Zyxin is up-regulated during megakaryocytic differentiation of human UT-7/c-mpl cells.

To characterize genes involved in megakaryocytic commitment, we compared expression profiles of bipotent cells (UT-7/c-mpl) with those of the same cells induced to differentiate towards megakaryopoiesis in the presence of TPO. Using cDNA arrays, we showed that 12 out of 2260 genes changed their expression level after 6h of TPO stimulation. One of these genes encodes for zyxin, a cytoskeleton protein component. Zyxin is up-regulated at the mRNA and protein levels in UT-7/c-mpl cells in response to TPO confirming the reliability of the cDNA array technology. Similarly, when CD34 positive cells were induced to differentiate into megakaryocytes, zyxin mRNA was accumulated. Furthermore, when megakaryocytes were allowed to spread on fibrinogen, formation of stress fibers and lamellipodia was induced and zyxin was localized at the picks of actin stress fibers. These results suggest an important role for zyxin during megakaryocytic differentiation and more precisely in the regulation of the integrin mediated adhesion process in megakaryocytes.

Targeting the kinesin Eg5 to monitor siRNA transfection in mammalian cells.

RNA interference, the inhibition of gene expression by double-stranded RNA, provides a powerful tool for functional studies once the sequence of a gene is known. In most mammalian cells, only short molecules can be used because long ones induce the interferon pathway. With the identification of a proper target sequence, the penetration of the oligonucleotides constitutes the most serious limitation in the application of this technique. Here we show that a small interfering RNA (siRNA) targeting the mRNA of the kinesin Eg5 induces a rapid mitotic arrest and provides a convenient assay for the optimization of siRNA transfection. Thus, dose responses can be established for different transfection techniques, highlighting the great differences in response to transfection techniques of various cell types. We report that the calcium phosphate precipitation technique can be an efficient and cost-effective alternative to Oligofectamine in some adherent cells, while electroporation can be efficient for some cells growing in suspension such as hematopoietic cells and some adherent cells. Significantly, the optimal parameters for the electroporation of siRNA differ from those for plasmids, allowing the use of milder conditions that induce less cell toxicity. In summary, a single siRNA leading to an easily assayed phenotype can be used to monitor the transfection of siRNA into any type of proliferating cells of both human and murine origin.

Thrombopoietin induces histidine decarboxylase gene expression in c-mpl transfected UT7 cells.

The leukemic cell line UT7 is endowed with both megakaryocyte and basophil differentiation potential, as judged by its capacity to respond to PMA by displaying megakaryocytic and basophilic markers and to produce histamine by neosynthesis. Herein, we addressed the question whether the biological activities characteristic of basophil differentiation were still induced when c-mpl-transfected UT7 cells received a specific megakaryocytic differentiation signal delivered by thrombopoietin (TPO). Surprisingly, we found that histamine synthesis did effectively occur in response to the growth factor. This activity was not associated with megakaryopoiesis since it was not detected in megakaryocytes generated from CD34(+) cells cultured in the presence of TPO. Comparing different c-mpl-transfected cell lines, we found that the amount of histamine generated in response to TPO correlated with their responsiveness to PMA, but not with their level of c-mpl expression, thus revealing an intrinsic basophil differentiation potential. Both PMA- and TPO-induced histamine synthesis was reduced by PKC and MEKs inhibitors, indicating that the induction occurred through a common signalling pathway.

The granulocyte colony-stimulating factor receptor supports erythroid differentiation in the absence of the erythropoietin receptor or Stat5.

To evaluate the functional conservation of signal transduction mechanisms between haematopoietic receptors and to characterize the molecules activated in this phenomenon, we introduced granulocyte colony-stimulating factor receptor (G-CSFR) cDNA into mouse fetal liver cells using a retroviral vector. In semi-solid medium assays, G-CSFR-infected cells gave rise to all types of colonies [granulocyte-macrophage (GM), megakaryocyte (MK) and mixed lineage (GEMM) colony-forming units (CFU) and erythroid burst-forming units (BFU-E)] in the presence of G-CSF alone. The direct effect of G-CSF on erythroid differentiation of G-CSFR-transduced erythroid progenitors was demonstrated by the development of erythroid colonies using G-CSFR-expressing Lin- cells cloned at one cell per well in liquid culture in the presence of G-CSF. Interestingly, while Stat5, but not Stat3, was activated in erythroid cells in response to erythropoietin (EPO), both were activated in erythroid and granulocytic cells stimulated by G-CSF. Furthermore, G-CSF induced the growth of erythroid colonies from G-CSFR-expressing fetal liver cells from EPO receptor-/- (EPO-R-/-) or Stat5a-/- Stat5b-/- mice, demonstrating that erythroid differentiation can occur in the absence of EPO-R or Stat5. These data show that forced expression of G-CSFR allows G-CSF-dependent multilineage proliferation and differentiation of haematopoietic progenitors and rescues EPO-R-/- erythroid cells. While G-CSF induces Stat5 activation in G-CSFR-expressing erythroid cells, this activation is not necessary for the terminal erythroid differentiation induced by G-CSF.

