The tetrapeptide AcSDKP specifically blocks the cycling of primitive

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1994 84: 1534-1542
The tetrapeptide AcSDKP specifically blocks the cycling of primitive
normal but not leukemic progenitors in long-term culture: evidence
for an indirect mechanism
JD Cashman, AC Eaves and CJ Eaves
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The Tetrapeptide AcSDKP Specifically Blocks the Cycling of Primitive
Normal But Not Leukemic Progenitors in Long-Term Culture:
Evidence for an Indirect Mechanism
By J.D. Cashman, A.C. Eaves, and C.J. Eaves
In the present study,we investigated the ability of the tetrapeptide NAc-Ser-Asp-Lys-Pro-OH(AcSDKP), a repotted inhibitor of primitive hematopoietic cells, to influencethe proliferative behavior of primitive normal and chronic myeloid
leukemia (CML) progenitor cells in the adherent layer of
long-term cultures (LTCs). Addition of a50 ng/mL of AcSDKP
t o LTCs of normal cells at the time of the regular weekly
half-medium changeselectivelyand reversibly decreased
the proportion of high proliferative potential erythroid and
granulopoietic progenitors in the adherent layer that were
in S-phase without changing their numbers, but had no effect on either the cycling activity or number of analogous
(neoplastic) cells in the adherent layer of CML LTCs. Specificity of the effect of AcSDKP on primitive normal progenitors was demonstrated by thefinding that a similar addition
of either the control peptide, AcSDKE, or 100 ng/mL of tumor
necrosis factor-a (TNF-a, which contains the SDKP sequence), or SDKP itself (at 300 ng/mL) did not inhibit the
proliferation of primitive normal progenitors in LTC adherent
layers. Incorporation of 230 ng/mL of AcSDKP (but not the
related control peptide, AcSDKE) directly into methylcellu-
lose cultures of normal marrow cells resulted in a dose-dependent suppression of colony formation, which was not
seen in similar studies with CML marrow or after removal of
adherent cells from normal marrow. Additional experiments
showed that the inhibitory effect of AcSDKP on primitive
normal progenitor cycling in theLTC system could be overcome by the simultaneous addition of macrophage inflammatory protein-1/3 (MIP-1/3); an antagonist of MIP-la. The
apparent differential effect of AcSDKP on primitive normal
and CML progenitors may thus be a secondary consequence
of the dtferential responsiveness of these cells to MIP-la
(or another molecule antagonized by MIP-1/31. whose production or releaseby adherent marrow cells is inducible by
AcSDKP.Such a mechanism may offer a method for obtaining localized increases in vivo of cytokines like MIP-la,
suggesting novel and perhaps less toxic strategies for protecting primitive normal progenitors during repeated treatesments with cycle-active chemotherapeutic agents where
calating the dose of drug given would be desirable.
0 199J by The American Society of Hematology.
T
cells in vivo, because these cells are endogenously supported
and regulated in the LTC system by mesenchymal fibroblastlike cells that appear ontologically related to the cells that
constitute the marrow microen~ironment.~”
For example,
both high proliferative potential erythropoietic and granulopoietic cells that are normally quiescent in vivo, are also
maintained in a similar state in the fibroblast-containing adherent layer of LTCs, unless these are perturbed by the addition of any of a variety of indirect-acting, as well as directacting, cytokines following eachweekly change of the
medium.*”’ Incontrast, the same types of progenitors located
in the nonadherent fraction (or in cultures in which no fibroblasts are present) proliferate c o n t i n u ~ u s l y . ~ ~ ’ ~
Such observations suggest that the adherent layer of LTCs
serve as a source of both inhibitors and stimulators of primitive hematopoietic cell cycle progression; the local balance
of which can be altered by cytokine manipulation. We have
previously identified transforming growth factor-p (TGF-p)
as one of the endogenously produced inhibitors that contributes to the proliferation arrest of primitive normal hematopoietic cells in the adherent layer of unperturbed LTCS.’’
More recent studies indicate that endogenously produced
macrophage inflammatory protein-a (MIP-la), or a related
chemokine whose activity may likewise be blocked by MIPlP,l4may have a similar function and acts in LTCs inconcert
with TGF-P.I5Less is known about the identities of endogenous factors that mediate the supportive/stimulatory functions of the adherent layer. Multiple candidates have been
suggested, including interleukin-6 (IL-6), granulocyte CSF
(G-CSF), and GM-CSF alone, or in ~ o m b i n a t i o n ~ ~and
“~”
additional possibilities exist, including factors like interleukin-l 1 (IL-1
Steel factor,* and the ligand for flk-2/flt3” that marrow fibroblasts are also known (or thought) to
produce. Recent results demonstrating thathuman clonogenic progenitor productionin vitro canbe supported for
HE PRODUCTION OF mature blood cells continues
throughout adult life, and involves coordinated
changes in cell phenotype that span many cell generations.
