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Blood, Vol. 95 No. 10 (May 15), 2000:
pp. 3001-3009
PLENARY PAPER
From the Cattedra di Ematologia, Istituto di Ostetricia e
Ginecologia, Universita' Cattolica del Sacro Cuore, Rome, Italy.
Primitive, proliferating hematopoietic progenitors (defined as
cytokine low-responding primitive progenitors; CLRPP), isolated from
human CD34+ cells, expressed endoglin (CD105) and produced transforming growth factor-
The molecular mechanisms underlying the survival and
function of primitive precursors during hematopoiesis are largely
unknown. Several studies have shown that bcl-2, c-myc, and p53 are
involved in the regulation of survival and proliferation of human
cells.1-3 The transcriptional regulation of bcl-2 and c-myc
in the hematopoietic system is, in part, under the control of various
hematopoietic cytokines and their receptors, which concomitantly exert
well-known differentiating signals.4-7 Less is known about
the regulation of p53 in hematopoietic progenitors.2 The
response of hematopoietic precursors to cytokines determines a
stage-specific molecular profile that is characterized by the
expression of considerable levels of bcl-2, p53, and c-myc in more
primitive precursors while differentiating progenitors down-modulated
the expression of these genes.8 In harmony with these
findings, concomitant expression of bcl-2, p53, and c-myc genes has
been suggested by Ryan et al9 as being the molecular state
by which precursors survive and proliferate and maintain stable
hematopoietic functions, as revealed by their studies on
bcl-2/c-myc/p53 cotransfected DP16-1 murine erythroleukemia cells. In
reference to this, a key aspect for a better understanding of
hematopoiesis is represented by the identification of the putative intrinsic mechanisms through which primitive precursors maintain their
specific functional/molecular profile despite the presence of powerful
differentiating stimuli. The present study examined the role of
autocrine levels of the cell cycle inhibitor10
transforming growth factor- Isolation of CD34+ hematopoietic precursors
Functional identification and isolation of primitive hematopoietic
progenitors
Culture of CLRPP in the presence or the absence of
anti-TGF- Flow cytometric analysis of TGF-  1 were detected
by flow cytometry in isolated and cultured CLRPP. Up to
1.5 × 105 cells were mixed with 5 µL of a
phycoerythrin-conjugated anti-CD105 (CD105 PE) mAb (clone SN6; Serotec,
Oxford, UK) and incubated for 30 minutes at room temperature in the
dark. Then, cells were washed twice with phosphate-buffered saline
(PBS)-1% bovine serum albumin (BSA) and analyzed by flow cytometry.
Before detection of intracellular TGF-1 by flow cytometry, aliquots
of cells were preincubated for 4 hours in complete growth medium
containing 10 µg/mL brefeldin-A to block intracellular transport and
enhance cytokine accumulation in the Golgi complex. After washings with PBS-1% BSA, aliquots of up to 1 × 105 cells were
resuspended in 100 uL of fixing solution (Fix and Perm, Caltag,
Burlingame, CA) and incubated for 15 minutes at room temperature; after
centrifugation with PBS-1% BSA, cells were resuspended in 100 µL of
permeabilizing solution (Fix and Perm, Caltag) containing pretitrated
saturating amounts of anti-TGF-1 mAb (clone 9016.2; 0.1 µg for
each test; R&D Systems). Cells labeled with anti-TGF-1 were
subsequently incubated with a PE-conjugated goat antimouse
immunoglobulin (Immunotech, Marseilles, France) with a ratio of 1:40 to
cell suspension. After extensive washings, cells were analyzed by flow
cytometry. Staining experiments for flow cytometric detection of
TGF-1 protein were carried out omitting cell fixation and
permeabilization to estimate the amount of TGF-1 bound on cell
membrane following extensive washings of serum-free and
serum-containing cultures. For the simultaneous assessment of CD34 and
bcl-2 expression, up to 1 × 105 isolated and
cultured CLRPP were first stained with pretitrated dilutions of
PerCP-conjugated anti-CD34 mAb (1:10 to cell suspension; clone 8G12;
Becton Dickinson, Mountain View, CA) for 30 minutes at room
temperature, followed by fixation with Ortho Permeafix solution (Ortho
Diagnostics, Raritan, NJ) for 40 minutes at room temperature. After
washings in PBS-1% BSA, cells were incubated with saturating amounts
of a PE-conjugated antihuman bcl-2 mAb (1:10 to cell suspension; clone
bcl-2/100; Pharmingen, San Diego, CA) for 30 minutes at 4°C. After
washings in PBS-1% BSA, cells were kept on ice until flow cytometry