Inhibition of erythroid differentiation and induction of megakaryocytic differentiation by thrombopoietin are regulated by two different mechanisms in TPO-dependent UT-7/c-mpl and TF-1/c-mpl cell lines.

Thrombopoietin (TPO) regulates megakaryocytic (MK) maturation and platelet production. Molecular and cellular mechanisms of the TPO-induced MK differentiation are not totally understood. In order to develop cellular models to study these mechanisms, we introduced c-mpl into UT-7 and TF-1 cells by means of a retroviral vector and compared the effects of TPO on these two cell lines. UT-7 and TF-1 cell lines are two factor-dependent leukemic cell lines with an erythroid and MK phenotype. They proliferate in response to IL-3, GM-CSF and EPO, but not to TPO. The erythroid differentiation of both cell lines can be markedly increased by EPO. Several UT-7/c-mpl and TF-1/c-mpl cell clones which express different levels of the c-mpl protein (Mpl) were obtained and all became TPO-dependent for their proliferation. The UT-7/c-mpl clones, but not the TF-1/c-mpl clones, were capable of undergoing MK differentiation in response to TPO. This was demonstrated by the increase in MK markers (GPIIb, GPIIIa, GPIb alpha, GPIX and vWF), the appearance of cytoplasmic alpha-granules, intracellular membranes resembling demarcation membranes which were immunologically labeled with an GPIIb/IIIa anti-antibody, and a small percentage of polyploid cells (8N and 16N). In contrast, TPO inhibited the erythroid program of differentiation (glycophorin A, beta-globin and EPO receptor) as well as the differentiative activity of EPO in both UT-7/c-mpl and TF-1/c-mpl clones. It is noteworthy that the differentiative effect of EPO in TF-1/c-mpl cells was associated with an increase in GATA-1 transcripts which was totally suppressed by TPO. Overall the effects of TPO are the same as those of phorbol myristate acetate (PMA) which also induces MK differentiation and inhibits erythroid differentiation. These results suggest that: (1) Mpl expression is necessary but not sufficient for induction of MK differentiation; and (2) induction of Mk differentiation and inhibition of erythroid differentiation by TPO involve different signaling pathways; the pathway involved in the inhibition of erythroid differentiation might be related to a downregulation of GATA-1 expression in TF-1 cells.

Protein kinase C mediates the mitogenic action of thrombopoietin in c-Mpl-expressing UT-7 cells.

Protein kinase C (PKC) has been implicated in signal transduction events elicited by several hematopoietic growth factors. Thrombopoietin (TPO) is the major regulator of megakaryocytic lineage development, and its receptor, c-Mpl, transduces signals for the proliferation and differentiation of hematopoietic progenitors. In this study we have examined the effect of TPO on the subcellular distribution of PKC (a measure of enzyme activation) in a growth factor-dependent pluripotent hematopoietic cell line that was engineered to express the c-Mpl receptor (UT-7/mpl). In addition, we have assessed the significance of this activation for the induction of both mitogenesis and differentiation. Using a PKC translocation assay, TPO was found to stimulate a time- and dose-dependent increase in the total content of PKC activity present in the membrane fraction of UT-7/mpl cells (maximum increase = 2.3-fold above basal level after 15 minutes with 40 ng/mL TPO, EC50 = 7 ng/mL). Accordingly, a decrease of PKC content in the cytosolic fraction was observed. Immunoblot analysis using PKC isotype-specific antibodies showed that TPO treatment led to a marked increase of the Ca2+/diacylglycerol-sensitive PKC isoforms alpha and beta found in the membrane fraction. In contrast, the subcellular distribution of these isoforms did not change after treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF). Exposure of UT-7/mpl cells to the selective PKC inhibitor GF109203X completely inhibited the PKC activity associated to the membrane fraction after TPO treatment, and blocked the mitogenic effect of TPO. In contrast, GF109203X had no effect on the TPO-induced expression of GpIIb, a megakaryocytic differentiation antigen. Downregulation of PKC isoforms alpha and beta to less than 25% of their initial level by treatment with phorbol 12,13-dibutyrate also abolished the TPO-induced mitogenic response, but had no significant effect when this response was induced by GM-CSF. Taken together, these findings suggest that (1) TPO stimulates the activation of PKC, (2) PKC activation mediates the mitogenic action of TPO, and (3) PKC activation is not required for TPO-induced expression of megakaryocytic surface markers.

