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Stem Cells, Vol. 18, No. 2, 102-111, March 2000
© 2000 AlphaMed Press

Release from Quiescence of Primitive Human Hematopoietic Stem/Progenitor Cells by Blocking Their Cell-Surface TGF-ß Type II Receptor in a Short-Term In Vitro Assay

Nicolas Fortunela, Jacques Hatzfelda, Sergueï Kisseleva, Marie-Noëlle Moniera, Karin Ducosa, Angelo Cardosob, Pascal Batarda, Antoinette Hatzfelda

a Laboratoire de Biologie des Cellules Souches Somatiques Humaines, Centre National de la Recherche Scientifique, Villejuif, France;
b Division Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

Key Words. Hematopoiesis • Stem/progenitor cell • HPP-Q in vitro assay • Quiescence • TGF-ß receptor

Jacques Hatzfeld, Ph.D., UPR 1983, 7, rue Guy Môquet, 94800 Villejuif, France. Telephone: 33-1-49-58-33-16; Fax: 33-1-49-58-33-15; e-mail: hatzfeld{at}infobiogen.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Genetic alterations of the signaling cascade of transforming growth factor-ß (TGF-ß) are often associated with neoplastic transformation of primitive cells. This demonstrates the key role for this pleiotropic factor in the control of quiescence and cell proliferation in vivo. In the high proliferative potential-quiescent cell (HPP-Q) in vitro assay, the use of TGF-ß1 blocking antibodies (anti-TGF-ß1) allows the detection within two to three weeks of primitive hematopoietic cells called HPP-Q, which otherwise would not grow. However, the possibility of triggering cell proliferation by blocking the cell-surface TGF-ß receptors has not been investigated until now. We have tested here the efficiency of a blocking antibody against TGF-ßRII (anti-TGF-ßRII) on CD34+CD38– hematopoietic cells, a subpopulation enriched in primitive stem/progenitor cells, and compared its effect with that of anti-TGF-ß1. About twice as many HPP colony-forming cells were detected in the presence of anti-TGF-ß1 or anti-TGF-ßRII, compared to the control (p < 0.02). Moreover, anti-TGF-ßRII was as efficient as anti-TGF-ß1 for activating multipotent HPP-granulocyte erythroid macrophage megakaryocyte and HPP-Mix, bipotent HPP-granulocyte-macrophage (GM) and unipotent HPP-G, HPP-M and HPP-BFU-E. We therefore propose the use of anti-TGF-ßRII to release primitive cells from quiescence in the HPP-Q assay. This strategy could be extended to nonhematopoietic tissues, as TGF-ß1 may be a pleiotropic regulator of somatic stem cell quiescence.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The most primitive cells of the hematopoietic system are largely in a quiescent state in adults. They require an appropriate stimulation to express their high proliferative potential [1-4]. This characteristic renders particularly difficult the development of rapid in vitro stem cell assays. Quiescence may be due either to the absence of mitogens or the presence of growth inhibitors which block these primitive cells in G0/G1 phase of the cell cycle.

One important physiological regulator of hematopoietic stem/progenitor cell quiescence is transforming growth factor-ß1 (TGF-ß1). Indeed, this factor is well-known for its antiproliferative effect on the most primitive cells of both murine [5-10] and human hematopoietic systems [11-18]. TGF-ß1 is a 25-kDa protein produced by bone marrow stromal cells [19-21] and hematopoietic progenitors which then contribute to their own cell cycle control through an autocrine process [14].

Several cell surface proteins have been identified for their ability to bind TGF-ß1. Among these, two subfamilies of transmembrane serine/threonine kinases called the type I and type II TGF-ß receptors (TGF-ßRI and TGF-ßRII) are required for signal transduction in mammalian cells [22-25]. The role of these receptors in TGF-ß1 signaling is now well established. TGF-ß1 first binds to TGF-ßRII, which is a constitutively active kinase. TGF-ßRI is then recruited by the TGF-ß/TGF-ßRII complex and phosphorylated by TGF-ßRII. Phosphorylation allows TGF-ßRI to propagate the signal to downstream substrates, such as the intracellular Smad proteins [26, 27]. The first member of the Smad family proteins was the product of Drosophila gene and was called Mad for "Mother against dpp". Three Mad homologues were identified in C. elegans and called Sma because their mutation causes small body size. Then homologues were described in vertebrates and named Smads for Sma/Mad related [27].

