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S C Clark

Publications and source records attributed to S C Clark.

At least 73 records · Page 4Linked to original sources
PubMed1992

Clonal proliferation of murine lymphohemopoietic progenitors in culture.

We have used a two-step clonal culture system to unequivocally demonstrate that individual primitive lymphohemopoietic progenitor cells have the capacity for differentiation along either the myeloid or the B-lymphoid lineage. Highly enriched murine marrow cells were plated individually in culture by micromanipulation in the presence of pokeweed mitogen-stimulated spleen cell conditioned medium, erythropoietin, steel factor (SF), and interleukin (IL) 7. Forty-five percent of the single cells formed primary colonies expressing multiple hemopoietic lineages. When aliquots from individual colonies were replated in secondary methyl cellulose culture containing SF and IL-7, 41% of the primary colonies gave rise to lymphocyte colonies. Cells of the lymphocyte colonies were blast-like and B220+, sIg-, Mac-1-, Gr-1-, Ly-1-, L3T4-, Ly-2-, and CD3-. Thirty to 70% of the cells were Thy-1+. mu-chain mRNA was detected in most of the cells by in situ hybridization with an antisense RNA probe. When lymphocyte colonies derived from a single cell were pooled and individually injected into scid mice, donor-type IgM was measurable in the serum of mice and spleens contained donor-type B cells. We then carried out initial screening of growth factors to identify growth factors that might replace pokeweed mitogen-stimulated spleen cell conditioned medium in the primary culture. Combinations of two factors that included SF plus IL-6, IL-11, or granulocyte colony-stimulating factor were all effective in the primary culture in the maintenance of the B-lymphoid potential. Interestingly, IL-3 could neither replace nor act synergistically with SF to support the lymphoid potential of the primary cultures. Our observations demonstrate that many primitive progenitors previously believed to be myeloid-committed also possess B-lymphoid potential. This culture system should prove valuable for elucidation of the mechanisms regulating early stages of lymphohemopoiesis.

Animals↗
PubMed1992

Enhancement of murine hematopoiesis by synergistic interactions between steel factor (ligand for c-kit), interleukin-11, and other early acting factors in culture.

Entry into the cell cycle of dormant hematopoietic progenitors appears to be regulated by multiple synergistic factors, including interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), IL-11, and the ligand for c-kit, which is also known as steel factor (SF). We have tested the effects of these and other hematopoietic factors on the proliferation of partially enriched dormant murine progenitors in the presence and absence of serum. In serum-containing cultures, SF and IL-11 interacted to support the formation of multilineage colonies; the level of colony formation was comparable with the colony formation supported by other effective two-factor combinations. In serum-free cultures, colony formation supported by two factors was significantly less than that in serum-containing culture and the most effective two-factor combination in serum-free culture was SF plus IL-3. In serum-free cultures, three-factor combinations consisting of SF, IL-3, and one of IL-6, G-CSF, or IL-11 yielded colony formation that was comparable with that seen in serum-containing cultures. These studies indicate that IL-11 belongs to a group of early-acting hematopoietic synergistic factors that now includes IL-6, G-CSF, and IL-11. In contrast, SF is unique among the synergistic factors in that it interacts either with growth factors such as IL-3 or GM-CSF or with synergistic factors such as IL-6, IL-11, or G-CSF.

Animals↗
PubMed1992

Growth factor requirements for survival in G0 and entry into the cell cycle of primitive human hemopoietic progenitors.

In this study we have isolated populations of dormant human hemopoietic progenitors by two different approaches. First CD34+ cells isolated by panning were further separated on the basis of absence of HLA-DR expression by using fluorescence-activated cell sorting. Second, CD34+ HLA-DR- cells were isolated by nonadherence to soybean agglutinin, negative immunomagnetic bead selection with lineage-specific antibodies, and two-color cell sorting. Progenitors in either cell population were unable to form colonies in the presence of interleukin (IL)-3 alone but yielded a substantial number of colonies, including multilineage colonies, in the presence of combinations of IL-3 and IL-6. Similarly, IL-3 plus any one of the other synergistic factors, including granulocyte colony-stimulating factor, IL-11, leukemia inhibitory factor, and steel factor, effectively supported colony formation from CD34+ HLA-DR- progenitors. Sequential observation of colony formation from single CD34+ HLA-DR- cells provided definitive evidence that the synergistic factors trigger cell divisions of dormant cells. Studies with delayed addition of factors to the cultures provided evidence that this population of cells also requires IL-3 or granulocyte/macrophage colony-stimulating factor (GM-CSF) to survive even while dormant. In contrast, none of the synergistic factors were able to replace IL-3 or GM-CSF in this function. These findings confirm and extend the model that multiple factors with overlapping functions operate both independently and in combination to regulate early stages of hemopoiesis.