Thrombopoietin does not induce lineage-restricted commitment of Mpl-R expressing pluripotent progenitors but permits their complete erythroid and megakaryocytic differentiation.

In this study, we examined the in vitro and in vivo effects of forced expression of Mpl-R (the thrombopoietin receptor) on the progeny of murine hematopoietic stem cells. Bone marrow cells from 5-FU-treated mice were transduced with retroviral vectors containing the human Mpl-R cDNA, or the neomycine gene as a control. After 7 days cocultivation on virus-producer cells, GpE86-Mpl-R or Gp86-Neo, the types of hematopoietic progenitor cells responding to thrombopoietin (TPO) were studied by clonogenic assays. Mpl-R-infected cells gave rise to CFU-GEMM, BFU-E, CFU-MK, but not CFU-GM while Neo-infected cells produced only megakaryocytic colonies. In addition, when nonadherent cells from GpE86-Mpl-R cocultures were grown with TPO as the only stimulus for 7 days, a marked expansion of CFU-GEMM, BFU-E, and CFU-MK was observed, while no change in CFU-GM number was seen. Erythroid and megakaryocytic maturation occurred in the presence of TPO while a block in granulocytic differentiation was observed at the myeloblast stage. The direct effects of TPO on Mpl-R-transduced progenitor cells were demonstrated by single cell cloning experiments. To analyze the effects of the constitutive expression of Mpl-R on the determination of multipotent progenitors (CFU-S) and long-term repopulating stem cells, Mpl-R- or Neo-infected cells were injected into lethally irradiated recipient mice. No difference was seen in (1) the number of committed progenitor cells contained in individual CFU-S12 whether colonies arose from noninfected or Mpl-R-infected CFU-S; (2) the mean numbers of progenitor cells per leg or spleen of mice reconstituted with Mpl-R- or Neo-infected cells, 1 or 7 months after the graft; and (3) the blood parameters of the two groups of animals, with the exception of a 50% reduction in circulating platelet counts after 7 months in mice repopulated with Mpl-R-infected bone marrow cells. These results indicate that retrovirus-mediated expression of Mpl-R in murine stem cells does not modify their ability to reconstitute all myeloid lineages of differentiation and does not result in a preferential commitment toward the megakaryocytic lineage.

Ectopic expression of the erythropoietin receptor in a murine interleukin-6-dependent plasmacytoma cell line (TEPC-2027) confers proliferative responsiveness to erythropoietin.

To compare the signal transduction pathways used by erythropoietin (Epo) and interleukin-6 (IL-6), the cDNA for the murine Epo receptor (Epo-R) was introduced into an IL-6-responsive plasmacytoma cell line (TEPC-2027) by retrovirally mediated gene transfer. G418-resistant clones were amplified in IL-6 and studied for their ability to grow and differentiate in response to Epo. Epo-R synthesized from the viral gene showed the same affinity for Epo as did the receptor on erythroid cells; however, the numbers of Epo receptors expressed on the cell membrane varied among clones. After a delay of 3 to 5 days in the presence of Epo, all the clones studied proliferated as well in response to Epo as in response to IL-6. In response to IL-6, Stat3 was activated and JunB mRNA was accumulated, whereas in response to Epo, Jak2 and Stat5 were activated and JunB mRNA was not accumulated in Epo-R-expressing TEPC (Epo-R/TEPC) cells. These results suggest that Epo and IL-6 transduced their proliferative signals through different pathways. Further studies showed that, in Epo-R/TEPC cells, Epo neither induces the synthesis of erythroid-specific mRNA nor modifies the synthesis of gamma 1 lg heavy chain, suggesting that ectopic expression of the Epo-R in plasmacytoma cells does not modify their differentiative potential. The data show that Epo induces a proliferative response without differentiation providing a new cellular model for evaluating molecular events specific for proliferation.