Regulation of hematopoiesis is thus typically visualized in
terms of the responses of a hierarchy of cells to extracellular
factors that either directly or indirectly control their proliferation, differentiation, viability, and location. Many of these
factors are believed to be produced by fixed cellular elements
of the marrow where their actions are local. This maybe
due, in part, to the physiologic maintenance of levels of
factor production that are of limited effectiveness, although
additional mechanisms, including the production of membrane-bound forms of some growth factors (eg, colony-stimulating factor-l [CSF-l]’ and Steel facto?), and the ability
of others to bind to extracellular matrix components (eg,
granulocyte-macrophage-CSF [GM-CSFI3) have also been
described. Long-term marrow cultures (LTCs) have been
particularly useful in the identification of candidate cytokines
that may control the proliferation of primitive hematopoietic
From the Terry Fox Laboratory, British Columbia Cancer Agency,
and Departments of Medical Genetics, Medicine, and Pathology,
University of British Columbia, Vancouver, Canada.
Submitted October 11, 1993; accepted May 9, 1994.
Supported by the National Cancer Institute of Canada. C.J.E. is
a Terry Fox Cancer Research Scientist of the National Cancer Institute of Canada.
Address reprint requests to C.J. Eaves, PhD, Terry Fox Laboratory, 601 W 10th Ave, Vancouver, British Columbia V52 IW, Canada.
The publication costsof this article were defrayedin part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8405-06$3.00/0
1534
Blood, Vol 84,
No 5 (September l), 1994: pp 1534-1542
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INDIRECT REGULATORY ACTION OF AcSDKP
many weeks as effectively when cocultured with various
types of murine fibroblasts, as with the complex array of
cell types present in the adherent layer of primary human
marrow LTCs, indicate that at least L-6, G-CSF, GM-CSF,
and Steel factor are not essential?.’’
In this study, we have used the LTC system to analyze
the activity of another candidate inhibitor of primitive hematopoietic cells, the tetrapeptide NAc-Ser-Asp-Lys-Pro
(AcSDKP). It has been previously reported that this tetrapeptide can block the proliferation of clonogenic cells freshly
isolated from normal marrow and cord blood:0,21 can cause a
transient decrease in nonadherent cell and progenitor output
when added repeatedly to normal LTCS,” and is detectable
In
in
experiments with marrow from patients with
chronic myeloid leukemia (CML), no effect of AcSDKF’ on
the cycling of freshly isolated neoplastic progenitors was
observed.*’ We have recently shown that the deregulated
proliferative activity of primitive CML progenitors, seen
both in vivo” and in LTCs,” may be explained by their
selective unresponsiveness to the inhibitory effect of ME”
la.’5It was, therefore, of interest to determine if AcSDKP
addition to LTCs would mimic the effects of M P - l a and
TGF-fi on normal cells in a system that shows many of the
regulatory characteristics of the marrow microenvironment,
and, if so, whether the primitive subsets of neoplastic progenitors in LTCs of CML cells would prove responsive or unresponsive to AcSDKP addition. The present study was undertaken to investigate these possibilities.
MATERIALS AND METHODS
Reagents. The tetrapeptide AcSDKF’, its unacetylated form
SDKF’, and a control peptide (AcSDKE) were synthesized and purifiedto homogeneity by high-performance liquid chromatography
(HPLC) at the Microsequencing Centre of the University of Victoria,
Victoria, Canada. The purity of all three peptides was established
by amino acid analysis, as well as by mass spectrometry and HPLC.
They were stored in concentrated form at 4°C until just before addition to either methylcellulose cultures or LTCs, as indicated. Highly
purified (>95%) recombinant human “-la
expressed by
transfected Chinese hamster ovary cells was obtained as a gift from
S . Wolpe (Genetics Institute, Cambridge, MA). The final material
was stored at -20°C in a proprietary buffer designed to minimize
aggregation, and was diluted in myeloid LTC medium (Stemcell
Technologies, Vancouver, Canada) just before addition to the cultures. Recombinant human MIP-1P in the form ofa
cDNA
transfected COS cell culture supernatant was also a gift from S .
Wolpe. Purified recombinant human MIP-1P was purchased from
R & D Systems, (Minneapolis, MN). Purified recombinant human
tumor necrosis factor-a (TNF-a) was purchased from R & D Systems, and high specific activity 3H-thymidine (25 Ci/mmol) was
purchased from Amersham (Oakville, Canada).
Cells. Normal bone marrow (BM) aspirate cells were obtained
either as leftover cells from allogeneic transplants or as cadaveric
material stored frozen at - 135°C until use.25Peripheral blood (PB)
and BM cells from Philadelphia chromosome (Ph)-positive chronicphase CML patients were obtained at diagnosis, or as part of routine
follow-up procedures. All CML patients whose cells were used in
these experiments had elevated white blood cell counts (>50 x lo9/
L) at the time of study. Both normal and leukemic samples were
obtained with informed consent according to guidelines approved
by the Clinical Screening Committee for Research and Other Studies
Involving Human Subjects of the University of British Columbia.