We previously proposed the use of TGF-ß1 antisense oligonucleotides or blocking antibodies to improve the efficiency of short-term in vitro assays (two to three weeks) to detect the hematopoietic stem/progenitor cell compartment [14, 16]. This approach allows the release from quiescence of a subpopulation of human primitive stem/progenitor cells which we term high proliferative potential-quiescent cells or HPP-Q cells, as they possess the potential for generating in vitro very large colonies or clones with more than 105 cells, but are maintained in a quiescent state by low, physiological, concentrations of TGF-ß1 [18].

A possible alternative approach to release these primitive hematopoietic cells from TGF-ß1-dependent quiescence was to neutralize the biologic activity of their cell surface TGF-ßR. For this purpose, we used a blocking antibody which specifically blocks human TGF-ßRII. As detailed in this report, this new strategy was tested with success on human umbilical cord blood (UCB) cells with the CD34+CD38– phenotype, a cell subpopulation which is known to be highly enriched in primitive hematopoietic stem/progenitor cells [28].


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
CD34+ Cell Preparation
UCB samples were collected immediately after delivery using the method of Brossard et al. [29]. Informed consent was obtained before sample collection. Mononuclear cells of less than 1.077 g/ml density were isolated by centrifugation on Ficoll-Hypaque (Seromed Biochrom KG; Berlin, Germany). Hematopoietic progenitors expressing the CD34 antigen were purified using the magnetic cell sorting system MACS (Miltenyi Biotec; Bergisch Gladbach, Germany). The procedure provided a high recovery of CD34+ cells. Purity of the samples routinely exceeded 98%.

Cell Sorting
Double staining of CD34 and CD38 antigens was performed using the following procedure: CD34+ cells were suspended in phosphate-buffered saline/bovine serum albumin (PBS/BSA) (0.2%) and incubated with an anti-CD34 fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (mAb) (mouse IgG1; 8G12 clone; Becton Dickinson; San Jose, CA) and an anti-CD38 phycoerythrin (PE)-conjugated mAb (mouse IgG1; HB-7 clone; Becton Dickinson) for 30 min at 4°C and washed twice in PBS/BSA (0.2%). Irrelevant FITC- and PE-mouse IgG1 mAbs (Becton Dickinson) were used as negative controls to evaluate the background signal. A dual-parameter diagram displaying FITC (CD34) and PE (CD38) fluorescence was then generated. A sorting window for CD34+ cells expressing undetectable levels of CD38 antigen (CD34+CD38– cells) was defined on the basis of the negative controls. CD34+ and CD34+CD38– cells were sorted using a Vantage fluorescence-activated cell sorter (FACS Vantage; Becton Dickinson). After sorting, purity of the samples was above 99.5%.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
CD34+ and CD34+CD38– cells were washed twice in PBS after sorting. Total RNA was then prepared using the RNABle® extraction kit (Eurobio; Paris, France), as specified by the manufacturer, and resuspended in 1 mM EDTA. Single-stranded cDNA were synthesized by primer extension using random hexanucleotides (Boehringer; Mannheim, Germany) and avian myeloblastosis virus (AMV) RT (Promega; Madison, WI); RNA samples were supplemented with 1.74X Taq polymerase buffer (Promega), 6 mM MgCl2, 870 µM dNTPs, 0.045X random hexanucleotides (Boeringer), and 174 U/ml AMV RT (Promega) in a total volume of 23 µl. RT was performed in a PTC-200 Peltier Thermal Cycler (MJ Research, Inc.; Watertown, MA) using the following program: 10 min at 23°C, 60 min at 42°C, 5 min at 95°C and then 1 min at 99°C. Five µl of cDNA were used for each amplification reaction. Each cell sample was analyzed for TGF-ß1, TGF-ßRI and TGF-ßRII mRNA expression. We amplified the housekeeping gene GAPDH mRNA as a positive control, and included a negative control for each PCR experiment. Negative controls consisted of evaluating the product of RT when RNA was omitted. PCR reactions were performed in a PTC-200 Peltier Thermal Cycler (MJ Research, Inc.) using the following conditions: 1X Taq polymerase buffer, 60 µM dNTPs, 725 nM upstream and downstream primers, and 24 U/ml Taq DNA polymerase (Promega) in a total volume of 40 µl. For GAPDH, TGF-ß1 and TGF-ßRI cDNA amplification, one PCR cycle was constituted by 30 sec at 94°C, 30 sec at 61°C and then 60 sec at 72°C. For TGF-ßRII cDNA amplification, one PCR cycle was constituted by 30 sec at 94°C, 30 sec at 57°C and then 60 sec at 72°C. Amplification was performed during 35 and 36 cycles for GAPDH and TGF-ß1, respectively, and during 38 cycles for TGF-ßRI and TGF-ßRII. PCR products were electrophoresed in 3% agarose gels (Eurogentec; Seraing, Belgium) and visualized under an ultraviolet light after staining with ethidium bromide.