Stromal cells maintain the radioprotective capacity of CFU-S during retroviral infection.

Retroviral vectors provide an efficient means to introduce genes into hematopoietic stem cells. In order to develop retroviral infection protocols which preserve the radioprotective capacity of CFU-S, we designed a clonal hematopoietic reconstitution assay. In this assay, single CFU-S-derived derived colonies from bone marrow cells of 5-FU-treated mice were tested for their capacity to prevent radiation-induced mortality. Three parameters which may modify stem cell potential were tested in infection protocols using a retroviral vector containing the gene for neomycin resistance: (1) the partition of stem cells between the adherent and nonadherent fraction; (2) the replacement of the packaging cell line by a "competent' stromal cell line; and (3) the effects of G418 selection. All CFU-S having radioprotective capacity were found in the adherent fraction when the packaging cell line or the stromal cell line (MS-5) chosen for its capacity to maintain long-term bone marrow culture were used during the co-culture. The neo resistance gene was transduced into CFU-S with the same efficiency using co-culture with the packaging cell line or co-culture with the MS-5 cell line plus viral supernatant. However, in the presence of MS-5, a much higher proportion of CFU-S (70% versus 30%) had radioprotective properties, suggesting an important role for the stromal cells in the maintenance of hematopoietic reconstituting ability. Finally, G418 selection, even for a limited period (24 h), significantly decreased the radioprotective capacities of CFU-S (56% versus 18%). Subsequently, hematopoietic reconstitution by single CFU-S was quantified in recipient mice. The progeny of CFU-S were found at a significant level in the blood, spleen and bone marrow in 38% and 15% of mice, 1 and 3 months after transplantation, respectively. These results demonstrate that we have substantially improved the infection protocol. Under these conditions of infection, it is possible to conserve CFU-S properties and to transduce a gene into a stem cell with short-term hematopoietic reconstitution potential.

Constitutive expression of GATA-1 interferes with the cell-cycle regulation.

GATA-1, mainly expressed during erythroid differentiation, has been shown to regulate the genes specifically expressed in the late stages of erythropoiesis and to protect erythroid cells from apoptosis, suggesting that it might interfere with the cell cycle. By expressing the retrovirally transduced human GATA-1 cDNA in NIH3T3 fibroblasts, we have shown that GATA-1 alone was unable to transactivate its erythroid-specific target genes in these nonerythroid cells. However, GATA-1 expression had a dramatic effect on the proliferation of these fibroblasts. The cloning efficiency of the GATA-1-expressing fibroblasts was maintained but their S phase was greatly elongated and their G1 and G2/M phases were reduced, impairing substantially their proliferation. When cultured at low serum concentrations for 48 hours, GATA-1-expressing fibroblasts failed to accumulate in the G0/G1 phases but did not become serum independent. GATA-1-expressing fibroblasts expressed D1, A, and B1 cyclin mRNAs under conditions of serum starvation or at confluence, whereas these cyclin mRNAs were downregulated in the parental NIH3T3 cells cultured under the same conditions. Moreover, these effects of GATA-1 expression on proliferation were not limited to NIH3T3 cells, since different clones of hGATA-1 virus-infected FDCP-1 cells, a murine interleukin-3-dependent hematopoietic cell line, had a slower growth rate than control cells. Based on these data, we hypothesize that GATA-1 plays a role in the regulation of the cell cycle during terminal erythroid differentiation.

Inhibition of the erythropoietin-induced erythroid differentiation by granulocyte-macrophage colony-stimulating factor in the human UT-7 cell line is not due to a negative regulation of the erythropoietin receptor.

