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1.
Interferon inhibits uptake of the radiolabeled queuine analog, rQT3, into cultured human fibroblasts. Simultaneous exposure to 10 nM phorbol-12,13-didecanoate (PDD) potentiates interferon-induced inhibition of rQT3 into cultured fibroblasts. All three major classes of human interferon tested affected uptake similarly, with fibroblast derived beta-interferon being more effective in dose response than gamma or alpha interferons. This suggests that endogenous production of interferon by cultured cells, such as that observed during a low grade viral infection, inhibits queuine uptake and may subsequently lead to a decreased level of queuine modified transfer RNA. Queuine-hypomodified transfer RNA has been implicated in growth control, differentiation and neoplastic transformation.  相似文献   

2.
R W Trewyn  H B Gatz 《In vitro》1984,20(5):409-415
The tumor promoter phorbol 12,13-didecanoate (PDD) significantly altered the growth properties of early passage normal human skin cells in vitro in culture medium supplemented with elevated concentrations of selected amino acids. Continuous treatment of cells with 10(-7) or 10(-8) M PDD resulted in a 5 to 10-fold increase in saturation density at early passages followed by a long-term two- to fourfold increase. The PDD-treated cultures remained in exponential growth at cell densities greater than 10-fold higher than the control cultures. Removal of PDD from the culture medium while the cells were at a high cell density resulted in a return to near-normal saturation density by the subsequent passage. Anchorage independent growth of normal human cells in methylcellulose was also promoted by PDD in a dose dependent manner, with prior subculturing in the presence of PDD being required for maximal colony formation. The structural analog 4 alpha-phorbol 12,13-didecanoate failed to elicit similar cellular responses.  相似文献   

3.
It has been suggested that the rate of queuine uptake into cultured human fibroblasts is controlled by phosphorylation levels within the cell. We show that the uptake of queuine is stimulated by activators of protein kinase C (PKC) and inhibitors of protein phosphatase; while inhibitors of PKC, and down-regulation of PKC by chronic exposure to phorbol esters inhibit the uptake of queuine into cultured human fibroblasts. Activators of cAMP- and cGMP-dependent kinases exert no effect on the uptake of queuine into fibroblast cell cultures. These studies suggest that PKC directly supports the activity of the queuine uptake mechanism, and that protein phosphatase activity in the cell acts to reverse this. Regardless of the modulation of uptake rate, the level of intracellular queuine base saturates in 6 h. However, there is still an effect on the incorporation rate of queuine into tRNA of fibroblast cultures even after 24 h. We now show that the incorporation of queuine into tRNA in cultured human fibroblasts by tRNA-guanine ribosyltransferase (TGRase) is also stimulated by activators of PKC and inhibitors of protein phosphatase: while inhibitors of PKC decrease the activity of this enzyme. These studies suggest that PKC supports both the cellular transport of queuine and the activity of TGRase in cultured human fibroblasts, and that protein phosphatase activity in fibroblasts acts to reverse this phenomenon. A kinase-phosphatase control system, that is common to controlling both intracellular signal transduction and many enzyme systems, appears to be controlling the availability of the queuine substrate and the mechanism for its incorporation into tRNA. Since hypomodification of transfer RNA with queuine is commonly observed in undifferentiated, rapidly growing and neoplastically transformed cells, phosphorylation of the queuine modification system may be critical regulatory mechanism for the modification of tRNA and subsequent control of cell growth and differentiation.  相似文献   

4.
An endothelial cell (EC) growth factor isolated from bovine brain stimulates in vitro growth of human umbilical vein endothelial cells, and permits long term serial propagation. In the presence of increasing concentrations of EC growth factor, confluent cultures of early (CPDL less than or equal to 20) and late (CPDL greater than 20) passage human endothelial cells exhibit an increased incorporation of 3H-glucosamine and Na235SO4 into the glycosaminoglycans (GAG), hyaluronic acid, chondroitin, chondroitin-4-sulfate, dermatan-4-sulfate, and chondroitin-6-sulfate. An increase in both labelled sulfated and nonsulfated GAG was observed in the cytosol, membrane, secreted and extracellular matrix fractions. In contrast, endothelial cells grown in the presence of EC growth factor contained decreased amounts of labelled heparan sulfate than cells grown without EC growth factor. Confluent cultures of early passage cells had significantly more labelled GAG but significantly less heparan sulfate than cultures of late passage cells on a per cell basis. Extracellular matrix from early passage cells contained about two- to seven-fold more labelled GAG than extracellular matrix from late passage cells, but only about half as much labelled heparan sulfate. Cell adhesion was enhanced when cells were grown in the presence of EC growth factor as compared to adhesion of cells grown without EC growth factor. Conversely, trypsin-mediated detachment of cells grown in the presence of growth factor was inhibited as compared to detachment of cells grown in medium without EC growth factor. The composition of the extracellular matrix influenced incorporation of labelled GAG into extracellular matrix. Early passage cells grown to confluence on a matrix from late passage cells incorporated significantly less labelled GAG into extracellular matrix than when grown to confluence on matrix from early passage cells. Incorporation of labelled GAG into extracellular matrix was significantly higher when late passage cells were grown on a matrix from early passage endothelial cells than when grown on matrix from late passage cells. We conclude that EC growth factor selectively stimulates incorporation of isotopic precursors into GAG in cultures of early and late passage endothelial cells but inhibits incorporation of radiolabel into heparan sulfate; early passage cells contain more GAG but less heparan sulfate than late passage cells, extracellular matrix controls the amount of GAG and heparan sulfate incorporated into matrix.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

5.
Summary The tumor promoter phorbol 12,13-didecanoate (PDD) significantly altered the growth properties of early passage normal human skin cells in vitro in culture medium supplemented with elevated concentrations of selected amino acids. Continuous treatment of cell with 10−7 or 10−8 M PDD resulted in a 5 to 10-fold increase in saturation density at early passages followed by a long-term two- to fourfold increase. The PDD-treated cultures remained in exponential growth at cell densities greater than 10-fold higher than the control cultures. Removal of PDD from the culture medium while the cells were at a high cell density resulted in a return to near-normal saturation density by the subsequent passage. Anchorage independent growth of normal human cells in methylcellulose was also promoted by PDD in a dose dependent manner, with prior subculturing in the presence of PDD being required for maximal colony formation. The structural analog 4α-phorbol 12,13-didecanoate failed to elicit similar cellular responses. This work was supported by grants from the Air Force Office of Scientific Research, Department of Defense (AFOSR-80-0283) and the National Cancer Institute, Department of Health and Human Services (P-30-CA-16058).  相似文献   

6.
7.
PLC/PRF/5 hepatoma cells cultured with a tumor promoter teleocidin showed polygonal cellular appearance with many vacuole-like structures, and reduced both c-myc mRNA level and growth rate. These teleocidin effects were partly mimicked by sodium butyrate but not by a protein kinase C stimulant 1-oleoyl-2-acetylglycerol(OAG). Protein kinase C inhibitor 1-(5-isoquinolinyl-sulfonyl)-2-methyl-piperazine(H7), calmodulin-dependent protein kinase antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide(W7) and topoisomerase II inhibitor novobiocin failed to inhibit the effects of teleocidin. These results may suggest the presence of still unknown biochemical pathways which mediate the actions of teleocidin.  相似文献   

8.
Serum, phorbol 12,13-didecanoate (PDD) and 1-oleoyl-2-acetoy-sn-glycerol (OAG) stimulated O2- release in human histiocytic leukemia U937 cells. The kinetics of O2- release caused by PDD but not by serum or OAG in growing cells differed from those in resting cells. Both the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl) 2-methylpiperidine (H-7) and calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) reduced the superoxide generation induced by these stimuli. H-7 inhibited the O2- release either from growing or resting cells but the effect of W-7 varied according to the growth phase. From these results, it is suggested that activation of protein kinase C and calmodulin-dependent process has an important role in O2(-)-release induced by serum, OAG and PDD, and that the mechanism for PDD-induced O2(-)-release is different in growing and resting cells.  相似文献   

9.
Early passage normal diploid Syrian hamster (SH) fetal cell cultures contain a transient subpopulation of contact-insensitive (CS-) cells which lack density-dependent inhibition of cell division. The size of this CS- subpopulation decreases during in vitro passage by conversion of the CS- cells to contact-sensitive (CS+) cells. Approximately 10-15 population doublings after the frequency of the CS- cells has declined to below 0.001%, mass cultures cease proliferating and exhibit cellular senescence. Cultures with higher initial numbers of CS- cells exhibit longer in vitro proliferative life spans than cultures with smaller initial numbers of CS- cells. Active tumor promoting phorbol esters (12-O-tetra-decanoyl-phorbol-13-acetate [TPA] and phorbol-12,13-didecanoate [PDD]) retard the decline in the proportion of CS- cells during in vitro passage, while the inactive tumor promoting phorbol ester, 4 alpha-phorbol-12,13-didecanoate (4 alpha PDD) has no effect on the rate of loss of the CS- cells. In addition, continuous treatment from secondary culture with TPA or PDD extends by approximately twofold the in vitro proliferative life span of SH fetal cell cultures. Treatment must, however, begin at passage 1 or 2 when the CS- cells are still present. After the proportion of the CS- cells has decreased to less than 0.001% as in passage 6 cultures, promoters have no effect on the life span of the culture. This finding that exposure to promoters results in both a prolonged maintenance of the CS- cellular subpopulation, as well as an extension of in vitro proliferative life span, suggests that the conversion of CS- cells to CS+ cells is involved in the mechanism of in vitro senescence.  相似文献   

10.
Protein kinase C modulation of queuine uptake in cultured human fibroblasts   总被引:2,自引:0,他引:2  
Protein kinase C modulates the activity of a highly specific uptake mechanism for queuine in cultured human fibroblasts. Activators of protein kinase C induce an increased uptake rate for the radiolabeled analog of queuine, rQT3. The protein kinase C inhibitors, H-7, staurosporine and sphingosine all induced a dramatic decrease in the uptake rate of rQT3. This suggests that protein kinase C is tied to efficient cellular uptake of queuine. Uptake is prerequisite to the modification of transfer RNA with queuine. Perturbation of queuine-modified transfer RNA levels has been associated with neoplastic transformation, differentiation and growth control.  相似文献   

11.
Treatment of hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient human promyelocytic leukemia (HL-60) cells with 6-thioguanine results in growth inhibition and cell differentiation. 6-Thioguanine is a substrate for the tRNA modification enzyme tRNA-guanine ribosyltransferase, which normally catalyzes the exchange of queuine for guanine in position 1 of the anticodon of tRNAs for asparagine, aspartic acid, histidine, and tyrosine. During the early stages of HGPRT-deficient HL-60 cell differentiation induced by 6-thioguanine, there was a transient decrease in the queuine content of tRNA, and changes in the isoacceptor profiles of tRNA(His) indicate that 6-thioguanine was incorporated into the tRNA in place of queuine. Reversing this structural change in the tRNA anticodon by addition of excess exogenous queuine reversed the 6-thioguanine-induced growth inhibition and differentiation. Similar results were obtained when 8-azaguanine (another inhibitor of queuine modification of tRNA that can be incorporated into the anticodon) replaced 6-thioguanine as the inducing agent. The data suggest a primary role for the change in queuine modification of tRNA in mediating the differentiation of HGPRT-deficient HL-60 cells induced by guanine analogs.  相似文献   

12.
Regulation of lipid synthesis from acetate in human diploid fibroblast cultures has been studied at various passage levels and at different stages of cell growth. When cultures were transferred to lipid free medium, a stimulation of [14C]acetate incorporation into lipid occurred within three to six hours after removal of exogenous lipid. In early passage cultures, this stimulation was observed whether cells were transferred to protein-free medium or medium supplemented with delipidized serum protein. However, in late passage cultures the presence of delipidized serum protein was required for the stimulation of lipid synthesis. When logarithmically dividing and stationary phase cultures were compared, the cultures in log phase showed stimulation of acetate incorporation into lipid in the presence or absence of delipidized serum protein, whereas in the stationary cultures the delipidized serum protein was required. When cultures were partially synchronized by a thymidine block, stimulation of acetate incorporation into lipid in the blocked cells only occurred in the presence of delipidized serum protein; in released cells stimulation occurred in protein free medium. When inhibition of lipid synthesis from acetate was compared in young vs. old or dividing vs. stationary cultures, however, no differences were observed. The data indicate the response of diploid fibroblast cultures to change in exogenous lipid is dependent on passage level and state of growth.  相似文献   

13.
Summary Two in vitro passages of a human endometrial adenocarcinoma continuous cell line (RL95-2), an early (subcultured <30 times) and a late passage (subcultured >200 times) have provided an interesting model to study the growth, morphologic, and invasive properties of endometrial tumors. The early passage, which has been shown to be estrogen-receptor positive, has characteristics closely resembling a primary tumor, whereas the estrogen receptor negative late passage exhibits several features of the metastatic phenotype. Compared to the early passage cells, the late passage cells were less serum dependent, formed foci, demonstrated a faster rate of growth (due to their shorter doubling times), and attained higher saturation densities. The late passage cells also displayed an altered morphology which was accompanied by alterations in the distribution of F-actin. Even though early and late passages showed similar invasive potential in an in vitro invasion assay, the late passage cells, by virtue of their several transformed characteristics, maintain distinctive properties compared with their early passage counterparts.  相似文献   