1535
Normal and CML BM cells were prepared for colony assays by
ammonium chloride lysis of contaminating red blood cells and, after
washing, were diluted in complete methylcellulose culture medium
to give a find concentration of 2 X io5or 5 X 104 ceh/n& respectively. Cell suspensions for use in colony assays of cultured marrow
cells before and after adherent cell depletion were obtained from
the nonadherent fraction of 10-day-old LTCs initiated with normal
BM. For each experiment, the nonadherent cells from several such
cultures established from a single inoculum of marrow were pooled,
centrifuged on Ficoll-Paque (Pharmacia Biotech, Baie d’Urfe, Quebec, Canada) to isolate the light density ( ~ 1 . 0 7 7@cm3) fraction,
and the cells then washed and either cultured directly in methylcellulose at lo5 cells/mL, or first placed in tissue culture flasks (3 X lo6
cells/lO cm2) in Iscove’s medium with 20% fetal calf serum (FCS)
and incubated overnight at 37°C to obtain an adherent cell-depleted
fraction.
LTCs. Conventional LTCs were established by placing aliquots
of 2.5 X lo7 fresh unprocessed normal BM aspirate cells into 60mm tissue culture dishes in 8 mL of myeloid LTC medium (Stemcell
Technologies). This consists of an enriched a-medium supplemented
with 12.5% FCS, 12.5% horse serum, and
moVL 2-mercaptoethanol. Freshly dissolved hydrocortisone sodium hemisuccinate was
added immediately before use to give a final concentration of
moVL. LTCs were incubated in a humidifiedatmosphere of 5% COz
in air at 37°C for the first 3 to 4 days, and thereafter at 33°C. At
the end of the first 10 days, after 14 days, and weekly thereafter,
half of the medium and half of the nonadherent cells were removed,
and an equal volume of fresh LTC medium was added as previously
described.26
For experiments in which LTCs were initiated on preestablished
feeders, the latter were obtained from conventional LTCs initiated
with normal BM, and incubated for at least 2 to 3 weeks to generate
confluent adherent layers. These cells were then trypsinized, irradiated in suspension with 15 Gy of 250 kVp X-rays, and subcultured
into new tissue culture dishes at lo6 cells per 60-mm dish or 3 X
lo5 cells per 35-mm dish in 8 or 2.5 mL of LTC medium, respectively.” Normal or neoplastic hematopoiesis was then initiated by
seeding 8 X lo6 light-density normal BM cells or 6,000 to 10,000
CD34WLA-DR’” (FACS-sorted) light-density normal BM cells
(also gated for low-forward and low-orthogonal light scatter properties as previously described6) or 2.5 X lo7 light-density CML PB
cells onto normal marrow feeder layers. These were then placed at
33°C and used for progenitor cycling studies at the time of the first
(day lo), second (day 14), or third (day 21) half-medium change.
The presence of the preestablished feeders eliminated the 3 weeks
otherwise required for the formation of an adherent layer able to
inhibit the cycling of primitive normal clonogenic cells (eg, see
Table 1).
Methylcellulose msuys. Methylcellulose cultures containing 3
U/mL of highly purified (80,000 U/mg) human erythropoietin and
10% (voUvo1) agar-stimulated human leukocyte-conditioned medium (LCM) (Stemcell Technologies) were used to assay for clonogenic cells in most experiments. The exceptions were those involving
assays of cells from LTCs initiated with CD34+DRloWcells, where
the LCM in the methylcellulose cultures was replaced with 50 ng/
mL of Steel factor (Amgen), and 20 ng/mL each of L-6 (Immunex,
Seattle, WA) GM-CSF (Sandoz, Basel, Switzerland), G-CSF (Amgen. Thousand Oaks, CA), and L - 3 (Sandoz). This change in factors
used to stimulate colony formation had no effect on the ’H-thymidine
suicide values calculated for high and low proliferative potential
clonogenic progenitors identified using the same scoring criteria
originally developed for LCM-containing assays, and results have
therefore been pooled (Table 1).
3H-thymidinesuicide procedure. After trypsinization of the adherent layer,” cells were washed twice in Iscove’s medium, diluted
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1536
CASHMAN, EAVES, ANDEAVES
Table 1. Simultaneous Addition of MP-1p and AcSDKP to Normal
LTCs Allows the Activation of Primitive Progenitors inthe
Adherent Layer
% Kill of Primitive Progenitors
After Exposure to 'HAddition
None
AcSDKP
62
2
50
45
MIP-18
AcSDKP + MIP-10
9
2 1
2 13
-t 15
2
Percent kill values for all primitive progenitors (ie, primitive BFU-E
and primitive CFU-GM combined) in the adherent layer were determined 2 to 3 days after the first half-medium change, ie, 10 to 12 days
after initiating the cultures, and are based on actual colony counts (in
the control assays) of 18 to 113 colonies. In two experiments, these
were initiated from light-density normal BM cells, and in two from
FACS-sorted CD34' HLA-DR'"" light-density normal BM cells. None
refers to a regular half-medium change. AcSDKP was added to give
a final concentration of 200 ng/mL and MIP-1P to give a concentration
of 300 ng/mL. Results from all four experiments were similar, and
have therefore been combined. Values shown are the mean 2 SEM
of the individual percent kill values from these four experiments.The
total number of primitive progenitors per LTC adherent layer relative
to LTCs given a regular half-medium change only (=100%)was 230%
If- 70%, 210% 2 3096, and 230% 2 70% for AcSDKP, MIP-18, and both
combined, respectively.
