Stimulatory effects of neopterin on hematopoiesis in vitro are mediated by activation of stromal cell function

The pteridine neopterin (NP) was shown to be produced by monocytes and is known to be a useful marker of immunological activation, although, its biological activity is still unclear. Recently, we found that intravenous administration of NP increased the numbers of blood leukocytes, and granulocyte-macrophage progenitor cells (CFU-GM) in the bone marrow and spleens of mice. In order to elucidate the mechanism whereby NP stimulates hematopoiesis, the effects of NP on hematopoietic stem cell proliferation and differentiation in vitro were studied using a long-term bone marrow culture (LTMC) system with cloned stromal cell line, MS-5. Adding NP to the LTMC increased the numbers of cells in total, CFU-GM and colony-forming unit in spleen (CFU-S). NP also increased the number of CFU-GM in a soft agar culture system, but it did not enhance CFU-GM colony formation when target bone marrow cells were semi-purified (T, B and adherent cell-depleted bone marrow cells) and cultured in this system, suggesting that NP did not directly affect the proliferation of hematopoietic progenitors. Conditioned medium obtained from NP-treated stromal cells had much greater colony-stimulating activity than that obtained from untreated stromal cells. Furthermore, NP treatment stimulated the production of IL-6 and GM-CSF by stromal cells. All these findings suggest that NP stimulates hematopoietic cell proliferation and differentiation in vitro by activating stromal cell function.     

IKAROS isoform 6 enhances BCR-ABL1-mediated proliferation of human CD34+ hematopoietic cells on stromal cells

The BCR-ABL1 induces chronic myelogenous leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL). Recent studies revealed high ratios of loss of the IKZF1 gene which encodes IKAROS in BCR-ABL1+ ALL and lymphoblastic crisis (LBC) of CML. However, little is known about the cooperativity between the aberrant IKAROS and BCR-ABL1 in primary human hematopoietic cells. We investigated the effects of expression of BCR-ABL1 and/or IK6, a natural dominant negative isoform of IKAROS, on proliferation and differentiation of human CD34+ cord blood cells with or without human bone marrow-derived stromal cells which support early B cell differentiation. Cell proliferation was remarkably enhanced by co-expression of BCR-ABL1 and IK6, with reduced expression of glycophorin A and increased expression of CD41, especially on stromal cells, compared with expression of BCR-ABL1 alone that resulted in expansion of erythroid progenitors. Interestingly, p190BCR-ABL1 showed higher dependency on stromal cells to stimulate cell growth with IK6, than p210BCR-ABL1. Furthermore, the cooperation was found to depend on direct cell adhesive interaction of hematopoietic progenitors with stromal cells. These findings suggest that IK6 and BCR-ABL1 synergistically contribute to leukemogenesis in human bone marrow stromal microenvironment, and may provide a clue to elucidate the mechanisms of leukemogenesis of Ph+ ALL and CML-LBC.     

PU.1 supports proliferation of immature erythroid progenitors

Despite normal levels of erythropoiesis in PU.1(-/-) embryos, PU.1(-/-) fetal hematopoietic progenitors are unable to establish sustained erythropoiesis in the adult bone marrow. This study demonstrates that PU.1(-/-) fetal erythroid progenitors are synergistically expanded by TPO plus SCF, but not combinations of EPO plus SCF, IL-3 or GM-CSF. The EPO defect is not corrected by a constitutively active variant of EPOR. Microarray analysis identified several candidate PU.1 target genes known to affect cytokine signaling and gene regulation in the erythroid lineage. These data suggest that PU.1 plays an important role in regulating the proliferation of immature erythroid progenitors.     

CD44 and Hyaluronan Binding by Human Myeloid Cells

The CD44 cell surface molecule has been shown to be the principal cell surface receptor for hyaluronan (or hyaluronic acid), a glycosaminoglycan component of marrow extracellular matrix. However, its affinity for hyaluronan is not constitutive, since it depends on the cell type, the stage of differentiation and on activation by external stimuli including certain anti-CD44 antibodies and phorbol esters. Except for a few lymphoid cell lines, hematopoietic cells do not spontaneously bind hyaluronan and initial studies reported that, contrary to lymphocytes, myeloid cells could not be activated to bind hyaluronan.

