responsive, the
dorsolateral lobe less responsive, and the ventral lobe the least responsive.
[159].
The expression of both known estrogen receptor subtypes in adult human
and rodent prostate is now well established, with expression of ERα
described primarily in a subset of stromal cells and ERβ restricted to the
ductal epithelium [186-188]. Although the newly discovered ERβ shares
many of the functional characteristics of ERα, the molecular mechanisms
regulating the transcriptional activity of ERβ may be distinct from those of
ERα. For example, the growth effects of estrogens during fetal development
are mediated primarily by ERβ in the human prostate, which can be
immunodetected in the nuclei of nearly 100% of epithelial and in the
majority of stromal cells throughout gestation. However, ERα has been
shown to contributes to postnatal glandular development [156].
Estrogen plays an important role both in prostate physiology and
pathophysiology. The developing prostate is particularly sensitive to
estrogenic exposure. During prostate morphogenesis, elevated levels of
endogenous (maternal or excess local production) or exogenous
(diethylstilbestrol or environmental chemicals) estrogens induce permanent
changes in prostate growth in rodents. Fetal and neonatal exposure to
estrogens results in pathological and functional changes of the prostate [189].
High-dose of testosterone together with estradiol stimulates prostatic
carcinogenesis in adult male rats [190]. In mice, these effects are doserelated as low-dose estrogen exposure may increase the adult prostate size
whereas high-dose exposure reduces prostate size [189]. An increase in AR
levels has been associated with low-dose estrogen-induced increases in
prostate size [190]. Neonatal exposure of rodents to high doses of estrogen is
known to permanently imprint the growth and function of the prostate and
predispose the gland to hyperplasia and severe dysplasia analogous to PIN
with aging [160]. Following neonatal exposure of rats to high doses of
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estrogen on days 1-5 of life, a permanent reduction in prostate growth and
responsiveness to androgen occurs relative to a reduction in AR expression
in adult animals [165]. Moreover, exogenous estrogen administration in adult
rodents leads to squamous metaplasia of the AP [157, 159]. As mentioned
earlier, development of squamous metaplasia has been shown to be mediated
through stromal ERα [160, 161].
INTERACTIONS BETWEEN PROLACTIN AND ESTROGENS IN THE
PROSTATE GLAND
Estrogen are known to act directly on pituitary lactotrophs and indirectly on
the hypothalamic dopaminergic system and several studies suggest that
neonatal estrogen treatment can induce long-term alterations in pituitary
synthesis and release of PRL [191-193]. Moreover, estrogens are wellknown to promote PRL release resulting in elevated PRL levels systemically
[78, 79]. It is thus quite possible that the prostate effects of estrogen
imprinting are in fact partially PRL-mediated. Furthermore, PRL is able to
stimulate expression of both ERα and ERβ in corpus luteum and decidua
during pregnancy [194-196] as well as stimulate estradiol binding activity or
mRNA levels in the mammary gland [197] and liver [198]. In the prostate,
effects of estrogen treatment appear to be in part mediated by increased PRL
levels [199], something that is further demonstrated in the aforementioned
dysplastic prostate model of estrogen-treated Noble rats [142].
ACTION OF OTHER PEPTIDE HORMONES AND GROWTH FACTORS IN
THE PROSTATE
Growth factors regulate cellular growth, differentiation and apoptosis. In
addition to steroid hormones, an array of positive and negative growth
factors controls the balance between cell proliferation and apoptosis in the
prostate. Several oncogene products that contribute to neoplastic
proliferation have been found to be homologues to growth factors, growth
factor receptors, or molecules in the signal-transducing pathways of these
receptors. There are numerous growth factor families that have been
implicated in normal, neoplastic and malignant prostate growth and it is far
beyond this thesis to review the action of all reported hormones and growth
factors. The in the literature mentioned growth factors include, the IGF
family, EGF, TGF, FGF family, platelet-derived growth factor (PDGF) and
VEGF, which all are the main stimulatory regulators of proliferation in the
prostate [200]. Furthermore, the pituitary hormones, GH and luteinizing
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Karin Dillner
hormone (LH), play physiologically significant roles in the normal prostate,
either alone or synergistically with androgens [201]. Nevertheless, the
involvement of these hormones in the development of BPH and prostatic
carcinoma is an issue that needs to be addressed.
The TGF-family is the main inhibitory regulator of proliferation acting on
the epithelial cells. However, recent studies have demonstrated proliferative
and anti-apoptotic effects of TGFβ in stromal cells [202].
Altogether, the growth factors exert autocrine and paracrine effects upon
stromal and epithelial cells and interact with other factors and binding
proteins to control prostate growth [203].
FUNCTIONAL GENOMICS IN THE STUDY OF THE
PROSTATE GLAND
The network of action of different hormones and growth factors on the
prostate gland and their involvement in prostate pathophysiology are
unquestionable complex. The recent completion of the human [204], and the
draft of the mouse [205], genome sequence together with the improvement
of high-throughput technologies, such as gene expression profiling, will
hopefully provide a basis for rational determination of which pathways and
molecular targets that are appropriate to further study. The unveiling of a
detailed genetic map of the main species and models of prostate research
promise to dramatically increase our understanding in the genetic basis of
prostate disorders together with the basic mechanism of the action and
involvement of hormones and growth factors for the induction of prostate
disease.
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AIMS OF THE THESIS
The overall aim of this thesis was to study the consequences of chronic
exposure to extraordinary high levels of PRL in the development of prostate
hyperplasia as well as to characterize the molecular mechanisms present in the
hyperplastic prostates.
The specific aims were:
• To investigate the role of circulating androgen in the promotion of
prostate hyperplasia in PRL transgenic mice with transgene onset
during early prostate development (Paper I)
• To characterize a new PRL-transgenic model of prostate hyperplasia in
which the PRL transgene was overexpressed specifically in the prostate
with onset at puberty (Paper III)
• To compare the ductal development in two models of prostate
hyperplasia; one with fetal onset and the other with pubertal onset of the
PRL transgene expression (Paper III)
• To evaluate the use of differential gene expression analysis in
characterization of the molecular mechanisms of the prostate
hyperplasia in PRL transgenic mice (Paper II and IV)
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METHODOLOGICAL CONSIDERATIONS
TRANSGENIC ANIMALS
The establishment of the transgenic technology has introduced new and
invaluable techniques to study and understand the function of a specific gene
in biological processes. There are basically two types of transgenic animals
based on the technique used to generate them. The first method to be
established in 1980 was the microinjection technique allowing overexpressing
of a gene product by injection of foreign DNA into a one cell mouse zygote
[206]. The incorporation of the foreign DNA is completely random using this
approach and it is only possible to overexpress a gene product and not to
mutate a certain gene. In contrast, the other embryonic stem cell (ES)-cell
technique (also known as gene knockout) was established in 1987 and this
method made it possible to interact with the mouse genome at a specific
position and to mutate a specific gene [207]. A wide range of transgenic and
knockout mouse models have now been established and further technical
improvements have made both temporal and spatial overexpression/gene
deletion possible. These accomplishments have given unique insights into the
specific biological properties and functions of specific genes and furthermore
provided valuable models for investigating the functional in vivo role of target
genes.
In this thesis we utilized two different transgenic mice models. The rat PRL
transgene where used in both constructs, in parallel with two different
promoters to direct spatial (where) and temporal (when) expression of the
transgene, resulting in two different PRL transgenic mouse models. In paper
I and II, the metallothionein (Mt) promoter was used to drive the PRL
transgene. The Mt gene is expressed in virtually all cell types. Activation of
the Mt-1 promoter during the early embryonic stage is well described, with
abundant expression already by day 12 of gestation reported [208, 209].
Thus, the PRL expression was considered general in the Mt-PRL transgenic
mice. In contrast to the general expression of a transgene, a cell-specific
promoter can be used that direct the expression of the transgene to a certain
cell type. In paper III and IV, the probasin (Pb)-PRL transgenic mice were
utilized. The construct of these mice include the minimal Pb promoter to
direct the expression of the rPRL transgene to the epithelial cells of the DP,
LP, and VP [210]. Pb is an androgen-dependent basic secretory protein,
abundantly localized in the lumen and acinal regions of the rat prostate
epithelium [211]. Studies have demonstrated that the Pb minimal promoter
(458 bp) can target heterologous gene expression specifically to the prostate
- 30 -
in a developmentally and hormonally regulated fashion [210]. In contrast to
the Mt-1 promoter, activation of the Pb promoter is androgen dependent, and
it is thus activated by the increasing androgen levels seen in late prepubertal
stage in the animals [210]. The Pb promoter is consequently not active until
after the most essential period of ductal morphogenesis in the neonate
prostate gland has occurred [4].
The integration of the transgene in the genome is considered a random event
and the number of copies inserted can not be regulated. In the majority of
zygotes injected, the integration will occur at a single position on one of the
chromosomes. As a consequence, the resulting animal will be heterozygous
for the integrated transgene. To rule out the possibility that the transgenic
phenotype is being a consequence of a heterozygous mutation introduced by
the integration of the transgene, it is preferable to generate more than one
line of transgenic animals, allowing comparisons.
Identification of transgenic animals takes place in several steps. The founder
animals are first identified at the DNA level. Lines of transgenic animals are
then generated from founders and expression of the transgene is characterized
at RNA or protein level. Detection of transgenic expression in the desired
tissues denotes the successful establishment of a new transgenic animal. The
founder animals are analyzed at the DNA level by obtaining a tail biopsy at
two weeks of age followed by DNA preparation. Either southern blot
hybridization or PCR, using one primer located in the promoter and the other
in the structure gene of the construct, is used to identify the transgene.
Southern blot verification is more reliable and therefore preferred at least in
the identification of founder animals. Thereafter, PCR screening is accepted
for identification of the transgene in the subsequent transgenic offspring
generated from the founder animals.
In case of cDNA constructs, it is important to allow discrimination between
the mRNA expression generated by the transgene and the contaminating
cDNA construct. This can be done by introducing intron sequences in the
DNA construct. Moreover, it is important to be able to distinguish between
mRNA expression and protein production of the transgene and the
corresponding endogenous gene’s products.
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Karin Dillner
APPROACHES TO GENE EXPRESSION ANALYSIS
During the last ten years, the development of more and more powerful
techniques for differential gene expression studies have provided entirely new
insights into molecular mechanisms underlying biological processes. In this
thesis, we applied two different methods, cDNA RDA and cDNA microarray
analysis, to molecular characterize the prostate hyperplasia of PRL transgenic
mice and those two methods will be discussed in more detail in the following
chapter.