to IO6 cells/mL in Iscove's medium (no FCS) and twoI - m L aliquots,
incubated for 1 hour at 37°C in an environment of 5% CO2 in air
to restabilize the cells ata fixed temperature and pH (=7.2). To one
of these aliquots,a small volume containing20 pCi of high-specific
activity 'H-thymidine was added and both aliquots ofcells incubated
at37°C in anatmosphereof 5% CO2 in air foranadditional 20
minutes.Excesscoldthymidinewasimmediatelyaddedtoboth
tubes, and thecells washed twice before being plated in methylcelluIO5 original
losemediumat
a finalconcentrationequivalentto
adherent cells/mL, assuming no losses during the entire procedure.
Differences in the number of colonies obtained in assays of the 'Hthymidine-treatedcellscomparedwiththecontrolcellsareexpressedaspercent kill values? These represent the proportion of
clonogenic cells (of thetype designated) that werein S-phase at the
time of harvesting the cells from the LTCs.
Colonies were evaluated according to their size and morphology
to determine the stage
of maturation of thedifferent typesof progenitor cells being assessed,
as described previously?'* Theterms primitiveburst-formingunit-erythroid(BFU-E)andcolony-forming
unit-granulocyte macrophage (CFU-GM) refer to progenitors that
are quiescent in normal adult
marr0w,2~and give rise to colonies
containingmorethaneightclustersofhemoglobinizederythroid
cells and more than500 granulocytes and macrophages, respectively.
The corresponding terms mature BFU-E and CFU-GM refer to the
progenitors of all smaller colonies containing more than two
clusters
of erythroid cells or more than 20 granulocytes and macrophages,
respectively.
RESULTS
AcSDKP addition to n o m 1 LTCs blocks the activation
of primitive progenitors in the adherent layer. The ability
of AcSDKP to influence the cycling statusof primitive normal clonogenic cells was evaluated by adding varying concentrations of this tetrapeptide (from 20 to 300 ng/mL) at
the time of the routinehalf-medium change to3- or 4-week-
old primary LTCs established from normal BM cells. As
canbeseeninFig
1, addition of 100 ng/mLormore of
AcSDKP was sufficient to consistently prevent the known
ability of a fresh medium change to stimulate the primitive
erythroid and granulopoietic progenitorsin the adherent
layer to enter S - p h a ~ e In
. ~ contrast, at no concentration did
AcSDKP have any effect on theproliferative activity of the
morematureprogenitorscoexisting
in thesame adherent
layers, and assessed at the same time. Similar concentrations
of the control peptides, AcSDKE and SDKP, added to parallel cultures in the same experiments had no
effect on the
proliferative status of any type of clonogenic cell. The total
number of primitive progenitors detected in controlcultures,
given a routine half-medium change, was not significantly
different fromthe number of progenitors detected in cultures
treated with any of the tetrapeptides (Table 2). This latter
result, together with the apparent specificity of the AcSDKP
effect for primitive progenitors, and the demonstration that
these could be
reactivated on plating in methylcellulose, indicate that none of the tetrapeptide preparations were directly
cytotoxic. Sincetheactions of AcSDKP appeared to be
stage-specific, rather thanlineage-specific,
as previously
shown also for the effects of the LTC adherent layer, itself,
and for the two factors thus far
identified as mediators of its
inhibitory activity, ie, TGF-P and M P - l a (or some other
factor whoseactivity can beneutralized by MIP-1P),K.'5 data
for both typesof primitive progenitors in subsequent
experiments were pooled before calculation of percent kill values.
Tumor necrosis factor-cu (TNF-a) contains the SDKP sequence, and TNF-ct has been found to inhibit colony formation in semisolid culture systems containing certain accessory cellszs~29although stimulatory effects ofTNF-(U on
primitive hematopoietic
cells
have
also
been
It
was, therefore, of interest to investigate if the addition of
TNF-ato
normal LTCsmightsimulatethe
effects of
AcSDKP. Table3 shows the results of two suchexperiments.
In both, TNF-a was added to 4-week-old
primary LTCs,
established with normal BM to give a final concentration in
the medium of 100 ng/mL, using the same protocol as for
the tetrapeptide experiments described earlier(Fig 1). However, in this case, the added cytokine had noeffect on either
the proliferative behavior or numbers of the primitive progenitors in the adherent layer (Table 3). Addition of TNFa also did not affect the number or cycling status
of the
more mature progenitor cells present in these cultures (data
not shown).
Evidence that the inhibitory action of AcSDKP on normal
progenitors m a y involve accessory cells.The demonstration that adult rat hepatocytes are responsive to theinhibitory
effects of AcSDKP
in
but
not
in
vitro,33 suggested
that at least some of the effects of this tetrapeptide could
involve interactions with intermediatecells. To testthis possibility, varying concentrations of
AcSDKP were incorporated into methylcellulose assays of freshly isolated normal
BM cells or normal BM cells that had been in LTC for 10
days, and theresults compared with those obtainedin assays
of the same cells from which the adherent cells had been
rigorously removed. As can be seen in Fig 2, a significant
and reproducible dose-dependent inhibition of both erythroid
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1537
INDIRECTREGULATORYACTIONOFAcSDKP
100
I
.
I
Y
80
-A
BFU-E
-
CFU-GM
t
CI
c
Q)
0
\
L
&
Q)
e
-20
!
0200
I
I
I
I
I
300
100
0
100
200
300
Concentration of Tetrapeptide (ng/ml)
Fig 1. Effect of adding AcSDKP (circles), AcSDKE (squares), and SDKP (triangles)to normal LTC on the cycling status of the primitive (high
proliferative potential) BFU-E (A) andCFU-GM (B) preaentin the adherent layer. Datafor the response of mature CFU-GM (shaded circles, B)
to AcSDKP for thesame cell suspensions is also shown in (B). Conventional LTCI were establishedwith normal BM. After 4 weeks, tetrapeptides
were added with the weekly change of half of the medium. Adherent layer cells were harvested 2 to 3 days later, exposed to ’H-thymidine
lor not), and plated in methylcelluloseassays to determine percentkill values. Valuesshown are the maan 2 SEM from a total of 16 experiments.
The numbersof colonies scored in all of the control essays (no exposure to 3H-thymidine)of each group ranged from nine to 289 for primitive
BFU-E, five to 175 for primitive CFU-GM, and 51 to 677 for mature CFU-GM.
and granulopoietic colony formation occurred in assays of
both sources of unseparated normal BM cells. This effect
was most pronounced on the more primitive types of granulopoietic progenitors (eg, at 300 ng/mL survival of primitive
CFU-GM in normal marrow was 40% -C 4% v 73% 5 6%
for mature Cm-GM, and 61% -C 6% for primitive BFU-E
v 70% 5 10% for Cm-E), and was apparent in assays that
contained 2 3 0 ng/mL of AcSDKP, a concentration similar
to that required to block the activation of primitive normal
progenitors in LTC adherent layers (Fig 1). Interestingly, the
unacetylated tetrapeptide, SDKP, which was inactive in the
LTC system, showed the same antiproliferative effect as its
active acetylated derivative in clonogenic assays of unseparated fresh BM cells. However, the other control peptide,
AcSDKE, had no effect, as had also been the case in LTCs.
Removal of adherent cells completely abrogated the inhibi-
tory action of AcSDKP on colony formation by primitive
normal progenitors (Fig 3), suggesting that the inhibitory
effect of this tetrapeptide on hematopoietic cells in cultures
containing adherent cells may be indirectly mediated.
AcSDKP addition to CML LTCs has no effect on the prolqeration of primitive leukemic progenitors. Primitive CML
progenitors show an increased turnover relative to their normal counterparts, both in vivo, and in LTCs containing a
preestablished normal marrow adherent layer.” This heightened proliferative activity does not appear to be due to the
operation of either autocrine or paracrine mechanisms,” or
to the acquisition of a decreased sensitivity to the inhibitory
activity of TGF-P.” We have, therefore, postulated the existence of other cytokines that would mimic the action and
Table 3. TNFw Does Not Block the Activation Into S-Phase of
Primitive Progenitors in the Adherent Layer of Normal LTCs
Table 2. Addition of Tetrapeptides to LTCs Has No Effect on
Primitive Hematopoietic Cell Numbers
No. of Primitive Progenitors per LTC
Adherent Layer (% of controls1
Addition
(et timeof the half-medium
change)
Mean ? SEM
No. of
Experiments
AcSDKP
AcSDKE
SDKP
150 2 30
130 2 30
120 2 30
12
3
7
Values shown are expressed as a percent of values measured in
control LTCs given a regular half-medium change only. Data are from
the same experiments shown in Fig 1 for LTCs to which 200 to 300
mglmL ofeither AcSDKP or AcSDKE was added, or to which300 mg!
mL SDKP was added.