Because CD44 plays an important role in the initial phases of hematopoiesis, as shown by experiments using blocking anti-CD44 monoclonal antibodies, its capacity to mediate adhesion of primitive myeloid cells has been investigated. It was found that CD44 could mediate spontaneous adhesion to hyaluronan of immature myeloid cell lines KG1, KG1a, and TF1, which serve as a model for hematopoietic progenitors. However, despite expressing high amounts of CD44, no more than 15% of bone marrow progenitors could adhere to hyaluronan. Recent experiments have shown that a very important feature of CD44 is its capacity to be rapidly activated by certain antibodies and cytokines (GM-CSF and KL) from a low affinity to a high affinity state for hyaluronan. These data shed light on striking similarities in the functional regulation of CD44 and of the two integrin receptors VLA-4 (a4b1), and VLA-5 (a5b1), which are also expressed on hematopoietic progenitors. The relevance of these data to the regulation of normal hematopoiesis and mobilization of CD34+ progenitors in the view of cell grafting is analyzed. In addition, we show that in idiopathic myelofibrosis, the amount of hyaluronan is markedly increased in the extracellular matrix from the myeloproliferative spleen. Considering that the production of cytokines is enhanced in this disease, we discuss whether CD44-hyaluronan interaction may have a role in the pathophysiology of this myeloproliferative syndrome.     

In vitro characterization of the hematopoietic system in pediatric patients with acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) has been recognized as a hematologic neoplasia that originates at the level of a primitive lymphoid stem/progenitor cell. To date, however, the biology of the hematopoietic system in this disorder is still not fully understood. In the present study, we have determined the progenitor cell content (including myeloid, erythroid and multipotent progenitors) in 14 children with ALL and followed the proliferation kinetics of these cells in Dexter-type long-term marrow cultures. We have also characterized some aspects related to the composition and function of the hematopoietic microenvironment developed in vitro. All patients included in this study showed extremely reduced levels of progenitor cells (median of 6.2% of the levels found in normal marrow). Proliferation of these cells in long-term cultures was markedly deficient, since they showed very low numbers - compared to normal cultures - and reached undetectable levels after only a few weeks. Regarding the microenvironment developed in vitro, whereas normal marrow samples contained a median of 8 fibroblastic progenitors/10(5) marrow cells and the stromal cell layers developed in culture contained a median of 341000 adherent cells per well, ALL marrow samples showed no fibroblastic progenitors and the numbers of adherent cells were 21% of those in normal cultures. Interestingly, the levels of TNFalpha and IL-6 in ALL culture supernatants were significantly increased, compared to normal cultures. Bone marrow samples from all 14 children were also analyzed once they reached a complete clinical and hematological remission. Myeloid, erythroid and multipotent progenitor cell levels were significantly increased, compared to patients at diagnosis, and proliferation of myeloid progenitors in long-term cultures was also improved. In contrast, proliferation of erythroid progenitors showed no difference to that in cultures from patients at diagnosis. The numbers of fibroblastic progenitors and adherent cells were significantly increased, compared to patients at diagnosis, and TNFalpha and IL-6 levels returned to normal. In summary, in the present study, we have demonstrated significant in vitro alterations of the hematopoietic system, both in terms of its composition and function, in pediatric patients with ALL. Importantly, most of these alterations are corrected, at least partially, after chemotherapy.     

Effect of lymphokine-activated killer cell fraction on the development of human hematopoietic progenitor cells

Lymphokine-activated killer (LAK) cells from cultures of human peripheral blood mononuclear cells with recombinant interleukin-2 (rIL-2) have been clinically used in adoptive immunotherapy for cancer patients. To study their influence on human hematopoiesis, the LAK cell fraction was cocultured with marrow nonphagocytic cells from normal subjects in an assay system of hematopoietic progenitors. The fraction suppressed colony growth from relatively mature erythroid progenitors in a dose-dependent manner. Although unactivated cells, which were produced without IL-2, augmented the growth of early erythroid progenitors, the LAK cell fraction did not. This fraction suppressed colony growth from mature granulocyte-macrophage progenitors (day 7 CFU-GM) especially with an 18-h preincubation prior to coculture. It also suppressed both immature granulocyte-macrophage progenitors (day 14 CFU-GM) and multipotential hematopoietic progenitors. The suppressive effects were observed on colony growth from autologous marrow cells as well as allogeneic marrow cells. The suppression of day 7 CFU-GM colony growth by supernatants due to preincubation with marrow cells and the LAK cell fraction suggested that the humoral factor contributes to the suppression by the LAK cell fraction. These data suggest that the LAK cell fraction suppresses the development of human hematopoietic progenitor cells.     

Decrease of circulating hematopoietic progenitor cells During interleukin-2 treatment is associated with an increase of vascular cell adhesion molecule-1, a critical molecule for progenitor cell adhesion

Administration of interleukin-2 (IL-2) to cancer patients has been shown to transiently decrease the number of circulating hematopoietic progenitor cells, but the mechanism of this phenomenon is unknown. Recently, the interaction of vascular adhesion molecule-1 (VCAM-1) with leukocyte very late antigen-4 (VLA-4) has been demonstrated to play a crucial role in the adhesion of progenitor cells to bone marrow stromal elements. Cytokine induced upregulation of VCAM-1 leads to increased binding of progenitor cells to stromal cells in vitro, and inhibition of this interaction by monoclonal antibodies is associated with marked progenitor cell mobilisation in vivo. In the present study we serially determined peripheral blood progenitor cell numbers during IL-2 treatment (10 courses) in 6 cancer patients and determined in parallel levels of soluble VCAM-1 as a surrogate marker for the in vivo activation of this molecule. Our data indicate that continuous intravenous administration of IL-2 for 5 days leads to a marked decrease of circulating progenitor cells associated with a substantial increase of circulating VCAM-1. Circulating myeloid progenitor cells (CFU-GM) dropped from a mean value of 167 +/- 187 / ml pre IL-2 to 16 +/- 15 / ml on day 3 (p < 0.01). Similarily, mean erythroid progenitors (BFU-E) decreased from 282 +/- 204 / ml before IL-2 administration to 86 +/- 61 / ml on day 3 (p < 0.005). In contrast, soluble VCAM-1 rose from a mean value of 1814 +/- 451 ng/ml before to 4607 +/- 736 ng/ml at the end of IL-2 therapy (p < 0.0001). Sera from IL-2 treated patients did not inhibit hematopoietic colony formation from normal bone marrow. These results suggest redistribution and increased adhesion of progenitor cells to stromal and/or endothelial elements during IL-2 via the VCAM-1/VLA-4 interaction as a possible mechanism for the decrease of circulating progenitor cells during IL-2 therapy.     

Notch ligand Delta-1 differentially modulates the effects of gp130 activation on interleukin-6 receptor alpha-positive and -negative human hematopoietic progenitors

Interleukin (IL)-6 plays pleiotropic roles in human hematopoiesis and immune responses by acting on not only the IL-6 receptor-alpha subunit (IL-6Ralpha)(+) but also IL-6Ralpha(-) hematopoietic progenitors via soluble IL-6R. The Notch ligand Delta-1 has been identified as an important modulator of the differentiation and proliferation of human hematopoietic progenitors. Here, it was investigated whether these actions of IL-6 are influenced by Delta-1. When CD34(+)CD38(-) hematopoietic progenitors were cultured with stem cell factor, flt3 ligand, thrombopoietin and IL-3, Delta-1, in combination with the IL-6R/IL-6 fusion protein FP6, increased the generation of glycophorin A(+) erythroid cells but counteracted the effects of IL-6 and FP6 on the generation of CD14(+) monocytic and CD15(+) granulocytic cells. Although freshly isolated CD34(+)CD38(-) cells expressed no or only low levels of IL-6Ralpha, its expression was increased in myeloid progenitors after culture but remained negative in erythroid progenitors. It was found that Delta-1 acted in synergy with FP6 to enhance the generation of erythroid cells from the IL-6Ralpha(-) erythroid progenitors. In contrast, Delta-1 antagonized the effects of IL-6 and FP6 on the development of monocytic and granulocytic cells, as well as CD14(-)CD1a(+) dendritic cells, from the IL-6Ralpha(+) myeloid progenitors. These results indicate that Delta-1 interacts differentially with gp130 activation in IL-6Ralpha(-) erythroid and IL-6Ralpha(+) myeloid progenitors. The present data suggest a divergent interaction between Delta-1 and gp130 activation in human hematopoiesis.     

Effect of angiotensin II and angiotensin(1-7) on hematopoietic recovery after intravenous chemotherapy

PURPOSE: Previous studies have shown that angiotensin peptides stimulate the proliferation of hematopoietic progenitors in vitro, promote survival after exposure to lethal irradiation as well as accelerate the recovery of white blood cells (WBC), i.e., lymphocytes, monocytes and neutrophils, and platelets. These changes in the level of formed elements in the blood after irradiation was thought to be due to increases in the numbers of bone marrow progenitors including myeloid, erythroid and megakaryocyte progenitors by the action of angiotensin peptides. In view of these findings, the effect of angiotensin peptides on recovery after chemotherapy was assessed. MATERIALS AND METHODS: The effect of angiotensin II (AII) and angiotensin(1-7) (A1-7) on the recovery of WBC and platelets in the blood, as well as the number of myeloid, erythroid and megakaryocyte progenitors in the bone marrow and the number of myeloid progenitors in the blood after intravenous administration of chemotherapeutic drugs was assessed in a mouse model. RESULTS. In initial studies, subcutaneous administration of 10 or 100 microg/kg per day of AII starting either 2 days before or 2 days after intravenous administration of 5-fluorouracil (5FU) accelerated WBC recovery (return to baseline between 7 and 14 days). Further, consistent with previous observations, the number of myeloid progenitors in the bone marrow and blood was increased after systemic administration of angiotensin peptides. The comparability of A(1-7) and AII in their effect on hematopoietic recovery after chemotherapy was shown in subsequent studies. Daily administration of both AII and A(1-7) increased platelet numbers in the peripheral blood and myeloid, erythroid and megakaryocyte progenitors in the bone marrow. As 5FU is not a stem cell toxin, these studies were repeated with administration of A(1-7) initiated before or after intravenous cyclophosphamide. Following treatment with A(1-7) before cyclophosphamide the numbers of circulating WBC initially increased and then decreased starting on day 14. Following treatment with A(1-7) 2 days after cyclophosphamide the numbers of WBC and the numbers of myeloid progenitors increased in the peripheral blood and bone marrow. CONCLUSIONS: These findings suggest that angiotensin peptides accelerate hematopoietic recovery in multiple cellular lineages after chemotherapy, perhaps through an increase in the number of early hematopoietic progenitors.     

Survival of B lineage leukemic cells: signals from the bone marrow microenvironment

Stromal cells are an essential component of the bone marrow microenvironment that regulate development of immature hematopoietic progenitor cells. Through production of soluble cytokines, and signaling through adhesion molecule interactions, stromal cells impact survival, proliferation, and differentiation of hematopoietic progenitor cells. Similarities between normal pro-B and pre-B cells and B lineage acute lymphoblastic leukemic (ALL) progenitors have been well characterized which provide a model for investigation of the mechanisms by which ALL cells respond to bone marrow microenvironment signals. In addition to providing survival signals to B lineage ALL during initiation of disease, the bone marrow has long been recognized as a "sanctuary site" for leukemic cells during traditional chemotherapy. In the current review, mechanisms by which stromal cells contribute to leukemic cell survival, and the potential impact on treatment efficacy, are discussed. A growing appreciation of the significance of the bone marrow microenvironment in the progression of ALL, and further investigation of the signaling between leukemic progenitors and stromal cells, may contribute to novel treatment strategies aimed at enhancing sensitivity of ALL cells to currently available chemotherapeutic agents.     

The effect of tumor necrosis factor on normal human hematopoietic progenitors

Tumor necrosis factor-alpha (TNF-alpha), a product of activated macrophages that is cytotoxic to tumor cells, could be used to purge tumor cells from bone marrow before autologous bone marrow transplantation for hematologic malignancies and/or solid tumors. To determine whether exposure to TNF-alpha would have an inhibitory effect on hematopoietic progenitors, we incubated normal human bone marrow with a wide range of concentrations of recombinant human TNF. In order to mimic the conditions that would be used in bone marrow purging, bone marrow cell suspensions were incubated with TNF in doses ranging from 500 to 100,000 U/ml for 24 hours, and were assayed for colony formation in agar. We noted a dose-dependent inhibition of total colony-forming units (CFU) at days 7 and 14, with 50% inhibition occurring at 60,000 U/ml of TNF. TNF exerted a differential effect on CFU so that colony formation by erythroid (CFU-E), multipotential (CFU-GEMM), and macrophage (CFU-M) progenitors was suppressed to a greater extent than that by granulocyte progenitors (CFU-G). However, even after preincubation with TNF at high doses such as 100,000 U/ml, the inhibitory effects of TNF could be abolished by washing cells before culturing. This study demonstrates that hematopoietic precursors survive treatment with TNF at doses that have been shown to be cytotoxic to tumor cells. Although TNF has a significant inhibitory effect on the growth of erythroid, multipotential, and macrophage progenitors in vitro, this effect depends on continuous exposure to TNF for more than 24 hours. Thus, TNF may be useful as a bone marrow purging agent against tumor cells, with relative sparing of normal marrow elements.     

Bone morphogenetic protein 9 is a potent synergistic factor for murine hemopoietic progenitor cell generation and colony formation in serum-free cultures.

The effect of the recently cloned cytokine bone morphogenetic protein 9 (BMP-9) on colony formation and generation in vitro clonable hematopoietic progenitors (CFU-C) in serum-free liquid cultures (LC) of both normal and post-5-fluorouracil murine bone marrow cells was studied in the presence of various other cytokines. In LC, BMP-9 concentrations of 100 ng or more per ml led to complete inhibition of Steel Factor (SF) + interleukin-11 (IL-11) or IL-12 supported CFU-C generation, which was partly abrogated when IL-3 was additionally included. We found this inhibitory effect of BMP-9 to be mediated by an increased TGF-beta1 elaboration and TGF-beta1 mRNA expression in bone marrow cells with increasing BMP-9 concentrations. In the presence of neutralizing antibodies (Ab) against TGF-beta1, BMP-9 concentrations of 3 ng or higher synergized with IL-3, SF+IL-3, SF+IL-11/12, or IL-3+SF+IL-11/12 to increase CFU-C generation. Similarly, high BMP-9 concentrations dramatically inhibited primary colony formation induced by SF+IL-11/12, whereas in the presence of TGF-beta1 neutralizing Ab only 3 ng or more BMP-9 per ml stimulated both the time of colony appearance, the colony size and colony numbers in the presence of IL-3, M-CSF, GM-CSF, SF, SF+Flt3-L, SF+IL-3, SF+IL-11/12 or IL-3+SF+IL-11/12. BMP-9 neither stimulated CFU-C generation nor colony formation as a single factor, nor did it synergize with thrombopoietin (Tpo), erythropoietin (Epo), Flt3-L, IL-11, IL-12 or G-CSF. The effect of BMP-9 on its target cells was direct as demonstrated using single-sorted stem cells. These observations demonstrate that BMP-9 plays a dual role in regulating proliferation of primitive hemopoietic progenitor cells. Thus, in addition to its ability to enhance TGF-beta1 elaboration in bone marrow cells, it acts as a potent synergistic activity that is different from SF, Flt3-L, IL-11 or IL-12. BMP-9 mRNA was exclusively detected in the liver of adult mice, whilst no expression was found in stromal cell lines propagated from day-16 fetal liver or neonatal or adult bone marrow. 125I-BMP-9 bound specifically to a high percentage of blast cells in lineage-depleted post-fluorouracil bone marrow cells and to megakaryocytes in normal and post-fluorouracil bone marrow, indicating that BMP-9R are expressed on these cells. The dissociation between the site of BMP-9 production and its target cells in the bone marrow makes BMP-9 a hemopoietic hormone.     

Newer Options for Treating Drug-Resistant (MDR+) Cancer Cells using Photoradiation Therapy

Emergence of drug resistance with conventional cytotoxic therapy is a major challenge towards the curability of many cancers, especially in patients undergoing autologous BMT with ex-vivo purged hematopoietic support. We have explored the potential role of photoradiation therapy in purging hematopoietic stem cells of various hematological malignancies. Benzoporphyrin derivative, monoacid ring A (BPD-MA), dihematoporphyrin ether (DHE), and MC-540 were evaluated for the “ex-vivo” purging of residual tumor cells from autologous bone marrow (BM) grafts. BPD-MA and DHE photosensitizing activity was tested against two human large cell lymphoma cell lines and colony forming-unit leukemia (CFU-L) derived from patients with acute myelogenous leukemia (AML). In mixing experiments four log elimination of tumor cell lines was observed after 1 hr of incubation with BPD-MA or DHE followed by white light exposure. By comparison, using the same concentration of BPD-MA or DHE, the mean recovery of normal BM progenitors was 4-5.2% for granulocyte-macrophage colony forming unit (CFU-GM) and 5-9.8% for burst forming unit erythroid (BFUE).

The T lymphoblastic leukemia cell line CEM and its vinblastine (VBL)-resistant subline CEM/VBL₁₀₀. along with the acute promyelocyte leukemia cell line HL-60 and its vincristine (VCR)-resistant subline HL-60/VCR, were also tested. Our results demonstrated the preferential cytotoxicity of BPD-MA and DHE toward neoplastic cell lines and CFU-L from AML patients. In addition, DHE was slightly more effective in purging tumor cells expressing the p-170 glycoprotein. These results suggest that photoradiation would be useful for “ex-vivo” purging of malignant cells. Other methods to deal with decreasing drug resistance are also detailed.     

The developmental program of murine erythroleukemia cells

Hematopoietic stem cells (HSCs) or early progenitors respond to external stimuli in bone marrow and differentiate into cell-restricted lineages of blood cells of limited life span. In leukemias, however, early hematopoietic progenitors self-renew themselves, fail to respond to differentiation signals, and do not undergo programmed cell death (apoptosis). The basic mechanisms of differentiation and apoptosis of leukemia cells have been the long-term objective of our work. By exploiting widely studied murine and human leukemic cell systems as models of hematopoietic cell differentiation, we explored the mechanisms by which pharmaceutical agents initiate differentiation in leukemic systems. In this article, we present the developmental program of MEL cells with emphasis given on the role of commitment to terminal maturation. Commitment is initiated via inducer-receptor-mediated processes and leads to discrete patterns of expression of several genes that contribute to growth arrest at the G1 phase, expression of differentiated phenotype, and differentiation-dependent apoptosis (DDA). Overall, MEL erythroid cell differentiation represents a developmental program with a highly coordinated set of processes that is "triggered" by an inducer and functions via a network of genes and proteins interacting with each other harmonically to give birth to lineage-restricted phenotype.     

Chromatin remodeling gene SMARCA5 is dysregulated in primitive hematopoietic cells of acute leukemia.

We identified a subset of genes involved in chromatin remodeling whose mRNA expression changes in differentiating mouse erythroleukemia (MEL) cells. We furthermore tested their mRNA expression patterns in normal and malignant CD34+ bone marrow cells. SMARCA5, imitation switch gene homologue, was rapidly silenced during in vitro erythroid differentiation of MEL cells whereas it was up-regulated in CD34+ hematopoietic progenitors of acute myeloid leukemia (AML) patients. Moreover, SMARCA5 mRNA levels decreased in AML CD34+ progenitors after the patients achieved complete hematologic remission. We detected high levels of SMARCA5 mRNA in murine bone marrow and spleen and monitored its expression in these hematopoietic tissues during accelerated hematopoiesis following hemolytic anemia induced by phenylhydrazine. SMARCA5 expression levels decreased after the onset of accelerated erythropoiesis. Our data suggest that both in vitro and in vivo induction of differentiation is followed by down-regulation of SMARCA5 expression. In CD34+ AML progenitors over-expression of SMARCA5 may thus dysregulate the genetic program required for normal differentiation.     

Transformation of early erythroid precursor cells (BFU-E) by a recombinant murine retrovirus containing v-erb-B

Avian erythroblastosis virus (AEV) is a replication-defective retrovirus that transforms erythroid and fibroblast cells in vitro and in vivo. The transforming ability of AEV is due primarily to the oncogene v-erb-B. A recombinant murine retrovirus has been constructed by inserting a chimeric gag-v-erb-B gene into a Moloney murine leukemia virus based vector. This retrovirus was used to examine v-erb-B-induced transformation of murine hematopoietic cells. Infection of murine primary fetal liver, adult bone marrow or adult spleen cells with the recombinant virus generated large hemoglobinized erythroid colonies in the absence of exogenous growth factors. Generation of such colonies usually requires the presence of erythropoietin (Epo) and interleukin-3 (IL-3). These growth-factor independent colonies were shown to be derived from early (BFU-E) and not late (CFU-E) erythroid progenitor cells, and the effect was not attributable to growth factors elicited by the virus-producing cell lines. In order to confirm that the recombinant virus was responsible for this transformation of BFU-E to growth factor independence, bone marrow cells from post 5-fluorouracil treated mice were infected and used to repopulate lethally-irradiated mice. Growth factor-independent BFU-E were obtained in up to 30% of day-13 spleen colonies and it was shown by DNA analysis that cells from these colonies contained integrated provirus. Our results indicate that v-erb-B transforms early erythroid progenitors to growth factor independent growth and subsequent differentiation to erythrocytes -a process that normally requires Epo plus either IL-3 or granulocyte-macrophage colony stimulating factor (GM-CSF).     

Hematopoietic progenitor cells from patients with myelodysplastic syndromes: in vitro colony growth and long-term proliferation

It is known that the levels of hematopoietic progenitor cells (HPC) are greatly reduced in the majority of patients with myelodysplastic syndromes (MDS). To date, however, only limited information exists on the growth kinetics of these cells in long-term marrow cultures (LTMC), particularly in terms of erythroid and multipotent progenitors. In the present study, we have determined the HPC content in the bone marrow of 12 MDS patients and followed the proliferation kinetics of myeloid (including granulocyte, macrophage and granulocyte macrophage), erythroid (including early and late) and multipotent progenitor cells in LTMC throughout a 7-week culture period. Both the non-adherent and adherent fractions of the cultures were analyzed, so we were able to look at progenitor cells in suspension and those that physically associated to the stromal cell layer developed in culture. All 12 patients were grouped based on their FAB subtype and the in vitro growth of the HPC was analyzed accordingly. The results presented here indicate that in the majority of MDS patients, pronounced deficiencies exist both in the content and the long-term proliferation of marrow HPC. Such deficiencies were particularly evident for multipotent progenitors and those committed to the erythroid lineage, in which alterations in the maturation process also seem to be present. Our results suggest that, at least in some patients, HPC--besides showing an impaired proliferative capacity--lose their ability to adhere to the stromal cell layers developed in culture. RA patients showed the less affected in vitro HPC growth, whereas HPC from RAEB and RAEB-t showed a markedly deficient growth in culture. Interestingly, myelopoiesis was significantly increased in cultures of CMML patients. These results give some new insights into the biology of MDS-derived HPC.     

Myeloma phenotype: clues to disease origin and manifestation.

Phenotypic analysis of myeloma cells has had a major impact on our understanding of the development of the disease. Heterogeneity in the expression of lineage- and differentiation-associated antigens has helped delineate a circulating clonal premyeloma cell compartment coexpressing CD19 and CD11b. These cells can be stimulated in vitro to proliferate and differentiate into the mature myeloma cells. Other studies have demonstrated the involvement of very early bone marrow B lymphocytes, which could be differentiated into myeloma cells through a CD10-positive intermediate stage. These data suggest that myeloma originates in the bone marrow and is mobilized through the circulation to and from extramedullary sites, probably lymph nodes, which are required for their development. Subsequently, these cells return to the bone marrow or soft-tissue sites, using adhesion molecules for homing to sites that can provide the stimuli for expansion and maturation. Development of myeloma and disease manifestation are governed by a network of cytokines. Among the cytokines, IL-6 has been promoted as the major myeloma growth factor. Recent findings indicate that, whereas myeloma cells have the ability to express both the IL-6 and its receptor gene, their ability to respond to the cytokine is minimal. The requirement in vitro for both IL-3 and IL-6 for the stimulation of premyeloma cell proliferation and differentiation suggests a role for IL-6 in affecting differentiation of myeloma progenitors and the involvement of an earlier hematopoietic progenitor. Frequent association with myeloid dysplasia and neoplasia and expression of multiple hematopoietic lineage-associated markers forward the hypothesis that myeloma originates in a hematopoietic stem cell.