ISSN:0021-9541    

DOI:10.1002/jcp.1041480302

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe B‐myb cDNA has extensive sequence similarities to the c‐myb protooncogene, but, at variance with c‐myb, it is expressed in cells other than hematopoietic cells. In this paper, we show that (1) B‐myb is expressed in mouse, human, and hamster fibroblasts; (2) B‐myb mRNA levels are growth‐regulated in both fibroblasts and peripheral blood mononuclear cells; (3) by its mode of growth regulation (peak of expression, behavior in G1‐specific temperature sensitive (ts) mutants and in the presence of cycloheximide), B‐myb can be classified, like c‐myb, thymidine kinase, PCNA, and others, as a late growth‐regulated gene; (4) B‐myb mRNA levels decrease when HL‐60 cells are induced to differentiate; and (5) the increase in mRNA levels in serum‐stimulated cells is only partially explained by an increase in rate of transcription. The possibility that the B‐myb gene may be the equivalent in fibroblasts and epithelial cells of the c‐myb proto‐oncogene of h

ISSN:0021-9541    

DOI:10.1002/jcp.1041480303

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe human lymphocyte‐specific tyrosine kinase gene,Ick, is transcribed from two distinct promoters, resulting in two classes of transcripts (type I and II) differing in their 5′ untranslated regions. The steady‐state levels of the type I and IIIcktranscripts were measured in a variety of lymphoid and non‐lymphoid human tumor cell lines by S1 nuclease mapping and by a sensitive assay system using the polymerase chain reaction. Human thymocytes and all the leukemic T cell lines tested express both type I and IIIcktranscripts, albeit at different relative levels. Peripheral blood T cells express mainly type IIIcktranscripts, whereas two colonic carcinoma lines, COLO 201 and COLO 205, express exclusively type IIcktranscripts. Treatment of the leukemic T cell lines, P30/OKUBO and Jurkat, by the phorbol esters tetradecanoylphorbol acetate (TPA) or phorbol dibutyrate (PDB) results in the down‐regulation of the type I, and the up‐regulation of the type II,Icktranscript levels. The effect of PDB on the in vitro differentiation of Jurkat cells, and the expression ofIcktranscripts, is reversible. The modulation ofIcktranscript levels in TPA‐treated Jurkat cells is not due to differential RNA stability, suggesting that the twoIckpromoters are utilized differentially during T cell differentiation. The leukemic T cell line, Jurkat, may thus serve as a model for the elucidation of molecular mechanisms that regulateIcktranscription and T cell di

ISSN:0021-9541    

DOI:10.1002/jcp.1041480304

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractCoordinate production of interleukin‐1β (IL‐1β) and granulocyte macrophagecolony stimulating factor (GM‐CSF) or IL‐6 by the blast cells of acute myeloblastic leukemia (AML) and normal peripheral blood leukocytes have been previously reported (van der Shoot et al.:Blood74:2081–2087, 1989; Bradbury et al.:Leukemia4:44–47 1990a,British Journal of Haematology16:(in press), 1990b; Rodriguez‐Cimadevilla et al.: Blood 76:1481–1489, 1990; Schindler et al.: Blood 75:40–47, 1990). In the present study, we show that IL‐6 production by AML blasts is up‐regulated by endogenously produced IL‐1α. Neutralization of the endogenous source of IL‐1 results in a significant decrease in IL‐6 production, as determined by ELISA. Conversely, exposure of AML blasts to IL‐1α results in a significant increase in IL‐6 production in 10 of 16 patient samples. Antibodies against IL‐1α and ‐β also cause a drastic decrease in IL‐6 and GM‐CSF gene expression by the cells, suggesting that cytokine gene expression in AML blasts is driven, at least in part, by endogenous IL‐1. The biologic significance of IL‐6 production in culture of AML blasts has been addressed using a neutralizing antibody against IL‐6. Our data indicate that IL‐6 is important for the survival of clonogenic blasts in culture. In contrast, the survival of the total population of blasts is IL‐6‐independent, as assessed by the integrity of cellular DNA, even in the presence of anti‐IL‐6. These observations are consistent with the view that AML blasts might be organized as a lineage, with comparable hierarchy as in normal hemopoiesis and, perhaps, increased heterogeneity despite a homogenous appearance (McCulloch and Till:Blood Cells 7:63‐77, 1981; Buick and McCulloch:Control of Animal Cell Proliferation.Academic Press, New York, vol. 1, pp. 25–57, 1985). Buick and McCulloch have identified a subpopulation of AML clonogenic cells with stem‐cell‐like properties, and suggested that the majority of blasts may have undergone a determination‐like step. Our data indicate a marked difference in IL‐6 requirement for cell survival between precursors and the majority of blasts, suggesting that IL‐6 responsiveness may decrease followin