Antigens, CD↗
PubMed1992

Human interleukin-9 supports formation of a subpopulation of erythroid bursts that are responsive to interleukin-3.

We have investigated the biological activities of recombinant human interleukin-9 (IL-9) on enriched hematopoietic progenitors, alone or in combination with other cytokines, including Epo, G-CSF, IL-3, and GM-CSF, under serum-containing and serum-free cultures. IL-9 alone did not support colony formation. However, IL-9 plus Epo induced erythroid burst (BFU-E) formation derived from peripheral blood (PB) progenitors. Delayed addition experiments demonstrated that a part of bone marrow (BM) derived BFU-E, which seems to be immature, only responded to IL-9 and formed erythroid bursts. The burst-promoting activity (BPA) of IL-9 was confirmed using neutralizing aIL-3, aGM-CSF, and aIL-9 antisera and serum-free culture. IL-9 supported a part of BFU-E population that respond to IL-3, which was almost identical to the number of BFU-E supported by GM-CSF. IL-9 had no additive effect on erythroid and mixed colony formation supported by IL-3. In contrast, IL-9 showed an additive effect on erythroid burst formation supported by GM-CSF in serum-free culture. These data suggest that IL-9 and GM-CSF act on distinct IL-3-responsive BFU-E population. In addition, delayed addition experiment clearly demonstrated that IL-9 supports survival and the early stage of proliferation of BFU-E. These results led us to propose that IL-9 possibly acts as a BPA and selectively supports a subpopulation of early class of BFU-E that respond to IL-3.

Cell Division↗
PubMed1992

Composition and functional integrity of the in vitro hemopoietic microenvironment in acute myelogenous leukemia: effect of macrophage colony-stimulating factor.

In the present work, we have investigated the composition and hemopoietic supportive capacity of eleven normal and six acute myelogenous leukemia (AML) marrow-derived stromal adherent layers, established in the absence or in the presence of recombinant human colony-stimulating factor 1 (rhCSF-1, macrophage colony-stimulating factor). Two of six AML adherent layers were deficient in composition (i.e., no confluency, reduced numbers of macrophages and fibroblastic progenitors, and no fat cell formation), resulting in reduced CSF-1 production and a poor hemopoietic supportive capacity (assessed by the ability of an irradiated stroma to sustain the growth of myeloid, erythroid, and multipotential progenitors derived from a second innoculum of normal bone marrow). Four out of six AML adherent layers showed levels of macrophages, fibroblastic progenitors, fat cells, and CSF-1 similar to those observed in adherent layers from normal bone marrow; however, their capacity to sustain normal hemopoiesis was still significantly reduced. The deficient hemopoietic supportive capacity of all AML adherent layers correlated with the presence of a soluble activity in the culture supernatant that inhibited hemopoietic colony formation. Addition of rhCSF-1 during the establishment of AML adherent layers significantly increased their hemopoietic supportive capacity. In contrast, the hemopoietic supportive capacity of normal adherent layers was reduced by rhCSF-1. The opposite effects of rhCSF-1 on the hemopoietic supportive capacity of normal and AML adherent layers correlated with the levels of the soluble inhibitory activity, that is, increased levels in cultures containing rhCSF-1-treated normal adherent layers, and slightly reduced levels in cultures of rhCSF-1-treated AML layers. These results indicate that, despite a morphologically normal composition in most cases (four out of six), the hemopoietic microenvironment developed in long-term marrow culture (LTMC) from all AML marrows analyzed has a deficient hemopoietic supportive capacity, due, at least in part, to the production of hemopoietic inhibitor(s). Such a deficiency can be partially overcome by establishing the stroma layers in the presence of rhCSF-1.

Bone Marrow↗
PubMed1992

Immunogenotypes and clonal culture analysis in B-precursor acute lymphoblastic leukemia.

