摘要:

AbstractIn mouse L cells, relatively low doses of UV light (e.g., about 35 J/m2) induced the rapid breakdown of the molecules of many RNA species transcribed shortly before irradiation. This included 28S, 18S, 5.8S, and 5S rRNA, U1, U2, U3, U4, and U5 small nuclear RNA, but not the main band of transfer RNAs or 7SL RNA. At higher UV doses, an RNA band that contains tRNAleuwas also degraded rapidly after UV irradiation. RNA molecules synthesized long before irradiation (e.g., 22 h for small RNAs, 4 h for large rRNAs) were not affected. Our results suggest that the maturation and/or assembly into fully mature ribonucleoprotein particles of several small RNA species is not completed 4 h after transcription. The effect of UV radiation occurred in mouse L cells, but not in human HeLa or KB cells. In a previous report, L cells were transformed by DNA transfection with two mouse U1b RNA genes, named U1.1 and U1.2. We observed now that, in L cells transformed with the U1.2 gene, the ratio of radioactivity in the apparent U1b and U1a RNA precursors after 5 min of labeling was about 20 times higher than (a) this ratio in briefly labeled L cells that had been transformed with the U1.1 gene, and (b) the ratio of radioactive mature U1b and U1a RNA after 20 h of chase in L cells transformed with the U1.2 gene. These results suggest that very high levels of U1b RNA are transcribed from the exogenous U1.2 gene copies, followed by the rapid degradation of most of these transcripts.

ISSN:0021-9541    

DOI:10.1002/jcp.1041410102

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

年代:1989

数据来源: WILEY

摘要:

AbstractTransforming growth factor‐beta (TGF‐β) and fibroblast growth factor (FGF) are two growth factors that will modulate chondrocyte growth and matrix synthesis. Here we report that these two growth factors act in a synergistic fashion to suppress the synthesis of type II collagen by embryonic chicken sternal chondrocytes. Treatment of chondrocytes with 20 ng/ml TGF‐β or 100 ng/ml FGF (acidic or basic) results in a 60–70% suppression of expression of the pro α1 chain of type II collagen. By comparison, when chondrocytes are exposed to a combination of 1 ng/ml TGF‐β and 10 ng/ml FGF, a complete suppression of type II collagen synthesis was observed. Epidermal growth factor (EGF), platelet‐derived growth factor (PDGF), or insulin‐like growth factor‐1 (IGF‐1) produce no suppression of synthesis either individually or in combination with TGF‐β. The decreased expression of the protein results from a decrease in the steady‐state level of the mRNA transcript coding for type II procollagen, as indicated by a northern analysis. Finally, chondrocytes transfected with a plasmid carrying the CAT gene driven by the collagen II promoter/enhancer sequence displayed high levels of CAT activity when cultured in control media, but treatment of the cells with a combination of the two growth factors resulted in a dramatic reduction of CAT activity, indicating diminished promoter activity. These results suggest that both TGF‐β and FGF can down‐regulate transcription of the collagen II gene through regulatory DNA sequences in the promoter and/or enhancer region. In addition, the finding of synergy suggests that these two growth factors may

ISSN:0021-9541    

DOI:10.1002/jcp.1041410103

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

年代:1989

数据来源: WILEY

摘要:

AbstractThis study reports on the effects of heparin, basic and acidic fibroblast growth factors (bFGF and aFGF, respectively), and transforming growth factor type‐e (TGFe) on the growth of a human adrenocortical carcinoma cell line, SW‐13. Heparin has previously been shown to inhibit growth in several cell types, including smooth muscle cells, certain fibroblasts, and epithelial cells, and to modulate the effects of fibroblast growth factors. Whereas bFGF and aFGF bind tightly to heparin and elute from a heparin‐Sepharose column with 2 M NaCl and 1.6 M NaCI, respectively, TGFe binds to heparin with lower affinity and can be eluted from heparin‐Sepharose column with 0.5 M NaCI. TGFe is a polypeptide unrelated to FGF, is present in neoplastic and nonneoplastic tissues, and stimulates the growth of certain epithelial cells and fibroblasts in soft agar and monolayer. Since the growth of SW‐13 cells is stimulated by TGFe and by bFGF, we hypothesized that heparin would inhibit the growth of SW‐13 cells by binding to these growth factors and that the effects of heparin could be overcome with the addition of either growth factor. Our experiments confirmed that heparin inhibits the growth of SW‐13 cells. A dose‐dependent growth inhibition was observed in both monolayer and soft agar. The inhibition in monolayer was partially reversed upon heparin withdrawal. The effects of heparin in both monolayer and soft agar were at least partially overcome by TGFe and by basic or acidic FGF. Overall protein synthesis does not appear to be affected by heparin as measured by [35S]methionine uptake. In contrast, epidermal growth factor (EGF) and insulin‐like growth factor I (IGF‐I) were unable to overcome heparin‐induced inhibition both in monolayer and in soft agar. Heparin also inhibited [3H]thymidine incorporation in AKR‐2B and partially inhibited AKR‐2B cell stimulation by TGFe; however, it further potentiated the already potent stimulation by bFGF. We propose that heparin, TGFe, bFGF, and aFGF modulate the growth of SW‐13 cells and possibly of other epithelial cells in complex ways and that heparin‐like substances present in the extracellular matrix play an important role in the

ISSN:0021-9541    

DOI:10.1002/jcp.1041410104

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

年代:1989

数据来源: WILEY

摘要:

AbstractRetinoic acid dramatically increases the size of domes in confluent MDCK monolayers in a hormonally defined medium (medium K‐1). After 4–5 days of retinoic acid treatment, enlarged domes began to appear in confluent MDCK monolayers. After 7days with 3 × 10−7M retinoic acid, the majority of the domes in the monolayers were between 27 and 80 × 10−3μ M2in area, whereas in control medium the majority of the domes were between 0 and 9 × 10−3μm2in area. The dependence of the retinoic acid effect on prostaglandin E1(PGE1) was examined. In normal MDCK cells, the effects of retinoic acid on dome size were observed only in medium K‐1 supplemented with PGE1. This observation indicated that retinoic acid did not elicit its effects simply by stimulating PGE production. In contrast, in monolayers of PGE1‐independent MDCK cells, retinoic acid treatment resulted in an increase in dome frequency even in medium K‐1 lacking PGE1This observation can be explained by the elevated cyclic adenosine monophosphate (cAMP) levels in these PGE1‐independent MDCK cells. Dibutyryl cAMP‐resistant MDCK cells, which normally do not form domes in medium K‐1, were also studied. Remarkably, the dibutyryl cAMP‐resistant MDCK cells were observed to form domes at a significant frequency when medium K‐1 was supplemented with retinoic acid. However in medium K‐1 lacking PGE1,an effect of retinoic acid on dome formation by dibutyryl cAMP‐resistant MDCK monolayers was not observed. The inability of dibutyryl cAMP‐resistant MDCK cells to form domes in medium K‐1 has previously been attributed to their decreased cAMP‐dependent protein kinase activity. The stimulatory effects of retinoic acid on dome formation may possibly be due to an increase in the activity of a particular cAMP‐dependent protein

ISSN:0021-9541    

DOI:10.1002/jcp.1041410105

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

年代:1989

数据来源: WILEY

摘要:

AbstractSn‐1,2‐diacylglycerols (DAG) and ionized‐free calcium can act as intracellular second messengers for cell activation. Traditionally, T‐lymphocyte activation is assessed by measurements of DNA synthesis or lymphokine production, but these responses require several days to occur and involve multiple intermediary regulatory steps. In contrast, we have found that T‐lymphocytes demonstrate rapid enhancement of A‐(alanine‐favoring) system amino acid uptake when treated with DAG or ionomycin. A 30–40% increase in the initial velocity of uptake (vi) of the synthetic A‐system specific amino acid, methylamino‐isobutyric acid (MeAIB), was measured following 5 min of exposure to DAG or ionomycin. The viwas enhanced 60% from 12 to 19 μmol/liter cell water per min after 30 min exposure of T‐cells to optimal concentrations of dioctanoylglycerol (30 μM), oleoylacetylglycerol (30 μM), or ionomycin (5 μM) (P<.01 for each agent). A 50‐fold excess of non‐radioactive MeAIB inhibited 80% of [14C]MeAIB uptake in both unstimulated and stimulated cells, indicating that uptake remained largely carrier‐mediated on treatment with these agents. Cycloheximide, 100 μg/ml, inhibited protein synthesis but did not block the A‐system amino acid transport enhancement induced by DAG or ionomycin. The DAG‐induced increase in the viwas blocked 40% with 100 μM H‐7, an inhibitor of protein kinase C. H‐7 treatment did not inhibit the ionomycin‐induced A‐system enhancement. A marked increase in cytoplasmic free calcium was measured when T‐lymphocytes were exposed to ionomycin but not on DAG exposure, and the A‐system effect of ionomycin but not DAG was blocked by extracellular EGTA. These data are compatible with two pathways for rapid enhancement of A‐system amino acid uptake in T‐lymphocytes. DAG stimulation is mediated via protein kinase C whereas ionomycin produces an A‐system effect of similar magnitude independent of

ISSN:0021-9541    

DOI:10.1002/jcp.1041410106

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

年代:1989

数据来源: WILEY

摘要:

Abstract2‐Mercaptoethanol (2‐ME) exerts several effects on murine lymphocytes in culture that might explain its ability to enhance survival and growth of these cells. The uptake of the essential amino acid cystine and consequently the maintenance of intracellular glutathione levels are enhanced by 2‐ME. Furthermore, 2‐ME (even in the disulfide form) causes lymphocytes to release thiols into the culture medium. These effects might protect the cells from oxidative damage. The additional cystine provided by treatment of lymphocyte cultures with 2‐ME might also allow adequate protein synthesis to support survival and/or growth. This study was conducted to assess the relative importance of the antioxidant and protein synthesis effects of 2‐ME. As expected, 2‐ME increased cystine uptake at all concentrations that enhanced growth and survival, but four nonthiol antioxidants that enhanced growth and/or survival either did not substantially affect cystine uptake or decreased it and did not affect the release of cystine or its products. The results presented here demonstrate that antioxidant protection is necessary and sufficient for lymphocyte survival and that cystine uptake in untreated lymphocytes is sufficient to support the protein synthesis needed for survival and limited growth. However, we also noted that concentrations of 2‐ME that stimulated maximal growth more than doubled protein synthesis as measured at 8 hr. Thus the portion of the effects of 2‐ME not accounted for by antioxidant action could be accounted for by enhanced

ISSN:0021-9541    

DOI:10.1002/jcp.1041410107

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe effects of the tumor necrosis factor (TNF), and a second pleiotropic cytokine interferon‐gamma (IFN), were examined in a line of human myeloblastic leukemia cells (ML‐1). By itself, TNF causes ML‐1 to differentiate along the monocytic pathway. The cells exhibit an increase in Fc receptors and acquire the morphological characteristics of maturing phenotype. They remain viable and continue to proliferate (at ≥ 50% of the control growth rate) even with 102–104units/ml TNF. IFN alone has similar effects, causing an increase in Fc receptors but little cytotoxicity. In contrast to either cytokine alone, the combination of TNF plus IFN causes a cessation of proliferation and extensive cell death. Cytotoxicity occurs in a synergistic fashion; it requires the simultaneous presence of both cytokines, occurring with concurrent but not sequential exposure. These different responses, differentiation (TNF alone) and cytotoxicity (TNF + IFN), occur with a similar range of doses (∼ 102–104units/ml) and in a similar time frame (beginning on day 2). In other cell types, IFN can augment either the differentiation‐inducing or the cytotoxic effect of TNF. In ML‐1, the combined application of TNF plus IFN results in a shift from differentiatio

ISSN:0021-9541    

DOI:10.1002/jcp.1041410108

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

年代:1989

数据来源: WILEY

摘要:

AbstractCellular ATP levels are determined by the rates of ATP production and ATP hydrolysis. Both phenomena are affected by ischemia. Mitochondrial enzymes are damaged, inhibiting this organelle's ability to make ATP. Mitochondria are also uncoupled by ischemia and have the ability to hydrolyze ATP. We designed a series of experiments to determine whether decreased production or increased hydrolysis of ATP was the primary effect of mitochondrial damage. Rat hearts were subjected to 45 min of warm ischemia in order to induce irreversible cell damage. ATP or ADP was injected into cuvettes containing mitochondria isolated from normal myocardium or myocardium damaged by ischemia. Luciferin‐luciferase, which fluoresces in the presence of ATP, was also added to the tubes as an indicator of ATP levels. Mixtures of uncoupled and coupled mitochondria were made and compared with the mitochondria damaged by ischemia. The results showed that mitochondria damaged by prolonged ischemia hydrolyze ATP more rapidly than normal mitochondria; however, normal mitochondria can easily compensate for increased ATP hydrolysis when in mixture with equal amounts of uncoupled mitochondria. These data suggests that the low cellular levels of ATP following irreversible ischemia are primarily due to decreased ATP synthesis and not to increased hydrolysi

ISSN:0021-9541    

DOI:10.1002/jcp.1041410109

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

年代:1989

数据来源: WILEY

摘要:

AbstractQuantification of changes in levels of c‐fosRNA was used as an indicator of the presence of functional responses to nerve growth factor in several human nonneuronal cell lines which have previously been shown to express high levels of NGF receptors. Four Ewing's sarcomas, one Wilm's tumor, and one melanoma were examined. Of these cell lines, the Ewing's sarcoma IARC‐EW1 showed greatly increased levels (10–20‐fold) of c‐fosRNA after 1 hour of exposure to NGF. Except for the melanoma line, the other tumor lines exhibited small, but reproducible, elevation of c‐fosRNA expression. In IARC‐EW1 cells, this induction was analyzed for kinetics, dose‐response, and suppression by selective inhibitors of NGF action. The results indicate that these cells bear high‐affinity receptors for NGF, which utilize signal pathways similar to NGF receptors on PC12 cells. Thus, we report new types of cells with functional responses to NGF and indicate that these may constitute a new model which will usefully complement those presently used for studying the mechanism

ISSN:0021-9541    

DOI:10.1002/jcp.1041410110

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

年代:1989

数据来源: WILEY

摘要:

AbstractTransforming growth factors beta (TGF‐β) inhibit the growth of a variety of cell types, including lymphocytes. The immunosuppressive effects of TGF‐β have been attributed to the interference of these molecules with the interleukin‐2 (IL2)‐driven component of lymphocyte proliferation. In order to elucidate in more detail the effects of TGF‐β on IL‐2‐induced proliferation, we investigated the effects of porcine transforming growth factor beta 1 and 2 (pTGF‐β1 and 2) on the IL‐2‐driven proliferation of a murine IL‐2‐dependent T‐lymphocyte line (CTLL). The results showed that pTGF‐β1 and 2 decreased3H‐thymidine incorporation in CTLL cells in a dose‐dependent fashion (maximum decrease of 75–85%). Combined‐time kinetic analysis of the effects of pTGF‐β on3H‐thymidine incorporation, cell growth, and cell‐cycle distribution (monitored as DNA content distribution) revealed that, in the first 48 h of culture, pTGF‐β1 increased the doubling time from 11.4 to 19.2 h without significantly affecting the cell‐cycle distribution of CTLL cells. After 96 h of culture in the presence of pTGF‐β1, cells started to accumulate in G0/G1, although at this time point 30% of the pTGF‐β1‐treated cells were still in S‐G2/M. Furthermore, during the first 48 h, neither the expression of the 55 kd chain of the IL‐2 receptor (IL‐2R) nor the expression of the transferrin receptor (TfR) was affected by TGF‐β. After 72 h of culture in the presence of pTGF‐β1, the expression of the IL‐2R and TfR was decreased. The data suggest that in CTLL cells TGF‐β initially slows the progression of cells in all phases of cell cycle. In addition, the initial TGF‐β‐mediated decrease of IL‐2‐induced3H‐thymidine incorporation and cell pr

ISSN:0021-9541    

DOI:10.1002/jcp.1041410111

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

年代:1989

数据来源: WILEY