ISSN:0021-9541    

DOI:10.1002/jcp.1041480305

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractRecently, a novel cytokine designated stem cell factor (SCF) was isolated from medium conditioned by buffalo rat liver cells and proved to be the ligand forc‐kit.We have examined the effects of recombinant rat SCF alone and in various combinations with interleukin‐3 and interleukin‐4 on murine mast cell colony formation in methylcellulose culture. As a source of connective tissue‐type mast cells (CTMC), we used peritoneal mast cells. No individual factor supported colony formation by purified peritoneal mast cells. When cells were grown in combinations of two factors, significant mast cell colony growth was seen. When cells were grown in the presence of three factors, not only the number of colonies was increased but also the colonies were larger. Mast cells in these colonies contained safranin‐ and berberine sulfate‐positive cells, but the proportions of positive and negative cells varied depending on the factor combinations. We then examined the effects of these factors on proliferation of bone marrow‐derived mast cells (BMMC) by replating pooled mast cell colonies. As a single factor, only interleukin‐3 supported mast cell colony formation. Combinations of two of the three factors supported mast cell colony formation. However, the most impressive synergism was seen again with the combination of the three factors. Not only was the number of colonies increased, but there was a significant increase in size. These results indicate that SCF is an important factor for the proliferation of bot

ISSN:0021-9541    

DOI:10.1002/jcp.1041480306

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractHuman hematopoietic cells can be maintained in vitro for many weeks in the absence of exogenously provided hematopoietic growth factors if an adequate stromal cell containing adherent layer is present. We have now extended the use of this type of long‐term culture (LTC) system to create a model of perturbed hematopoiesis in which human tumor cells that constitutively produce a variety of factors are co‐cultured together with normal human marrow cells. In the present study, we used the human bladder carcinoma cell line (5637) because these cells were known to produce not only a variety of factors active directly on hematopoietic cells but also factors that can stimulate hematopoietic growth factor production by human marrow stromal cells. Analysis of mRNA extracted from the adherent layer and measurement of growth factor bioactivity in the medium of established LTC of human marrow containing irradiated 5637 cells, showed increased levels of interleukin‐1 and ‐6, as well as granulocyte and granulocyte‐macrophage colony‐stimulating factor production by comparison to control cultures. As in normal cultures, high proliferative potential clonogenic hematopoietic cells were found almost exclusively in the adherent layer of these co‐cultures, but these primitive cells were maintained in a state of continuous turnover, in contrast to control cultures where the same cell types showed the expected oscillation between a quiescent and a proliferating state following each weekly change of the medium. A similar perturbation of primitive progenitor cycling was achieved by adding medium conditioned by 5637 cells twice a week to otherwise normal LTC. The presence of irradiated 5637 cells in the LTC or the addition of 5637 conditioned medium also resulted in modest (2‐ to 3‐fold) but sustained increases in the total hematopoietic progenitor population, as well as in the final output of terminally differentiated granulocytes and macrophages. These findings indicate that primitive hematopoietic cells in LTC can be kept in a state of continuous activation for many weeks by appropriate endogenous or exogenous hematopoietic growth factor provision and that this does not necessarily lead either to their rapid exhaustion or to a large amplification in output

ISSN:0021-9541    

DOI:10.1002/jcp.1041480307

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractWe have shown in previous studies that metastatically‐competent variant subpopulations (B5, C1) derived from a non‐metastatic murine mammary adenocarcinoma (SP1) have a pronounced growth advantage over their non‐metastatic tumor cell counterparts in primary tumors. As a result, primary tumors can be progressively overgrown by cells having the competence to spread elsewhere in the body. This occurs despite any evidence to indicate an intrinsic in vivo growth rate advantage of the metastatic cells when grown as isolated populations. This suggested that cell‐cell interactions between metastatic and non‐metastatic tumor populations may be involved in the metastatic cell growth dominance process. Evidence was therefore sought for growth factors released by SP1 cells which could preferentially stimulate the B5 or C1 variants and thereby mediate this cell‐cell interaction process. We found that cocultures of SP1 and C1 or B5 cells with irradiated C1, B5, or SP1 “feeder” cells showed significant stimulation of C1 and B5 by SP1 “feeder” cells. Cell growth stimulation in response to EGF, TGF‐α, TGF‐β1, bFGF, PDGF, NGF, IGF‐1, or IGF‐2 demonstrated that only TGF‐β1 could duplicate this effect. A repeat of the coculture experiment in the presence of specific neutralizing anti‐TGF‐β antibodies was therefore undertaken and this was found to markedly reduce the stimulation of C1 or B5 cells by irradiated SP1 cells. Conditioned media from the SP1 and C1 cell lines was quantitated for TGF‐β activity and contained 4.5 ng/ml and 2.0 ng/ml, respectively. However, the majority of the TGF‐β released by SP1 cells was found to be spontaneously active, whereas 70% of the TGF‐β released by C1 cells was in its latent form. Scatchard analysis revealed approximately four times the number of TGF‐β receptors, of similar type and affinity, present on C1 as compared with SP1 cells. The in vitro results support the hypothesis that active TGF‐β released by SP1 cells may stimulate the proliferation of metastatic variant cells in a paracrine like fashion. In vivo evidence for this was obtained by showing that coinjection of irradiated SP1 cells could selectively stimulate tumor growth of viable C1 cells and this effect was markedly diminished by neurtralizing polyclonal anti‐TGF‐β antibodies. Taken together, the results suggest a novel role for TGF‐β in clonal evolution of malignant tumor growth and as a molecular mediator of tumor cell‐tumo

