摘要:

AbstractIn an attempt to better establish the relationship between polyamine levels and the differentiation of embryonal carcinoma cells, we have examined the ability of α‐difluoromethylornithine (DFMO), a known inducer of differentiation in one embryonal carcinoma cell line, to stimulate the differentiation of embryonal carcinoma cells from a variety of cell lines. Differentiation was monitored using a variety of criteria including morphological alterations and changes in biochemical and antigenic parameters. Depend ng on their response to difluoromethylornithine, three classes of cell lines could be identified, those which (1) differentiate extensively, (2) differentiate poorly, and (3) fail to differentiate. Three different classes of embryonal carcinoma cell lines reflect differential changes in polyamine levels resulting from inhibition of ornithine decarboxylase enzyme activity by DFMO. The specific cell lines which exhibit large decreases in both ornithine decarboxylase activity and polyamine levels also show extensive differentiation. The cell lines which show only moderate decreases in enzyme activity and polyamines differentiate poorly while the cell lines which fail to respond to DFMO in that polyamines do not drop below the threshold level necessary to induce differentiation fail to differentiate. These studies suggest that decreases in intracellular polyamines induce EC cell differentiation in vit

ISSN:0021-9541    

DOI:10.1002/jcp.1041220102

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

年代:1985

数据来源: WILEY

摘要:

AbstractWe analyzed the release of activities capable of stimulating the in vitro growth of human hemopoietic progenitor cells by long‐term cultured T cell growth factor (TCGF)‐dependent human T lymphocytes. Seven cell lines tested produced colony‐stimulating activity (CSA) as well as burst‐promoting activity (BPA). The CSA stimulated primarily the growth of the cells forming colonies after 14 days of incubation. In addition the supernatants from these seven Tcell lines showed the ability to induce the in vitro growth of mixed granulocyte, erythroid, megakaryocyte, macrophage colonies (CFU‐GEMM). The release of hemopoietic factors did not depend on the presence of accessory cells or phytohemagglutinin or serum during the incubation for factor production. In six of the T cell lines the majority of the cells were reactive to the OKT 8 monoclonal antibody (MoAb), whereas one cell line contained mostly OKT 4+ cells. Suppressor activity was detected in three tested OKT 8+ cell lines, while the one OKT 4+ displayed helper activity. All cell lines produced hemopoietic factors with equal efficiency. These results indicate that factors affecting human hematopoiesis are produced by normal T lymphocytes in long‐term culture and this property is not related to the helper or suppressor activity of the cul

ISSN:0021-9541    

DOI:10.1002/jcp.1041220103

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

年代:1985

数据来源: WILEY

摘要:

AbstractWhen the disulfide of 2‐mercaptoethanol (ESSE) is added to the medium of cultured Chinese hamster ovary (CHO) cells, a time and concentration dependent release of 2‐mercaptoethanol to the medium is observed. The reduction of ESSE to 2‐mercaptoethanol by cells is a saturable process, the rate being approximately 50 nmoles of 2‐mercaptoethanol per mg cell protein for an hour upon exposure to 250 μM ESSE. Reduction rate of ESSE by cells attached to a substratum is independent of glucose and insulin for periods up to 4 hours. However, in detached cells, swirled in suspension, addition of glucose and insulin is necessary in order to obtain a linear reduction rate of ESSE. The rate limiting enzyme in the sterol biosynthetic pathway, 3‐hydroxy‐3‐methyl‐glutaryl Coenzyme A reductase (E.C. 1.1.1.34), is inhibited by ESSE when isolated from CHO cells but total nonsaponifiable lipids synthesis from [2−14C]‐acetate in intact cells is not affected by ESSE at concentrations up to 500 μM. Cytosolic reduced glutathione can spontaneously exchange disulfide bonds with ESSE and thus prevent it from inhibiting the reductase. Cultured cells respond to ESSE administration by elevating their total and acid‐soluble glutathione levels. The use of ESSE as a perturbant of the GSH Statu

ISSN:0021-9541    

DOI:10.1002/jcp.1041220104

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

年代:1985

数据来源: WILEY

摘要:

AbstractIn order to study the effect of glucose on the differentiation of cultured human colon cancer cells, a subpopulation of HT‐29 cells was selected for its capacity to grow in the total absence of sugar. These cells (GIc−cells) exhibit, after confluency, an enterocytic differentiation, in contrast to cells grown with glucose (Glc+cells), which always remain undifferentiated. The differentiation is characterized by a polarization of the cell layer with apical brush borders and tight junctions, and by the presence of sucrase‐isomaltase. The differentiation of Glc−cells is reversible: the addition of glucose to postcon‐fluent cultures of Glc−cells results in an inhibiting effect on the expression of sucrase‐isomaltase; switching growing cultures of Glc−cells to the Glc+medium for several passages results in a progressive reversion to the undifferentiated state, which is completed after seven passages. The dedifferentiation process is associated with a parallel, passage‐related, increase in the rates of glucose consumption and lactic acid production, and decreases of intracellular glycogen content, which return to the values of the undifferentiated original Glc+cells. The values of these metabolic parameters are correlated, at each passage, with the degree of dedifferentiation of the cells. When these dedifferentiated cells, after having been cultured in Glc+medium for 20 passages, are switched back to the Glc−medium, they readily grow without mortality, and reexpress the same enterocytic differentiation as the parent Glc−cells. These results show that the capacity of this subpopulation to grow and differentiate in the absence of sugar is a stable characteristic. They further suggest that glucose metabolism interferes with the program of different

ISSN:0021-9541    

DOI:10.1002/jcp.1041220105

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

年代:1985

数据来源: WILEY

摘要:

AbstractAngiotensin I‐converting enzyme (ACE) activity was measured in lysates of cloned and uncloned cultures of bovine fetal aortic endothelial cells. The expression of ACE activity in these cells was complex, and influenced by subcultivation, cell density, serum, cumulative population doublings, and clonal heterogeneity. The ACE specific activity at any point in the in vitro lifespan was determined, at least in part, by interaction of these culture variables. After subcultivation to subconfluent densities, cellular ACE specific activity decreased markedly and did not reach detectable levels until cells attained confluent densities. The use of different suppliers' lots of serum in the growth medium resulted in different cellular ACE specific activities. The ACE specific activity decreased as cultures were serially subcultivated, but remained detectable throughout the lifespan, suggesting a linkage between the proliferative history of an endothelial cell and its remaining capacity to express ACE. Increased ACE activity was observed when cells at the end of their lifespan were cultured at high densities. Cloned strains behaved similarly to the uncloned parent culture, except that they exhibited a wide range of ACE specific activitie

ISSN:0021-9541    

DOI:10.1002/jcp.1041220106

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

年代:1985

数据来源: WILEY

摘要:

AbstractThe S‐100 protein level in mouse neuroblastoma (N18TG‐2 and NIE‐115), rat glioma (C6, C6BU‐1, and C6V‐1), and hybrid (NG108‐15, 140‐3, 141‐B, NBr10A, NBr20A, NCB20, and NX3IT) cells was determined with a sensitive enzyme immunoassay system that uses a rabbit antibody to bovine brain S‐100 protein. S‐100 protein was detected in glioma but not in neuroblastoma cells. All seven hybrid cells derived from neuroblastoma and glioma or other types of cells were found to possess a very little or undetectable S‐100 protein. The induction of S‐100 protein level in prestationary phase cultures of glioma C6BU‐1 cells was examined by forskolin, which was a highly specific activator of adenylate cyclase of the cells and produced morphological differentiation. After incubation with 10 μM forskolin for 48 hr, the S‐100 protein level increased 2–2.5‐fold in C6BU‐1 glioma cells whose mean control level was 60 ± 26 ng/mg protein (± SD). The forskolin induction of S‐100 protein in the cells was dose dependent, and the concentration of forskolin required for 50% activation of S‐100 protein was about 0.6 μM. The increase by forskolin was initiated from 10–15 hr after incubation with it and was inhibited with cycloheximide and actinomycin D. In NG108‐15 hybrid cells the induction of S‐100 protein was also observed by forskolin as well as prostaglandin (PG) E1plus theophylline which are known to activate adenylate cyclase of the cells. The results indicate that S‐100 protein biosynthesis is genetically controlled in these clonal cells, and that S‐100 protein can be regulated in

ISSN:0021-9541    

DOI:10.1002/jcp.1041220107

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

年代:1985

数据来源: WILEY

摘要:

AbstractConfluent cultures of the MDCK kidney epithelial cell line exhibit dome formation, a result of transepithelial fluid transport influenced by cell‐cell and cell‐substratum interaction. Dome formation was inducible by hexamethylene bisacetamide (HMBA) or dimmethylformamide (DMF), compounds known as inducers of cell differentiation (Lever, 1979b). Analysis of the incidence of the dome‐forming phenotype in colonies derived nonselectively from the MDCK cell line suggested that inducers recruit an increased fraction of the cell population to express dome formation. Variant MDCK cell lines were isolated which differed from the parental line in response to inducers while retaining cuboidal epithelial morphology. In five independently isolated and cloned MDCK variants, dome formation was not inducible by DMF and only marginally increased by HMBA. This phenotype was also associated with increased cell adhesiveness to a plastic substratum. Results from cocultivation experiments suggested that the DMF‐unresponsive phenotype of variant cells may be partially overcome by cell‐cell contact with wild‐type cells. Sodium pump transport activity assessed by ouabain‐sensitive Rb+uptake was partially inhibited by HMBA and by DMF in a “wild‐type” inducer‐responsive clone. By contrast, DMF did not inhibit ouabain‐sensitive Rb+uptake in DMF‐unresponsive variant clones, and sodium pump inhibition by HMBA was greatly diminished. This close correspondence between altered sodium pump modulation by inducers in variant clones and their altered dome‐forming response reinforces our previous conclusions (Kennedy and Lever, 1984) that sodium pump modulation is closely associated with mechanisms of inducer action. Taken together, these findings implicate cell‐cell interaction, cell‐substratum interaction and sodium pump modulation in regulation of the differentia

ISSN:0021-9541    

DOI:10.1002/jcp.1041220108

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

年代:1985

数据来源: WILEY

摘要:

AbstractMeasurements were made of the electrophysiological and cAMP response to changes in extracellular [Ca2+] and to hormone application in a bone cell clone. Both transient and long‐term electrophysiological responses were studied. An increase in extracellular [Ca2+] usually resulted in a transient hyperpolarization of about 60‐sec duration. In addition, increases in extracelllular [Ca2+] from 0.9 to 1.8 mM and from 1.8 to 3.6 mM resulted in long‐term hyperpolarization and increased potential fluctuations. Increasing bathing [Ca2+] until the membrane potential reached the K+equilibrium level resulted in a significant decrease in fluctuations. Addition to the bathing medium of quinine, a putative blocker of the Ca2+‐dependent K+channel, resulted in long‐term depolarization of the mean membrane potential, and a long‐term decrease in potential fluctuations. Addition of Mg2+, a mild antagonist of Ca2+entry into the cell, produced transient depolarization and reduction of potential fluctuations. These effects suggest that the potential fluctuations reflect cytoplasmic [Ca2+] fluctuations via Ca2+‐dependent K+membrane channels. Under an extracellular[Ca2+] of 1.8 mM, the application of prostaglandin E2(PGE2), isoproterenol, and parathyroid hormone produced no significant effect on mean membrane potential or on the sustained potential fluctuations, but PGE2did significantly raise intracellular cAMP. Under an increased bathing [Ca2+], significant changes in mean potential and fluctuations did occur in response to PGE2, but not in response to the other hormones, while the PGE2effect on cAMP was not greatly changed. Hyperpolarizing transients of about 30‐sec duration occurred in response to all of the hormones, particularly at an extracellular [Ca2+] of 3.6 mM. Thus, there are both transient and long‐term electrophysiological responses to hormone application, with only the long‐term response correlated with the production of cAMP. These electrophysiological responses may represent separate transient and long‐term calcium transport responses t

ISSN:0021-9541    

DOI:10.1002/jcp.1041220109

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

年代:1985

数据来源: WILEY

摘要:

AbstractWhen randomly proliferating rat 3Y1 fibroblasts were treated with sodium butyrate, more than 90% of their cells were arrested reversibly with a 2C DNA content at least 12 h before the G1/S boundary. When cells synchronized in the early S phase were treated with butyrate, approximately 70% of all cells were arrested with a 4C DNA content. The arrests in both G1 and G2 phases by the single inhibitor suggest that the two phases share a common mechanism. The ability of cells to undergo mitosis on time was quickly lost with time of arrest in the G2 phase. Upon removal of the inhibitor, the cells arrested with a 4C DNA content entered a new S phase without intervening mitosis. The tetraploid cells thus produced kept proliferating as fast as diploid cells. These results suggest that the inhibition of the normal G2 traverse is somehow responsible for the formation of the proliferative polyploid cells.

ISSN:0021-9541    

DOI:10.1002/jcp.1041220110

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

年代:1985

数据来源: WILEY

摘要:

AbstractThe extracellular ionized calcium and magnesium requirements for lectin‐induced lymphocyte DNA synthesis were measured in a serum‐free system. The use of this system permitted measurements of the ionized calcium and magnesium concentrations with ion‐selective electrodes. Maximal DNA synthesis was observed at 270 μ ionized calcium and at 100 μ ionized magnesium in phytohemagglutinin‐treated lymphocytes. Lymphocyte DNA synthesis was much more sensitive to reduction of external ionized calcium than to reduction of ionized magnesium. In calcium‐free medium (ionized calcium 25 μM), DNA synthesis was reduced by 90%, but in magnesium‐free medium (ionized magnesium concentration 7 μM) DNA synthesis was reduced by only 30%. Fifty percent of DNA synthesis stimulated by phytohemagglutinin (PHA) and concanavalin A (Con A) was observed at external ionized calcium concentrations of 97 and 43 μM, respectively. When lymphocytes were stimulated with PHA and the external calcium was chelated with EGTA, 50% inhibition of DNA synthesis was observed at 98 μM ionized calcium. This value agreed well with the free calcium required for PHA activation of DNA synthesis (97 μM). Cytoplasmic calcium, measured with the fluorescent probe Quin 2, increased following lectin exposure if the extracellular ionized calcium concentration was greater than 80 μM. No increase in cytoplasmic calcium could be detected in lectin‐treated lymphocytes below 80 μM extracellular ionized calcium, although substantial DNA s

ISSN:0021-9541    

DOI:10.1002/jcp.1041220111

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

年代:1985

数据来源: WILEY