Protection of mice against lethal doses of 1 beta-D-arabinofuranosylcytosine by pluripotent stem cell inhibitors.

The aim of this work was to study whether an inhibitor of pluripotent stem cell (CFU-S) recruitment, which we have shown previously to be able to increase the number of CFU-S after a fractionated treatment with 1-beta-D-arabinofuranosylcytosine, could increase the survival of mice given injections of lethal doses of the same drug. Two protocols of 1-beta-D-arabinofuranosylcytosine treatment were used in two different mouse strains, which both killed the mice within a week. An inhibitor of CFU-S was prepared by dialysis from fetal calf marrow, and a first step of purification was made by chromatography on Sephadex G-10. When given injections 2 hr before the drug, the number of surviving mice was increased significantly with the dialysate; fractions separated by chromatography appeared to be more effective to increase the animal survival. These preliminary results indicate that a factor of low molecular weight (below M.W. 3500) extracted from fetal calf marrow is able to protect animals during 1-beta-D-arabinofuranosylcytosine treatment. The inhibitor seems to be specific for CFU-S, without any inhibiting effect on tumor cell kinetics in vitro. If the absence of species specificity found for higher to lower species is confirmed for the lower to the higher species, then this inhibitor could be an effective tool during cancer chemotherapy.

Prevention of hematotoxic side effects of cytostatic drugs in mice by a synthetic hemoregulatory peptide.

The application of certain cytostatic drugs causes the recruitment of pluripotent hemopoietic stem cells (CFU-S) into active proliferation. Further application of the drug(s) may then lead to severe and long lasting disturbances of hemopoiesis. We investigated if the hemoregulatory peptide pGlu-Glu-Asp-Cys-Lys (HP5b) could be used to inhibit stem cell recruitment and consequently to protect mice against the toxicity of repeated high doses of 1-beta-D-arabinofuranosylcytosine (ara-C). CFU-S recruitment (induced by injecting a single dose of 900 mg/kg ara-C) was prevented by either treating the bone marrow of these mice in vitro with 1 x 10(-7) M/liter HP5b, or by injecting 0.6 microgram HP5b (10(-9) mol, 30 micrograms/kg) at -2, +2, and +6 h relative to the ara-C injection. Multiple high dose ara-C applications (4 x 900 mg/kg at 0, 7, 24, and 30 h) lead to proliferative activation of CFU-S and resulted in the death of 90% of the mice within 7-9 days. Reconstitution of the hemopoietic system by a bone marrow transplant given after ara-C application decreased the mortality to about 45%, indicating the nonhematological component of ara-C toxicity. A single injection of HP5b (30 micrograms/kg at 26 h, when few CFU-S were found in S phase) decreased the mortality to 59%, not significantly different from the transplanted group. Inactive peptides given instead of HP5b had no protective effect. HP5b did not change the ara-C sensitivity of transformed cell lines (HL-60, Raji, Friend), even not in such cases (myeloid cell lines) where it had a direct inhibitory effect on the cells (e.g., HL-60). These results suggest that HP5b may be used as a myeloprotector in cancer chemotherapy by keeping hemopoietic stem cells out of cycle during the most hazardous treatment phase. Its lack of species specificity, its low toxicity, its high selectivity for hemopoiesis, the small size, as well as the availability through standard synthetic techniques may be of advantage for its clinical use.

Inhibitory peptides in hematopoiesis.

This paper will review the present knowledge on two small chemically unrelated peptides, the pentapeptide pGlu-Glu-Asp-Cys-Lys (pEEDCK) and the tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP, Seraspenide) focusing on 1) their inhibitory effects on normal hematopoiesis, 2) their effect on malignant cells, especially leukemic cells, and 3) their potential clinical use as marrow protectors during cancer therapy.

Long-term effects of high doses of cytosine arabinoside on pluripotent stem cells (CFU-S).