ISSN:0021-9541    

DOI:10.1002/jcp.1041480308

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractMutant forms of the p53 gene have been shown to cooperate with an activated ras gene in transforming primary cells in culture. The aberrant proteins encoded by p53 mutants are thought to act in a dominant negative manner in these assays. In vivo data, however, reveal that where p53 has undergone genetic change in tumors, both alleles have been affected. We previously identified a case of human acute myelogenous leukemia (AML) in which both alleles of the p53 gene had undergone independent missense mutations (at codons 135 cys to ser and 246 met to val). In these blasts, p53 mutations appear to be acting recessively. We have assayed the transforming potential of these p53 mutations, as well as that of another mutation at codon 273, also identified in a human neoplasm. Both mutations from the AML blasts (codon 135 and codon 246) confer transforming ability on the mutant protein. While transformation assays may define functionally different subsets of p53 mutations, the overexpression phenotype of mutants in this assay may not accurately reflect the pathological effects of p53 mutations in vivo.

ISSN:0021-9541    

DOI:10.1002/jcp.1041480309

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractWe studied the effect of transforming growth factor‐beta 1(TGF‐β 1) on colony formation of leukemic blast progenitors from ten acute myeloblastic leukemia (AML) patients stimulated with granulocyte colony‐stimulating factor (G‐CSF), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), interleukin‐3 (IL‐3), interleukin‐6 (IL‐6), or interleukin‐1β (IL‐1β). These CSFs and interleukins by themselves stimulated the proliferation of leukemic blast progenitors without adding TGF‐β 1. G‐CSF, GM‐CSF, and IL‐3 stimulated blast colony formation in nine patients, IL‐6 stimulated it in five, and IL‐1β stimulated in four. TGF‐β 1 significantly reduced blast colony formation stimulated by G‐CSF, GM‐CSF, or IL‐6 in all patients. In contrast, TGF‐β 1 enhanced the stimulatory effect of IL‐3 on blast progenitors from three cases, while in the other seven patients TGF‐β 1 reduced blast colony formation in the presence of IL‐3. To study the mechanism by which TGF‐β 1 enhanced the stimulatory effect of IL‐3 on blast progenitors, we carried out the following experiments in the three patients in which it occurred. First, the media conditioned by leukemic cells in the presence of TGF‐β 1 stimulated the growth of leukemic blast progenitors, but such effect was completely abolished by anti‐IL‐1 β antibody. Second, the addition of IL‐1 β in the culture significantly enhanced the growth of blast progenitors stimulated with IL‐3. Third, leukemic cells of the two patients studied were revealed to secrete IL‐1 β and tumor necrosis factor‐α (TNF‐α) constitutively; the production by leukemic cells of IL‐1 β and TNF‐α was significantly promoted by TGF‐β 1. Furthermore, the growth enhancing effect of TGF‐β 1 in the presence of IL‐3 was fully neutralized by anti‐IL‐1 β antibody. These findings suggest that TGF‐β

ISSN:0021-9541    

DOI:10.1002/jcp.1041480310

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractAlthough hematopoietic growth factors influence renewal and differentiation of blast progenitors in acute myelogenous leukemia (AML), morphological maturation of leukemic blasts is thought a rare event, even when cultured in the presence of appropriate growth stimulants. However, light microscopic observation may not be sufficient to clarify precisely the effects of hematopoietic growth factors on the morphological differentiation of leukemic blasts. In this study, using cell culture techniques and electron microscopic cytochemistry for platelet peroxidase (PPO), we studied the effects of interleukin‐3 (IL‐3) and interleukin‐6 (IL‐6), both of which are considered to play an important role in normal megakaryocytopoie‐sis, on the growth and differentiation of blast cells from two patients with childhood acute megakaryoblastic leukemia (AMKL). In both of the two cases, IL‐3 stimulated leukemic colony formation in methylcellulose culture, whereas IL‐6 showed little such activity. However, in suspension culture, IL‐6 was active in promoting megakaryocytic differentiation, although incomplete, as detected by increase in the number of PPO‐positive cells, some having demarcation membrane‐like structure. This effect was evident in culture with IL‐6 alone in one patient, but it was detectable only when IL‐6 was used in combination with IL‐3 in the other patient. In contrast, IL‐3 alone stimulated differentiation towards myeloid but not megakaryocytic lineage. These results indicate that IL‐3 and IL‐6 have a distinct role in leukemic megakaryocytopoiesis (IL‐3 stimulates growth, whereas IL‐6 promotes morphological differentiation) and that cooperation between these two cytokines functions most effectively for megakaryocytic differentiation of AMKL cells in a fashion similar to th

ISSN:0021-9541    

DOI:10.1002/jcp.1041480311

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY