摘要:

AbstractThe course of the differentiation and proliferation of the human erythroid burst‐forming units (BFU‐E) to colony‐forming units (CFU‐E) was directly investigated using a combination of highly purified BFU‐E, a liquid culture system, and the following clonal assay. Highly purified human blood BFU‐E with a purity of 45–79% were cultured in liquid medium with recombinant human erythropoietin (rEP) and recombinant human interleukin‐3 (rlL‐3) to generate more differentiated erythroid progenitors. The cultured cells were collected daily for investigating the morphology, the increment in the number of cells and the clonality. Ninety percent of purified BFU‐E required not only rEP but also rlL‐3 for clonal development. By 7 days of liquid culture, the total cell number increased 237 ± 20‐fold above the starting cells, while erythroid progenitors increased 156 ± 74‐fold. As the incubation time in liquid culture increased, the cells continuously differentiated in morphology. Replating experiments with rTEP combined with or without rlL‐3 showed the following: (1) The number of erythroblasts that were part of erythroid colonies decreased with accompanying erythroid progenitor differentiation and proliferation. (2) As the incubation time in liquid culture increased, erythroid progenitors had a graded loss of their dependency on rlL‐3 and a complete loss of dependency was observed after 3 days of liquid culture. At that time 85% of the erythroid progenitors gave rise to colonies of more than 100 erythroblasts which were equivalent to mature BFU‐E. These studies provide a quantitative assessment of the loss of lL‐3 dependency by BFU‐E and indicate that the size of the generated erythroid colonies and their lL‐3 requirement correlate

ISSN:0021-9541    

DOI:10.1002/jcp.1041490102

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

年代:1991

数据来源: WILEY

摘要:

AbstractAlthough the precise intracellular function(s) of the polyamines remain incompletely defined, a myraid of evidence now shows that the polyamines must accumulate or be maintained at a specific intracellular concentration in order for all mammalian cells to grow or divide. The initial step in polyamine biosynthesis normally involves the decarboxylation of ornithine by the enzyme ornithine decaboxylase (ODCase E.C. 4.1.1.17) to yield putrescine. Increases in the steady‐state level of intracellular ornithine have been reported to markedly alter the accumulation of the polyamines following stimulation of Reuber H35 Hepatoma cells with 12‐O‐tetradecanoylphorbol‐β‐acetate (TPA) in the presence of serum (Wu and Byus:(Biochem. Biophys. Acta804:89–99, 1984)) Wu et al.: (Cancer Res. 41:3384–3391, 1981). We wished to determine whether or not incubation of H35 hepatoma cells with exogenous ornithine would result in a stimulation of DNA synthesis following treatment with the mitogens TPA and insulin. For these studies, H35 cells were maintained under serum‐free conditions for 2–3 days in order to obtain synchronous cultures suitable for analysis of the level of DNA synthesis. Cultures treated in this manner were highly viable, maintained similar growth rates, and possessed the equivalent levels of intracellular ornithine and polyamines as the serum‐containing cultures. Arginine levels, however, were approximately twofold higher following culture under serum‐restricted conditions for 3 days. The addition of exogenous ornithine (0.5 mM) was accompanied by a 4–5‐fold increase in intracellular steady‐state ornithine levels and by a 6–8‐fold increase in the presence of TPA and ornithine. In a manner identical to the serum‐containing cultures (Wu and Byus (1984)) the addition of TPA and exogenous ornithine to the serum‐free cells caused a dose‐dependent increase in intracellular putrescine (up to 5‐fold) and a concomitant decrease in ODC activity in comparison to stimulation with TPA alone. The addition of TPA led to a 3–5‐fold increase in the incorporation of tritiated thymidine into DNA. In the presence of exogenous ornithine, TPA‐induced DNA synthesis was further stimulated more than twofold in a dose‐dependent manner. Insulin (10−10–10−8M) proved to be more efficacious as a mitogen in the H35 cells and led to greater stimulation of DNA synthesis than TPA. Insulin alone also resulted in a higher steady‐state level of ornithine and putrescine in comparison with TPA alone. However, ornithine addition to the culture medium was not accompanied by any further increase in the insulin‐mediated elevation in DNA synthesis. The data is discussed in relation to the selective ability of mitogens to alter the flux through extracellular and intracellular pools of ornithine as required to supply sufficient polyamines to support maximal rates of DNA synthesis. The results furthers support the suggestion that the high levels of intracellular putrescine observed following TPA + ornithine might enhance any of a number of the specific or unique transductive events employed by TPA, in c

ISSN:0021-9541    

DOI:10.1002/jcp.1041490103

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

年代:1991

数据来源: WILEY

摘要:

AbstractOur recent evidence for the requirement of endogeneous histamine in IL‐3‐induced proliferation of day‐8 CFU‐S has prompted us to investigate whether or not GM‐CSF, which shares with IL‐3 the ability to stimulate bone marrow histamine synthesis, could also affect the cell cycle status of CFU‐S via this mediator. We show herein that recombinant GM‐CSF alone fails to trigger day‐8 CFU‐S into S phase, but supports their survival. However, in the same experimental conditions, GM‐CSF in combination with IL‐1 induces a CFU‐S proliferation similar to that obtained in response to IL‐3, while IL‐1 by itself has no effect on this biological activity. We further provide evidence that this phenomenon is completely abolished: (i) by preventing GM‐CSF‐induced histamine synthesis by α‐FMH, the specific inhibitor of histidine decarboxylase (HDC), or (ii) by blocking the binding sites of H2histamine receptors with their specific antagonist oxmetidine. Similar results are obtained when progenitor‐enriched bone marrow cells are used instead of the unfractionated population. In addition, we provide an argument in support of a histamine receptor modulation by GM‐CSF that could explain the lack of effect of factor‐induced histamine on day‐8 CFU‐S cell cycling. Indeed, the entry of these progenitors into S phase that is normally promoted by dimaprit, a specific histamine H2receptor agonist, is abolished by a preincubation with GM‐CSF. Taken together, our data support the conclusion that IL‐1 makes CFU‐S sensitive to GM‐CSF‐induced endogeneous histamine that will trigger them into cell cycle, while GM

ISSN:0021-9541    

DOI:10.1002/jcp.1041490104

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

年代:1991

数据来源: WILEY

摘要:

AbstractCaco‐2 cells, which express spontaneous enterocytic differentiation at confluency, is one of the most relevant in vitro models for the study of differentiation and regulation of intestinal functions. However, these cels are normally cultured in the presence of 15–20% serum which renders extremely complex the identification of the factors involved in the regulation of both proliferation and differentiation. This study has been devoted to the establishment of chemically defined culture conditions which can sustain growth and differentiation of Caco‐2 cells. The replacement of serum by ITS (insulin, transferrin, and selenium) allowed for normal structural and functional differentiation of cells as revealed by the establishment of cell polarity and the expression of brush‐border membrane enzyme markers (sucrase, maltase, lactase, alkaline phosphatase, γ‐glutamyltransferne, aminopeptidase N, and dipeptidyl‐dipeptidase IV), although the levels of sucrase activity were lower in ITS‐supplemented medium. Coating petri dishes with either type IV collagen or basement membrane proteins (Matrigel) did not improve the differentiation of cells, brush‐border membrane enzyme activities being, in fact, lower when the cells were grown on these substrata. When triiodothyronine (T3, 5 × 10−8M) was added to the ITS‐supplemented medium, disaccharidase and alkaline phosphatase activities were significantly increased while γ‐glutamyltransfrase activity was diminished by T3and stimulated by epidermal growth factor (1.6 × 10−6M). On the other hand, hydrocortisone (HC, 106M) did not modify disaccharidase and peptidase activities. These data clearly show that Caco‐2 cells can be maintained in serum‐free medium and that this system allows the study of the factors involved in the regulation of the differe

ISSN:0021-9541    

DOI:10.1002/jcp.1041490105

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

年代:1991

数据来源: WILEY

摘要:

AbstractTransforming growth factor(β)(TGFβ) treatment of rat osteoblast‐rich calvarial cells or of the clonal osteogenic sarcoma cells, UMR 106‐01, resulted in dosedependent inhibition of plasminogen activator (PA) activity, and increased production of 3.2 kb mRNA and protein for PA inhibitor‐1 (PAI‐1). Although tissue‐type PA(tPA) protein was not measured, TGF(β)did not influence production of mRNA for tPA. Production of 2.3 kb mRNA for urokinase‐type PA (uPA) was also increased by TGF(β)in a dose‐dependent manner. The effects of TGFβ on synthesis of mRNA for PAI‐1 and uPA were maintained when protein synthesis was inhibited, and were abolished by inhibition of RNA synthesis. Although uPA had not been detected previously as a product of rat osteoblasts, treatment of lysates of osteoblast‐like cells with plasmin yielded a band of PA activity on reverse fibrin autography, corresponding to a low Mr form of uPA. Untreated conditioned media from normal osteoblasts or UMR 106‐01 cells contained no significant TGFβ activity, but activity could be detected in acidified medium. Treatment of conditioned media with plasmin resulted in activation of approximately 50% of the TGFβ detectable in acidified media. The results identify several effects of TGFβ on the PA‐PA inhibitor system in osteoblasts. Net regulation of tPA activity through the stimulatory actions of several calciotropic hormones and the promotion of PAI‐1 formation by TGFβ could determine the amount of osteoblast‐derived TGFβ activated locally in bone. Stimulation of osteoblast production of mRNA for uPA could reflect effects on the synthesis of sc‐uPA, a precur

ISSN:0021-9541    

DOI:10.1002/jcp.1041490106

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

年代:1991

数据来源: WILEY

摘要:

AbstractIn this paper we show that in C3H10T1/2 mouse fibroblasts, the inducibility of c‐fos mRNA by heat shock or serum addition is strongly dependent on the cell's past. Four hours after a heat shock, a time point where the induced c‐fos mRNA has disappeared, c‐fos mRNA could not be induced again by a second heat shock. Four hours after serum addition, by which c‐fos was induced, a second serum addition also failed to induce c‐fos mRNA again. When, however, serum was added 4 hours after heat shock or heat shock was given 4 hours after serum addition, levels of c‐fos mRNA could be enhanced again. The induction by serum of c‐fos mRNA levels in thermotolerant cells might be related to their increased stimulation of DNA synthesis as compared to

ISSN:0021-9541    

DOI:10.1002/jcp.1041490107

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

年代:1991

数据来源: WILEY

摘要:

AbstractVasculotropin/vascular endothelial cell growth factor (VAS/VEGF) is a newly purified growth factor with a unique specificity for vascular endothelial cells. We have investigated the interactions of VAS/VEGF with human umbilical vein endothelial cells (HUVE cells).125I‐VAS/VEGF was bound to HUVE cells in a saturable manner with a half‐maximum binding at 2.8 ng/ml. Scatchard analysis did show two classes of high‐affinity binding sites. The first class displayed a dissociation constant of 9 pM with 500 sites/cell. The dissociation constant and the number of binding sites of the second binding class were variable for different HUVE cell cultures (KD= 179 ± 101 pM, 5,850 ± 2,950 sites/cell). Half‐maximal inhibition of125I‐VAS/VEGF occurred with a threefold excess of unlabeled ligand. Basic fibroblast growth factor (bFGF) and heparin did not compete with125I‐VAS/VEGF binding. In contrast, suramin and protamin sulfate completely displaced125I‐VAS/VEGF binding from HUVE cells. VAS/VEGF was shown to be internalized in HUVE cells. Maximum internalization (55% of total cell‐associated radioactivity) was observed after 30 min.125I‐VAS/VEGF was completely degraded 2–3 hr after binding. At 3 hr, the trichloroacetic acid (TCA)‐soluble radioactivity accumulated in the medium was 60% of the total radioactivity released by HUVE cells. No degradation fragment of125I‐VAS/VEGF was observed. Chloroquine completely inhibited degradation. VAS/VEGF was able to induce angiogenesis in vitro in HUVE cells. However, it did not significantly modulate urokinase‐type plasminogen activator (u‐PA), tissue‐type plasminogen activator (t‐PA), plasminogen activator inhibitor (PAI‐1), and tissue factor (TF). Prostacyclin production was only stimulated at very high VAS/VEGF concentrations. Taken together, these results indicate that VAS/VEGF might be a potent inducer of neovascularization resulting from a direct interaction with endothelial cells. The angiogenic activity seems to be independent of the plasmi

ISSN:0021-9541    

DOI:10.1002/jcp.1041490108

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

年代:1991

数据来源: WILEY

摘要:

AbstractMutants of Chinese hamster ovary cells were selected for resistance to a 3 hour exposure to 4 μ/ml N‐methyl‐N'‐nitro‐N‐nitrosoguanidine and tested for glutathione (GSH) levels. Six of eight clones that survived the initial treatment had reduced GSH levels ranging from 26 to 61% of wild‐type values. These eight cell lines were tested for their susceptibility to a drug conjugate in which methotrexate (MTX) is disulfide‐linked to poly(D‐lysine) (MTX‐SS‐PDL) to test their capacity to cleave the endocytosed disulfide bond and release free MTX from this otherwise undegradable drug conjugate. We had shown that wild‐type cells were killed by ∼1 × 10−7M MTX given as free drug, as MTX‐poly(L‐lysine) or as MTX‐SS‐PDL, but were not affected by MTX‐poly(D‐lysine). All six lines with abnormally low levels of GSH were resistant to MTX‐SS‐PDL. The variants with the lowest levels of GSH (MNR‐5) and MNR‐10 were tested further and showed near‐normal sensitivity to MTX and MTX poly(L‐lysine). As expected, both lines were hypersensitive to melphalan. They were, however, normally sensitive to diphtheria toxin and ricin, indicating that some cleavage opf the interchain disulfides in these protein toxins occurs even when cellular GSH is abnormally low. The lesser GSH requirement for toxin a

ISSN:0021-9541    

DOI:10.1002/jcp.1041490109

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

年代:1991

数据来源: WILEY

摘要:

AbstractWithangulatin A is a newly identified in vitro topoisomerase II inhibitor isolated from the Chinese antitumor herbPhysalis angulata.In vivo, it was found to be cytotoxic, capable of suppressing general protein synthesis and of inducing the synthesis of a small set of proteins including those generated by heat‐shock treatment. The 70 kDa protein generated by withangulatin A was unequivocally identified as the heat‐shock protein70 (HSP70) since both proteins migrated to the same position on two‐dimensional polyacrylamide gels, could be recognized by a monoclonal antibody to human HSP70, and exhibited identical peptide maps. The induction of protein synthesis by withangulatin A was regulated at the transcriptional level since it was aborted in cells pre‐treated with actinomycin D. However, the initiation of this process did not require de novo protein synthesis since it was not affected by cycloheximide. Other cellular effect of withangulatin A was alterations of protein phosphorylation including an enhancement of phosphorylation of a 32 kDa protein which was also detected in the heat‐shocked cells. Morevoer, this process was observed within 7.5 min after the initial heat treatment which is much faster than the onset of HSP synthesis. Therefore, increased phosphorylation of the 65 kDa protein may represent on of the earliest signals generated by both heat‐shock and withangulatin A and may be involved in the upstream regulation of heat‐shock resp

ISSN:0021-9541    

DOI:10.1002/jcp.1041490110

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

年代:1991

数据来源: WILEY

摘要:

AbstractSince both heat and sodium arsenite induce thermotolerance, we investigated the differences in synthesis and redistribution of stress proteins induced by these agents in Chinese hamster ovary cells. Five major heat shock proteins (HSPs; Mr 110, 87, 70, 28, and 8.5 kDa) were preferentially synthesized after heat for 10 min at 45.5°C, whereas four major HSPs (Mr 110, 87, 70, and 28 kDa) and one stress protein (33.3 kDa) were preferentially synthesized after treatment with 100 μM sodium arsenite (ARS) for 1 hr. Two HSP families (HSP70a,b,c and HSP28a,b,c) preferentially relocalized in the nucleus after heat shock. In contrast, only HSP70b redistributed into the nucleus after ARS treatment. Furthermore, the kinetics of synthesis of each member of HSP70 and HSP28 families and their redistribution were different after these treatments. The maximum rates of synthesis of HSP70 and HSP28 families, except HSP28c, were 6–9 hr after heat shock, whereas those of HSP70b and HSP28b,c were 0–2 hr after ARS treatment. In addition, the maximum rates of redistribution of HSP70 and HSP28 families occurred 3–6 hr after heat shock, whereas that of HSP70b after ARS treatment was significantly less than that after heat treatment. These results suggest that heat treatment but not sodium arsenite treatment stimulates the entry of HSP70 and HSP28 families into the n

ISSN:0021-9541    

DOI:10.1002/jcp.1041490111

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

年代:1991

数据来源: WILEY