analysis. In some growth experiments, CLRPP were cultured for 6 days
and then evaluated for the expression of CD15, CD11b, CD41a, and
glycophorin-A differentiation antigens. Up to
2.5 × 105 cells were incubated with 10 µL of
anti-CD41aPE (Caltag) or antiglycophorin-A PE (Caltag) for single-color
analysis or costained with 10 µL of anti-CD15FITC
(Immunotech)/CD11bPE (Caltag) for dual-color analysis. In all staining
experiments, background fluorescence was assessed using isotype-matched
fluorochrome-conjugated irrelevant mouse immunoglobulins (Immunotech,
Becton Dickinson, Pharmingen, Caltag) in direct assays or substituting
the specific mAb with isotype-matched irrelevant mouse immunoglobulins
(Immunotech) in indirect assays. Cell cycle status of CLRPP was
evaluated before culture and every 9 hours as follows. Cells were
incubated for 30 minutes at 4°C with 1 mL of DNA staining buffer
(0.5% Nonidet P-40, 10 mg/mL propidium iodide -PI-, 11.25 Kunitz units
RNAse, and 0.1% sodium azide, Sigma Chemical), protected from light, and then analyzed by flow cytometry. Calculations of cell cycle compartments were performed on a 1024-channel histogram with ModFit LT
software (Verity Software House, Topsham, ME) for DNA content analysis.
Finally, CFDA-SE fluorescence (green fluorescence) was analyzed by flow
cytometry and the Proliferation Wizard option of ModFit LT software
(Verity Software House) in 72-hour-cultured CLRPP as a measure of cell
replication rate of these cells. All samples were run through a FACScan
flow cytometer (Becton Dickinson).
Cloning and LTC-IC assays Colony-forming cells (colony-forming unit granulocyte macrophage, CFU-GM; burst-forming unit-erythroid, BFU-E) were evaluated from isolated and cultured CLRPP by cloning assays, as previously described.13 The incidence of LTC-IC in control and study cultures was assayed using limiting dilution analysis and hematopoietic long-term culture on supportive stromal layers consisting of the genetically engineered murine stromal cells M2-10B4 (kindly provided by Connie Eaves, Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, Canada), as previously described.13Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) The messenger RNA (mRNA) expression levels of p53, c-myc, p21WAF1/Cip1, and bcl-2 were evaluated by RT-PCR on isolated and cultured CLRPP. RNA extraction was carried out with the RNeasy mini kit (Quiagen, Hilden, Germany) and RNA obtained from 20 000 cells was reverse transcribed, as previously described.11 Two microliters of these complementary DNA (cDNA) products were amplified with 1 U of Ampli Taq Gold (Perkin Elmer/Roche, Branchburg, NJ) in the presence of the specific primers for the mRNA of interest, together with the aldolase-A primers,14 used as internal control. Phytohemagglutinin (PHA)-stimulated lymphocytes were used as positive controls because they were previously shown to express detectable levels of all the markers studied in the present work. PCR reactions were carried out in the Gene Amp PCR system 9600 (Perkin Elmer) using 1 U of Ampli Taq Gold DNA polymerase in 5 mM MgCl2. A first incubation of 10 minutes at 95°C, 45 seconds at 65°C, and 1 minute at 72°C was followed by 27 cycles for p53 and by 28 cycles for c-myc, p21WAF1/Cip1, and bcl-2 performed as follows: 45 seconds at 95°C, 45 seconds at 65°C, and 1 minute at 72°C. The conditions were chosen so that none of the RNAs analyzed reached a plateau and so that the 2 sets of primers used in each reaction did not compete with each other. The PCR products were loaded onto 2% agarose gels and stained with ethidium bromide. Each set of reactions included a no-sample negative control. The ratio between the sample RNA to be determined and aldolase-A was calculated to normalize for initial variations in sample concentration and as a control for reaction efficiency. The sequences of the specific primers for bcl-2, aldolase A, c-myc, and p53 were previously described.14-16 The following primers were used for p21: 5'-CCCAGTGGACAGCGAGC-3' and 5'-ACTGCAGGCTTCCTGTGGGC-3', which yielded a 499 bp product. All oligonucleotide primers were synthesized by Pharmacia Biotech (Uppsala, Sweden). Analysis of GATA-1 and PU.1 expression levels was performed essentially as previously described, using-actin as an internal control.17 PCR products were
electrophoresed and transferred to Hybond membranes (Amersham
International, Buckinghamshire, UK), which were then hybridized to the
specific cDNAs. Images of the resulting radiographic films were
acquired as described below. All values were normalized to their
respective -actin internal control.
Preparation of the tissue lysates and Western blotting analysis Isolated or cultured CLRPP (1 × 106) were lysed in 20 mmol/L Tris-HCl pH 7.4, 0.1 mol/L NaCl, 5 mmol/L MgCl2, 1% NP-40, 0.5% sodium deoxycholate, 50 mmol/L NaF, 2 mmol/L Na vanadate, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 2 µg/mL leupeptin, and 2 kallikrein inhibitor units/mL aprotinin. The protein concentration was determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). Each protein sample (80 µg) was separated on a 5% to 15% polyacrylamide gradient sodium dodecyl sulfate (SDS) gel and electroblotted on polyvinyldene fluoride (PVDF) membranes (Millipore, Bedford, MA). Membranes were incubated with 6% nonfat dry milk in 1 × TBST (0.1 mol/L Trizma Base, 0.15 mol/L NaCl, 0.05% Tween 20, pH 7.4) for blocking and then with the primary antibody in 3% nonfat dry milk in 1× TBST. After incubation with the appropriate secondary antibody (goat antirabbit or goat antimouse horseradish peroxidase [HRP] conjugated, Bio-Rad), detection was performed with the ECL-Plus system (Amersham International) and the blots were exposed to X-AR-5 OMAT Kodak films. The blots were used up to 4 times by stripping at 50°C for 30 minutes in 100 mmol/L 2-mercaptoethanol, 2% SDS, and 62.5 mmol/L Tris-HCl pH 6.7, followed by extensive blocking and reprobing with a different antibody. The following antibodies were used for Western blotting: anti c-myc (clone N-262), p21 WAF1/Cip1 (clone C-19), p16Ink4 (clone C-20), bcl-xL (clone L-19), and bax (clone N-20) rabbit polyclonal antibodies from Santa Cruz Biotechnology Inc (Santa Cruz, CA); anti-p27KIP1 (clone G173-524), Rb (clone G3-245), and p107 (SD9) mouse mAb were all from Pharmingen. Anti-TGF-1 mouse mAb
(clone 9016.2) was from R&D Systems.
Image analysis and quantification Images of the RT-PCR ethidium bromide-stained agarose gels, radiographic films, and Western blotting PVDF membranes were acquired with a Cohu CCD camera and quantification of the bands was performed by Phoretix 1 D (Phoretix International, Newcastle upon Tyne, UK). Band intensity was expressed as relative absorbance units. The bar graphs in the figures show the mean and standard deviation of all experiments. All values reported in the text and figure are ratios of relative absorbance units.Data analysis Comparisons between control and study cultures were performed by unpaired 2-tailed t test. A P < .05 was considered significant.
Expression of CD105 (endoglin) and CD34/bcl-2 and autocrine
production of TGF- 1 and TGF-3 receptor complex) (Figure 1C).
