DNA Alterations in Human Aberrant Crypt Foci and Colon

DNA Alterations in Human Aberrant Crypt Foci and Colon
Cancers by Random Primed Polymerase Chain Reaction
Liping Luo, Biaoru Li and Theresa P. Pretlow
Cancer Res 2003;63:6166-6169.
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[CANCER RESEARCH 63, 6166 – 6169, October 1, 2003]
Advances in Brief
DNA Alterations in Human Aberrant Crypt Foci and Colon Cancers by Random
Primed Polymerase Chain Reaction1
Liping Luo, Biaoru Li, and Theresa P. Pretlow2
Institute of Pathology [L. L., T. P. P.] and Department of Biochemistry [B. L.], Case Western Reserve University, Cleveland, Ohio 44106
Abstract
Colon cancers are the result of the accumulation of multiple genetic
alterations. To evaluate the role genomic instability plays during tumor
development, we compared DNA fingerprints of 44 aberrant crypt foci
(ACF; the earliest identified neoplastic lesion in the colon), 23 cancers, and
normal crypts generated by random primers with PCR. The PCR products, separated by PAGE and viewed after silver staining, demonstrate
altered fingerprints for 23.3% of the ACF and 95.7% of the cancers. In
this first study of human ACF with this approach, the finding of altered
DNA fingerprints in these microscopic lesions suggests that genomic
instability can occur very early in human colon tumorigenesis.
Introduction
ACF3 (Fig. 1A) are the earliest neoplastic lesions that can be
detected microscopically in whole mounts of human colonic mucosa
(1, 2). The prevalence of ACF is increased with familial adenomatous
polyposis and colorectal cancer (1, 3, 4). Demonstrations of monoclonality (2) and similar genetic alterations (5, 6) in ACF suggest that
ACF are precursors of cancer in human colon. Colorectal tumorigenesis is a stepwise process that involves multiple genetic alterations (7).
Mismatch repair deficiency gives rise to microsatellite instability that
characterizes hereditary nonpolyposis colorectal cancer. Microsatellite instability is also found in about 15% of sporadic colorectal
cancers (8) and a similar proportion of ACF (9, 10). However, most
colorectal cancers have multiple chromosomal abnormalities and a
high frequency of loss of heterozygosity that are thought to be the
result of general chromosomal instability or “CIN” (11). The RAPD
method, which amplifies random DNA fragments with single primers
of arbitrary nucleotide sequence, provides genomic profiles without
prior sequence information and has been used to detect and localize
allelic alterations in colon cancer (12–14). By comparing RAPD
fingerprints of human ACF and colon cancers with those of normal
crypts, genomic alterations were found in 22 of 23 colon cancers and
in 10 (23.3%) of 43 ACF analyzed.
Materials and Methods
Samples. Human colon specimens were collected in 4°C saline by the
Tissue Procurement Core Facility of the Comprehensive Cancer Center of
Case Western Reserve University and University Hospitals of Cleveland.
Strips of grossly normal mucosa (located between 4 and 28 cm from the
cancer; mean, 14 cm) were separated from the submucosa, snap-frozen flat
over liquid nitrogen, and stored at ⫺195°C. ACF and two samples of normal
crypts were collected under a dissecting microscope as described by Bird et al.
Received 6/2/03; revised 7/25/03; accepted 8/5/03.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported in part by NIH Grants CA66725 and CA43703.
2
To whom requests for reprints should be addressed, at Institute of Pathology, Case
Western Reserve University, 2085 Adelbert Road, Cleveland, OH 44106. Phone: (216)
368-8702; Fax: (216) 368-1278; E-mail: tpp3@cwru.edu.
3
The abbreviations used are: ACF, aberrant crypt foci; RAPD, random amplified
polymorphic DNA.
(15) from the same specimen (average area evaluated, 11 cm2) of colonic
mucosa fixed for 30 min in 70% ethanol and stained with 0.2% methylene
blue. From the same patient, cancer samples with ⬎50% malignant cells were
obtained from frozen sections adjacent to H&E-stained sections of tumor. In
the first experiment, ACF, normal crypts, and tumor from 23 different patients
were amplified by PCR with three to eight random primers, depending on how
much DNA was available. In the second experiment, 21 ACF and matching
normal crypts from 16 additional patients were amplified with 10 random
primers. Two samples of normal crypts were used for each patient. One sample
contained the same number of crypts as were in the ACF; the second sample
contained twice as many crypts to see whether DNA concentration altered the
fingerprint pattern. All 39 patients had colorectal cancer; 18 had Dukes’ stage
B, 16 had stage C, and 5 had stage D cancer. The patients ranged in age from
34 to 98 (67 ⫾ 13) years old. The ACF had 52 ⫾ 36 (range, 13–150) aberrant
crypts per focus and covered an area of 1.68 ⫾ 1.16 mm2 (range, 0.39 – 4.95
mm2); 10 (23%) ACF were from the right colon, and 34 (77%) were from the
left colon.
Arbitrarily Primed-PCR Amplification. Samples of ACF, normal crypts,
and tumors were suspended in 1⫻ PCR buffer [10 mM Tris-HCl (pH 9.0), 2%
formamide, 50 mM KCl] that contained 200 ␮g/ml proteinase K (Fisher
Scientific, Pittsburgh, PA) and 0.5% Tween 20. After incubation of the sample
at 42°C for 24 h, the proteinase K was inactivated at 95°C for 10 min. The
extracted DNA was cooled to 4°C in an ice bath and used for PCR without
purification.
PCR-based amplification of random DNA segments with single primers of
arbitrary nucleotide sequence (12, 16) was used to detect genetic changes. The
primers chosen for our studies were 10 –24 nucleotides in length, had a G⫹C
content between 46 and 67%, and contained no palindromic sequences (Table
1). Some of the individual primers were combined, as noted below, to generate
additional fingerprints as demonstrated previously (16). PCR was carried out
in a volume of 50 ␮l that contained 0.4 ␮M primer, 20 to 100 ng genomic
DNA, 2 mM MgCl2, 250 ␮M each dNTP, and 1 unit of Taq polyperase (Fisher
Scientific) in 1⫻ PCR buffer. In the first experiment with samples from 23
patients, two-stringency PCR was performed with primers P2, P3, P4, P5, P6,
P1⫹P2, P2⫹P6, and P4⫹P6. PCR amplifications were carried out in a thermal
cycler (MJ Research, Inc., Watertown, MA) for 10 cycles of low stringency
(95°C for 30 s, 36°C for 40 s, and 72°C for 30 s) followed by 30 cycles of high
stringency (95°C for 1 min, 50°C for 1 min, and 72°C for 1 min). In the second
experiment with 21 samples of ACF and normal crypts, two-stringency PCR
was carried out for the primers (P2, P5, P6, and P1⫹P2) that gave the clearest
RAPD fingerprints in the first experiment. An additional six primers (PGKB,
P2⫹P10A, P2⫹P10B, P2⫹FO3, P4⫹FO3, and P5⫹FO3) were used to amplify these 21 samples for 40 cycles (95°C for 1 min, 36 – 40°C for 1 min, and
72°C for 2 min). Six ␮l of PCR products were mixed with loading buffer and
separated in 6% polyacrylamide denaturing gel in a sequencing gel electrophoresis apparatus (Model S2; Life Technologies, Inc., Gaithersburg, MD)
with 60 W for 3 h. The gels were viewed after silver staining (17).
Semiquantification of PCR Results. The mean absorbance of each PCR
band was evaluated with Kodak 1D Image Analysis Software (Scientific
Imaging Systems; Eastman Kodak Co., New Haven, CT). A single band
(marked “S” in Figs. 1 and 2), that appeared in all of the lanes with near equal
intensities, was chosen as a standard band for each patient. The density of each
band in a lane was standardized against this S band by forming a ratio of the
bands. An allelic ratio was then determined for each band in the ACF (A) or
tumor (T) lane by dividing the standardized density of the ACF or tumor band
by the standardized density of the same band in the normal sample(s), e.g.,
Ta:Ts/Na:Ns. When two normal samples were evaluated for the same patient,
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ALTERED DNA FINGERPRINTS IN HUMAN ACF
Table 1 Arbitrary primers used in RAPD
Primer
Sequence 5⬘–3⬘
Reference
P1
P2
P3
P4
P5
P6
F03
P10A
P10B
PGKB
CTT GCG CGC ATA CGC ACA AC
AAC CCT CAC CCT AAC CCC AA
AAC CCT CAC CCT AAC CCC GG
CCC CAC CGG AGA GAA ACC
GAT AGC CAG CAC AAA GAG AGC TAA
CGA CCG TGT TTT GCA AAC AGA TGT
CCG GCT ACG G
ACG GTA CAC T
ACG GTA CAC G
CCT ACA CGC GTA TAC TCC
(13)
(13)
(22)
(23)
(23)
(23)
(24)
(12)
(12)
(16)
an average of the two standardized values was used. An allelic ratio of 2 or
greater was considered a gain of a band; an allelic ratio of 0.5 or less was
considered a loss of a band. This is in the same range as used by us and others
to determine allelic loss (2, 18).
Results
Reproducible RAPD fingerprints from multiple patients (Fig. 1)
were generated by PCR amplification of genomic DNA with each
random primer or random primer pair. Samples 4004233 (Fig. 1C)
and 4003641 (Fig. 1D) have similar RAPD fingerprints with multiple
bands between 100 and 500 bp when amplified with the primer pair
P4⫹P6. For sample 4004233 (Fig. 1C), there are multiple changes
that occur in both the ACF and tumor, and additional alterations (gain
of bands at a2 and b) that occur only in the tumor when the RAPD
bands are compared with those from normal crypts. For sample
4003641 (Fig. 1D), there are different alterations in both the ACF and
the tumor; i.e., there is a loss of a band at d in the ACF that is not seen
in the tumor, and there are two alterations in the tumor (a gain of a
band at a1 and a loss of a band at g) that are not seen in the ACF.
In addition, each random primer or random primer pair generated a
unique RAPD fingerprint for each patient (Fig. 2). Amplifications
were successful for 43 of 44 ACF samples; 10 (23.3%) of 43 ACF
showed a gain and/or loss of RAPD bands compared with the fingerprints of the corresponding normal crypts (Table 2; Figs. 1 and 2).
One of the ACF (from patient 4002483) showed RAPD alterations
with three primers (Table 2). For three ACF, somewhat similar
alterations of RAPD fingerprints were observed both in the ACF and
cancer samples, compared with fingerprints of corresponding normal
crypts from these same patients (Fig. 1C, discussed above; Fig. 2, A
and C). For the ACF in Fig. 2C, there was an additional loss of a band
at allele a that was not seen in the tumor. For most ACF, the genomic
changes detected in the ACF differed from those seen in the corresponding cancer samples (Fig. 1, C and D and Fig. 2, B, E, and F). In
the second experiment, one ACF shows both the gain and loss of DNA
bands (Fig. 2D), whereas two ACF (Fig. 2, G and H) show only a loss
of DNA bands when their RAPD fingerprints are compared with the
fingerprints from normal crypts (tumor samples not done). It is interesting that the sizes of the ACF with altered bands vary widely, from
13 crypts to 109 crypts, i.e., the smallest ACF with 13 crypts is
included but not the largest ACF with 150 crypts. In fact, 7 of the 10
ACF with altered bands have less than 52 crypts, the average size of
the 44 ACF analyzed. The age, sex, location in the colon, and stage of
colon cancer did not influence the occurrence of altered bands in the
ACF that were analyzed.
As illustrated (Figs. 1 and 2), cancer samples generally showed
more altered bands per RAPD fingerprint than did the ACF with the
same primer. All 23 cancer samples were successfully amplified, and
22 (95.7%) of 23 cancers showed altered fingerprints when compared
with the fingerprints of their corresponding normal crypts. The cancer
samples also had more altered RAPD fingerprints per primer than did
the ACF (Fig. 3); 17 tumors exhibited genomic alterations with two or
more random primers. The total number of altered RAPD fingerprints
per successful amplification was low for ACF (12 of 309 or 3.9%)
compared with cancer samples (77 of 134 or 57.5%; P ⬍ 0.01). The
main genetic alterations are gain and/or loss of RAPD bands that are
observed both in ACF and cancer samples compared with the fingerprints of normal crypts. In addition to these quantitative alterations,
we also observed qualitative changes in the relative intensity of DNA
bands between those from ACF or cancer samples and those from
normal crypts (Fig. 1D, allele d in tumor).
Discussion
To our knowledge, this is the first report of altered DNA fingerprints in human ACF, the earliest identified neoplastic lesions in the
colon (2). Genome-wide alterations identified with RAPD in 23.3% of
Fig. 1. DNA fingerprints from two different
patients (4004233 shown in C and 4003641 shown
in D) amplified with the same random primer
(P4⫹P6). A, an ACF with 48 crypts from 4004233
that was used for the DNA fingerprint in C (Lane
A). B, frozen section of cancer stained with H&E
from 4004233 that was used for the DNA fingerprint in C (Lane T). C and D, DNA fingerprints of
normal crypts (Lane N), ACF (Lane A), and cancers (Lane T) from patients 4004233 and 4003641,
respectively. Arrows show the altered DNA bands,
i.e., ACF and/or tumor bands with allelic ratios of
0.5 or less or with allelic ratios of 2.0 or greater;
numbers at left, length in bp. In C (patient
4004233), losses of DNA bands for alleles c, d, e,
f, and g (allelic ratios of 0.2– 0.5) are observed in
both ACF and tumor; gains of DNA bands for
alleles a2 and b (allelic ratios 2.4 and 3.0, respectively) are seen only in tumor. In D (patient
4003641), there is a loss of allele d (allelic ratio,
0.3) only in the ACF, whereas the tumor shows a
gain of a band at a1 (allelic ratio, 2.6) and a loss of
a band at g (allelic ratio, 0.5).
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ALTERED DNA FINGERPRINTS IN HUMAN ACF
Fig. 2. Genomic fingerprints of normal crypts (Lane N), ACF (Lane A), and cancers (Lane T), obtained with primers described in Tables 1 and 2, from eight different patients (Table
2). Arrows show the altered DNA bands, i.e., ACF and/or tumor bands with allelic ratios of 0.5 or less or with allelic ratios of 2.0 or greater. Similar gain of a DNA band is seen in
both ACF and cancer (allelic ratios 2.6 and 2.3, respectively) from the same patient in column A. In column B, only the tumor shows a gain of a DNA band at a (allelic ratios 2.4 for
Lane T and 1.5 for Lane A) and only the ACF shows a gain of a DNA band at b (allelic ratios 2.0 for Lane A and 1.6 for Lane T). Both the ACF and tumor show the gain of multiple
bands in C, but only the ACF shows the loss of a band at a (allelic ratio 0.4). The ACF in D shows the gain of DNA bands at alleles a and b (allelic ratios, 2.1 and 2.9) and the loss
of bands at alleles c and d (allelic ratios, 0.5). In E, the gain of DNA bands are seen in alleles a, b, c, d, f, g, i, and j (allelic ratios, 2.1–3.6) in the cancer, whereas the loss of DNA
bands in alleles e and h (allelic ratios, 0.5) are seen in the ACF. In F, the loss of a DNA band at allele a (allelic ratio, 0.3) is seen in the ACF with the gain of bands at alleles b, c,
and d (allelic ratios, 3.1– 8) in the cancer. Multiple losses of DNA bands in ACF were observed in G (allelic ratios, 0.2– 0.4) and H (allelic ratios, 0.3– 0.5).
ACF suggest that chromosomal instability is a very early event and
might play a crucial role during the development of some colorectal
cancers. There are previous reports of chromosomal instability as
early as the polyp stage (19, 20), and some have suggested that genetic
instability is required for the development of tumors (discussed in
Refs. 20 and 21). Although RADP was not used to identify genomic
alterations in those polyp studies, Peinado et al. (13) used two arbitrary primers to identify genetic alterations in a large number of
human colorectal cancers and a few polyps. By cloning and further
analysis of the altered RAPD bands in tumors, they demonstrated that
these altered bands are the result of losses or gains of DNA sequences
in the original tumor (13). As compared with colorectal tumors, the
smaller numbers of altered bands per human ACF and the smaller
proportion of ACF with genomic alterations support the role of ACF
as early precursors of some colorectal cancers. Luceri et al. (14), with
21 random primers, found genomic alterations in 16 of 16 colon
tumors and 7 of 10 ACF induced in rats with azoxymethane. The
finding of more alterations in rat ACF, compared with our study with
human ACF, could be attributable to species differences, the use of
more and/or different primers in the rat study, and/or the presence of
more advanced lesions in rats treated with a carcinogen.
The advantages of RAPD for our studies are that it requires only
small amounts of DNA to generate genome-wide fingerprints that can
show multiple alterations and it does not require prior knowledge of
DNA sequences. Two-stringency PCR was used in our first study as
used previously in the study of human colon tumors (13). The initial,
low-stringency condition allows a large number of hybridizations to
take place throughout the genome; the second, high-stringency condition allows only the segments that closely match the primer to be
amplified further. In addition we added primers in pair-wise combinations, which produce distinct genomic fingerprints different from
those generated with either single primer alone (16). Whereas these
conditions produced fingerprints that demonstrate multiple differences between normal mucosa and 95.7% of our cancer samples, they
did not reveal as high a proportion (7 of 22, or 32%) of altered human
ACF as had been reported in the rat study (14). In hopes of improving
our results in the second experiment with 21 additional ACF, we used
only four primers from our first experiment and added six new
primers with a single stringency (12). This time only 3 of 21 or 14.3%
of the ACF showed alterations. From these very limited experiments,
it appears that the two-stringency PCR with the original primers,
especially P4⫹P6 and P5, was more effective with our very small
Table 2 Human ACF with altered RAPD fingerprints
Patient
4002298
4002483
4002518
4005933
4002393
4004233
4003641
4004689
4006039
93-04-W219
2
No. of crypts in ACF
Size of ACF (mm )
Dukes’ stage
Age
Sex
Primer
Alteration of DNA bands
Gel in Fig.
13
58
0.39
1.78
Left
Left
B
C
75
57
F
M
0.5
3.25
1.17
2.28
1.18
1.2
0.61
3.23
Left
Right
Left
Left
Left
Left
Right
Left
B
C
B
C
C
C
D
C
70
87
41
68
52
76
63
71
M
M
M
M
F
F
F
F
Gain
Gain
Gain
Loss
Gain/Loss
Gain/Loss
Loss
Loss
Loss
Loss
Loss
Loss
2A
2B
20
109
41
48
40
33
26
65
P2
P5
P2⫹P6
P1⫹P2
P4
P2⫹F03
P4⫹P6
P4⫹P6
P4⫹P6
P4⫹P6
P5
P5
Location in colon
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2C
2D
2E
1C
1D
2F
2G
2H
ALTERED DNA FINGERPRINTS IN HUMAN ACF
Fig. 3. Percentage of altered RAPD profiles when ACF and colon cancers from the
same patients were successfully amplified with each of the listed random primers or
primer pairs.
samples of human DNA. Consequently, our results likely underestimate the amount of chromosomal instability in human ACF.
The wide range of sizes of ACF, from 13 to 109 crypts, with altered
fingerprints, also argues that chromosomal instability occurs very
early in colon tumorigenesis. Some ACF (Fig. 2, A and C) have gains
of DNA bands that are similar to those seen in the tumor samples from
the same patient. These results suggest that these alterations observed
in the tumors occurred early in the ACF and persisted in the final
cancer. One ACF and tumor (Fig. 1C) show multiple similar losses of
DNA bands, but the tumor shows additional alterations. This supports
the hypothesis that ACF are early precursors that gain additional
alterations to become cancer. However, several ACF (Figs. 1D and 2,
B, C, E, and F) show losses or gains of DNA bands that are not seen
in the cancers from the same patients. One possible explanation is that
these changes observed in the ACF do not contribute to tumorigenesis,
i.e., these ACF are not likely to persist. A second equally plausible
explanation is that each tumor develops independently along its own
unique pathway, and not every change observed in each ACF will be
observed in all tumors.
In summary, the observations of altered fingerprints in microscopic
lesions known as ACF suggest that chromosomal instability can occur
very early in colon tumorigenesis and may be a driving factor of this
process. Future studies of larger numbers of ACF and cancers with
RAPD might aid in finding the earliest molecular changes that occur
in colon tumorigenesis.
Acknowledgments
We thank Karen Stiffler and Erin Vittori for their technical assistance.
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