Primers were designed according to sequences published previously (Table 1Go). Their specificity was verified using the Blast sequence homology program.


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  		Table 1. Oligonucleotide primers for RT-PCR
 
Growth Factors and Neutralizing Antibodies
Recombinant human (rHu) interleukin 3 (IL-3), rHuIL-6, rHuIL-11, and rHuGM-CSF were generously provided by the Genetics Institute (Cambridge, MA). rHu erythropoietin (EPO), rHu steel factor (SF), and rHuG-CSF were supplied by Valbiotech (Paris, France) and rHuTGF-ß1 by R&D (Abingdon, UK). Anti-TGF-ß1 was raised in chicken (StemBio Research; Villejuif, France). This polyclonal antibody specifically neutralizes the biological activity of human TGF-ß1 and was used at 5 µg/ml. Anti-TGF-ßRII was produced in goat (R&D). It was purified by affinity chromatography and specifically neutralizes human TGF-ßRII bioactivity. It was used at 5 µg/ml.

Colony Assay
CD34+ cells were cultured in semisolid methylcellulose SB*1d medium containing 30% fetal calf serum (StemBio Research; Villejuif, France) in the presence of the following cytokines: IL-3, IL-6, IL-11, steel factor (SF), G-CSF, GM-CSF and EPO. Depending on the culture condition, anti-TGF-ß1, anti-TGF-ßRII or irrelevant antibodies were added to this control medium, as specified.

For each experiment and culture condition, cells were plated in triplicate in 35 mm suspension culture dishes. Dishes contained 150 sorted cells in 1 ml medium. Cultures were incubated at 37°C in a fully humidified atmosphere with 5% CO2. Colonies were identified and counted after 21 days of culture under an inverted microscope, as already described [30, 31].

Statistical Analysis
The paired Student's t-test was used to determine the statistical significance of differences between means, calculated from several (n) experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Detection of the mRNAs Coding for TGF-ß1 and TGF-ßRI and TGF-ßRII in CD34+ and CD34+CD38– Cells
Using RT-PCR, we first detected an expression of TGF-ß1, TGF-ßRI and TGF-ßRII mRNAs in the total CD34+ cell population, which includes both primitive and more mature progenitors (Fig. 1Go). To focus this study on the most primitive hematopoietic compartment, we further used the CD38 membrane antigen as a marker to separate the CD34+ population into a more immature and quiescent subpopulation believed to contain the stem cell compartment (CD34+CD38–) and a fraction of later commited progenitors (CD34+CD38+), as first described by Terstappen et al. [28]. In our experiments, a sorting gate for CD34+CD38– cells was defined to isolate the 5% of CD34+ cells with the lowest level of cell-surface CD38 antigen labeling (Fig. 2Go). TGF-ß1, TGF-ßRI, and TGF-ßRII mRNAs were detected in this subpopulation highly enriched in primitive stem/progenitor cells (Fig. 3Go), which suggests that these cells are not only responsive to TGF-ß1 but are able to produce it. Moreover, it is interesting to note that we performed a similar analysis at the single-cell level using a nested PCR method, and we confirmed the capacity for one single CD34+CD38– cell to express TGF-ß1, TGF-ßRI, and TGF-ßRII mRNAs [32]. These molecular data are in agreement with a previous in vitro functional study where we demonstrated, using antisense oligonucleotides in single-cell experiments, the existence of an autocrine loop involving TGF-ß1 for the control of normal human hematopoietic stem/progenitor cell quiescence [14].



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  		Figure 1. Detection of TGF-ß1, TGF-ßRI, TGF-ßRII, and GAPDH mRNAs in CD34+ cells by RT-PCR. The results presented are from one typical experiment out of three. (A) PCR products for TGF-ßRI (224 bp), TGF-ßRII (465 bp) and GAPDH (445 bp). (B) PCR products for TGF-ß1 (284 bp) and GAPDH (445 bp).

 


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  		Figure 2. Analysis of CD38 antigen expression on CD34+ cells by flow cytometry. A sorting gate corresponding to CD34+ cells expressing a low or undetectable level of CD38 was defined on the basis of a negative control and used to isolate a cell subpopulation, the phenotype of which is termed CD34+CD38–. One typical labeling profile is shown, where the CD34+CD38– cell fraction represented about 5% of the total CD34+ population.

 


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  		Figure 3. Detection of TGF-ß1, TGF-ßRI, TGF-ßRII and GAPDH mRNAs in FACS-sorted CD34+CD38– cells by RT-PCR. The results presented are from one typical experiment out of three. (A) PCR products for TGF-ßRI (224 bp), TGF-ßRII (465 bp) and GAPDH (445 bp). (B) PCR products for TGF-ß1 (284 bp) and GAPDH (445 bp).

 
Comparative Effects of Anti-TGF-ß1 or Against Anti-TGF-ßRII on the Cloning Efficiency and Cell Proliferation of CD34+CD38– Progenitors
FACS-sorted CD34+CD38– hematopoietic cells were cultured at low density (150 cells/ml) to minimize any TGF-ß1 paracrine effect in a semisolid medium containing a combination of cytokines (IL-3, IL-6, IL-11, G-CSF, GM-CSF, SF, and EPO) optimized for primitive progenitors and able to sustain a wide range of hematopoietic differentiation. This medium allows the obtention of colonies from early progenitors but does not stimulate the growth of quiescent cells of the stem cell compartment, which remain mostly unresponsive to cytokines in classic short-term liquid or semisolid in vitro assays. In contrast, we previously described that an identical medium, to which antisense oligonucleotides or antibodies neutralizing autocrine, paracrine and endogenous TGF-ß1 were added, was able to release these primitive cells from quiescence [14, 16, 18, 33]. To assess whether a similar growth-inducing effect could be obtained by the inactivation of the cell-surface TGF-ßR, we have compared the efficiency of anti-TGF-ß1 or against anti-TGF-ßRII, which is the first effector of the TGF-ß1-signaling cascade. After 21 days of culture, the resulting colonies were identified and classified into two categories: large colonies generated by primitive high proliferative progenitors (HPP-CFC) and small colonies from later progenitors of low proliferative capacity.

Anti-TGF-ßRII, as well as anti-TGF-ß1, showed a significant positive effect on the cloning efficiency of CD34+CD38– progenitors (Fig. 4Go). For a total of 450 cells plated in three Petri dishes, only 109.0 ± 30.3 total colony-forming cells (CFC) were detected in the control medium (CM), whereas 153.3 ± 31.2 total CFC were obtained in the presence of anti-TGF-ßRII and 154.0 ± 38.6 in the presence of anti-TGF-ß1 (mean ± SD calculated from three independent experiments). This represented a total CFC increase of 43.0 ± 12.7% and 42.1 ± 5.0% in the presence of anti-TGF-ßRII and anti-TGF-ß1, respectively, compared with the CM (p < 0.05; n = 3).



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  		Figure 4. Effect of anti-TGF-ß1 or anti-TGF-ßRII blocking antibodies on colony formation by CD34+CD38– cells. FACS-sorted CD34+CD38– cells were plated in triplicate at 150 cells/dish in semi-solid medium. The control medium (CM) was supplemented with anti-TGF-ß1, anti-TGF-ßRII or irrelevant antibodies. Colonies were examined and identified after 21 days of culture. Results are detailed for total colonies (CFC), large colonies generated from primitive high proliferative potential colony-forming cells (HPP-CFC) and small colonies generated from later low proliferative progenitors. Data are expressed as relative colony numbers in comparison with the control condition. The values correspond to mean ± SD calculated from three similar independent experiments (*p < 0.05; ** p <0.02, using the paired Student's t-test).

 
More precisely, the growth-inducing effect of anti-TGF-ßRII and anti-TGF-ß1 was targeted to the most primitive HPP-CFC. The number of HPP-CFC detected for a total of 450 cells plated was 46.7 ± 20.0 in CM, but reached 91.7 ± 32.3 in the presence of anti-TGF-ßRII and 101.3 ± 38.6 in the presence of anti-TGF-ß1. This represented a 103.0 ± 22.9% increase of the number of HPP-CFC for anti-TGF-ßRII and a 121.4 ± 15.6% increase of HPP-CFC for anti-TGF-ß1, compared with the CM (p < 0.02; n = 3). In contrast, the two antibodies did not increase the number of smaller colonies derived from later progenitors (p > 0.010; n = 3). As a consequence of these effects, after 22 days of culture, we found an increased number of total cells in dishes containing anti-TGF-ßRII or anti-TGF-ß1 (Fig. 5Go). Irrelevant antibodies had no effect either on cloning efficiency or cell proliferation (Figs. 4 and 5GoGo).



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  		Figure 5. Effect of anti-TGF-ß1 or anti-TGF-ßRII blocking antibodies on cell proliferation. FACS-sorted CD34+CD38– cells were plated in triplicate at 150 cells/dish in semisolid medium. The control medium (CM) was supplemented with anti-TGF-ß1, anti-TGF-ßRII or irrelevant antibodies. After 22 days of culture, colonies were pooled and resuspended in liquid medium. Total cell proliferation was evaluated in each dish by counting using a Malassez chamber. Data are expressed as a proliferation index between day 1 and day 22 (total number of cells counted at day 22/number of plated cells). The values correspond to means ± SD of triplicate determinations for one typical experiment out of three.

 
Comparative Efficiency of anti-TGF-ß1 and anti-TGF-ßRII on the Release from Quiescence of Primitive CD34+CD38– Stem/Progenitor Cells with Various Potentials of Differentiation
To further characterize the effects of anti-TGF-ßRII and anti-TGF-ß1 on stem/progenitor cells, the results were detailed for primitive erythro-myeloid progenitors (HPP-granulocyte erythroid macrophage megakaryocyte [GEMM] and HPP-Mix), early erythroid (HPP-BFU-E) and HPP-GM and myeloid progenitors (HPP-GM, HPP-G and HPP-M). As the most primitive multipotent HPP-GEMM and HPP-Mix cells correspond to a rare stem/progenitor cell subpopulation, it was therefore necessary to collect data from a larger panel of cells to obtain a representative view of the effects of anti-TGF-ßRII and anti-TGF-ß1. For this reason, colony numbers stained from three independent experiments with determinations performed in triplicate were cumulated to obtain a total of 1,350 CD34+CD38– cells plated (Fig. 6Go).



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  		Figure 6. Effect of anti-TGF-ß1 or anti-TGF-ßRII blocking antibodies on colony formation by various types of primitive stem/progenitor cells contained in the CD34+CD38– subpopulation. FACS-sorted CD34+CD38– cells were plated as previously described in a control medium (CM) and in the presence of anti-TGF-ß1, anti-TGF-ßRII or irrelevant antibodies. Colonies were examined and identified after 21 days of culture. The values correspond to cumulative numbers of colonies obtained in nine dishes per culture condition, which represent a total of 1,350 CD34+CD38– plated cells for each condition. (A) Total number of primitive high proliferative potential colony-forming cells detected (HPP-CFC). (B) Primitive HPP erythro-myeloid progenitors (HPP-GEMM and -Mix). (C) Primitive HPP erythroid progenitors (HPP-BFU-E). (D) Primitive HPP myeloid progenitors (HPP-GM, -G and -M).

 
Anti-TGF-ßRII, as well as anti-TGF-ß1 efficiently activated the growth of these various stem/progenitor cell types. However, we observed that the growth-inducing effects of anti-TGF-ßRII and anti-TGF-ß1 were relatively more important for the most primitive multipotent stem/progenitor cells. Indeed, the two antibodies allowed the detection of about four times more HPP-GEMM and HPP-Mix, compared to the control (Fig. 6BGo), two times more HPP-BFU-E (Fig. 6CGo) and two times more HPP-GM, HPP-GM, and HPP-M (Fig. 6DGo). Irrelevant antibodies showed no effect on colony formation (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The aim of this study was to evaluate whether anti-TGF-ßRII could be used as a tool to release from quiescence a subpopulation of primitive hematopoietic stem/progenitor cells, in a short-term in vitro assay.

The efficiency of TGF-ß1 antisense oligonucleotides or anti-TGF-ß1 to trigger the cell-cycling of early hematopoietic progenitors has been previously described. Anti-TGF-ß1 was shown to stimulate the in vitro clonal growth of human hematopoietic progenitors [14, 16, 18, 34] in methylcellulose colony-forming assays or single-cell liquid cultures. This has been confirmed in the murine system [7-9]. Anti-TGF-ß1 was also used to prolong or reactivate human primitive progenitor cell proliferation in stroma-supported long-term culture systems [19]. However, the possibility of releasing hematopoietic stem/progenitor cells from TGF-ß1-dependent quiescence by blocking their cell surface TGF-ß receptors had not been investigated until now.

The HPP-Q cell assay that we have developed consists of a comparison between a control medium and an identical medium to which an inhibitor of the TGF-ß1-signaling cascade is added. This later culture condition is termed HPP-Q medium. The control medium reveals easily activated progenitors, as does a classic mixed colony assay. In addition to these progenitors, the HPP-Q medium activates quiescent stem/progenitor cells with a high proliferative potential. These primitive cells which we call HPP-Q can be quantified after only two to three weeks by the difference between the number of large colonies obtained in the two culture conditions [18]. It is important to note that this assay is easier to interpret if cells are plated at very low density in methylcellulose semisolid medium (150 cells/ml) as we did here, or in single-cell liquid cultures (not shown). These conditions are better adapted to an optimal development of the macroscopic colonies or clones generated by HPP-Q cells and they prevent the effect of paracrine TGF-ß1.

In this study, we show that a similar growth-inducing effect on primitive quiescent hematopoietic stem/progenitor cells is obtained whether anti-TGF-ß1 or anti-TGF-ßRII blocking antibodies are used. When added separately to clonal cultures of human CD34+CD38– cells, the two antibodies increased significantly primitive stem/progenitor cell growth. Indeed, about twice as many HPP-CFC were detected in culture conditions containing anti-TGF-ß1 or anti-TGF-ßRII, compared with the control medium (Fig. 4Go) (p < 0.02). More precisely, anti-TGF-ßRII was as efficient as anti-TGF-ß1 for activating primitive erythro-myeloid (HPP-GEMM and -Mix), erythroid (HPP-BFU-E) and myeloid progenitors (HPP-GM, -G, and -M) (Fig. 5Go). According to these results, we therefore propose the use of anti-TGF-BRII as an alternative strategy to reveal primitive quiescent hematopoietic cells in the HPP-Q assay.

Of more general interest, the data presented in this report provide convincing elements which suggest that a loss of responsiveness to TGF-ß1 may be one critical event leading to malignant transformation. Indeed, our results demonstrate that the inactivation of TGF-ßRII allows early hematopoietic stem/progenitor cells, which are normally quiescent, to escape from cell-cycling inhibition. The physiological relevance of this mechanism has been clearly shown in the hematopoietic system by the description of abnormalities in the expression of TGF-ßR in proliferative syndromes including early myeloid [35, 36] and lymphocytic leukemia [37, 38]. In these pathologies, a selective advantage is given to the tumor cells by the loss of TGF-ßRI or TGF-ßRII expression. A loss of sensitivity to the growth-inhibitory effect of TGF-ß1 due to an inactivation of TGF-ßRII has also been described for T-cell lymphoma [39, 40]. Active TGF-ß1 present in the bone marrow microenvironment is able to exert a negative control on the growth of normal hematopoietic progenitors, but not on leukemic cells which have overcome TGF-ß1 regulatory signals.

It is interesting to note that the same phenomenon is observed in various types of nonhematopoietic cancers. Indeed, mutations or genetic defects resulting in a lack of TGF-ßRI or TGF-ßRII function are associated with the acquisition of a transformed phenotype in several types of cancers including colon cancers [41, 42], gastric cancers [43, 44], prostate cancers [45], pancreatic cancers [46], thyroid tumors [47], hepatic tumors [48, 49], retinoblastoma [50, 51], and lung adenocarcinoma [52]. Moreover, the importance of TGF-ß signaling for the control of normal somatic cell proliferation has been demonstrated in the case of skin keratinocytes [53, 54], cells of the mammary gland, lung and exocrine pancreas [55, 56], using transgenic mice expressing a dominant-negative mutant TGF-ßRII. In addition, downstream elements of the TGF-ß signal transduction pathway have also been identified as potential targets for oncogenic transformation. Indeed, mutations or somatic alterations resulting in a disruption of the Smad signaling cascade are observed in several tumor cells resistant to the growth-inhibitory effect of TGF-ß1 [57]. In these cells, insensitivity to TGF-ß1 is either due to an inactivation of the TGF-ß signal transducers Smad2 and Smad4 [58–60], or to an enhanced expression of the TGF-ß1 signaling inhibitor Smad6 [61].

In the present report, we show that the inhibition of the antiproliferative TGF-ß1 signaling pathways in human hematopoietic stem/progenitor cells, using blocking antibodies against TGF-ß1 or TGF-ßRII, allows quiescent cells to escape from cell-cycling inhibition. Unlike the case of cancers, in which the loss of sensitivity to TGF-ß1 is due to a genetic alteration and is therefore irreversible, the use of blocking antibodies or antisense oligonucleotides in the HPP-Q assay allows the transient abolishment of the negative control exerted by TGF-ß1 on normal stem/progenitor cell proliferation. It is known that antibodies or oligonucleotides are rapidly degraded, when added to cell cultures. However, this treatment is sufficient to activate, within hours, quiescent cells which then grow and differentiate in response to the cytokines present in the culture medium. As proof of this, in experiments where anti-TGF-ß1 antibody was tested on hematopoietic CD34+ progenitors, we observed that its effect was similar whether cells were pretreated for only 12 h before plating in semisolid medium or whether the antibody was maintained or added repeatedly throughout the culture period (unpublished results). These data are particularly important, as they show that the large clones or colonies observed in the HPP-Q condition result from the activation of primitive stem/progenitor cells which remain "silent" in the control condition, and not from an increase in colony size in response to a continuous stimulation of cell division by anti-TGF-ß1 during the two to three weeks of culture. Moreover, we know that when cells divide, mature and differentiate, they progressively loose their sensitivity to the growth inhibitory effect of low concentrations of TGF-ß1 (unpublished results).

Taken together, these in vitro and in vivo studies emphasize the importance of understanding the role of the genes involved in the TGF-ß signal transduction pathways. This would help to solve various problems linked to the control of normal and malignant somatic cell proliferation, in which TGF-ß1 plays a key role. First, progress in the knowledge of the mechanism by which somatic stem/progenitor cell proliferation is regulated will allow stimulation of the growth of cells which normally do not easily divide in vitro, as for normal primitive hematopoietic cells in the HPP-Q system [14, 18, 62]. Second, transfection of the wild-type TGF-ßRI, TGF-ßRII or Smad genes would reduce the tumorigenic capacity of cancer cells by reestablishing the responsiveness to TGF-ß1 of cells which have escaped from its growth inhibitory signals [63-68].


   Acknowledgments
 
We are indebted to Dr. Mary Osborne for her critical reading of the manuscript, Dr. Jane Olsson for her help in the design of oligonucleotide primers and Barbara Fortunel for her excellent photographic work. This study was supported by the European Contract No. BIO4-CT96-0646 and the Centre National de la Recherche Scientifique (CNRS). N.F. and K.D. were recipients of fellowships from the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche (MENESR) and from Association pour la Recherche sur le Cancer (ARC). S.K. was supported by the Direction des Recherches Etudes et Techniques (DRET).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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accepted for publication February 10, 2000.




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