The human pluripotent UT-7 cell line is growth factor-dependent for proliferation and differentiation. We have previously shown that (1) granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Epo) induce a myeloid and erythroid pattern of differentiation, respectively; (2) GM-CSF acts predominantly over Epo for cell differentiation; (3) GM-CSF induces a rapid downmodulation (4 hours) of Epo receptors (Epo-R) at the mRNA and binding site levels; and (4) in contrast, Epo has no effect on GM-CSF receptor (GM-CSF-R) expression. These results suggested that UT-7 cell commitment or differentiation may be directed by a hierarchical action of growth factors through an early and rapid transmodulation of growth factor receptors. To test this hypothesis, we introduced and expressed the murine Epo-R (muEpo-R) in UT-7 cells using a retroviral strategy. Two retroviral vectors were constructed: one carrying the neomycin resistance gene, and another carrying a mouse Epo-R cDNA devoid of its regulatory untranslated 3' sequence placed under the transcriptional control of the viral long terminal repeat element (LTR) and the neomycin resistance gene. Three UT-7/Epo-R infected clones (12, 6, 10) and one UT-7/neomycin clone (Neo) were selected in medium containing G418. After growth factor deprivation (18 hours), Epo-Rs were expressed at the same level (approximately 6,000 receptors per cell) in all four clones 12, 6, 10, Neo, and in parental UT-7 cells, and exhibited similar affinity (0.1 to 0.2 nmol/L). Cross-linking experiments showed that Epo is associated with three proteins of about 66, 85, and 100 kD in cells of parental UT-7, as well as in cells of clones 10 and 12. An inhibitory antibody directed specifically against the human Epo-R (huEpo-R Ab) abolished almost completely the cross-linking on parental UT-7 cells, but not on cells of clone 12, demonstrating that more than 90% cell surface Epo-Rs were of murine origin. The presence of GM-CSF significantly reduced the number of Epo-Rs expressed on parental UT-7 cells, but not on cells of clones 12, 10, and 6. HuEpo-R Ab inhibited Epo-induced parental UT-7 cell growth, but not that of cells of clone 12, suggesting that the muEpo-R is able to induce human UT-7 cell proliferation. When cells of clone 12 were switched from a medium containing GM-CSF to one with Epo, cell surface glycophorin A (GPA) was induced, as in parental UT-7 cells without inhibition by the huEpo-R Ab, demonstrating that the muEpo-R is also able to transduce a differentiation signal in human cells. However, in cells of clones 12, 6, 10 and Neo, as well as in parental UT-7 cells, the induction of GPA by Epo was inhibited by GM-CSF. This finding demonstrates that, although GM-CSF does not downregulate muEpo-R binding sites on UT-7/muEpo-R infected clones, it still inhibits the effects of Epo on cell differentation. Therefore, hierarchical regulation induced by growth factors for cell commitment or differntiation more likely acts downstream of cell surface receptors at either the signal transduction or transcriptional levels.

Pluripotent stem cells constitutively expressing a normal erythropoietin receptor give rise to normal hematopoiesis in lethally irradiated recipient mice.

The cellular mechanism by which the stem cell differentiates toward an individual myeloid lineage is unknown. To determine whether lineage-specific cytokines are involved in stem cell determination, murine bone marrow cells were infected with a retroviral vector carrying a murine erythropoietin receptor (EpoR) cDNA. Infected marrow cells were transplanted into lethally irradiated syngeneic recipient mice, and the effect of Epo was studied on EpoR-expressing pluripotent stem cell determination. The graft contained, among myeloid cells, around 100 CFU-S12, half of which were retrovirally infected. One month after grafting, the bone marrow of mice reconstituted with EpoR-infected cells contained 50 times more infected multipotent progenitors than mice reconstituted with control bone marrow cells. However, this number returned to normal 45 days after the graft. No variation was observed in peripheral blood, bone marrow, and spleen cellularities or in committed progenitors in the bone marrow and in the spleen when Neo or EpoR reconstituted mice were assayed. When Epo was delivered into reconstituted mice one month after grafting, Epo had no differential effect in EpoR or Neo reconstituted mice. This study emphasizes the in vivo Epo proliferative response of multipotent progenitors expressing a normal EpoR gene and shows that, in vivo as in vitro, the differentiation of these multipotent progenitors is not preferentially oriented toward erythropoiesis.

Murine pluripotent hematopoietic progenitors constitutively expressing a normal erythropoietin receptor proliferate in response to erythropoietin without preferential erythroid cell differentiation.

Erythropoietin (EPO) is a prime regulator of the growth and differentiation of erythroid blood cells. The EPO receptor (EPO-R) is expressed in late erythroid progenitors (mature BFU-E and CFU-E), and EPO induces proliferation and differentiation of these cells. By introducing, with a retroviral vector, a normal EPO-R cDNA into murine adult bone marrow cells, we showed that EPO is also able to induce proliferation in pluripotent progenitor cells. After 7 days of coculture with virus-producing cells, bone marrow cells were plated in methylcellulose culture in the presence of EPO, interleukin-3, or Steel factor alone or in combination. In the presence of EPO alone, EPO-R virus-infected bone marrow cells gave rise to mixed colonies comprising erythrocytes, granulocytes, macrophages and megakaryocytes. The addition of interleukin-3 or Steel factor to methylcellulose cultures containing EPO did not significantly modify the number of mixed colonies. The cells which generate these mixed colonies have a high proliferative potential as shown by the size and the ability of the mixed colonies to give rise to secondary colonies. Thus, it appears that EPO has the same effect on EPO-R-expressing multipotent cell proliferation as would a combination of several growth factors. Finally, our results demonstrate that inducing pluripotent progenitor cells to proliferate via the EPO signaling pathway has no major influence on their commitment.

Infection with a Kirsten-retrovirus can induce a multiplicity of tumorigenic phenotypes in the interleukin-3-dependent FDC-P1 cells.

The identification of ras oncogenes in both human and animal tumors as well as in preleukemic and precancerous lesions suggests that activated ras genes participate in neoplastic development, yet the precise role of ras oncogenes in leukemogenesis is not clear. To assess the functional role of ras genes in tumorigenesis, we introduced with a retroviral vector either a wild-type (Gly-12) or a mutant (Val-12) Kirsten ras cDNA into the cells of a factor-dependent myeloid cell line, FDC-P1. FDC-P1 cells are nontumorigenic and their proliferation is dependent on either interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). The Ki-Val 12-infected FDC-P1 cell population is still strictly IL-3-dependent but has acquired the ability to survive up to 72 hours in the absence of growth factor and to form tumors in nude mice. These tumors are easily established into cell lines that are clonal and show a multiplicity of phenotypes with respect to their growth factor dependence. These results suggest that, in contrast with the overexpression of a normal Ki-ras, Ki-ras oncogene can efficiently promote the tumorigenic conversion of FDC-P1 cells. However, the clonality of the tumors as well as the distinct phenotypes indicates that other genetic events are required for tumorigenicity. Therefore, in FDC-P1 cells, an activated ras gene acts as a dominant oncogene through the induction of tumor progression. Finally, in this simple experimental system we observed a multiplicity of tumorigenic phenotypes which are reminiscent of those observed in patients with acute myeloid leukemia.

Leukaemia inhibitory factor is necessary for maintenance of haematopoietic stem cells and thymocyte stimulation.

Leukaemia inhibitory factor (LIF) has a variety of effects on different cell types in vitro, inhibiting the differentiation of embryonic stem cells and promoting the survival and/or proliferation of primitive haematopoietic precursors and primordial germ cells. Here we show that LIF-deficient mice derived by gene targeting techniques have dramatically decreased numbers of stem cells in spleen and bone marrow. Injection of spleen and marrow cells from these mice promotes long-term survival of lethally irradiated wild-type animals, however, showing that the LIF- stem cells remain pluripotent. The numbers of committed progenitors are also reduced in the spleen but not the bone marrow, suggesting that stem cells interact differently with the splenic and medullary microenvironment. Heterozygous animals are intermediate in phenotype, implying that LIF has a dosage effect, and defects in stem cell number can be compensated by exogenous LIF. LIF thus appears to be required for the survival of the normal pool of stem cells, but not their terminal differentiation.

Further studies on the biological activities of the CFU-S inhibitory tetrapeptide AcSDKP. II. Unresponsiveness of isolated adult rat hepatocytes, 3T3, FDC-P2, and K562 cell lines to AcSDKP. Possible involvement of intermediary cell(s) in the mechanism of AcSDKP action.

Because the molecular mechanisms of the tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP; an inhibitor of spleen colony-forming unit [CFU-S] DNA synthesis) are difficult to study on bone marrow due to the scarcity of CFU-S in this tissue, we sought a pure cell population responsive to the molecule in vitro. Although growth factor-stimulated DNA synthesis in primary culture of hepatocytes and Balb/c 3T3 cells can be inhibited by transforming growth factor beta (TGF beta) and interferon alpha/beta (IFN[alpha/beta], respectively, neither hepatocytes nor 3T3 cells were found to be sensitive to AcSDKP. DNA synthesis in stimulated murine FDC-P2 cell lines and in human K562 cell lines also remained unchanged after exposure to the tetrapeptide. The fact that hepatocytes do respond in vivo to AcSDKP implies the existence of intermediary cell(s) involved in AcSDKP action in vivo that are lacking in hepatocyte culture. Whether intermediary cell(s) are implicated in the inhibitory action of AcSDKP on CFU-S entry into DNA synthesis is now being investigated.

Mock retroviral infection alters the developmental potential of murine bone marrow stem cells.

Retroviral vectors were used to introduce an activated ras gene into murine pluripotent hemopoietic stem cells. We attempted to reconstitute the hemopoietic system of lethally irradiated mice with isolated spleen colonies obtained in vivo after injection of infected bone marrow cells. Spleen colonies derived from infected bone marrow were inefficient in promoting long-term survival of irradiated hosts. This loss of reconstitutive capacity of spleen colonies was not due to the retroviral infection per se but to the in vitro culture of spleen colony precursors. Incubation for 24 h in the presence of fetal calf serum and interleukin-3 without virus-producing cells was sufficient to abolish completely the reconstitutive capacity of spleen colonies while maintaining both self-renewal and pluripotential capacities of spleen colony precursors. These results show that the in vitro manipulation of stem cells that is included in current protocols for retroviral infection can modify the developmental potential of these cells. This finding clearly indicates that the use of retroviral vectors can introduce a bias in the analysis of hemopoiesis.

Further studies on the mechanism of CFU-S determination--I. Pluripoietin(s) responsible for CFU-S determination toward granulopoiesis after fractionated doses of Ara-C.

Fractionated doses of Ara-C orient CFU-S differentiation towards granulopoiesis and megakaryocytopoiesis in vivo in contrast to the preferential channelling towards erythropoiesis after a similar dose of Ara-C given as a single injection. In this paper, we show that pluripoietin found in the serum of mice treated with fractionated doses of Ara-C is responsible for the choice of CFU-S differentiation towards granulopoiesis. Therefore, we confirm our previous results involving single doses of Ara-C, that humoral factors can commit CFU-S preferentially towards one of the cell lineages. It is not as yet known whether there is a specific pluripoietin for each cell lineage or whether the same pluripoietin has a different effect according to its concentration in the organism. The difference in response of CFU-S commitment to single and fractionated doses of Ara-C is not due to the difference in the kinetic status of CFU-S:CFU-S are cycling after both treatments.

Further studies on the mechanism of CFU-S determination--II. Feedback regulation.

In previous papers, we have demonstrated that the determination of CFU-S differentiation is under the control of humoral factors, which we have called pluripoietins. The activity of these regulators varies according to the experimental protocol. The aim of this work was to determine the mechanisms involved in the regulation of pluripoietin secretion after each treatment. The results suggest that the production of pluripoietin(s) is under the control of a feedback mechanism, originating, at least in part, in the progenitor compartments. After one dose of 20 mg of Ara-C, CFU-S determination is channelled towards erythropoiesis when the BFU-E compartment is more depleted than the GM-CFC compartment. During fractionated doses of Ara-C, at the time of the third injection, GM-CFC survival is lower than BFU-E survival and CFU-S orient their differentiation towards granulopoiesis. After bleeding, there is no difference of survival between the two compartments and CFU-S determination is not changed. These results seem to indicate that CFU-S determination is modified in order to restore normal homeostasis of the hemopoietic tissues by preferentially replenishing the more depleted compartment. This is most likely achieved by the secretion of pluripoietin(s) that vary in either their nature or their concentration according to the events occurring in the more mature compartments.

Further studies on the mechanism of CFU-S determination--III. CFU-S cell lineage determination is not influenced by GM-CSF, Epo, multi-CSF nor WEHI 3B conditioned medium.

CFU-S proliferation is under the control of factors such as stimulators and inhibitors whereas commitment of CFU-S is controlled by pluripoietins. We have studied the effects in vitro of Epo, GM-CSF, multi-CSF and WEHI 3B conditioned medium in order to elucidate their eventual role on CFU-S proliferation and cell lineage determination. Neither Epo nor GM-CSF are able to induce the entry of CFU-S into cycle. Multi-CSF and WEHI 3B conditioned medium (similar to interleukin 3) stimulate CFU-S into DNA synthesis. However, we were able to show that the stimulator we have studied is different from IL 3. Epo, GM-CSF, multi-CSF and WEHI 3B CM have no effect on CFU-S commitment. Therefore, these four hemopoietic regulators are different from the pluripoietins we have previously described and have no influence on CFU-S determination.