The purpose of this work was to investigate the long-term effects of high doses of cytosine arabinoside (Ara-C) on the pluripotent stem cell (spleen colony-forming units; CFU-S) compartment in mice. Studies were carried out on mice that survived the administration of repeated high doses of Ara-C (HDAra-C) with or without the injection of a partially purified CFU-S inhibitor or a bone marrow graft. The following features were examined 1, 1.5, and 5-7 months after treatment: CFU-S number, proliferative ability, and differentiation into different lineages. The results indicate that these parameters, which were severely disturbed soon after drug administration, returned to control levels within a month and remained unchanged as compared to age-matched controls for the following 6 months. Therefore, HDAra-C, given alone or with a CFU-S inhibitor or prior to bone marrow grafting, did not seem to induce long-lasting damage of the CFU-S compartment. However, our studies cannot eliminate the possibility of some residual stromal damage or some impairment of other properties of stem cells. It would be of importance to further clarify these points because HDAra-C are now used in the treatment of leukemias and prior to bone marrow transplantation.

Comparison of the inhibitory effect of AcSDKP, TNF-alpha, TGF-beta, and MIP-1 alpha on marrow-purified CD34+ progenitors.

The aim of this study was to compare the inhibitory effect of the tetrapeptide AcSDKP, tumor necrosis factor-alpha (TNF-alpha), which contains the sequence of the peptide, transforming growth factor-beta (TGF-beta), and macrophage inflammatory protein-1 alpha (MIP-1 alpha) on sorted CD34+ cells using both proliferation and clonogenic assays. Although a short treatment with any of the molecules decreased the growth of colony-forming unit granulocyte/macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-E) progenitors (except for TNF-alpha as it is a greater inhibitor for CFU-GM), further experiments using a 6-day liquid culture in the presence of a combination of growth factors (recombinant human interleukin-3 [rhIL-3], IL-6, IL-1 beta, GM colony-stimulating factor [GM-CSF], G-CSF, erythropoeitin [Epo], and stem cell factor [SCF]) allowed us to determine a number of differences between their effects: 1) TGF-beta and TNF-alpha induced a stronger decrease in the proliferation and clonogenicity of CD34+ subsets than MIP-1 alpha and AcSDKP, 2) the dose-response curves appeared different, and 3) contrary to TGF-beta and TNF-alpha, AcSDKP and MIP-1 alpha required repeated addition to induce inhibition. Therefore, our data clearly show that while the inhibitory effect of TNF-alpha and AcSDKP appeared to be different, there is a close similarity in the effect of AcSDKP and MIP-1 alpha on normal human progenitor response to the combination of growth factors used.

Photoprotection of normal human hematopoietic progenitors by the tetrapeptide N-AcSDKP.

The tetrapeptide AcSDKP (Ser-Asp-Lys-Pro) is a reversible inhibitor of normal human hematopoietic progenitor growth. In this paper, we report that preincubation of bone-marrow mononuclear cells (MNC) with AcSDKP at 10(-10) M for 20 hours protects the granulocyte-macrophage colony-forming unit (CFU-GM) progenitors against photofrin II-mediated phototherapy. This protective effect was observed after short-term exposure to photofrin (2.5 micrograms/mL) and irradiation by high-energy doses at 514 nm. Nevertheless, AcSD-KP, which has no effect on leukemic cell proliferation, does not protect the HL-60 and K-562 leukemic cell lines against photosensitization.

The influence of interleukin (IL)-4, IL-13, and Flt3 ligand on human dendritic cell differentiation from cord blood CD34+ progenitor cells.

Culturing cord blood CD34+ cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-alpha for 12 days, and stem cell factor (SCF) for 5 days, resulted in a 40- +/- 26-fold expansion in cell numbers, with 38 +/- 20% dendritic cells (DCs). Interleukin (IL)-4 and IL-13, which share properties, were examined first. Adding either one to the former baseline condition beginning on day 0 halved cell growth while the percentage of DCs increased to 60-70%, resulting in unchanged DC yields. Delaying use of IL-4 or IL-13 to day 5 led to 25-fold cell expansion with approximately 80% DC, the yield of which was then twofold over that of baseline control cultures, while numbers of other cells decreased. IL-4 and IL-13 had no additive or antagonistic effect on DC generation. The effect of Flt3 ligand (FL), known to enhance proliferation of hematopoietic progenitors induced by other growth factors, was examined next. FL added alone induced DC in the same manner as SCF. Using both FL and SCF throughout the culture period enhanced total cell recovery fourfold above that of baseline control cultures on day 12 compared with > or =2.5-fold if either one was stopped on day 5. When both FL and SCF were used for 12 days, DC recovery was fivefold that of control cultures, whereas it was to three- to 3.5-fold when either one was stopped on day 5. A similar trend was noted for CD15+ cells, and, to a lesser extent, for CD14+ cells. Finally, using SCF and FL for 12 days, with IL-4 or IL-13 added from day 5 onwards, led to comparably enhanced cell yields relative to control cultures with approximately 60% DC. These data underline the need to use appropriate cytokine combinations and schedules to optimize generation of DCs from CD34+ progenitors. Associated with GM-CSF and TNF-alpha, IL-4 or IL-13 promotes differentiation and maturation of DCs over other myeloid cells. Under the same baseline conditions, FL appears to potentiate SCF throughout the culture period, inducing proliferation and development of DC as well as of other myeloid cells.

Thymosin beta4, inhibitor for normal hematopoietic progenitor cells.

Thymosin beta4 (Tbeta4), isolated from the calf thymus fraction 5, has a ubiquitous localization and plays a pleiotropic role in both the immune and nonimmune systems. Because it contains at its N-terminal end the sequence of a known inhibitor of hematopoiesis, the acetylated tetrapeptide Ac-N-Ser-Asp-Lys-Pro (AcSDKP, Goralatide), we have assayed Tbeta4 on human hematopoietic cells. We demonstrate that it inhibits normal bone marrow progenitor cell growth; indeed, it decreased the growth of both granulo-macrophagic and erythroid progenitors and reduces their percentage in S phase. Furthermore, we show that Tbeta4 reduces both the clonogenicity and the cell proliferation of purified CD34+ cells induced by a combination of seven growth factors. Although Tbeta4's inhibitory effect is very similar to that of AcSDKP, we demonstrate, using neutralizing antibodies and a truncated form of Tbeta4 devoid of the AcSDKP sequence, that the inhibitory effect of Tbeta4 is not mediated by the sequence AcSDKP. These data indicate that Tbeta4 is a novel inhibitor for human normal hematopoietic progenitors.

The tetrapeptide AcSDKP, an inhibitor of the cell-cycle status for normal human hematopoietic progenitors, has no effect on leukemic cells.

The tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) inhibits the entry into DNA synthesis of murine spleen colony-forming units (CFU-S) and protects these cells during chemotherapy. This synthetic peptide also inhibits the growth of normal human marrow progenitors granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E) and decreases their percentage in DNA synthesis at nanomolar concentration. In view of its clinical application as a marrow protector, we have investigated its effects on malignant cells. Studies were carried out on HL-60 cells and on fresh leukemic cells from patients with either chronic myeloid leukemia (CML) or acute myeloid leukemia (AML). Results showed that AcSDKP, whatever the doses used, did not modify the proliferation of both HL-60 cells and AML cells even when enhanced by stimulating factors such as interleukin 3 or granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, no change in the number and the percentage in S-phase of both HL-60 clonogenic cells and CML progenitors was observed. Our data clearly demonstrate that the tetrapeptide AcSDKP was ineffective on leukemic cells and therefore by acting selectively on normal progenitors represents a potent therapeutical agent for the protection of normal bone marrow progenitors during chemotherapy.

Tumor necrosis factor alpha in human long-term bone marrow cultures: distinct effects on nonadherent and adherent progenitors.

Although tumor necrosis factor alpha (TNF alpha) exerts a variety of activities on hematopoietic cells, suggesting it may have some potential therapeutic applications, its long-term effects on hematopoiesis are not well defined. Therefore, we took the advantage of long-term bone marrow cultures (LTBMCs) to evaluate the long-term role of TNF alpha on both the microenvironment and the hematopoietic progenitors. LTBMCs were inoculated with 100 U/ml of recombinant human TNF alpha (rhTNF alpha) either at the onset of the cultures (d0) or at day 21 (d21) when the adherent layer (AL) was already established. Then TNF alpha was added at each weekly medium change. The cellularity and the content of progenitors in both the nonadherent layer (NAL) and AL, the formation of the AL, and the presence of various cytokines in the supernatants were examined weekly. The data showed 1) a strong and durable inhibitory effect on total nonadherent cells; 2) a rapid and transient inhibition of NA progenitors, whereas adherent progenitors were lately affected; and 3) microenvironmental changes consisting of the disappearance of adipocytes and the secretion of high levels of interleukin 6. The results suggest that the inhibitory effects of TNF alpha on the NAL are in part counterbalanced by stromal modifications that in turn lead to a faster exhaustion of hematopoiesis.

Reversible inhibitory effects and absence of toxicity of the tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) in human long-term bone marrow culture.

The effects of acetyl-N-Ser-Asp-Lys-Pro (Ac-SDKP) in human long-term bone marrow cultures (LTBMCs) were assessed by measuring the number of progenitors and the development of stromal cells over a 6-week course. In a first set of experiments, AcSDKP was added weekly at each medium change. Under these conditions, no significant effect of the peptide was observed. In contrast, by adding AcSDKP daily at 10(-10) M, the growth of the progenitors of the non-adherent (NA) compartment was inhibited by about 35%. This inhibition was entirely reversible; after stopping the addition of the peptide at the fourth week, the number of progenitors returned to control level within 2 weeks. Conversely, AcSDKP did not significantly change the number of the progenitors present in the adherent layer. In addition, AcSDKP did not affect the formation of the stromal layer nor induce the secretion of cytokines, such as tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), granulocyte colony-stimulating factor (G-CSF), interleukin 3 (IL-3), or interleukin 6 (IL-6). Our results indicate that AcSDKP has inhibitory but reversible effects on NA progenitors and does not induce long-term modifications of the microenvironment, both of particular interest for its clinical application.

Inhibition of human bone marrow progenitors by the synthetic tetrapeptide AcSDKP.

The purpose of this work was to study the effects of a tetrapeptide, acetyl-N-Ser-Asp-Lys-Pro (AcSDKP), an inhibitor of spleen colony-forming unit (CFU-S) entry into DNA synthesis, on human progenitor cells. Normal human mononuclear cells were incubated with concentrations of the synthetic tetrapeptide ranging from 10(-12) to 10(-7) M for 1.5 and 24 h and then plated in methylcellulose in the presence of human placenta-conditioned medium and recombinant human erythropoietin. The proportion of progenitors in DNA synthesis was determined by the thymidine suicide assay. Incubation with AcSDKP for 24 h leads to a significant inhibition of granulocyte-macrophage colony-forming unit (CFU-GM) and erythroid burst-forming unit (BFU-E) growth and in some cases of erythroid colony-forming unit (CFU-E) growth. The inhibition, which was never greater than 50%, was obtained with 10(-10)-10(-9) M AcSDKP, whereas no effect was seen at higher concentrations. The percentage of CFU-GM, BFU-E, and CFU-E in DNA synthesis was significantly reduced in five consecutive patients after incubation of cells for 24 h with inhibitory doses of the peptide, indicating that it is active on cycling cells. Therefore, these studies provide the first evidence that the tetrapeptide AcSDKP, originally obtained from bovine marrow and now chemically synthesized, is able to inhibit the in vitro growth of human progenitors and to decrease their proportion in cell cycle.

Secretion of tumor necrosis factor-alpha by fresh human acute nonlymphoblastic leukemic cells: role in the disappearance of normal CFU-GM progenitors.

The disappearance of normal hematopoiesis during acute nonlymphoblastic leukemia (ANLL) is poorly understood. Several reports indicate that conditioned medium obtained from leukemic cells might inhibit the formation of normal hematopoietic progenitors. However, these blast-conditioned medium (BCM) inhibitory activities are not well characterized. In order to evaluate whether BCM might contain an activity inhibiting the growth of normal marrow progenitors, BCM from 13 consecutive patients with ANLL were tested on normal bone marrow in methylcellulose assays. In all the cases, a significant inhibition of the growth of granulocyte-macrophage colony-forming unit (CFU-GM) progenitors was observed, whereas erythroid burst-forming unit (BFU-E) progenitors were not affected. Further characterization of the BCM inhibitory activity showed using both a biological assay and RIA, the presence of tumor necrosis factor-alpha (TNF-alpha) in 10 out of 13 BCM. Northern blot analysis performed in six patients showed a correlation between the expression of TNF-alpha mRNA by leukemic cells and the presence of TNF-alpha in BCM. Moreover, the BCM inhibitory activity could be neutralized with an anti-TNF-alpha antiserum. These data indicate that leukemic cells express and release frequently TNF-alpha, which may therefore play an important role in the inhibition of granulopoiesis during leukemia.

High-level gene transfer to cord blood progenitors using gibbon ape leukemia virus pseudotype retroviral vectors and an improved clinically applicable protocol.

The best methods for transducing hematopoietic progenitor cells usually involve either direct co-cultivation with virus-producing cells or human stromal supportive cells. However, these methods cannot be safely or easily applied to clinical use. Therefore, we aimed at improving retrovirus-mediated gene transfer into hematopoietic progenitors derived from cord blood CD34+ cells using viral supernatant to levels achieved at least with direct co-cultivation and under conditions that are suitable for clinical applications. In a first set of experiments, CD34+ cells were infected with supernatant containing amphotropic retroviral particles carrying the nls-lacZ reporter gene and the effects of centrifugation, cell adhesion to fibronectin, and Polybrene on the transduction of both clonogenic progenitors (CFC) and long-term culture initiating cells (LTC-IC) were studied. Transduction efficiency was evaluated on the percentage and total number of progenitors expressing the beta-galactosidase activity. Results show that a 48-hr infection of CD34+ cells with viral supernatant combining centrifugation at 1000 x g for 3 hr followed by adhesion to fibronectin allows transduction levels for both CFC and LTC-IC to be reached that are as good as using direct co-cultivation. In a second set of experiments, CD34+ cells were infected using this optimized protocol with pseudotyped retroviral particles carrying the gibbon ape leukemia virus (GALV) envelope protein. Under these conditions, between 50 and 100% of CFC and LTC-IC were transduced. Thus, we have developed a protocol capable of highly transducing cord blood progenitors under conditions suitable for a therapeutical use.

High level of retrovirus-mediated gene transfer into dendritic cells derived from cord blood and mobilized peripheral blood CD34+ cells.

Dendritic cells (DCs), the most potent antigen-presenting cells, can be generated from CD34+ hematopoietic stem cells and used for generating therapeutic immune responses. To develop immunotherapy protocols based on genetically modified DCs, we have investigated the conditions for high-level transduction of a large amount of CD34+-derived DCs. Thus, we have used an efficient and clinically applicable protocol for the retroviral transduction of cord blood (CB) or mobilized peripheral blood (MPB) CD34+ cells based on infection with gibbon ape leukemia virus (GALV)-pseudotyped retroviral vectors carrying the nls-LacZ reporter gene. Infected cells have been subsequently cultured under conditions allowing their dendritic differentiation. The results show that using a growth factor combination including granulocyte-macrophage colony-stimulating factor plus tumor necrosis factor alpha plus interleukin 4 plus stem cell factor plus Flt3 ligand, more than 70% of DCs derived from CB or MPB CD34+ cells can be transduced. Semiquantitative PCR indicates that at least two proviral copies per cell were detected. Transduced DCs retain normal immunophenotype and potent T cell stimulatory capacity. Finally, by using a semisolid methylcellulose assay for dendritic progenitors (CFU-DCs), we show that more than 90% of CFU-DCs can be transduced. Such a highly efficient retrovirus-mediated gene transfer into CD34+-derived DCs makes it possible to envision the use of this methodology in clinical trials.

Differentiation pathways of pluripotent hemopoietic stem cells in long-term mouse bone marrow culture.

Differentiation pathways of CFU-S were examined in mouse bone marrow long-term culture system. E/G ratios of non-adherent CFU-S in this system increased from 2 to 7-9 by 2-3 weeks of culture and then fluctuated during the culture period. On the other hand, E/G ratios of adherent CFU-S were lower than that of non-adherent CFU-S, and always remained under 2. Some of the supernatants collected from culture flasks at weekly intervals increased E/G ratios of normal bone marrow CFU-S after 18-20 h of incubation. These results suggest that the differentiation of CFU-S is controlled by humoral factors, secreted from yet unknown cells in bone marrow, in this system.

Bone marrow protection.

During the Third International Conference on Negative Control of Hematopoiesis (Paris, April 1993), a special session was devoted to bone marrow protection. Myelotoxicity is often a limiting factor in the use of efficient doses of chemo- or radiotherapy. It appeared therefore of great importance to protect hematopoietic stem cells in order to allow the restoration of hematopoiesis and to prevent the occurrence of lethal aplasia. Several approaches to decrease the myelotoxicity of chemo- and/or radiotherapy were discussed: MDR gene therapy, use of chemical compounds (AS101, WR 2721), cytokines such as TNF alpha and IL-1 and small peptides, pEEDCK and AcSKDP. Interestingly, several of these molecules appear of interest to protect normal stem cells during marrow purging for autograft.

The tetrapeptide AcSDKP reduces the sensitivity of murine CFU-MK and CFU-GM progenitors to aracytine in vitro and in vivo.

The tetrapeptide Acetyl-Ser-Asp-Lys-Pro (AcSDKP) has been described as an inhibitor of CFU-S entry into DNA synthesis; as a result, its administration can protect mice against lethal doses of cytosine arabinoside (Ara-C). In the present study, we tested the protective effect of AcSDKP on CFU-MK and CFU-GM progenitor cells in mice treated at lower doses of Ara-C more relevant to human clinical situations. Firstly, we report for the first time that in vitro pre-incubation of murine BM MNC with AcSDKP at concentrations of 10(-10) and 10(-9) M for 48 h decreased CFU-MK, in parallel to CFU-GM, progenitor growth. This resulted in an increase of recovery of these progenitors after exposure to Ara-C. Secondly, we tested the effect of AcSDKP on progenitor cells in vivo in different conditions in Ara-C treated mice. We show that the administration of AcSDKP before starting Ara-C treatment resulted in a significant increase in progenitor CFU-GM, CFU-MK and mature MK numbers, 6 and 8 days after the first Ara-C injection. Interestingly, no difference was observed whether AcSDKP was started 24 or 48 h before Ara-C. In a protocol in which AcSDKP was administered for 8 days starting 48 h before Ara-C treatment, the dose did not appear to be critical at least within the range tested (4 vs. 40 micrograms/injection). In addition, the administration of AcSDKP at 64 micrograms/kg per injection for 5 days and stopping it 3 days before the end of Ara-C treatment, i.e. five instead of eight applications, further increased its protective effect. Thus our results demonstrate protective effect of AcSDKP for progenitors during a fractionated protocol of Ara-C treatment and indicates an importance of the dose and the schedule of administration of AcSDKP in designing future clinical trials.

Comparison of the effects of AcSDKP, thymosin beta4, macrophage inflammatory protein 1alpha and transforming growth factor beta on human leukemic cells.

We have compared the effects of AcSDKP, Thymosin beta4 (Tbeta4), MIP1alpha and TGFbeta on acute myeloid leukemia (AML) and B-lineage acute lymphoid leukemia (B-ALL) cells using liquid cultures in the presence of GM-CSF, IL-3 and SCF for AML cells and IL-3 and IL-7 for ALL cells. Each molecule was added daily and cell proliferation was evaluated on day 3 by thymidine incorporation. Whereas TGFbeta was found inhibitory in all the AML and B-ALL cases studied, MIP1alpha was inhibitory in 6/12 AML cases and had no effect on B-ALL cells. AcSDKP and Tbeta4 showed an inhibitory effect in a few cases but only at high doses which were inactive on normal cells. Thus, our study not only confirms the effect of TGFbeta, MIP1alpha and AcSDKP on AML cells but also provides new data concerning their effect on B-ALL and the possible inhibitory effect of AcSDKP at high doses. Furthermore, we show for the first time the effect of Tbeta4 on leukemic cells. Altogether, our data indicate differences of sensitivity of leukemic cells to negative regulators, some leukemias being inhibited by one or several of these molecules whereas others were unresponsive to all used. The clinical relevance of these observations still remains to be determined.

Heterogeneity of B lineage acute lymphoblastic leukemias (B-ALL) with regard to their in vitro spontaneous proliferation, growth factor response and BCL-2 expression.

The spontaneous proliferation and the effects of 8 various growth factors (GF) were evaluated on leukemic cells from 27 patients with B-lineage ALL. Two groups of ALLs were distinguished. ALLs from group I (21 patients) exhibited a low spontaneous proliferative rate and were stimulated by IL-3 + IL-7 +/- SCF and/or LIF, while ALLs from group II (6 patients) had a high spontaneous proliferative rate and did no longer require this combination of GFs for proliferation. No effect of bFGF, IGF-I, IL-10 and IL-11 alone or in combination, was observed. Such differences in the behaviour of B-ALLs indicated that the GF requirement of ALL blasts was not related to the presence of serum in the culture nor to the pattern of reactivity of ALL blasts for B lymphoid markers or CD34 antigen. Furthermore, we showed in 1/9 cases that high proliferation might be due to an overexpression of the bcl-2 proto-oncogene and to the acquisition of an autocrine secretion.