CDNA REPRESENTATIONAL DIFFERENCE ANALYSIS (RDA)
In paper II, we used the method of cDNA-RDA to identify differentially
expressed transcripts in the hyperplastic prostate of the Mt-PRL transgenic
mice compared to wildtype control littermates. RDA has been successfully
adapted to identify genes that are differentially expressed between two
populations of cells [212]. Representative cDNA fragments from each
population are first generated by restriction endonuclease digestion of cDNAs
followed by PCR amplification. The resulting mixtures, termed
‘representations’, are then subject to successive rounds of subtractive crosshybridization followed by differential PCR amplification. This leads to
progressive enrichment of cDNA fragments that are more abundant in one
population than the other. Figure 1 shows a schematic description of the RDA
procedure. The PCR products after each RDA round are termed differential
products (DP). Theoretically, consecutive DP should contain more stringently
selected gene fragments and less noise from non-differentially expressed
genes. To allow isolation of both up- and down-regulated genes, both samples
are used as tester and driver, respectively, in two parallel procedures. In paper
II, we aimed to identify both up- and down-regulated transcripts in the
hyperplastic prostates of Mt-PRL transgenic mice compared to controls, and
we therefore used both samples as tester and driver, in two parallel
procedures.
cDNA-RDA is a powerful technique for isolation of differentially expressed
genes, but it also has limitations in that not all of the differentially expressed
genes are necessarily enriched during the procedure. The lack of four-base
pair restriction sites in the messenger RNA may result in the generation of
<100% coverage of expressed genes in the representations. In contrast, the
restriction fragments may be too big for efficient amplification by PCR. The
PCR amplification step to generate the starting representations in the RDA
procedure is a very critical step for a successful RDA. In order to generate
representations that truly represent the original cDNA pool with respect to
- 32 -
Samples
PRL-transgenic
prostate
Control
prostate
mRNA
cDNA
Restriction
digestion
Linker
ligation
PCR
Tester
Driver
Digest and ligate
new linker
Digest
Representations
Mix, melt and
hybridize
Repeat
procedure
Tester : Tester
Tester : Driver
Driver : Driver
Exponential
amplification
Linear
amplification
No
amplification
PCR
Figure 1. A schematic description of the cDNA-RDA procedure. The first step is to
synthesize cDNA using purified mRNA as template. The double-stranded cDNA is cut with
a 4-basepair restriction enzyme. A linker, complementary to the generated overhangs, is
ligated onto the cDNA fragments. This generates a pool, which is amplified by PCR, using
primers complementary to the linker. This procedure generates a representation and one
such representation is made from each of the two mRNA pools to be compared. The linker
is then removed, using the same restriction enzyme as before. A new adapter is ligated onto
the tester fragments only. The tester is then mixed with driver in excess, the mix is heat
denatured and allowed to hybridize. A PCR amplification using primers complementary to
the new adapters is performed. During this step, only tester: tester hybrids are amplified
exponentially. Tester: driver hybrids are linearly amplified, and can be removed by
nuclease treatment. Driver: driver hybrids are not amplified at all. The procedure is then
repeated with increasing ratios of fresh driver. After a few rounds, distinct bands can be
visualized on an agarose gel. These bands are isolated, and the products are cloned into
vectors and characterized. Reproduced from Hubank et al [212].
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Karin Dillner
fragment distribution while avoiding a size bias, the PCR needs to be
carefully titrated for each sample.
The sensitivity of RDA remains to be defined. To allow detection of a
transcript, the relative differences in expression levels between tester and
driver populations are thought to be the major determinant. The flexibility of
the RDA methodology can be employed to overcome this issue, as variation
in the stringency of hybridization will influence the detection of small
differences in gene expression between tester and driver populations. By
lowering the stringency of subtraction (increase the amount of tester cDNA
relative to the driver cDNA), cDNA-RDA can enrich for genes with subtle
differences in gene expression [212]. However, too much driver cDNA can
cause insufficient enrichment of the targets, rendering differences invisible
whereas too little driver cDNA may cause insufficient exhaustion of common
(but differentially expressed) sequences in the tester cDNA, generating
background. The risk of cloning non-differentially expressed genes is
obviously also higher in the less stringent enrichment case.
Furthermore, the relative expression level of the corresponding gene may
affect the degree of enrichment on the cDNA-RDA. Not all differentially
expressed genes are equally enriched in the process, which favors fragments
with high levels of differential expression, especially if RDA is performed for
3-4 rounds (DP3 and DP4). There is an inverse relation between the degree of
enrichment for differentially expressed genes and the complexity of the output
of RDA. If RDA is performed for 1-2 rounds, a broader spectrum of
differentially expressed genes (including those with lower levels of
differential expression) are obtained together with many non-differentially
expressed genes, necessitating large-scale screening of the output, for example
by using microarray, to remove those transcripts.
Sequence analysis
To follow up the RDA output, sequence analysis of RDA clones were
performed by routine sequence analysis using cycle sequencing with dyelabeled nucleotides followed by running of purified products on an
automated sequencing machine. Subsequently, the Staden sequence analysis
package [213] was used for vector clipping, redundancy, and assembly
analysis. Sequences were annotated and given an accession number by
analyzing for homologies with published sequences in the non-redundant and
expressed sequence tags (EST) divisions of the public databases of NCBI
(National center for Biotechnology Information) by using the BLAST (N/X)
software [214]. More than 85% homology over at least 50 base pairs region
was required to annotating sequences based on homology to known genes or
- 34 -
ESTs. Clones that failed to match any existing database entry in BLAST
(N/X) search were denoted unknowns. Functional prediction was performed
in silico by using the information at UniGene, The Institute for Genomic
Research (TIGR)-EGAD, Online Mendelian Inheritance in Man (OMIM),
and Medline databases.
CDNA MICROARRAY ANALYSIS
The DNA microarray, also called chip, has become an important tool in gene
expression studies, monitoring RNA expression levels, but can also be utilized
to study mutations and polymorphisms at the DNA level. In this thesis, cDNA
micorarray technology was used to characterize the molecular mechanisms of
importance for the prostate hyperplasia in PRL-transgenic mice compared to
controls. However, we made use of this method in different ways in the two
studies. In paper II, we applied the cDNA microarray technology to verify the
cloned RDA products, isolated as differentially expressed between the MtPRL transgenic prostates compared to controls. In contrast, in paper IV, we
used the cDNA microarray technology to screen for differentially expressed
transcript in the Pb-PRL transgenic model of prostate hyperplasia.
Basically, there are two main types of microarrays. The first type is the one
composed of oligonucleotides which are synthesized in situ by
photolithography [215]. These chips are also available commercially and
form the basis of GeneChip™ technology sold by Affymetrix. The other type
of microarray, cDNA microarray, was originally developed by Brown and
colleagues at Stanford University [216]. This form of microarrays usually
comprises PCR-amplified inserts from cDNA clones representing known
genes and ESTs [217]. cDNA microarrays are generally used for
comparative analysis where the two samples to be compared are hybridized
onto a single chip. In contrast when using Affymetrix arrays, each sample is
hybridized on separate arrays. Although, this results in the use of increased
numbers of chips, it also provides the advantage that post hoc comparisons
not planned in the original experiment can be more easily made. Another
advantage of using short oligonucleotide probes on an array is the built-in
ability to distinguish close members of a gene family. However, the current
Affymetrix oligonucleotide expression platform is still significantly more
expensive than cDNA arrays and lack flexibility when it comes to producing
custom-designed arrays. Newer platforms using 50-70-mer spotted
oligonucleotides allow for rapid array design and implementation.
Experience with this technology is still limited, but it may offer the best
alternative.
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Karin Dillner
The microarray technology is advancing at an impressive rate and this thesis
will only describe the most important methodological characteristics. All steps
mentioned need to be carefully optimized for the successful application of
cDNA microarray analysis. Figure 2 shows a schematic description of the
DNA microarray procedure.
Array design and Printing
Customized cDNA microarrays are fabricated by first selecting the genes to
be printed/immobilized onto the array from public databases/repositories or
institutional sources. Control clones can help to validate the microarrayderived data. Selected cDNA clones may be spotted twice at different
locations on the chip to serve as “within slide” reproducibility controls. A set
of negative controls including repetitive DNA, polyA sequences, genomic
DNA and non-cross-reactive gene sequences from different organisms may
be utilized to ensure specific hybridization. In addition so-called spiking
controls (positive controls) may be used by adding RNA that will hybridize
specifically to spots included on the array. High throughput DNA
preparation is performed in either 96- or 384-well format by PCR
amplification of the selected clones/gene sequences. Subsequently, the DNA
is purified by ethanol precipitation and resuspended in an appropriate
“spotting” solution. Moreover, the purity of each gene is checked on an
Figure 2. Schematic of microarray experiments. From Duggan DJ et al. [218].
- 36 -
agarose gel. Spotting is carried out by a robot, which deposits a nanoliter of
PCR product onto an aminosilane-coated glass slide in serial order to
produce circular spots of about 90-200 µm in diameter. Spotted DNA is
cross linked to the matrix by ultraviolet irradiation and denatured by
exposure to heat.
Target preparation
The total RNA or mRNA samples, that are to be compared, are extracted from
two tissues or cell groups and labeled with different fluorescent dyes in a
reverse transcription reaction generating fluorescent dye-incorporated cDNA.
Most often, the cyanine-3, Cy3 (green), and Cy5 (red) dyes are used as they
have well separated emission spectra which enable efficient channel
separation in the signal detection. The labeling cDNA synthesis reaction is
rapid, but the bulky Cy-dye molecules may reduce the incorporation
efficiency of labeled nucleotides. In order to eliminate dye specific effects
caused by a labeling bias, resulting in an uneven labeling of the two dyes for a
specific gene sequences, a dye-swap design is recommended. Each
hybridization is then performed twice but with switched colors during
labeling. Finally, purification of samples is performed to remove
unincorporated dye. This is often performed by spin column purification.
The amount of total RNA required for one microarray experiment is
currently approximately 15 µg for each sample and this is considered one of
the bottlenecks in microarray analysis. Although a number of amplification
strategies have been developed, which aim to reduce the amount of starting
material, [219-222], the limitations of all these strategies are reproducibility
and unbiased amplification which is necessary to preserve the relative
expression levels from the two starting RNA samples that are to be
compared.
Hybridization
Hybridization of the labeled target is ideally linear (i.e. proportional to the
amount of labeled targets), sensitive so that low abundance genes are
detected, and specific so that probes hybridize only to the desired gene in the
complex target mixture. The large size of the cDNA probes is also helpful in
enabling stringent hybridization conditions and lowering cross-hybridization
of unrelated genes, although closely related gene families will still be able to
anneal to some extent. Procedures to reduce background (a step commonly
called pre-hybridization) include inactivation of free reactive groups on the
glass slide surface before hybridization. This can be performed either by
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Karin Dillner
chemical inactivation [223] or by treatment with biomolecules such as
bovine serum albumin (BSA) [224] to block the reactive groups. The
hybridization temperature and buffer will determine the stringency of the
hybridization. Salmon sperm DNA, polyA, tRNA, sodium dodecyl sulfate
(SDS), and Cot1 DNA are added to the hybridization to eliminate
nonspecific hybridization due to repetitive sequences [225]. After
hybridization, the chip is washed in multiple steps, to wash away disturbing
particles and loosely bound target DNA.
Image analysis and Normalization
The fluorescent signal of the hybridized probes is measured with a laser
scanner capable of detecting emission from the Cy3 and Cy5 channels
(showing green and red signals, respectively) to monitor the spots where target
DNA has bound. Laser intensity and detector gain should be adjusted to yield
images with non-saturated spots and approximately similar overall signal
intensities for the red and green channels. An overlay of the red and green
images will therefore allow a relative comparison, where the intensity of the
signals from the two different samples is directly correlated with the original
concentration of mRNA in the cell or tissue. Calculation of the expression
ratio for each clone (red/green channel), enables the assignment of upregulated, down-regulated, non-differentially or absent expression.
The image processing and subsequent data analysis from the microarray
experiments are crucial for extraction of useful information. Image analysis
in paper II and IV was performed by using GenePix Pro software. First, a
grid describing the array design is aligned on the image to localize and link a
clone identity to each spot. The software extracts intensity and background
measurement for each probe. Automatic flagging localizes absence of a spot
or very weak spots (≤1.4 (paper II) or ≤2 (paper IV) times above
background) and manual flagging is used to eliminate artifacts. The value of
the signal from each spot is calculated as the average intensity minus the
background.
To allow for inter-array comparisons, each array needs to be normalized to
remove systematic sources of variation. Normalization between the two
fluorescent images was performed using ‘LOWESS’ normalization method
in the SMA (Statistics of Microarrays Analysis) package [226, 227]. SMA is
an add-on library written in the public domain statistical language R [228]
and can be used to analyze simple replicated experiments. The LOWESS
(Locally Weighted Scatter Plot Smoother) algorithm performs a local fit to
the data in an intensity-dependent manner. The intensity value for each spot
- 38 -
is normalized based on data distribution in the immediate neighborhood of
the spot’s intensity. Bias in spatially defined sub-sets of the data can also be
compensated for by normalization strategies (‘Pin-wise LOWESS’) e.g.
when clear biases caused by pin-to-pin variations during array printing or
uneven hybridizations are observed.
Data Analysis and Statistical Evaluation
cDNA microarrays is now becoming used in a more or less standardized
fashion and it has become increasingly clear that simply generating the data
is not enough; one must be able to extract meaningful information about the
system being studied. Despite the combined efforts of biologists, computer
scientists, statisticians and software engineers, there is no one-size-fits-all
solution for the analysis and interpretation of genome-wide expression data.
There are now numbers of tools available for interpreting the data and
choosing among them is challenging.
The most basic question one can ask in a transcriptional profiling experiment
is which genes’ expression levels changed significantly. Highly abundant
genes with great differences in expression will normally not cause any
problems as they will display expression ratios above experimental noise and
measurement variations. However, for the detection of subtle expression
differences and low abundance genes, a statistically justified experimental
design and data evaluation is crucial.
The many sources of variation in a microarray experiment can be divided
into three different parts. First, the biological variation, which is intrinsic to
all organisms; it may be influenced by genetic or environmental factors, as
well as by whether the samples are pooled or individual. Second, the
technical variation, which might have been introduced to the samples during
the extraction, labeling or hybridization procedures. Third, measurement
error, which is associated with reading the fluorescent signals, which may be
affected by factors such as dust on the array. Technical replicates generally
involve a smaller degree of variation in measurements than the biological
replicates.
Replication is essential in experimental design because it allows accounting
for different sources of variability. It is more difficult to say how many
replicates should be done, although Lee et al indicates that three replicates are
sufficient to account for technical variability [229]. The ability to assess such
variability allows identification of biologically reproducible changes in gene
expression levels. Standard analyses of t-like tests assume that the data are
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Karin Dillner
sampled from normal populations with equal variances. Although log
transformation of the expression ratios can improve normality and help
equalize variances [230], ultimately the best estimates of the data’s
distribution come from the data themselves. Permutation tests, generally
carried out by repeatedly scrambling the samples’ class labels and computing
t statistics for all genes in the scrambled data, best capture the unknown
structure of the data [226, 231]. These types of tests do not assume normal
distribution of the data set. One advantage of permutation methods is that
they allow more reliable correction for multiple testing. The issue of multiple
tests is crucial, as microarrays typically monitor the expression levels of
thousands of genes.
In paper II and IV, we used the permutation-based statistical method,
Significance Analysis of Microarrays (SAM) software, adapted specifically
for microarrays. Today, SAM is a well accepted statistical method for
estimating the variability of the repeated experiment [231]. Briefly, SAM
assigns a score to each transcript on the basis of change in gene expression
relative to the standard deviation of multiple independent measurements.
Thereby, SAM allows selection of differentially regulated genes based on
estimation of the percentage of genes identified as differentially regulated by
chance, the so-called false discovery rate (FDR). To each of the genes in the
array a q-value is assigned. This value is similar to the familiar p-value and
measures the lowest FDR at which the gene is called significant.
Experimental design
The expression ratio obtained from a microarray experiment is relative, i.e. no
absolute values of the number of mRNA molecules per cell can be obtained.
The key issue in designing a cDNA microarray experiment is to decide
whether to use direct or indirect comparisons; that is, whether to make the
comparison within or between slides [232]. Figure 3a show a direct design
where the comparison of two different samples is made within one slide using
the same orientation of dye labeling. In this design, dye bias may affect the
final result [233, 234]. To avoid this, a technical replication may be performed
by comparing each sample using two arrays in a dye-swap design (Figure 3b).
If more than two samples are to be compared, a series of hybridizations that
can be correlated among them have to be performed and a so-called indirect
design has to be set up. A common strategy is to use a reference sample (e.g. a
pool of all samples, a common control or a zero time point) that is hybridized
to each array with one of the other sample (Figure 3c). Finally, the loopdesign may be applied, with sometimes very complex setups, which increase
the specificity in measurements and provides a more economical use of
resources. In this strategy every sample is compared to two other samples in a
- 40 -
fashion that finally relates all samples in a close loop where the number of
measurements per sample is automatically doubled (Figure 3c). Although,
several advantages of the loop design, the interpretation may be problematic to
a non-statistician. A concern is that this design is sensitive to failed
experiments. If one of the links in the loop is missing due to for example a
failed hybridization or too little starting RNA material, the entire set of
hybridizations will yield less valuable data.
The experimental design of the microarray experiments in paper II and IV
were according to the direct dye-swap experimental design of Figure 3b. This
design was used as we aimed to compare two different samples, the
hyperplastic prostates of PRL-transgenic mice versus the prostates of control
mice.
a.
b.
A
B
A
c.
d.
B
A
Reference
D
A
B
B
C
C
Figure 3. Experimental designs of cDNA microarray analysis. Letters represent RNA
samples, and arrows represent microarray hybridizations. A. Microarray design where the
two test samples (A and B) is directly compared. B. A variation of A using a dye swap for
each comparison. C. The standard reference design uses a single array to compare each
test sample to the reference RNA. D). Loop design.
Microarray databases
The importance of public access to microarray data and the possibility of
comparing different experiments using a common platform face a true
challenge. In an attempt to standardize the microarray procedures and data
handling, the international working group MIAME (the Minimum
Information About a Microarray Experiment) has been established to set up
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Karin Dillner
certain guidelines. These guidelines include details of: (1) how the
experiment was designed, (2) the design of the arrays or the name and
location of spots on arrays, (3) sample name, extraction and labeling, (4)
hybridization protocols, (5) methods for image measurements, and (6) the
controls used. Meanwhile, several local as well as public available data bases
have been created for gene expression data.
COMPARISONS BETWEEN CDNA RDA AND CDNA MICROARRAY
ANALYIS
In paper II, cDNA-RDA was used to isolate differentially expressed
transcripts in prostate of the Mt-PRL transgenic mice compared to controls.
In contrast, in paper IV, the cDNA microarray technology was used to
identify differentially expressed transcripts in prostatic hyperplasia of the
transgenic mice compared to controls. The similarity of these two methods
relies in the fact that both methods are applied to identify differentially
expressed transcripts between the two groups that are to be compared.
Depending of the aim of the study both of the methods can be successfully
used. The main difference between cDNA microarray and cDNA-RDA
analysis is that cDNA-RDA is a differential cloning method to isolate
differentially expressed transcripts, while cDNA microarray technology only
detect those sequences that have been previously identified and fixed to the
support matrices.
One might argue that when the human and mouse genome projects soon will
be completed, cloning methods such as RDA, will not be of particular use.
That is true for using the method as a pure cloning method, but not when it
comes to its use for identifying differentially expressed genes. One
advantage of RDA is that this method it is more sensitive than that of cDNA
microarray. The PCR amplification steps in the RDA procedure makes this
method superior in terms of identifying rare transcripts with large differential
expression. Even though the PCR amplification steps should make
differences greater, the RDA procedure may fail to detect abundant
transcripts that have small differential expression.
Although the method of cDNA microarray becoming more and more
standardized, this method is still very costly and consequently its use are
restricted to a small number of specialized laboratories. Moreover, most
laboratories are able to handle the bioinformatics associated with the
sequenced RDA output which is far from the sophisticated bioinformatics
following a cDNA microarray experiment.
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VERIFICATION STRATEGIES
cDNA microarray analysis
Coupling of RDA subtraction with microarray analysis creates an efficient
method for detection of unique, differentially expressed genes. The RDA
may be halted at an early round of subtraction, which lessens the loss of
differentially expressed cDNAs, perhaps due to PCR amplification
preferences, and maintains diversity. However, this probably also contribute
to a higher proportion of non-differentially expressed species in RDA output
compared to RDAs performed using a greater number of rounds. This more
complex output of RDA may be efficiently verified using cDNA
microarrays. This verification may be performed in two variants. First,
microarrays built with RDA-derived clones are hybridized to Cy3- and Cy5labeled RNA samples that served as starting material. This use of cDNA
microarray analysis might be inefficient for verification of low expressed
transcripts or to detect small expression differences. For those transcripts, an
alternative methodology that includes PCR amplification steps, such as RealTime RT-PCR, might need to be used. Alternatively, the arrayed RDA
output could be hybridized to Cy3- and Cy5-labeled RDA representations, as
well as with the differential products from the different RDA rounds (DP1,
DP2 etc). This will allow identification of those inserts that are differentially
represented in the starting populations and the subsequent DP products.
Clones that fail to hybridize at all to the representation targets but hybridize
differentially to the DP pools should likewise be selected for further
confirmation using an alternative method such as real-time RT-PCR as these
likely include rare transcripts that are truly differentially expressed. Those
clones that hybridize with the same intensity to the representation targets are
highly likely to be false positives, even if they show differential
hybridization with labeled DP targets. Therefore, the latter strategy might be
the most efficient in allowing the advantage of the sensitivity of the RDA
method in its ability to identify very rare and low abundant transcripts.
In paper II, we haltered the cDNA-RDA after two rounds of subtraction and
amplification rounds (DP2). The RDA output, DP2-hyperplastic and DP2control, was verified using cDNA microarray analysis. Cy3- and Cy5-labeled
RNA samples was used to verifiy the cDNA-RDA.
Real-time RT-PCR
The RT-PCR approach can be successfully applied to validate the results of
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Karin Dillner
any primary differential gene expression screening method once the
sequence of the candidate gene is known. The main three advantages of RTPCR is its sensitivity for the detection of low-abundance mRNA, the use of a
very small amount of starting RNA samples and the possibility of
discrimination between different splice forms of the transcript of interest.
The conventional RT-PCR technique is not considered to be quantitative, as
the final amount of PCR product is related not just to the initial template
concentration but also to primer-dimer accumulation, PCR product reannealing, and DNA polymerase binding to primers [235, 236] The
development of kinetic RT-PCR (often referred to as real-time RT-PCR) has
revolutionized the possibilities for quantitating mRNA [237]. In real-time
RT-PCR, the accumulation of PCR products is monitored at the end of each
cycle by fluorescence. During early cycles, the fluorescence is
indistinguishable from background, but after a subsequent number of cycles
the fluorescence increases exponentially. The PCR cycle number at which
the fluorescence crosses a threshold, which is within the exponential phases,
can be related to the amount of starting material; samples with more starting
template will achieve the threshold fluorescence level more rapidly than
those with less starting template.
There are two general methods for the quantitative detection of the amplicon:
(a) fluorescent probes and (b) DNA-binding agents. In the first “fluorescent
probes” method, a specific probe, the so-called “TaqMan” probe [238], for
the PCR product of interest is designed and labeled with a reporter dye at the
5’-end and a quencher dye at the 3’- end. During the extension phase of the
PCR, the TaqMan probe will be cleaved by the endonucleolytic activity of
the Taq polymerase, which allows the quencher and the reporter dye to be
separated, and fluorescence emitted from the reporter dye (Figure 4a). In the
second “DNA-binding agents” method, a non-sequence specific fluorescent
DNA-binding dye, such as SYBRGreen I, is used which possess the ability
to incorporate into double stranded DNA [239]. The unbound dye exhibits
little fluorescence in solution, but during elongation increasing amounts of dye
bind to the newly synthesized double-stranded DNA (Figure 4b). Its greatest
advantage is that it can be applied with any pair of primers for any target,
making its use less expensive than that of probe. While the sensitivity is
usually quite good, such dyes bind to any double stranded DNA and thus do
not distinguish between the PCR product of interest and alternate products,
primer-dimers, etc. The product of interest should therefore be validated in
assay development by stopping the kinetic PCR reaction after various
numbers of cycles and performing electrophoresis on the products in an
agarose gel. In addition, this problem can be overcome by generating a
melting curve of the amplicon [240].
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A. Fluorescent probes
B. DNA binding agents
i
i
FW Primer
Fluorophore
SYBR
Green I
Target
Quencher
RV Primer
ii
ii
Polymerase
Fluorophore
Quencher
Polymerase
Fluorescent dye
Figure 4. A. Fluorescent probes (e.g. TaqMan system). (i) After denaturation, primers
and probe anneal to the target. Fluorescence does not occur because of the proximity
between fluorophore and quencher. (ii) During the extension phase, the probe is cleaved
by the 5’ to 3’ enzymatic activity of Taq polymerase. Thereby quencher and fluorophore
are separated, allowing fluorescence emission from the reporter dye. FW, forward; RV,
reverse. B. DNA-binding agents (e.g. SYBR Green I). (i) The dyes free in the solution do
not emit fluorescence light. (ii) As soon as the SYBR Green binds to the dsDNA, target
fluorescence occurs.
Target RNA can be quantified using either absolute or relative
quantification. Absolute quantification determines the absolute amount of
target (expressed as copy number or concentration), whereas relative
quantification determines the ratio between the amount of target and a
reference transcript, usually a suitable housekeeping gene. This normalized
value can then be used to compare, for example, differential gene expression
in different samples. An important consideration is to ensure that the
housekeeping gene is expressed at constant levels in the two different
samples to be compared.
In paper IV, we verified a set of differentially expressed transcripts
subsequent to the identification using cDNA micorarray analysis by using
the SYBR Green real-time RT-PCR approach. To quantitate the target RNA
the relative quantification method was used with the acidic ribosomal
phosphoprotein PO (Arbp) as the internal standard. In this approach, the ratio
between the amount of target molecule and the internal standard molecule
within the same sample is calculated. This normalized value was
subsequently used to compare the relative expression ratio obtained with the
real-time RT-PCR method with that obtained in the cDNA microarray
analysis.
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Karin Dillner
ASSESSMENT OF APOPTOTIC ACTIVITY
In paper IV, the results from the microarray experiments indicated a
reduction in apoptotic activity in the Pb-PRL transgenic mice compared to
controls. Therefore we used two different apoptotic markers to assess
apoptotic activity between the prostates of transgenic and control mice.
Apoptosis is distinct from necrosis in both the biochemical and the
morphological changes that occur [241-245]. In contrast to necrotic cells,
apoptotic cells are characterized morphologically by compaction of the
nuclear chromatin, shrinkage of the cytoplasm and production of membranebound apoptotic bodies. Biochemically, apoptosis is distinguished by
fragmentation of the genome and cleavage or degradation of several cellular
proteins.
As with cell viability, no single parameter fully defines apoptosis; therefore,
it is often advantageous to use several different approaches when studying
apoptosis. Several methods have been developed to distinguish live cells
from early and late apoptotic cells and from necrotic cells. The method of
TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) is
widely used for detecting DNA nicks in apoptotic cells. An alternative
method is the detection of single stranded DNA (ssDNA) which signifies the
downstream event of DNA fragmentation. DNA fragmentation may be
detected in both histologically defined apoptotic cells and morphologically
intact apoptotic cells. The immunohistochemical method involving an
antibody specific for ssDNA protein in cells allows accurate assessment of
apoptosis [246, 247]. Furthermore, detection of ssDNA is considered more
apoptosis-specific than the widely used TUNEL method for detection of
DNA fragmentation and also detects apoptotic cells at an earlier stage than
TUNEL [248, 249].
Apoptosis is mediated by a proteolytic cascade. The caspases, a family of
cysteine proteases, play an essential role in the initiation, regulation, and
execution of the downstream proteolytic events occurring during apoptosis
[250-252]. Upon activation through proteolytic processing, caspases trigger
substrate proteolysis and other changes that result in chromatin condensation,
DNA fragmentation, and ultimately the apoptotic phenotype [251, 253, 254].
Caspase-3 is a key effector in the apoptosis pathway, amplifying the signal
from initiator caspases (such as caspase-8) and for apoptosis-associated
chromatin margination, DNA fragmentation, and nuclear collapse during
apoptosis [253]. The detection of activated caspase-3 could therefore be a
valuable and specific tool for identifying apoptotic cells in tissue sections,
even before all the morphological features of apoptosis occur.
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RESULTS AND TECHNICAL COMMENTS
PAPER I
Progressive prostate hyperplasia in adult Mt-PRL transgenic mice is not
dependent on elevated serum androgen levels
Transgenic mice overexpressing the rat PRL gene under control of the
ubiquitous Mt-1 promoter develop a dramatic prostatic enlargement with
parallel chronic hyperprolactinemia and elevated serum androgen levels.
Histologically the prostate enlargement is mainly characterized by an
expansion of the stromal compartment and areas of glandular hyperplasia with
an accumulation of secretory material [105]. In paper I, we aim to clarify the
role of circulating androgen levels in the promotion of abnormal prostate
growth in the adult Mt-PRL transgenic mouse prostate.
Separate groups of 12 weeks old animals (age-matched wild-type and MtPRL transgenic males) were surgically castrated followed by subcutaneous
implantation of slow-release testosterone pellets containing 7.5 mg
testosterone or placebo substance. The testosterone dose of 7.5 mg, was
chosen to give as normophysiological levels of testosterone as possible, and
was found to not significantly differ from the circulating testosterone serum
levels of wildtype controls. After 8 weeks of hormone/placebo pellet
treatment, animals were killed followed by serum sampling and prostate
dissection. As an additional control, prostates from age-matched groups of
non-treated wildtype and Mt-PRL transgenic mice were collected at both start
and endpoint of the experiment. Results revealed that progression of prostate
hyperplasia in adult Mt-PRL transgenic males was not affected by
normalization of circulating testosterone levels. Immunohistochemical studies
revealed a significantly increased proportion of AR positive epithelial cells
in all prostate lobes of the Mt-PRL transgenic compared to wild-type. The
increased distribution of epithelial AR remained high in the group of animals
that were castration and substitution to normophysiological androgen levels.
In addition, the Mt-PRL transgenic males possess more prominent stromal
AR positivity than wildtype controls.
The present study demonstrates that progressive prostate hyperplasia in adult
Mt-PRL transgenic mice is not dependent on the elevated serum androgen
levels present in the animals. In addition, our results suggest that prolonged
hyperprolactinemia results in changes in prostate epithelial and stromal cell
AR distribution. The increased AR distribution in both epithelial and stromal
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Karin Dillner
cells in the Mt-PRL transgenic prostate lobes may increase the androgen
sensitivity and thereby also influence the development of the observed
prostate phenotype.
Prolonged androgen treatment has no significant effect on prostate
growth in wildtype adult mice
The data about the importance of prolonged exposure to extraordinary high
levels of androgens on prostate growth in rodents is conflicting, showing both
unaffected prostate size [255] and induction of hyperplasia [256]. To
determine the long-term effects of elevated circulating androgen levels on the
prostate gland of wild-type male mice, a separate group of 12-week-old
wildtype mice were sham-operated and subcutaneously implanted with 30 mg
of testosterone slow-releasing pellet. The high dose of 30 mg testosterone was
selected to give the treated group of wildtype animals a comparable levels of
circulating testosterone as the Mt-PRL transgenic male mice have. After 8
weeks of treatment, prostates were dissected and serum samples obtained. On
average, these animals displayed a 4-fold increase in serum testosterone levels
compared to untreated wildtypes. These testosterone levels did not
significantly differ from levels found in Mt-PRL transgenic males. Prostate
wet weight in testosterone-treated wildtype did not significantly differ from
that in untreated wildtype males neither as separate lobe weight nor as total
organ weight. Histological appearance of the prostate lobes was not either
different from that observed in wildtypes. These findings establish that
prolonged androgen stimulation of adult male mice (C57BL/6JxCBA-strain)
has no significant effects on prostate growth or histological appearance. This
also supports the conclusions drawn from the results in castrated and androgen
substituted Mt-PRL transgenic males, indicating that the hyperplastic process
in transgenic prostate is not dependent on an elevated state of circulating
androgens.
Like the Mt-PRL transgenic mice, wildtype males treated with 30 mg of
testosterone exhibited significantly higher numbers of AR-positive epithelial
cells compared to untreated wildtypes. However in contrast to the Mt-PRL
transgenic mice, stromal AR content was unaffected in testosterone-treated
wildtypes.
Taken together, these results show that prolonged androgen stimulation of
young adult male mice has no significant effects on prostate growth or
histological appearance. These data further support the findings in castrated
and androgen substituted Mt-PRL transgenic males, that progression of
prostate hyperplasia is not dependent on elevated levels of circulating
androgens. Moreover, the results from the immunohistochemical analysis
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suggest that the prostate hyperplasia of Mt-PRL transgenic mice is not
primarily mediated via increased epithelial AR contents.
Comparison of post-castrational regression patterns in the prostate
lobes of Mt-PRL transgenic mice compared to wildtype mice
Involution of the prostate after androgen-deprival by testicular castration is a
well characterized process. Regression of the epithelial cell population,
through an active process of apoptosis, occurs rapidly after castration. To
establish the androgen dependency of this PRL transgenic prostate model,
separate groups of 12-week-old male Mt-PRL transgenic and wildtype mice
were castrated and subcutaneously implanted with placebo pellets. After 8
weeks, prostates were dissected and serum samples obtained. DLP and VP
were significantly reduced after castration, in both Mt-PRL transgenic and
wildtype prostates. In addition, similar histological appearance, with marked
loss of both glandular epithelium and interductal stroma, were observed in
both groups. Post-castrational VP weights in Mt-PRL transgenic males did
not significantly differ from those of wildtype, whereas post-castrational
DLP weight was significantly higher in Mt-PRL transgenic than in wildtype.
However, considering the small but significant weight difference already at
12 weeks of age, the relative rate of reduction in DLP weight after castration
was similar in Mt-PRL transgenic and wildtype, -66% and -78%,
respectively.
Altogether, these data show that androgens are clearly required for
maintaining the transgenic phenotype as demonstrated by the similar patterns
of prostatic regression seen in Mt-PRL transgenic and wildtype mice after
androgen withdrawal. In the DLP, some weight differences were maintained
after androgen-deprival; this difference may be attributable to the existing
difference in glandular size at the time of castration. This finding could also
partly be due to lobular differences in PRL responsiveness reported earlier
[171, 175].
PAPER II
Isolation of differentially expressed transcripts in the enlarged prostates
of Mt-PRL transgenic mice compared to controls
The objective of this study was to characterize the molecular mechanisms in
the prostate of importance for the prostate hyperplasia seen in Mt-PRL
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Karin Dillner
transgenic mice. Therefore, the method of cDNA representational difference
analysis (cDNA RDA) was used which allow identification of novel genes
that were differentially expressed in the enlarged prostates of the Mt-PRL
transgenic mice compared to controls. The cDNA RDA was performed on
prostatic tissue (DLP and VP) from mice of an age of four to six months. To
generate samples as representative as possible, reflecting the prostate
phenotype, RNA samples were pooled from four transgenic mice and five
control littermates, respectively. Representations, generated from control and
transgenic cDNA, respectively, were used as driver (control) and tester
(hyperplastic), or vice versa, to generate both up- and down-regulated
transcripts in the hyperplastic prostates compared to controls (see
METHODOLOGICAL CONSIDERATIONS). Two successive rounds of
subtraction and amplification were performed, generating libraries containing
two sets of difference products, DP2-hyperplastic and DP2-control. Upon gel
electrophoresis of the Sau3AI-cut RDA products of DP2-hyperplastic and
DP2-control, six distinct bands were visualized. To subclone as many
different products as possible from each library, each band were excised from
the agarose gel and subcloned individually. 384 bacterial colonies, 192 from
each RDA library, were picked, plasmid DNA was prepared followed by
routine sequencing. After sequence alignments, the sequences were analyzed
for homologies with published sequences in the non-redundant and EST
divisions of the public databases of NCBI. This reduced the complexity of the
RDA output so that the 384 clones sequenced, was reduced to 152 different
unique sequences having a length longer than 50 base pairs. 69 of these, 37
DP2-hyperplastic and 32 DP2-control, were identified as previously annotated
transcripts, whereas 83 were novel sequences not found in the public
databases (referred to as unknowns) at the time when the study was
performed.
Verification of the RDA output by using cDNA Microarray Analysis
To confirm that the obtained RDA difference products represented truly
differentially expressed transcripts, the 152 non-redundant RDA products
were selected for further verification using cDNA microarray analysis. 28 of
the different RDA products were printed in duplicates, at different locations
on the chip, to serve as “within slide” reproducibility controls (see
METHODOLOGICAL CONSIDERATIONS). The RDA-derived microarrays
were co-hybridized with labeled control and transgenic total RNA from a new
set of animals. In order to eliminate dye specific effects caused by a labeling
bias, dye-swap design of targeting labeling was used. The hybridizations were
performed four independent times, twice Cy3-labeling the control and Cy5labeling the hyperplastic total RNA, and twice with opposite colors (dye- 50 -
swapped). Probes rendering weaker signals than 1.4 times the background
were eliminated and not considered for further analysis. Using this criterion,
48 of the 152 uniquely printed RDA clones could be detected in the Mt-PRL
transgenic and control prostatic total RNA. To identify the significant
differentially expressed transcripts, the data from the four repeated microarray
experiments were statistically analyzed using the SAM algorithm [231].
Genes with average fold changes of more than 50% (correspond to a fold
change of 1.5) were counted as differentially expressed. With an estimated
FDR of less than 2%, 15 out of the 48 detected RDA products were identified
as differentially regulated (of which 5 were unknowns). In terms of fold
regulation, previous results from our laboratory have shown that this level can
be reproduced, as shown by independent validation using RNase protection
assay [257, 258]. Overall, the complexity of the RDA output could be largely
reduced by: i) the annotation step (from 384 to 152), ii) the detection
limitation of cDNA microarray technique (from 152 to 48), and iii)
verification step (from 48 to 15) – resulting in 15 significantly differentially
regulated transcripts, by an average fold-change of least 1.5, between the MtPRL transgenic and control prostates.
One might reflect on the low number of significantly differentially regulated
transcripts that were identified in this study. It has to be clarified that the
final outcome of RDA differential cloning method depends largely on how
extensive one makes the cloning. In our study, we cloned a number of 384
transcripts and certainly the more bacterial colonies that are cloned the
higher probability there is to isolate and cover the complete set of
differentially expressed transcripts that there are between the two groups that
are compared. This may contribute to missing out important transcripts.
Another reflection might be the obvious detection limitations of cDNA
microarray analysis. There are several possible explanations for the
relatively small number of detected transcripts. First, the ideal length of the
cDNAs to be printed on cDNA microarrays is approximately 1000 base
pairs. The lengths of our RDA products were between 200-500 base pairs,
which probably contribute to reduce the sensitivity for detection by
decreasing the hybridization. Second, a classical hybridization-based method
like that of cDNA microarray depends on the specific activity of probes.
Third, the PCR amplification steps in the RDA makes small expression
differences greater as well as enables detection of low expressed transcripts.
To enable verification of low expressed transcripts or small expression
differences, an alternative method including PCR amplification steps, such
as real-time RT-PCR, might need to be used. A further reflection is the
relatively few significantly differentially regulated transcripts that were
found at last. Most likely this is a consequence of the detection limitations of
the method of cDNA microarray, but there are also other possible
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Karin Dillner
explanations. A relative large proportion of the RDA output was expected to
be so-called “false positive” clones as the RDA procedure was haltered after
two rounds to. The reason for stopping the RDA subtraction and
amplification steps at an early round is to diminish the loss of differentially
expressed cDNAs and thereby maintain the RDA output diversity [259].
The verified differentially expressed RDA clones in hyperplastic versus
control prostates
In the present study, 152 non-redundant transcripts were differentially cloned
in the Mt-PRL transgenic prostate compared to control. Although, not all of
these transcripts could be detected and/or verified using cDNA microarray
analysis, we still think they together may contribute to an interesting result as
many of them are new transcripts to be cloned in the prostate. Therefore,
several of the 152 differentially expressed transcripts most likely hold
information of differentially expressed transcripts between the Mt-PRL
transgenic and control prostates which will be found to be significantly
differentially regulated if another verification method than cDNA microarray
is used.
Regarding the 10 annotated and verified differentially expressed transcripts,
a number of them gave interesting information of possible molecular
mechanisms involved in the development/ progression of the prostate
hyperplasia of Mt-PRL transgenic mice. Of particular interest were the upregulation of vimentin and the down-regulation of cytokeratin 8 which may
indicate the importance of the “embryonic reawakening theory” in the
development of the prostate phenotype of the Mt-PRL transgenic mice.
Furthermore, the down-regulation of aldose reductase may be a sign of
involvement of reduced apoptosis for development of the hyperplasia of the
Mt-PRL transgenic mice. In addition the down-regulation of the candidate
tumor-suppressor, the transcript coding for the RIL protein may further
contribute to the prostate phenotype of Mt-PRL transgenic mice.
In summary, the identified differentially expressed transcripts supports
molecular similarities between the prostate hyperplasia of the Mt-PRLtransgenic mice and human BPH. Furthermore, the finding of new prostate
hyperplasia related transcripts, both previously annotated and unknown
transcripts, might be of large use both as potential biomarkers and to
understand the underlying cause of benign growth of the prostate gland.
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PAPER III
Generation of transgenic mice overexpressing the PRL transgene
specifically in the prostate under normophysiological androgen levels
To address the role of local PRL action in the prostate, a new transgenic
mouse model (Pb-PRL) was generated using the prostate-specific rat
probasin (Pb) minimal promoter to drive expression of the rat PRL gene. PbPRL transgenic males developed a significant enlargement of both the DLP
and VP lobes evident from 10 weeks of age and increasing throughout
animal life span. In addition, the DNA content was measured in the prostate
gland at 20 weeks of age, showing a significant three-fold increase in the DLP
and VP, respectively, indicating a true hyperplasia with increased number of
cells.
Expression of the transgene was restricted to the prostate (DLP, VP, and AP)
and present from 4 weeks of age. Also, a weak expression of the transgene
could be observed in seminal vesicles at this age. Moreover, transgenic rPRL
was detectable at low levels in the circulation of transgenic animals from 10
weeks of age, most likely associated with the continuing increase in prostate
size. In contrast to the ubiquitous Mt-PRL transgenic mice, serum androgen
levels did not significant differ from that of wild-type mice at any time point.
The Pb-PRL prostate is histologically characterized by a significant stromal
hyperplasia and secretion-filled distended ducts and focal areas of epithelial
dysplasia. The glandular dysplastic foci had several morphological
characteristics in common with low-grade prostatic intraepithelial neoplasia
(PIN) lesions previously reported in other genetically engineered mouse
models [260]. No high-grade PIN or prostate tumor formation were detected
in Pb-PRL transgenic prostate. In addition, focal areas of mild to moderate
chronic inflammation, exhibiting stromal mononuclear (primarily
lymphocytes and macrophage) infiltrate, were frequently observed in both VP
and LP lobes in Pb-PRL transgenic mice.
Furthermore, immunohistochemical analysis revealed a significant increase
in stromal cell distribution of androgen receptors (AR) and estrogen
receptors alpha (ERα). In contrast, distribution of estrogen receptor beta
(ERβ) was nearly uniform in both Pb-PRL transgenic and wildtype prostate.
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Karin Dillner
Comparative analysis of prostate ductal branching morphogenesis and
quantitative analysis of prostate cellular composition in Mt-PRL and
Pb-PRL transgenic mice compared to controls
To reveal possible phenotypic differences in ductal architecture due to
different onset of transgenic rPRL expression, microdissection technique was
used to examine branching morphogenesis of individual lobes in Mt-PRL and
Pb-PRL transgenic prostate. Quantification was made by counting primary
urethral ducts as well as duct branchpoints and terminal ductal tips at 12
weeks of age. In 12-weeks-old Pb-PRL prostate, no statistically significant
differences were detected in the number of branch points per duct and the
number of ductal tips present in each lobe compared to wild-type controls.
However, marked ductal dilation and elongation was seen in the Pb-PRL from
an early age, and complete microdissection was not achievable in animals
over 20 week of age due to the formation of a densely fibrous interductal
stroma that abrogated its normally high susceptibility to collagenase. In
contrast, counting of ducts and tips in Mt-PRL VP and LP lobes at the same
age demonstrated a significant increase, with approximately a doubling in the
number of branching points and terminal tips compared to wildtype, whereas
the number of main urethral ducts remained unchanged. Like the Pb-PRL
transgenic prostate, the ducts were elongated and more dilated compared to
controls and microdissection was also prevented by formation of a densely
fibrous stroma in prostate lobes of older Mt-PRL animals.
Quantitative analysis of prostatic tissue cellularity demonstrated a marked
increase in the stromal to epithelial ratio in all lobes of both Mt-PRL and PbPRL transgenic prostates compared to controls. In wild-type controls, the
lobe-specific stromal:epithelial ratio varied between 1:2.5 and 1:10, whereas
in all lobes of Mt-PRL and Pb-PRL, transgenic prostate stromal and
epithelial cells were present in approximately equal numbers.
Overall, the Pb-PRL transgenic represents a new model for the study of PRL
effects in the prostate. Most significantly, the development of Pb-PRL
hyperplasia occurs mainly post-pubertally and in a setting of normal
androgen levels, thereby resembling the situation in the adult human
prostate. This study indicates the ability of PRL to promote, directly or
indirectly, ductal morphogenesis in the developing prostate and further to
induce abnormal growth primarily of the stroma in the adult gland in a
setting of normal androgen levels.
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PAPER IV
Global analysis of gene expression in the enlarged prostate lobes of PbPRL transgenic mice
The objective of this study was to characterize the molecular mechanisms
involved in the prostate hyperplasia seen in the Pb-PRL transgenic mice
overexpressing the PRL gene specifically in the prostate. Global changes of
gene expression were analyzed by using a cDNA microarray chip containing
about 6250 cDNA probes of rat and mouse origin. The gene expression
analysis was performed in DLP and the VP, separately. We have chosen to
denote genes as differentially regulated if their level of expression was
changed by 70% (corresponding to a fold change of 1.7) or more in a
statistically significant fashion. This level of change has previously been
shown to be valid when compared to other direct methods, such as Northern
Blot, ribonuclease protection assay, and RT-PCR [257, 258, 261, 262], as well
as in the present study. Of the 6250 cDNA clones analyzed, 2344 showed
hybridization in all independent determinations (DLP=2003 and VP=1962).
Of those, 266 non-redundant transcripts were found to be differentially
expressed (175 up-regulated and 91 down-regulated) in the enlarged prostate
of PRL transgenic mice compared to controls in at least one of the lobes. 159
were differentially expressed in the DL lobe (111 up and 48 down) and 224
differentially expressed in the VP (159 up and 65 down). Of those, 117
transcripts were commonly differentially expressed in both DLP and VP (95
up and 22 down). 84 of the 266 non-redundant differentially expressed
transcripts were transcripts with unknown function, identified as ESTs. The
differences between the gene expression of VP and DLP can reflect biological
differences and/or being a consequence of the detection sensitivity associated
with the technique of cDNA microarray. Consequently, we have not paid
specific attention to these differences.
Functional classification of the differentially expressed transcripts, based on
their known or suggested functions, revealed that virtually all cellular
processes were affected in the prostate hyperplasia compared to control
prostate. The two largest functionally categorized groups of both the DLP and
VP were those of signal transduction and cell tissue structure. Moreover, a
number of immune system-associated transcripts were found to be upregulated which is in line with previous observations of areas of mild to
moderate chronic inflammation, exhibiting stromal mononuclear (primarily
lymphocytes and macrophage) infiltrate, in both the Pb-PRL VP and LP
(Paper III).
Subsequential real-time RT-PCR, using mouse specific primers for the
orthologous mouse genes, did verify 10 of the differentially regulated
transcripts, indicating the validity of using cDNA microarray technology in
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Karin Dillner
combination with statistical methods for identifying differentially regulated
genes. Moreover, the verification using mouse specific primers for the
orthologous mouse genes to the cDNA clone of rat origin on the cDNA
microarray further supports the use of rat probes to measure orthologous
mouse genes.
Assessment of apoptosis activity in Pb-PRL transgenic and control
prostates
In order to assess possible differences in apoptotic activity in the Pb-PRL
transgenic and control prostates, two well accepted apoptosis markers where
used (activated caspese-3 and single stranded (ss) DNA). In the Pb-PRL
transgenic prostate, no activation of caspase-3 was detected using
immunofluorescence. In control prostate, distinct clusters of apoptotic
epithelial cells were occasionally detected in distal regions of the ductal
system. Furthermore, no detectable levels of ssDNA were present in any lobes
of Pb-PRL transgenic prostate using immunohistochemistry. In contrast,
control littermate prostate focally displayed distinct nuclear ssDNA
immunoreactivity in numerous epithelial cells, located almost exclusively in
the distal ductal regions of all the prostate lobe types.
Taken together, the ss-DNA and caspase-3 immunohistochemistry results,
clearly indicate an overall diminished apoptotic activity in all prostate lobes of
the Pb-PRL transgenic mice compared to controls.
Differentially expressed transcripts in the enlarged prostates of Pb-PRL
transgenic mice compared to control prostates
Interestingly, a number of the identified differentially expressed transcripts
in Pb-PRL transgenic compared to control prostate gave information of
possible molecular mechanisms involved in the development/progression of
the prostate hyperplasia. Among others a group of transcripts with proapoptotic activity were found to be down-regulated (Bok (Bcl-2-related
ovarian killer protein), CIPAR-1 (castration induced prostatic apoptosis
related protein-1) and Nuclear protein 1) in parallel with some transcripts
with anti-apoptotic activity that were up-regulated (clusterin (also known as
testosterone repressed prostate message-2 or sulfated glycoprotein-2) and
SARP-1 (Secreted apoptosis-related protein 1), in the Pb-PRL transgenic
prostate compared to controls. Together with the results of diminished
apoptotic activity in all prostate lobes of the Pb-PRL transgenic assessed by
immunohistohemistry, this likely indicates the importance of reduced
apoptosis activity in the pathogenesis of prostate hyperplasia in Pb-PRL
transgenic mice.
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Moreover, numerous differentially expressed transcripts, between the
enlarged prostates of Pb-PRL transgenic and control mice, were transcripts
associated with tissue remodeling. The increased stromal:epithelial ratio of
the Pb-PRL transgenic prostate, together with differential regulation of a
significant fraction of genes involved in tissue remodeling activity, including
synthesis and degradation of the ECM and changes in protease activity,
suggests that activation of the stroma is involved in the development of the
prostate phenotype. The obvious importance of the stromal compartment in
the development of the prostate phenotype supports the “embryonic
reawakening theory” of BPH etiology.
Overall, the differentially expressed transcripts identified in this study, show
many molecular similarities between the prostate hyperplasia of PRLtransgenic mice and human prostate pathology including both BPH and
prostate cancer.
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Karin Dillner
DISCUSSION
PRL is a factor which, alone or synergistically with androgens, exerts trophic
effects in the mature gland in rodents [201] and in human prostatic cells in
vitro [14], besides it’s all other demonstrated functions. Moreover, growthpromoting effects of PRL on the prostate are well known in rodents made
hyperprolactinemic by pituitary grafting [102-104, 109, 263]. To add to these
findings, is the ubiquitous transgenic expression of the rPRL gene in MtPRL male mice, demonstrating that long-term exposure to PRL leads to
prostate hyperplasia. In addition to chronic hyperprolactinemia, the Mt-PRL
transgenic mice display elevated serum androgen levels [105].
Extrapituitary production of PRL has raised interest in the past few years,
although its secretory control and functional relevance remain largely
unknown. Moreover, the recent detection of locally produced PRL in
prostate epithelium, together with the presence of the PRLR, have indicated
a possible auto/paracrine action of PRL in prostate tissue [93, 94]. In an
attempt to further explore the in vivo effects of enhanced PRL action in the
prostate gland, but without the possible systemic alterations resulting from a
prolonged hyperprolactinemic state, a prostate-specific PRL transgenic was
generated which allow us to study the role of PRL action locally in the
prostate. As shown in clinical reports, PRL may be elevated locally in BPH
tissue, without any significant increase in serum PRL levels [137]. Although
the contribution of local PRL production to circulating PRL levels is
presumably low, it may be sufficient to exert significant activity on its local
environment [264]. Local overexpression of the PRL transgene in the
prostate of Pb-PRL transgenic male mice results in a significant prostate
hyperplasia with a predominantly stromal phenotype. From this study we
concluded that local, rather than circulating, elevated PRL levels are
sufficient to induce prostate hyperplasia.
Ductal branching morpholology in the rodent prostate gland has been
extensively studied, showing that ductal formation is initiated around
embryonic day 15 and considered to be essentially completed at 4-5 weeks
(day 35) postpartum [4-6]. Due to the strong androgen-dependency of the Pb
promoter, expression of the PRL transgene was first detected at 4 weeks of
age. Thus, the PRL transgene is not expressed during the essential time
period of ductal development. Consequently, the PRL-induced prostate
hyperplasia of Pb-PRL transgenic mice arises from a normally developed
ductal structure, under normophysiological circulating androgen levels,
thereby resembling the situation in the adult human prostate.
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The use of prostate lobe microdissections can effectively demonstrate
changes in ductal structure resulting from neonatal exposure to
developmentally active factors. In paper III, the use of the microdisection
method revealed a significant increase of ductal morphogenesis in Mt-PRL
transgenic prostate compared to littermate controls, with approximately a
doubling in branching points and ductal tips evident in the DP, LP, and VP
lobes. Activation of the Mt-1 promoter during the early embryonic stage is
well described, with abundant expression already by day 12 of gestation
reported [208, 209]. In contrast to the Mt-PRL transgenic mice, the Pb-PRL
transgene expression initiates subsequent to the period when the ductal
formation and branching are essentially terminated. Consequently, the PbPRL prostate exhibited no significant changes in mature ductal architecture
when compared to controls. However, marked ductal dilation and elongation
was evident in both transgenic models, and the formation of a dense fibrous
and cellular interductal stroma appeared equally pronounced in the Pb-PRL
as in the Mt-PRL prostates. The differences in ductal architecture of the PRL
transgenic models can be explained by the temporal differences in expressin
of the transgene. Alternatively, the altered androgen status in the Mt-PRL
transgenic males may have an impact on the early ductal development in the
prostate. The involvement of elevated androgen levels in increased
branching morphogenesis has in fact been demonstrated previously in the VP
of hypogonadal mice, where a single neonatal dose of androgens caused an
increase in VP branching and lobe weight at adulthood [265]. However, in
another study neonatal castration experiments demonstrated that significant
branching morphogenesis occurs in the absence of androgens [7].
Furthermore, androgen replacement following neonatal castration results in
precocious ductal formation, but final numbers of ductal tips and
branchpoints do not exceed those seen in adult control males [7]. From this
study Donjacour and Cunha concluded that neonatal prostatic ductal
morphogenesis is sensitive to, but does not require, chronic androgen
stimulation. Taken together, these findings demonstrate that PRL can,
directly or indirectly through androgen stimulation, induce a significant
increase in neonatal prostate morphogenesis.
Comparative analysis of relative tissue areas and cellular area density
confirmed the histological similarities of the Mt-PRL and Pb-PRL transgenic
models, with marked expansion of the stromal compartment, resulting in a
marked increase in the stromal:epithelial ratio. The stromal phenotype is of
special interest because of its resemblance to human BPH which most
commonly also present a significant increase in primarily the stromal
compartment rather than epithelial [266, 267]. In addition, morphometric
quantitation of human BPH tissue firmly established a dominance of the
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Karin Dillner
stromal component, resulting in an increased stromal:epithelial ratio [21-24].
The implications of our findings in the PRL-transgenic mice models with
respect to a clinical setting have yet to be determined. Furthermore, in
symptomatic BPH patients, the stromal:epithelial ratio has been reported
significantly higher than in asymptomatic patients [22]. However, it is not
presently clear how the histological composition contributes to the
pathophysiology of clinical symptoms associated with BPH.
The histological resemblance of BPH and our PRL transgenic mice models,
regarding the importance of the stromal compartments for the development
of the prostate hyperplasia, is in line with the “embryonic reawakening
theory” of BPH etiology proposed by McNeal. This theory emphasizes that
BPH represents a reawakening of the embryonic and inductive potential of
prostatic stroma, which in turn induces hyperplastic changes in the
epithelium through stromal-epithelial interactions. Several studies have
proved the importance of the epithelial-stromal interactions both in normal
prostate development as well as the influence of abnormal reciprocal
interaction between epithelial cells and the embryonic mesenchyme or adult
stroma in the progression of neoplastic growth in the human prostate gland
[268]. Although the exact mechanisms of such tissue interactions are not
fully understood, there is growing evidence that they may operate through
cell-ECM interactions, remodeling of ECM and auto-/paracrine growth
factors [269].
Interestingly, the molecular patterns obtained from the gene expression
analysis using the methods of cDNA-RDA and cDNA microarray further
indicated a potential importance and possible activation of the stromal
compartment for the development and/or progression of the prostate
phenotype of the PRL transgenic mice. Although a few transcripts were
commonly found to be differentially expressed in both the Mt-PRL and PbPRL transgenic mice models, the more extensive nature of the cDNA
microarray, compared to that of the cDNA-RDA, enabled a greater insight
into the molecular mechanism behind the prostate phenotype of the Pb-PRL
than that of the Mt-PRL transgenic mice model. By this mean we do not
exclude the possible molecular similarities of these two models of inducing
the prostate phenotype, but a more extensive gene expression profiling of the
Mt-PRL transgenic mice await in order to be able to assign any more specific
molecular conclusions of this model’s phenotype. In addition, the differences
that these two models hold in terms of circulating androgen levels and ductal
morphogenesis further makes a direct comparison of these two models’
prostate phenotype and the identified differentially expressed transcripts
rather challenging.
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The broad molecular characterization made in the Pb-PRL transgenic mice
gave us interesting clues of possible molecular mechanisms of importance
for the prostate phenotype, including both the prostate hyperplasia and the
dysplastic lesions resembling PIN I and PIN II displayed in this mouse
model. Interestingly, numerous of the identified differentially expressed
transcripts are directly associated with stromal cells and the ECM proteins
that are secreted from stromal cells, including fibronectin, vimentin, laminin,
osteonectin and collagens. This may reflect altered activity of the prostatic
stromal cells in the prostates of the transgenic mice. In addition to their
structural role, ECM proteins have a pronounced influence on tissue
remodeling regulating cell growth, differentiation, communication, and
migration [269]. Moreover, degradation of the ECM, mediated by a variety
of proteolytic enzymes, such as matrix metalloproteinases (MMPs) and other
proteases, have significant roles in normal and pathological tissue
remodeling, including wound repair and tumorigenesis [51]. A set of
transcripts, including members of the families of cathepsins, MMPs and
TIMPs, were differentially regulated in the enlarged prostate of Pb-PRL
transgenic mice compared to controls. These transcripts may serve as
potential actors that modify the tissue homeostasis, possibly by changing the
reciprocal stromal-epithelial interactions, which eventually contributes to
promote the pathological tissue growth observed in the model. The
differential regulation of several of these candidate transcripts has previously
been associated with prostate disorders in both humans and animal models
indicating a relevance of these transcripts in the prostate pathogenesis of
PRL transgenic mice.
The phenomenon of tumorigenesis promotion by an activated stroma
(generation of a so-called “reactive stroma”) has previously been associated
with prostate pathology and other human cancers [52]. The reactive stroma is
characterized by ECM remodeling, elevated protease activity, increased
angiogenesis and an influx of inflammatory cells [52]. The list of
differentially regulated transcripts in the Pb-PRL transgenic prostate have
much in common with the processes involved in what is in the literature
described as characteristics of the reactive stroma (reviewed in [52]). The
up-regulation of vimentin together with a down-regulation of desmin,
suggest a myofibroblastic-like nature of the stroma cells, which is in line
with the described phenotype of reactive stroma. In addition, reactive stroma
cells typically express high levels of ECM components, such as collagen
type I and III, fibronectin and proteoglycans, as well as proteases that
degrade the ECM, observations that are in accordance with our present
results. One possible hypothesis might be that PRL influences the initial
induction of prostatic hyperplasia by modulating the stromal-epithelial
interaction that, in one way or the other, results in an activation of the stroma
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Karin Dillner
with the phenotypical features of reactive stroma, which fits well with the
stromal expansion as dominant feature of human BPH [21, 23, 24].
In contrast to the Pb-PRL transgenic mice, the Mt-PRL transgenic mice
exhibit an increased (in average about 3-fold) androgen serum level compared
to controls. Certainly, this could influence the phenotype of these mice, but in
what sense and to what degree is questionable. Nevertheless, significant
individual variations of circulating testosterone levels were seen in Mt-PRL
transgenic males (3.7-34 nmol/L of testosterone) without correlation to the
degree of prostate enlargement in the individual animals [105]. With the
intention to clarify the role of circulating androgen levels in the promotion of
abnormal prostate growth in the adult Mt-PRL transgenic mouse prostate, we
designed a castration and testosterone re-substitution study using age-matched
transgenic and wildtype mice (Paper I). The aim was to normalize the
circulating testosterone levels in young adult Mt-PRL transgenic males for a
prolonged time period of 8 weeks. From this study we concluded that elevated
serum androgen levels are not required for the progress of prostate hyperplasia
in adult Mt-PRL transgenic males. Furthermore, these findings are supported
by earlier reports in rodents using pituitary grafts, indicating a proliferative
effect of PRL on the prostate regardless of androgen status [102, 270].
The role of androgens as the causative factor for human BPH is debated.
However, they are certainly required to allow BPH development as indicated
by the facts that there are no reports of BPH occurring in castrated males.
This was in line with the observations in our castration study (8 week),
showing comparable post-castrational regressive changes in Mt-PRL
transgenic and control prostate (Paper I), clearly showing the androgensensitivity of the models. However, one should be aware of the differences in
long-term effects of androgen-withdrawal, causing prostate regression, and
the short-term effects of androgen-withdrawal, where PRL has been shown
to slow down the regression and act as a survival factor [107].
In rodents the effects of prolonged androgen treatment on prostate growth is
conflicting. Previous studies have showed both unaffected prostate size [255]
and induction of hyperplasia [256]. However, in paper I in this thesis, we
could not find any significant effect of prolonged androgen treatment on
prostate growth in wildtype adult mice. Moreover, the castration and resubstitution studies in the Mt-PRL transgenic mice demonstrated that
progressive prostate hyperplasia in adult Mt-PRL transgenic mice is not
dependent on elevated serum androgen levels. Furthermore, the Pb-PRL
transgenic mice display normophysiological serum androgens levels
throughout animal life span, which further support the hypothesis of ours
that the elevated circulating androgen levels are not responsible for the
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hyperplastic prostate phenotype seen in the Mt-PRL transgenic mice.
Prolonged hyperprolactinemia results in an increase in the prostate AR
distribution in our PRL transgenic mice models. Several modes of PRLinfluence on prostatic androgen sensitivity have been described earlier,
including up-regulation of ARs [175], increased activity of the enzyme 5alpha reductase [181] and increase in the uptake of testosterone into prostate
cells [183]. Conversion of testosterone to the more active androgen, DHT, is
primarily located to the stroma, due to the presence of type 2 5-alpha
reductase isoform in the stroma [271]. It is likely that 5-alpha-reductase
activity and AR distribution could play a role in the development of BPH.
The importance of stromal AR in the prostate is well known, and mediation
of some androgenic effects, such as ductal morphogenesis and epithelial
growth, has been proposed not to require intraepithelial AR [8, 272]. In our
study we demonstrated that testosterone treatment of adult wild-type mice
did not result in any significant prostate hyperplasia but resulted in an upregulation of epithelial AR, in contrast to unchanged stromal AR distribution
(Paper I). In both the Mt- and Pb-PRL transgenic models, we demonstrated
an increased precense of stromal AR, as detected by immunohistrochemical
analysis. Taken together, an increased stromal AR distribution may
contribute to the phenotype observed in our model. Interestingly, this
suggestion is further supported by the findings in the AR transgenic mice.
The AR transgenic mice, overexpressing the AR specifically in the prostate
secretory epithelium of DLP and VP, develop focal areas of intraepithelial
neoplasia, but no further progression into malignant lesions [154]. These
mice do not develop any signs of prostate hyperplasia, which the authors
suggested to be a result of the parallel increase of both proliferation and
apoptosis. Furthermore, regarding the similarities of the AR transgenic and
PRL transgenic mice in developing PIN formations, one might speculate that
the altered levels of epithelial AR are partially responsible. It would be very
interesting to make a parallel comparison of the AR distribution and
expression levels to further understand the similarities and differences
between these models.
In parallel with increased stromal AR, an increased stromal ERα content was
demonstrated (Paper III), although the relevance of this increase remains to
be determined. Recent work has established that both initiation and
progression of squamous metaplasia in the prostate after estrogen
administration are mediated through stromal ERα [158, 160]. Furthermore, a
distinct phenotype of focal epithelial hyperplasia in the VP has been reported
in aging mice lacking functional ERβ [162, 163], whereas no apparent
prostate pathology or enlargement has yet been reported in αERKO or
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Karin Dillner
αβERKO [155]. These findings are indicative of an anti-proliferative role for
epithelial ERβ and also suggest that an unbalanced stromal ERα action could
contribute to the phenotype observed.
In the normal adult prostate, a homeostasis appears to exist, whereby the
rates of prostatic cell growth and prostatic apoptosis are in equilibrium. A
changed balance between proliferative and apoptotic activity in the aging
prostate has been proposed as a mechanism for BPH as well as prostate
neoplasm formation and progression. Indeed, one of the proposed theories
behind the etiology of BPH, suggests an involvement of reduced rate of
apoptosis [42], based on the observations of reduced apoptotic activity in
BPH tissue compared to control [43, 44, 273]. Other studies have suggested
a potential role for the anti-apoptotic gene bcl-2 in BPH. In benign prostatic
tissues, bcl-2 expression is predominantly seen in basal epithelial cells and
has been associated with resistance to androgen ablation in BPH epithelium
[274]. The identified differentially expressed transcripts, both from the
cDNA-RDA and cDNA microarray analysis, gave us interesting insight into
possible molecular mechanisms that might contribute to the
development/progression of the prostate phenotype of the PRL-transgenic
mice. In addition to the large fraction of transcripts involved in the
aforementioned activation of the stromal cells, were transcripts associated
with apoptosis. To further validate those results, we used
immunohistochemical analysis for detection of two established apoptosis
markers, activated caspase-3 and presence of ssDNA. In the Pb-PRL
transgenic prostate, there were no immuno detectable levels of either
activated caspase-3 or ssDNA. In contrast, distinct clusters of apoptotic
epithelial cells were occasionally detected in distal regions of the ductal
system in all prostate lobes of control samples. These results clearly
indicated an overall diminished apoptotic activity in all prostate lobes of the
Pb-PRL transgenic mice compared to controls, results which correlate well
with the accepted participation of apoptosis in the BPH pathogenesis. In
addition, the results from the gene expression analysis in the Pb-PRL
transgenic mice demonstrate down-regulation of transcripts with proapoptotic activity and up-regulation of transcripts with anti-apoptotic
activity. Overall, this supports the importance of reduced apoptotic activity
in the pathogenesis of prostate hyperplasia in Pb-PRL transgenic mice.
Again, findings in the AR transgenic mice give interesting input to the
hypothesis of reduced apoptosis in the etiology of prostate hyperplasia. By
the presence of increased proliferation together with an elevated frequency
of apoptosis the authors explain the absence of hyperplasia phenotype in the
prostate of the AR-transgenic mice [154].
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The importance of PRL in regulation of apoptotic activity in the prostate is yet
to be determined. It is possible that PRL acts preferentially as a survival factor
rather than a growth factor. Earlier work has demonstrated PRL-induced
delay in castration-induced prostatic regression [108]. In line with this, PRL
was recently shown to significantly inhibit the androgen withdrawal-induced
apoptosis in DP and LP rat prostate cultures [107]. Moreover,
hyperprolactinemia has been shown to induce the synthesis of anti-apoptotic
Bcl-2 in rat prostate [275], which is in line with induced Bcl-2 expression in
human BPH tissue compared to control [274]. Furthermore, PRL has been
shown to inhibit TRAIL-induced apoptosis in the PC-3 prostatic cell line
[276]. TRAIL is a member of the TNF family that is known to induce
apoptosis in prostate cells [277]. This inhibition was suggested to be mediated
by increased phosphorylation of Akt/PKB, a critical regulator of cell survival.
The Akt pathway provides the survival signal that involves several proapoptotic proteins such as Bad [278, 279] and possible also other members of
the Bcl-2 family. PRL has been shown to possess an anti-apoptotic effect in
the rat decidua, and this was shown to involve inhibition of caspase-3
activity mediated by the Akt-pathway [280]. In that study, PRL was able to
down-regulate both caspase-3 mRNA levels as well as its activity. Taken
together, our results suggest an importance of reduced apoptotic activity in the
development of prostate hyperplasia in the PRL transgenic mice. The role of
PRL in this regulation needs further investigation, as well as the involvement
of diminished apoptotic activity for the development of prostate disease.
Overall, we conclude that the progression of the prostate hyperplasia in adult
Mt-PRL and Pb-PRL transgenic male mice does not require elevated
circulating androgen levels. Furthermore, the prostate phenotype of the two
PRL transgenic mouse models shares interesting histological characteristics
with human BPH. The use of differential gene expression technologies in our
studies has enabled us to find molecular similarities between the prostate
hyperplasia of the PRL transgenic mouse models and human prostate
disorders. Of particular interest is the potential significance of reduced
apoptosis for the development/progression of the prostate phenotype.
Another interesting observation is the importance and possible activation of
the stromal compartment for the development and/or progression of the
prostate phenotype of the PRL transgenic mice. There are striking
resemblance of the molecular pattern obtained in the PRL transgenic prostate
to that previously described in the literature as the “embryonic reawakening
theory” of BPH etiology and the theory of “reactive stroma” in prostate
cancer etiology.
The Pb-PRL transgenic model does resemble the situation in BPH better than
the Mt-PRL transgenic model since the prostate hyperplasia develops in a
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Karin Dillner
mature gland under normophysiological androgen levels. This in combination
with the molecular and histological similarities between the Pb-PRL
transgenic model and human prostate pathology illustrates the potential use of
this model as valuable tool in the study of BPH.
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CONCLUSIONS
•
Prolonged hyperprolactinemia (Mt-PRL) or prostate-specific PRL (PbPRL) overexpression leads to prostate hyperplasia
•
The PRL transgenic prostate is histologically characterized by a
prominent stromal hyperplasia with mild epithelial dysplastic features,
leading to an increased stromal/epithelial ratio. Accumulation of
secretory material is also a major characteristic.
•
Pb-PRL transgenic mice with normal circulating testosterone levels
develop prostate hyperplasia
•
Supraphysiological serum androgen levels are not required for the
progress of prostate hyperplasia in adult Mt-PRL transgenic mice and do
not induce prostate hyperplasia in androgen-treated wildtype mice.
•
PRL stimulates, directly or indirectly via increased androgen action,
prostate ductal morphogenesis in the developing prostate gland
•
Reduction of apoptotic activity might be involved in the development of
prostate hyperplasia in PRL transgenic mice
•
The changes in gene expression pattern seen in Pb-PRL transgenic
prostate suggest that activation of the stroma is important for the
development of prostatic hyperplasia
•
Histological and molecular similarities exist between the prostate
hyperplasia of PRL-transgenic mice and human prostate pathology,
including both BPH and prostate cancer
•
The use of differential gene expression analysis shows a great promise in
elucidation of molecular mechanisms behind the diseases of the prostate
gland
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Karin Dillner
ACKNOWLEDGEMENTS
I wish to express my sincere gratitude to everyone who has helped and contributed to this
thesis in one way or another. I would especially like to thank:
My outstanding supervisor and friend, Håkan Wennbo, for taking me on as a PhD student.
Your contribution to this thesis has been incredibly valuable due to your continuous support
and enthusiasm for this project, regardless of the physical distance between Stockholm and
Göteborg. Moreover, for all the superb dinners you have served during these years. You are
the absolutely best supervisor one can ever dream of and I hope we will always stay in
contact!
My supervisor, Gunnar Norstedt, for inviting me to work in your laboratory at the Center
for Molecular Medicine (CMM), Karolinska institutet. It has been an inspiring place to
work in and has enabled me to learn exciting differential gene expression methods and
bioinformatics. Furthermore, for interesting scientific discussions and sharing your
unlimited scientific visions.
My supervisor, Jan Törnell for giving me the opportunity to work at the Dept of
Physiology, Göteborg University. Olle G. Isaksson, for your generosity.
My co-author and colleague Jon Kindblom, for your important scientific input and
contribution in all possible ways to this thesis, and for guiding me as a PhD student
throughout these years. Furthermore, for all your work with the transgenic mice and for
your skilful handling with the knife, dissecting out the mouse prostates. Also for being a
splendid travel companion in Australia and in USA. I hope we will maintain our
collaboration.
Amilcar Flores-Morales, for distributing some of your infinite energy and teaching me
your enormous scientific knowledge in differential gene expression technologies and
accompanying bioinformatics. It has really been a pleasure to work with you.
See-Tong (Jacob) Pang, for being one of the few at CMM understanding the excitement in
hormonal regulation of the prostate. For answering all my clinical questions and for being a
great traveling companion in France and England. I hope we will keep in touch in the
future!
All former, present and associated members of GN’s group, CMM, for your scientifical
and non-scientifical contribution: Amilcar Flores-Morales, Christina von Gertten, Kåre
Hultén, Ingmarie Höidén-Guthenberg, Eva Johansson, Kristina Linder, Roxana
Merino, See-Tong (Jacob) Pang, Elizabeth Rico-Bautista, Nina Ståhlberg, Petra
Tollet-Egnell, Åsa Tellgren, Parisa Zarnegar – It has always been a pleasure to go to
work when you are all around creating a cheerful atmosphere!
All former and present colleagues at the Endocrine Division, Göteborg University,
especially: Håkan Billig, Jan Oscarsson and Staffan Edén for your contribution.
Moreover, Mohammad Bohlooly, Ola Brushed, Emil Egecioglu, Anders Friberg,
Fredrik Frick (baby elephant ☺), Maria Gebre-Medhin, Jenny Kindblom, Jon
Kindblom, Joakim Larsson, Daniel Lindén, Charlotte Ling, Emilia Markström, Bob
Ohlsson, Ruijin Shao, Klara Sjögren, and Louise Svensson for making me feel very
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welcome on my short visits at my home department, and some of you, for all the fun we
had traveling.
All the former and present members of ATCG and the Dept of Molecular Biology,
AstraZeneca R&D Mölndal, for all your help. A special thank to Harriet Thelander, for
your patience and assistance during my first year as a PhD student, and Maria Umaerus
for all your help in teaching me the technique of real-time RT-PCR.
My additional co-authors and co-workers: Chris Ormandy and Fiona Robertsson, Garvan
Institute in Sydney, Australia; Ruijin Shao and Charlotte Ling, Endocrine Division,
Göteborg University; Sophie Bernichtein and Vincent Goffin, INSERM, Paris, France;
Åke Pousette, Andrology Center, and Lena Sahlin, Dept of Woman and Child Health,
Karolinska institutet; Jan-Erik Damber, Dept of Urology, Sahlgrenska Hospital; Thank
you all for your fantastic help, interesting scientific discussions, great collaboration, and
providing me with tissues.
All former and present colleagues and friends at CMM, especially the members of Mats
Perssons’s group, Catharina Larsson’s group, Lars Terenius’s group, Georgy
Bakalkin’s group and Tomas Ekström’s group, for creating a great work environment.
For skillful and brilliant technical support: Kåre Hultén, Eva Johansson, and Britt
Masironi. For excellent secretarial service: Lena Olofsson at Göteborg University and
Christina Bremer, Delphi Post and Britt-Marie Witasp at Karolinska institutet.
Brita & Lasse, Eva, Nina Thérese and Åsa, for all your terrific dinners and all pleasant
time we have spent together, especially outside the CMM building.
Erika, Hanna, Linda, Sanna and Sara, for your support and all great laughs and
memories we have shared since our first day as undergraduates. Although we do not live in
the same cities anymore, I hope from all my heart that we will always stay updated and
keep in touch. Special thanks to Erika and Magnus for you endless hospitality during my
visits in Göteborg – it is always a pleasure to stay with such nice friends!
All my other friends outside the laboratory. In particular, my “bästis” Camilla, for your
long and valuable friendship.
The Family Larsson for your warm hospitality and your understanding when I left early in
the mornings to the lab on our short weekend visits.
My fantastic grand-mother, Anna – in every way you are my source of inspiration! My
parents Sven and Agneta, and my brother Fredrik, for always believing in me and
supporting me with all your love – it means so much to me!
Pär, my love, my life companion and my very best friend, for your endless support and
understanding. For driving me to work all mornings and being so patient waiting in the car
for hours when picking me up... You are the best and no one makes me as happy as you do!
This work was supported by grants from the Medical Faculty at Sahlgrenska Academy,
Swedish Society for Medical Research, Assar Gabrielsson’s Fund, Lars Hiertas Memorial
Fund, Foundation Clas Groschinsky's Memorial Fund, Wilhelm and Martina Lundgrens
Vetenskapsfond, King Gustav V Jubilee Clinic Cancer Research Foundation (Sahlgrenska
University Hospital), Konrad and Helfrid Johansson’s Foundation, Emil and Maria Palms
Foundation, Rådman och Fru Ernst Colliander’s Foundation, and AstraZeneca R&D.
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Karin Dillner
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Molecular Characterization of Prostate Hyperplasia in Prolactin Transgenic Mice Karin Dillner

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