Immunogenotypic changes in 32 patients with B-precursor acute lymphoblastic leukemia (ALL), including three patients with t(4;11) and 13 with t(9;22), were determined using immunoglobulin heavy (IgH) chain gene probe and T-cell receptor beta, gamma and delta chain gene probes. Clonogenic assay was performed in 12 of the 32 patients. In this study, four patients had a germline configuration of the IgH chain gene, showing a dissociation between phenotypic and genotypic expression; three patients had Philadelphia-positive (Ph+) ALL. The immunogenotypic manifestation in Ph+ ALL does not depend on whether the leukemia cells had rearrangement within the major breakpoint cluster region (major-BCR) DNA sequence or the leukemia cells had myeloid-associated antigens. Colony assay using various recombinant cytokines demonstrated that the leukemia cells from four of 12 patients formed colonies in response to myelopoietic stimulants; three of the four patients were major-BCR-rearranged Ph+ ALL. Notably, cells from one patient with Ph+ ALL formed colonies on the addition of granulocytic colony-stimulating factor. This indicates not only the biological heterogeneity of ALL cells but also that some of the characteristics of the cells are related to specific chromosome changes.

Adult↗
PubMed1991

Release of early human hematopoietic progenitors from quiescence by antisense transforming growth factor beta 1 or Rb oligonucleotides.

We have used antisense oligonucleotides to study the roles of transforming growth factor beta (TGF-beta) and the two antioncogenes, retinoblastoma susceptibility (Rb) and p53, in the negative regulation of proliferation of early hematopoietic cells in culture. The antisense TGF-beta sequence significantly enhanced the frequency of colony formation by multi-lineage, early erythroid, and granulomonocytic progenitors, but did not affect colony formation by late progenitors. Single cell culture and limiting dilution analysis indicated that autocrine TGF-beta is produced by a subpopulation of early progenitors. Antisense Rb but not antisense p53 yielded similar results in releasing multipotential progenitors (colony-forming unit-granulocyte/erythroid/macrophage/megakaryocyte) from quiescence. Rb antisense could partially reverse the inhibitory effect of exogenous TGF-beta. Anti-TGF-beta blocking antibodies, antisense TGF-beta, or Rb oligonucleotides all had similar effects. No additive effects were observed when these reagents were combined, suggesting a common pathway of action. Our results are consistent with the model that autocrine production of TGF-beta negatively regulates the cycling status of early hematopoietic progenitors through interaction with the Rb gene product.

Antigens, CD↗
PubMed1991

Synergistic interactions between interleukin-11 and interleukin-4 in support of proliferation of primitive hematopoietic progenitors of mice.

Interleukin-11 (IL-11) is a newly identified lymphohematopoietic cytokine originally derived from the primate bone marrow stromal cell line, PU-34. Separately, we reported that IL-11 augments IL-3-dependent proliferation of primitive murine hematopoietic progenitors in culture. We have now examined the synergistic interactions between IL-11 and IL-4 in support of colony formation from marrow cells of mice treated 2 days before with 150 mg/kg 5-fluorouracil. Neither recombinant human IL-11 nor murine IL-4 alone was effective in the support of colony formation. When the two factors were combined, there was major enhancement of colony formation, including that of multilineage colony-forming cells. Serial observations (mapping studies) of development of multipotential blast cell colonies indicated that the synergy between IL-11 and IL-4 is due in part to shortening of the dormant period of the stem cells, an effect very similar to that of IL-6 and granulocyte colony-stimulating factor. The combination of IL-11 and IL-4 may be useful in the stimulation of dormant hematopoietic stem cells in vivo.

Animals↗
PubMed1991

Effects of hematopoietic growth factors on the survival of primitive stem cells in liquid suspension culture.