Flow cytometric studies and Western blot analysis identified the
presence of a considerable amount of intracellular TGF-1 in CLRPP
with 100% of cells containing TGF-1 (Figure
2A and B). Staining experiments carried out
without cell fixation and permeabilization following extensive cell
washings revealed that anti-TGF-1 mAb does not react with CLRPP
cultured in serum-free or serum-containing medium, indicating the
absence of a detectable amount of TGF-1 bound on cell membrane,
which could interfere with the cytometric detection of the
intracellular protein. As revealed by a preliminary set of cultures,
the production of autocrine TGF-1 was maintained throughout the
whole culture period (72 hours) and also maintained in the absence of
serum (data not shown). Thus, all subsequent experiments were carried out in the absence of serum, using Bit H9500 serum substitute. As
previously reported, CLRPP coexpressed high levels of the CD34 progenitor antigen and considerable levels of the bcl-2 survival protein, with more than 50% of these cells consistently bcl-2+ (see
also Table 1 and Figure 5).
Growth, cloning ability, and LTC-IC content of cultured CLRPP with
or without TGF- 1 neutralizing antibody in a dose range from 0.1 to 20 µg/mL. After culturing CLRPP for 72 hours in serum-free conditions in
the absence of any antibody or in the presence of specific
anti-TGF-1 or irrelevant isotype-matched mouse immunoglobulins, a
6-fold increase was observed in the starting cell number (Figure 3; P > .05 for any experimental
condition as compared to controls), without any relevant effect on
CLRPP viability, as judged by flow cytometric propidium iodide
viability testing. Subsequent experiments carried out in the presence
of antisense or sense TGF-1 oligonucleotides in a dose range from 3 to 15 µmol/L produced similar results, with no significant
modification in cell number and viability as compared to untreated
controls (data not shown). However, cloning assays revealed that the
presence of a dose of anti-TGF-1 equal to or higher than 1 µg/mL
consistently decreased the frequency of CFU-GM and BFU-E, with the
recovery of 16% to 53% of cloning cells as compared to controls
(Figure 3; P < .0001 for a dose of anti-TGF-1  1
µg/mL, for both CFU-GM and BFU-E). The detrimental effect of TGF-1
neutralization on the cloning fraction was revealed both by CFU-GM and
by BFU-E reduction in the absence of any significant modification of
the size of recovered colonies. CLRPP cultures in the presence of
TGF-1 antisense oligonucleotides (15 µmol/L) produced a minor
reduction of cloning capacity as compared to anti-TGF-1-treated
cultures, with the recovery of an average of 70% (range
85-60%) of control colony number. We did not find any significant
variation of cloning ability when CLRPP were cultured in the presence
of irrelevant isotype-matched mouse immunoglobulins or TGF-1 sense
oligonucleotides. Table 2 shows
the average LTC-IC frequencies observed in 72- hour cultured CLRPP,
which revealed that the presence of anti-TGF-1 at the dose of 10 µg/mL produced an 85% reduction of LTC-IC content.
Bcl-2, p53, c-myc, and p21 gene expression in isolated and cultured CLRPP Isolated CLRPP showed considerable bcl-2, p53, c-myc, and p21 RNA levels. Expressed as the relative adsorbance ratios between the RNA levels of the gene of interest and that of aldolase-a, bcl-2, p53, c-myc, and p21 levels were, on average, 1.5, 1.4, 1.3, and 0.5, respectively. After 72-hour culture, CLRPP showed bcl-2, p53, c-myc, and p21 levels similar to starting cells (Figure 4). In the presence of anti-TGF-1
neutralizing antibody (10 µg/mL), a significant reduction of bcl-2
RNA levels was observed in the absence of any significant modification
of c-myc, p53, and p21 levels. On average, neutralization of TGF-1
reduced the bcl-2 relative adsorbance ratio from 1.4 to 0.3 in cultured
CLRPP. No gene expression modulations were observed in controls (10 µg/mL irrelevant isotype-matched mouse immunoglobulins). The presence of TGF-1 antisense oligonucleotides at the dose of 15 µmol/L reduced bcl-2 RNA levels to the average value of 1 (expressed as
relative adsorbance), as compared to control, TGF-1 sense, and
anti-TGF-1 mAb-treated CLRPP, which had average values of 1.4, 1.6, and 0.3, respectively. Parallel 72-hour CLRPP cultures, prepared in the
presence of 1 ng/mL TGF-1, showed that TGF-1 addition increased
bcl-2 RNA levels from 1.4 to 2.7, on average. This bcl-2 modulation by
exogenous TGF-1 was observed in the absence of significant effects
on cell proliferation (although a 20% reduction in cell number as
compared to controls was observed), p53, c-myc, and p21 expression
levels (data not shown), and CLRPP cloning ability (a slight decrease
in cloning cell frequency was observed in 3 of 6 samples treated with
TGF-1). However, the increase of bcl-2 expression by exogenous
TGF-1 was quite variable, with no significant variations in 2 samples of 6 studied (data not shown) and a very high increase in 2 samples of 6 (4-fold increase as compared to bcl-2 expression found in
controls; data not shown).
Coexpression of CD34/bcl-2 and expression of differentiation antigens on cultured CLRPP As revealed by RT-PCR and Western blot analyses, CLRPP had high levels of bcl-2. This result was confirmed by flow cytometry (Figure 5). CLRPP, cultured for 72 hours in the presence or absence of 10 µg/mL anti-TGF-1 neutralizing mAb, were
evaluated for the simultaneous expression of CD34 progenitor marker and
bcl-2 protein. Following culture, CLRPP maintained their CD34
expression both in control and anti-TGF-1-treated cultures while
the expression of bcl-2 protein was consistently and considerably
reduced in anti-TGF-1 treated CLRPP (68 ± 7% and
20 ± 5% bcl-2+ cells in control and anti-TGF-1-treated
CLRPP, respectively; P < .0001). Dual-color analysis showed
a certain correlation between bcl-2 and CD34 expression, so that the
dimmer CLRPP for bcl-2 in anti-TGF-1-treated cultures concomitantly
showed a certain down-modulation of CD34 antigen, suggesting a trend of
these primitive cells toward differentiation (the mean fluorescence
channel of CD34 staining decreased from the control value of 270 to the
value of 120 observed in anti-TGF-1 cultures, on average). Extension
of serum-free CLRPP cultures to 6 days revealed that the frequency and
the absolute number of nascent CD15+, CD11b+, and glycophorin-A+ cells
were 2-fold higher in anti-TGF-1-treated cells than in controls.
However, because of the primitive nature of CLRPP, the frequency of
CD15+, CD11b+, and glycophorin-A+ did not exceed 15%, 10%, and 5%,
respectively, in any condition tested. At this time, no mature
CD15+/CD11b+ and very rare CD41a+ cells were detected in control and
study cultures. No significant changes in CLRPP cumulative cell number were observed in study cultures as compared to controls.
Cell cycle analysis and tracking of cell division number of cultured CLRPP Cell cycle analysis was performed every 9 hours on CLRPP cultured in the presence or in the absence of 10 µg/mL anti-TGF-1. As detailed
in Figure 6, no significant variation of
CLRPP distribution into the different cell cycle compartments was
produced by the presence of anti-TGF-1 antibody at the dose of 10 µg/mL. Tracking of cell division number by CFDA-SE fluorescent dye
and flow cytometry demonstrated that TGF-1 neutralization was not
accompanied by any evident variation in the number of CLRPP divisions,
as indicated by the overlapping green fluorescence histograms obtained
in treated samples and their controls (Figure
7).
Western blot analysis of c-myc, bcl-2, bcl-xL, bax, and p16/p21/p27/p107/Rb cell cycle-related proteins during CLRPP growth experiments No significant change of c-myc, bax, bcl-xL, and p16/p21/p107/Rb cell cycle-related protein levels was observed in control and anti-TGF-1-treated CLRPP by Western blot analysis (Figures 8 and 9).
Moreover, the ratios between hyperphosphorylated and hypophosphorylated
Rb and p107 protein levels were comparable in control and study CLRPP
cultures (Figure 9). Western blot analysis confirmed a reduction of
bcl-2 levels in anti-TGF-1-treated CLRPP (Figure 8;
P < .01). A dramatic increase in p27 expression levels was
observed in anti-TGF-1-treated CLRPP with an average 5-fold increase as compared to control CLRPP (Figure 8; P < .0001)
GATA-1 and PU.1 transcription factor RNA level in CLRPP cultured in
the presence or absence of TGF- 1 neutralization on the differentiating status of
these primitive precursors. RNA analysis revealed that cells cultured
in the presence of anti-TGF-1 neutralizing antibody (10 µg/mL) had
GATA-1 and PU.1 RNA levels consistently higher than those observed in
control cultures (4.2 ± 0.8 versus 1.8 ± 0.2 and
13 ± 1.7 versus 2.5 ± 0.2, respectively,
P < .0001; Figure 10) despite
the maintenance of CD34 expression on their cell surface (Figure 5).
Molecular characterization of primitive hematopoietic progenitors Recent reports underlined the existence of molecular patterns associated with the undifferentiated status of hematopoietic progenitors, characterized by considerable expression of the survival protein bcl-xL and by the induction of bcl-2 expression after cytokine exposure.18 Studies on hematopoietic cell lines revealed that cotransfection of bcl-2, p53, and c-myc could guarantee prolonged survival and extensive proliferative ability to DP16-1 murine erythroleukemia cell line.9 This effect recalls the picture attributed to candidate stem cells and primitive precursors. In line with these observations, we recently demonstrated that primitive circulating hematopoietic precursors, functionally isolated following cytokine stimulation by tracking of cell division number, associate primitive functional properties with high expression of bcl-2, bcl-xL, c-myc, and p53.8 The above-mentioned population of primitive precursors (operationally defined as CLRPP) expressed intermediate levels of endoglin (CD105; a component of the TGF- receptor complex) and produced a considerable amount of autocrine TGF-1 (as revealed by flow cytometry and Western blotting). In the
light of these findings, we verified the existence of an autocrine loop
for TGF-1 in CLRPP and the role of this autocrine multifunctional cytokine in maintaining the functional properties and the molecular status of these cells.
Autocrine TGF- 1 is a well-known inhibitor of
primitive hematopoiesis19-21 and, when exogenously added,
delays cell cycle transition from the G0/G1 to
the S phase by inducing high levels of p15, p27, and p21
cyclin-dependent kinase (CDK) inhibitors22 and maintaining
retinoblastoma and p107 proteins in the hypophosphorylated
state.23,24 Furthermore, suppression or neutralization of
autocrine TGF-1 releases primitive blood progenitors from
quiescence.19,25,26 The present study indicated that
inactivation of the endogenous TGF-1 by specific neutralizing mAbs
significantly reduced the cloning ability of cultured CLRPP, decreasing
by more than half the colony-forming cells of all lineages and by 85%
primitive LTC-IC. We also found that TGF-1 neutralization resulted in the reduction of both bcl-2 RNA and protein levels. Using
TGF-1 antisense oligonucleotides, an appreciable, albeit less
evident, reduction both of bcl-2 expression and cloning cell number was
found. The lower effectiveness of antisense is not unexpected and the
cause could lie either in a suboptimal dose of oligonucleotides used (a
nontoxic dosage, as described by Hatzfeld et al19 ranging
from 3 to 15 µmol/L) or in the presence of an excess of secreted
maturating latent TGF-1 forms.27 Collectively, our data
fit well with studies on embryonic rat hippocampal neurons and mouse
neonate cortical neurons, which indicated that TGF-1 is responsible
for maintenance/induction of bcl-2 expression in these cells,
representing a mechanism for regulation of survival and protection from
stress produced by oxidative injury or removal of trophic
factors.28,29 Moreover, a recent report showed bcl-2 up-modulation in normal murine hematopoietic precursors and Ba/F3 cell
line following exposure to exogenous TGF-1.30 Bcl-2 is an essential survival factor for leukemic and normal hematopoietic progenitors, able to preserve cloning ability in cultured normal precursors.31,32 Our findings confirm and extend previous
studies on bcl-2 functions in hematopoietic cells31,32 and
link bcl-2 regulation to autocrine production of TGF-1 in primitive
progenitors. The specific influence of TGF-1 on bcl-2 expression was
revealed by the unaltered expression of p53, c-myc, bcl-xL, and bax in anti-TGF-1-treated CLRPP, compared to control CLRPP.
Effects of autocrine TGF- 1 neutralization does not cause detectable changes
in these cell cycle-regulating proteins. These results were confirmed
by the analysis of the cycling status and the number of cell divisions
of control and study cultures, which demonstrated that modulation of
bcl-2 gene occurred without any perturbation of progenitor cycling
activity. Hence, we can argue that the autocrine amount of TGF-1 is
unable, in the presence of a rich cocktail of stimulatory cytokines, to
negatively modulate cell cycle status while it contributes to the
maintenance of adequate bcl-2 expression in these cells, dissociating
its previously described cell cycle-related
activities19-26 from these newly observed survival functions. In fact, the aforementioned experimental evidence suggests that TGF-1 can exert distinct effects, inhibiting in a
dose-dependent manner cell cycle entry of quiescent precursors
21 and regulating survival when acting at autocrine levels on
cycling progenitors. Up-modulation of p27 protein in
anti-TGF-1-treated CLRPP was partially unexpected because this CDK
inhibitor is increasingly expressed in TGF-1-arrested mink Mv 1 Lu
epithelial cells.33 On the other hand, Ravitz et
al34 showed that exogenous TGF-1 induced DNA synthesis
in C3H10T1/2 mouse fibroblasts with a concomitant p27 down-modulation,
suggesting that different cell types may use these inhibitors in
different ways. A recent study suggested complex functions for p27
during hematopoietic differentiation, which include subcellular
redistribution and, possibly, switch of binding partners with the
consequent formation of either active or inactive cyclin/CDK complexes,
despite the presence of constant levels of total p27
protein.35 However, in line with previous studies that
showed an increase of total p27 levels on terminal differentiation,36 our present results suggest that p27
up-modulation in anti-TGF-1-treated CLRPP may be consequent to a
premature induction of differentiation produced by autocrine TGF-1
deprivation and may contribute to the loss of hematopoietic potential
observed in these precursors. Collectively, our findings fit well with the major hematopoietic defect observed in TGF-1 knockout mice in
which yolk sac anemia develops because of a 90% reduction of absolute
blood cell number.37 The occurrence of blood cell
hypoplasia in TGF-1 knockout mice rather than hyperplasia (as could
be expected due to the previously described inhibitory activity of
TGF-1 on hematopoietic cell proliferation19-21) supports
the hypothesis that, in vivo, TGF-1 also regulates hematopoietic
cell survival and differentiation. Hence, the hematopoietic defect of
TGF-1 knockout mice could result from the loss of multiple actions
exerted by TGF-1 on stem/progenitor cells in vivo, which translated
into continuous escape from quiescence of stem cells (with the
consequent exhaustion of the stem cell pool due to the absence of
TGF-1 inhibitory activity19,25,26) and premature death
and differentiation of cycling progenitors (due to the lack of these
newly observed survival functions of autocrine TGF-1).
Neutralization of autocrine TGF- 1-treated CLRPP was confirmed by a certain down-modulation of the CD34 antigen (whose expression was in part correlated to bcl-2) and by the appearance of a 2-fold higher number of
CD15+, CD11b+, and glycophorin-A+ cells in anti-TGF-1-treated cultures as compared to controls. Finally, the reduction of
LTC-IC frequency confirmed the relevant loss of hematopoietic potential in anti-TGF-1-treated CLRPP.
Submitted August 18, 1999; accepted January 4, 2000.
Supported in part by a grant from Associazione Italiana per la Ricerca sul Cancro (AIRC).
Reprints: Luca Pierelli, Servizio di Ematologia ed Emotrasfusione, Universita' Cattolica del Sacro Cuore, Largo A.Gemelli 8, 00168 Roma, Italy.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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| Copyright © 2000 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
1 is a cell
cycle-independent effect and influences their hematopoietic potential
Top
Abstract
Introduction
indicates the control; 
, anti-TGF-
,
anti-TGF-
, anti-TGF-
cells from all sources of hematopoietic
cells
is upregulated during granulocytic, but not monocytic, differentiation.
Blood.
1997;90:2591
precursors. Br J Haematol. In
press.