% Kill of Primitive Progenitors After
Exposure to ’H-Thymidine
Treatment
Mock feed
Regular feed
Regular feed + TNF-a
(100 n g h L )
Experiment 1
-
Experiment 2
59 (620)
2 (449)
40 (294)
41 (510)
51 (608)
Conventional normal BMLTCs were established and, after 4 weeks,
treated as indicated. Two or 3 days later, percent kill values for total
primitive progenitors (primitive BFU-E and primitive CFU-GM) in the
adherent layer were determined based on actual counts (in the control
assays) of 54 to 187 colonies. Values shown in parentheses are the
numbers of primitive progenitors per LTC adherent layer in each
group at the time of assessment.
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
1538
CASHMAN, EAVES, AND EAVES
120
1
BFU-E
l
CFU-GM
t
40
JA
0 200
I
100
1
I
300
B
0 200
1
I
100
I
300
t
CFU-GM
L
_"
"
ID
0 200
100
300
0 200
I
_"""
I
100
Concentration of Tetrapeptide (ng/ml)
primitive target cell specificity of TGF-/3 in LTCs of normal
cells, but that would be unable to regulate primitive CML
cells.L5Because AcSDKP appeared to have such an effect
on normal cells, it was of interest to determine if this tetrapeptide would have a similar action on primitive CML targets. This was initially examined in a series of experiments
with LTCs of CML cells using the same protocol as described above for LTCs of normal cells (Fig l), in which
AcSDKP was added at the time of a fresh medium change.
To approximate the conditions operating in the adherent
layer of these CML cultures as closely as possible to those
present in the 3- to 4-week-old LTCs of noqnd cells used
in Fig 1, and at the same time to ensure that all clonogenic
progenitors in the CML LTCs would be of neoplastic origin,
the latter were established by seeding PB cells from patients
with high white blood cell counts onto preestablished, irradiated, normal marrow feeder layers.
As
both the primitive erythroid and granulopoietic progenitors in the adherent layer of routinely maintained CML LTCs exhibited a consistently deregulated cycling behavior. The data for both types of progenitors in each
experiment were therefore pooledto derive 3H-thymidine
-A
l
I
300
Fig 2. Colony inhibition doseresponse curves for BFU-E (open
symbols, A and C) and CFU-GM
(closedsymbols, B and D) in
methylcellulose
assays
of
unfractionatedfreshnormalBM
cells (top pands) or 10-day-old
cuhred normal BM cdls (C and
D) containing various tetrapaptides. The tetraAesDKP
(circles),itsunacetylatedform,
SDKP (triangles), or the control
peptide AcSDKE (squares) were
each added directlyto the methylcellulose medium before plating to give final concentrations
ranging from 3 to 3001mL. Values shown arethe mean ? SEM
fromdifferentexperiments
(A
and B, n = 14, bottom panels, n
= 3) in which individual values
were normalized by exp-ng
each as a percent of the colony
countsobtainedincontrolcultures to which no peptide was
added.
suicide values for total primitive progenitors. These are
shown in Table 4. In three of the 10 experiments performed
(each with cells from a different CML patient), 100 ng/mL
of TNF-a was added with the half-medium change to one
of the cultures to investigate if this cytokine might also have
an inhibitory effect on primitive neoplastic progenitors in
the LTC setting. It can be seen that neither AcSDKP nor
TNF-a addition blocked the cycling or altered the numbers
of primitive or mature (data not shown) CML progenitors
in anyof these experiments. In each of these, 5 ng/mL of
TGF-/3 was added with the regular half-medium change to
one of the cultures to serve as a positive control. Previous
experiments of this type had shown that TGF-/3 can transiently arrest the cycling of primitive CML progenitors
without affecting their viability. As a result, they can subsequently be removed from treated LTCs, washed free of TGF/3, and plated in colony assays where they are then restimulated to divide and express their clonogenic potential at an
undiminished freq~ency.~'
Similar results were obtained in
the present series of experiments (Table 4).
Since primitive CML progenitors remain in cycle in LTCs
that have not been perturbed for 7 days under conditions
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1539
INDIRECT REGULATORY ACTION OF AcSDKP
120
-I
I
BFU-E
T
t
i-1
L
Fig 3. Lack
of
effect of
AcSDKP on colony formation by
BFU-E (open symbols, A) and
CFU-GM (closed svmbols.. BI. in
assays of adherent cell-depleted
10-day cultured normal BMcdls
(prepared as described in Materials and Methods). Assays
containedthe same tetrapeptides as
the in
experiments shown in Fig
2 (same symbols). Values (mean
2 SEMI were derived
thefrom
pooled results of nine different
experiments after normalization
to control values, as for Fig 2.
*O
p)
ca
60/*,
40
0
4."".
+._..."..._..."........ "-.."-.-"*-""
""""""-
I
1
C
p
CFU-GM
,
.
,
,
,l L
2 0 01 0 0
300
210000
300
Concentration of Tetrapeptide
(ng/ml)
where there is not an adequate positive stimulus to activate
their normal counterparts, we undertook a further experiment
to test if AcSDKP (andor TNF-a)might be able to inhibit
the cycling of primitive CML progenitors under this condition of minimal stimulation. Accordingly, another set of
CML LTCs were initiated. After 10 days, either 300 ng/mL
of AcSDKP or 100 ng/mL. of TNF-a, but no new medium
(or nothing), was added. The results were not different from
those shown in Table 4, ie, all categories of progenitors in
the adherent layer of all of these cultures were found to be
proliferating (with percent kill values ranging from a minimum of 41% to a maximum of 59%) when their cycling
status was assessed 2 days later.
Since the inhibitory action of AcSDKP on normal progenitors could be demonstrated in colony assays of unseparated
BM, the responsiveness of CML progenitors to AcSDKP
was also tested under these conditions. As shown in Fig 4,
fresh unseparated CML BM showed no decrease in either
erythroid or granulopoietic colony formation when AcSDKP
was included in the methylcellulose medium over a wide
range of concentrations (up to 30 times those that significantly decreased colony formation by normal progenitors in
assays of unseparated normal BM; Fig 2).
Evidence that the inhibitory action of AcSDKP in LTCs
may be indirectly mediated. In a recent series of experiments, we found that the addition of MIP-la to LTCs of
normal cells could, like the addition of TGF-P, block the
activation of both primitive erythroid and primitive granulopoietic progenitors in the adherent layer.15 However, unlike
TGF-P, addition of MIP-la to LTCs of CML cells was
unable to influence the cycling activity of the primitive neoplastic progenitors present in the adherent layer of these
cultures.I5Thus, the effects of AcSDKP, seen here in terms
of activity and target cell specificity, appear to closely parallel those previously documented for MIP-la. Because of the
findings that the effects of AcSDKP in colony assays of
normal progenitors involved accessory cells (Fig 2), and
because of the known ability of other cytokines to alter
MIP-la
it seemed possible that the inhibitory
activity attributed to AcSDKP might reflect its ability to
stimulate the release or availability of MIP-la (or a factor
with related activities). To test this hypothesis, we used the
strategy of adding exogenous MIP-1P (a known antagonist
of MIP-la and several related chemokines3') withAcSDKP.
These experiments followed the same general protocol used
initially to detect the ability of AcSDKP addition to block
Table 4. Addition of AcSDKP to CML LTCs Has No Effect on the Cell Cycle Status of Primitive Progenitors in the Adherent Layer
~~
____
~
~_________
% Kill of Primitive Progenitors After Exposureto 'H-Thymidine
Addition
54
36
None
61
TGF-P
42AcSDKP62
TNF-a
EXD 1
EXDZ
34
9 0
49
7
-
-
EXDJ
E X D ~
EEXXDD~~
EEE XXX DDD ~~~
0
4 0
3
-
-
-
EXD 10
0
4
0
-
-
72
61
28
Mean 5 SEM
54 t 3
122
51 t 4
54 -t 13
Percent kill values shown for all primitive progenitors (ie, primitive BFU-E and primitive CFU-GM combined) in the adherent layer of LTCs
were determined 2 to 3 days after various cytokine additions, and are based on actual counts (in the control assays) of 24 to 256 colonies.
These were made at the time of the first, second, or third weekly half-medium change as described in Materials and Methods. None refers to
a regular half-medium change. TGF-/3 was added togive a concentration of 5 ng/mL, AcSDKP to give a concentration of 200 or 300 ng/mL, and
TNF-a to give a concentration of 100 ng/mL. The total number of primitive progenitors per LTC adherent layer relative to LTCs given a regular
half-medium change only (=loo%) was 120% 2 20%. 140% 2 30%. and 190% 2 30% for addition of TGF-P, AcSDKP, and TNF-a, respectively.
Abbreviation: Exp, experiment.
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
1540
CASHMAN, EAVES, AND EAVES
120
110
BFU-E
4
"""""""-
90
I
i
70
60
1
I
I
I
B
'31
120
:
:
1
I
CFU-GM
]
1
1
,
1
,
60
0
2 0400600 0
800
CONCENTRATIONOFTETRAPEPTIDE
1000
(nglml)
Fig 4. Lack of effect of AcSDKP (solid symbols) on colony formation by BFU-E (A) and CFU-GM (B)in assays of unfractionated fresh
BM cells from seven patients with CML. Results for a control peptide
(AcSDKE, open symbols) are also shown. Values are the mean ?
SEM of the combined data for all experiments after normalization to
controls, as described in the legend to Fig 2.
primitive progenitor activation innormalBM
LTCs (Fig
1). However, in this case, additional groups of LTCs were
included, and to these, 300 ng/mL of MIP-1P both with and
without AcSDKP (at 200 ng/mL) were added to the new
medium. Results from the four experiments performed are
shown in Table 1. It can be seen that 300 ng/mL of MIP1P alone (without AcSDKP) had no effect on the activation
of primitive progenitor cycling induced by the addition of
fresh medium, but was sufficient to completely overcome
the ability of simultaneously added AcSDKP to block the
activation of these cells.
DISCUSSION
During steady-state hematopoiesis in vivo, the proportion
of cells in the most primitive progenitor compartments that
are in S-phase is low, often undetectable. This has been
attributed to the presence of a large G, population. Recently,
a large number of intracellular intermediates that control
the cell-cycle progression of mammalian cells have been
identified, and their role and mechanisms of action are beginning to be delineated in a variety of cell types, including
hematopoietic cells. Because of the evolutionary conservation of many aspects of these mechanisms, it is likely that
some of the unique features of hematopoietic stem-cell con-
trolwill be found tobe determined, at least in part, by
the spectrum of extrinsic cytokines to which these cells are
responsive. In fact, current evidence indicates that early hematopoietic cell differentiation involves changes, not only
in responsiveness to specific factors or factor combinations,
but also in the subsequent biologic response(s) they elicit.3x
Thus, an important aspect of studies of the physiologic regulation of primitive hematopoietic cells lies in the specificity
of the assays used to measure responsive target cells, as well
as the use of an experimental system that, at least, appears
to mimic the regulatory mechanisms operative in the marrow
in vivo.
In the present study, we have attempted to meet the first
requirement through the use of rigorously standardized colony assay culture components and colony sizing criteria.
These allow a reproducible distinction to be made between
classes of normal progenitors that differ dramatically in their
turnover rates bothin vivo andinLTCs.4 Because these
progenitors are not only characterized by marked differences
in the proliferative potential they manifest underdefined
conditions in vitro, but have also been shown to represent
sequential stages of development, they are referredtoas
primitive and mature.39There is thus a strong rationale for
interpreting the cycling changes found to characterize Phpositive progenitors, defined by similar criteria, as indicative
of the deregulation of a control mechanism normally operative on analogously definednormal cells. Consistent with
this is the well-documented expansion of the neoplastic clone
in patients with CML at all levels of clonogenic cell maturity,
as defined by their different proliferative potential^.'^
The observed parallels between the cycling behavior of
both normal and neoplastic hematopoietic cells in the adherent layer of LTCs, and in vivo provide support for the validity of the LTC system as an experimental model for investigating physiologically relevant control mechanisms. In this
report, we have confirmed (Fig 2) the ability of AcSDKP to
inhibit the generation of colonies of erythroblasts or granulocytes and macrophages from normal progenitors stimulated
to proliferate in semisolid media,*' and have demonstrated
the reversibility of this inhibition following removal of normal progenitors from the adherent layer of LTCs to which
AcSDKP had been added 2 to 3 days previously (Fig lj.
Moreover, the activity of AcSDKP in the LTC system
showed considerable structural specificity, in thatneither
TNF-a, which contains the SDKP sequence, nor a slightly
different tetrapeptide, AcSDKE, had similar effects. Our
present results further show that removal of adherent cells
can eliminate the inhibitory effect of AcSDKP incolony
assays of normal progenitors (Fig 3), and that addition of
excess MIP-1P, an antagonist of MIP-la and certain functionally related chemokines, can eliminate the effect we have
observed of AcSDKP in the LTC system (Table l). Given
the similarities in the target-cell specificity of MIP-la and
AcSDKP (inhibitory to primitive normal, but not to either
mature normalor primitive neoplastic clonogenic cells),
these findings suggest that at least one of the mechanisms
by which AcSDKP can elicit its effects is by stimulating
adherent cells to produce or release MIP-la (or some other
molecule antagonized by MIP- ID). However, preliminary
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
INDIRECT REGULATORY ACTION OF AcSDKP
studies have failed to show detectable changes in MIP-la
mRNA levels in the adherent layers of LTCs to which
AcSDKP was added, nor was immunoreactive M P - l a detectable in the supernatants of these cultures (Humphries
RK, Wolpe S, Cashman JD, et al, unpublished observations).
In summary, these findings support and extend a model
of primitive hematopoietic cell regulation in which the control of the proliferation of these cells is dependent on the
relative concentrations of stimulators and inhibitors to which
they are exposed. The involvement of secondary mediators
of inhibition shown here, as well as secondary mediators of
stimulation demonstrated previously,8 highlights the complexity of the mechanisms that need to be considered. In
addition, they reinforce the concept that reversible inhibition
of proliferation may be a major mechanism controlling primitive hematopoietic cell behavior in vivo, and is, itself, subject to independent regulation. Finally, these studies raise
the interesting possibility of investigating AcSDKP for its
clinical potential to optimize the therapeutic benefit achievable with cell cycle-specific chemotherapeutic agents by eliciting localized in vivo production of inhibitors of primitive
hematopoietic progenitor proliferation; one example of
which (MIP-la) has already been shown to be effective in
this regard in various animal model^.^.^'
ACKNOWLEDGMENT
The authors thank Dr S. Wolpe of the Genetics Institute (Cambridge, MA) for generous gifts of recombinant human M P - l a and
MIP-1P. The excellent technical assistance of Dianne Reid and the
secretarial expertise of Heather Calladine are also gratefully acknowledged.
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