We have examined the effects of 10 different growth factors either alone or in combination on colony-forming unit-spleen (CFU-S) and repopulating stem cell survival in vitro. Either interleukin-3 (IL-3), granulocyte-colony-stimulating factor (G-CSF), or IL-4 alone support CFU-S in vitro. The effects of IL-3 or G-CSF could be neutralized by adding antibodies against IL-3 or G-CSF, respectively. However, the effects of IL-4 could be neutralized with antibodies to IL-4 as well as with antibodies to IL-3 and G-CSF. The combinations of IL-3 and IL-6, IL-3 and G-CSF, IL-3 and IL-1 alpha, IL-3 and granulocyte-macrophage CSF (GM-CSF), and IL-4 and IL-6 acted synergistically to increase CFU-S number. Addition of macrophage inflammatory protein-1 alpha (MIP-1 alpha) to IL-3 and IL-6 inhibited the increase in CFU-S number. Repopulating stem cell function was measured in a competitive repopulation assay. Either IL-3 or IL-4 alone could preserve stem cell function in vitro. The combinations of IL-3 and IL-6, and IL-3 and G-CSF increased stem cell function approximately twofold. The combinations of IL-3 + G-CSF + IL-6, and IL-4 and IL-6 (both of which increased CFU-S number fivefold to 10-fold) decreased stem cell function approximately fourfold. These results demonstrate that certain combinations of growth factors can increase CFU-S number at the expense of stem cell function.

Animals↗
PubMed1991

Inhibition of hematopoiesis in normal human long-term marrow cultures treated with recombinant human macrophage colony-stimulating factor.

The effects of recombinant human macrophage colony-stimulating factor (rhCSF-1) in long-term marrow cultures (LTMC) established from normal bone marrow cells were examined. When added during the first 3 weeks of culture (every second day, at 15 ng/mL), rhCSF-1 strongly inhibited the growth of all hematopoietic progenitors analyzed (colony-forming unit-MIX [CFU-MIX], CFU-granulocyte macrophage [CFU-GM], CFU-M, CFU-G, burst-forming unit-erythroid). Paralleling the inhibition of progenitors was the complete loss of adipocytes from the stromal layer of rhCSF-1-treated cultures. The inhibitory effect of rhCSF-1 correlated in all instances with the accumulation in the supernatants of these cultures of an activity (different from CSF-1) that inhibited colony formation in semisolid cultures. When addition of rhCSF-1 was delayed 3 weeks, its inhibitory effects were significantly reduced, which correlated with reduced inhibitory activity detected in the supernatants. Analysis of CSF-1 concentration by radioreceptor assay confirmed that added rhCSF-1 increased culture CSF-1 levels and showed that the decreased inhibition observed when rhCSF-1 is added later in culture was not due to decreased CSF-1 levels at that point. In contrast, the ability of rhCSF-1 to inhibit hematopoiesis and accumulate inhibitory activity in LTMC correlated with its rate of utilization, much higher in the first 2 weeks of culture, when the stromal layer was being established, than later. These observations document the inhibitory effect of rhCSF-1 on all aspects of hematopoiesis conducted in cultures that simulate the hematopoietic microenvironment, demonstrate the importance of accessory/stromal cells in mediating the effects of rhCSF-1 in LTMC, and point to an inhibitory activity as the mediating agent.

Bone Marrow↗
PubMed1991

Induction of interferon gamma production by natural killer cell stimulatory factor: characterization of the responder cells and synergy with other inducers.

We previously reported that natural killer cell stimulatory factor (NKSF), a heterodimeric lymphokine purified from the conditioned medium of human B lymphoblastoid cell lines, induces interferon gamma (IFN-gamma) production from resting peripheral blood lymphocytes (PBL) and synergizes with interleukin 2 in this activity. In this study, we show that human NKSF induces IFN-gamma production from both resting and activated human PBL and from freshly isolated murine splenocytes. Human T and NK cells produce IFN-gamma in response to NKSF, but resting PBL require the presence of nonadherent human histocompatibility leukocyte antigens DR+ (HLA-DR+) accessory cells to respond to NKSF. The mechanism(s) by which NKSF induces IFN-gamma production results in accumulation of IFN-gamma mRNA, is insensitive to cyclosporin A, and synergizes with those mediated by phytohemagglutinin, phorbol diesters, anti-CD3 antibodies, and allogeneic antigens, but not by Ca2+ ionophores. The ability of NKSF to directly induce IFN-gamma production and to synergize with other physiological IFN-gamma inducers, joined with the previously described ability to enhance lymphocyte cytotoxicity and proliferation, indicates that this lymphokine is a powerful immunopotentiating agent.

Animals↗
PubMed1991

Growth factor requirements of childhood acute T-lymphoblastic leukemia: correlation between presence of chromosomal abnormalities and ability to grow permanently in vitro.

Cells from 10 cases of childhood acute T-lymphoblastic leukemia (T-ALL) were cultured in the presence of recombinant human interleukins (rhIL) or colony-stimulating factors (CSF) to analyze their growth factor requirements and differentiative potential. Although cells from most leukemic samples displayed a short-term proliferative response to several hematopoietic growth factors, only the ones featuring chromosomal translocations could be established as permanent cell lines. Two cell lines could be initiated only in the presence of IL-3 (TALL-103 and TALL-106), one in granulocyte-macrophage CSF (GM-CSF) (TALL-101), and one in IL-2 (TALL-104); only one cell line (TALL-105) was originated in the absence of growth factors. The TALL-101 and TALL-103 cell lines, derived from very immature T-ALL cases, underwent growth factor-dependent phenotypic conversion (lymphoid to myeloid). However, the T-cell receptor rearrangement and karyotype of the original leukemic clones were retained. In contrast, the TALL-104, -105, and -106 cell lines which originated from more mature T-ALL cases, maintained a T-lymphoblastic phenotype regardless of the growth factors in which they were expanded. These data demonstrate in vitro the aggressive nature of T-ALL cases bearing chromosomal abnormalities, and indicate that the lineage commitment of the malignant clone depends on its stage of maturation in T-cell ontogeny.

Adolescent↗
PubMed1991

Human interleukin-9: genomic sequence, chromosomal location, and sequences essential for its expression in human T-cell leukemia virus (HTLV)-I-transformed human T cells.

We have isolated the genomic sequence of human interleukin-9 (IL-9) based on its sequence homology with a human IL-9 cDNA isolated from human T-cell leukemia virus (HTLV)-I-transformed T cells by expression cloning. The entire genomic sequence has been determined and the gene consists of five exons and four introns. The human IL-9 gene is mapped to the long arm of human chromosome 5 at band 5q31-32, a region found to be deleted in a number of patients with acquired 5q- abnormalities and hematologic disorders. Several blocks of transcriptional control sequences have been identified at the 5'-flanking region of the human IL-9 gene that may play an important role in the control of IL-9 gene expression. The 5'-regulatory region of the human IL-9 gene also contains sequences identified in the 5'-flanking regions of other cytokine genes mapped to the long arm of human chromosome 5, including IL-3, IL-4, IL-5, and granulocyte-macrophage colony-stimulating factor and other T-cell growth factor genes including IL-2 and IL-6. The IL-9 gene is constitutively expressed in the HTLV-I-transformed human T cells and the expression of IL-9 in these cells can be further induced by 12-O-tetradecanoyl phorbol 13-acetate. Transient transfection analysis using the plasmid containing the 5'-flanking region of IL-9 gene upstream from the firefly luciferase ciferase report gene indicated that the 0.9-kb Smal-Sacl fragment of the IL-9 gene contains sequences required for the constitutive and activated expression of IL-9 gene in HTLV-I-transformed cells. These results will now allow us to study the regulatory mechanism of IL-9 gene expression in normal and leukemic human T cells.

Amino Acid Sequence↗
PubMed1991

Direct and synergistic effects of interleukin 11 on murine hemopoiesis in culture.

We have examined the effects of a stromal cell-derived cytokine designated interleukin 11 (IL-11) on the proliferation of murine hemopoietic progenitors in methylcellulose culture. COS cell-conditioned medium containing IL-11 supported formation of granulocyte/macrophage colonies and a small number of multilineage colonies including blast cell colonies in cultures of marrow cells from normal mice. When tested with marrow cells harvested 2 days after injection of 5-fluorouracil at 150 mg/kg, IL-11 enhanced interleukin 3-dependent colony formation, whereas IL-11 alone supported only scant colony formation. Serial observations (mapping studies) of cultures of post-5-fluorouracil spleen cells indicated that the mechanism of the synergistic effect of IL-11 is to shorten the dormant period of stem cells, an effect very similar to that of interleukin 6. When pooled blast cells were plated into medium containing IL-11 and erythropoietin, only macrophage colonies were observed. Thus, IL-11 can directly support the proliferation of committed macrophage progenitors and, and like interleukin 6 and granulocyte colony-stimulating factor, act synergistically with interleukin 3 to shorten the Go period of early progenitors.

Animals↗