14.
The secretory protein profiles of early and late passage cultures of human fibroblasts were compared using polyacrylamide gel electrophoresis. In comparison with early passage cell cultures (40-50% lifespan completed), late passage (greater than 80% of lifespan completed) cell cultures exhibited enhanced production of several peptides in the Mr range 55-60,000. One of those peptides had an apparent molecular weight of Mr = 55,000 and was constitutively present in the late passage cell conditioned medium. Late passage cell cultures synthesized the Mr = 55,000 peptide in the presence or absence of fetal bovine serum. Serum did not enhance its production by early passage cells. Further, production of the peptide was not induced in early passage cell cultures whose proliferation was arrested either by serum starvation or by contact inhibition. Pulse chase studies demonstrated that the peptide appears in the culture medium within 60 min of labeling. There was no evidence that it is derived via degradation of other proteins present either in early passage or late passage cell conditioned media. Further, the production of the 55,000 dalton peptide did not appear to be regulated by factors present in conditioned media. The peptide was detected in the conditioned media produced by late passage cultures of several different cell strains.  相似文献   

15.
The exposure of human fibroblasts (HF) aging in vitro to heat shock resulted in an attenuated expression of the heat shock-inducible HSP70. When late passage cells were cultured in the continuous presence of serum, we observed a reduced accumulation of the cytoplasmic polyadenylated HSP70 mRNA. The levels of HSF1 activation and nuclear HSP70 mRNA were comparable to those of early passage cells (M. A. Bonelli et al., Exp. Cell Res. 252, 20-32, 1999). When late passage cells were serum-starved overnight, we observed a reduced activation of HSF1 and a decreased level of HSP70 mRNA during heat shock. However, at 37 degrees C the levels of HSF1 differed little between late passage HF and early passage cells, irrespective of the presence of serum. Interestingly, during heat shock a marked decrease in the level and, consequently, in the binding activity of HSF1 was noted only in serum-starved, late passage HF. The decrease in the level of HSF1 was counteracted by back addition of serum to the cells during heat shock. Addition of the specific proteasome inhibitor MG132 blocked a decrease in HSF1 during heat shock, maintaining levels observed in late passage cells and HSF1 activity comparable to that of early passage HF. The recovery of the level and activity of HSF1 observed in late passage HF incubated in the presence of MG132 suggests that heat shock unmasks a latent proteasome activity responsible for HSF1 degradation.  相似文献   

16.
Transforming growth factor-beta 1 (TGF-beta 1) is a potent growth inhibitor for many cell types. On fibroblasts, TGF-beta 1 has been shown to inhibit human platelet-derived growth factor (PDGF)-induced mitogenicity. The mechanism implicated in this growth inhibition is unknown. In this work, we show on human bone marrow fibroblasts that TGF-beta 1, which inhibited PDGF-BB mitogenicity, was able to block PDGF-BB-induced early events such as polyphosphoinositide (PtdIns 4,5-P2, PtdIns 4-P, and PtdIns) breakdown and Ins 1,4,5-P3 formation. No significant modification by TGF-beta 1 of PDGF-BB binding (n1 = 200,000 vs. n2 = 195,000 sites per cell with TGF-beta 1; Kd1 = Kd2 = 0.5 x 10(-9) M) and of internalization kinetics was observed. In addition, TGF-beta 1 was shown to inhibit PDGF-BB receptor autophosphorylation either in intact cells or in partially isolated membranes and to partially inhibit PDGF-R tyrosine kinase activity. Since a dephosphorylation mechanism through protein phosphatases could be implicated, we used okadaic acid, a potent inhibitor of type 1 and 2A serine/threonine phosphatases and showed that okadaic acid restored PDGF-receptor autophosphorylation on tyrosine residues. Based on these data, we suggest that an alternative regulatory mechanism of PDGF tyrosine phosphorylation seems to involve serine/threonine phosphatase activation.  相似文献   

17.
The adjuvant Corynebacterium parvum, when administered intravenously during an ongoing alloimmunization, induces alloantigen-specific splenic suppressor cells which inhibit primary and secondary in vitro sensitizations. We have previously shown that these cells produce a soluble suppressor factor in culture. We now further characterize this factor and its mechanism of action. Release of this suppressive factor is dependent upon specific restimulation of the splenic suppressor cell with the sensitizing alloantigen for 24-48 hr in culture. The suppressor factor inhibits primary, but not secondary, in vitro sensitizations in an antigen-specific, genetically unrestricted manner. The suppressive activity is not absorbed by passage through immunoadsorbent columns containing anti-mouse immunoglobulin. The factor does not lyse tumor cells bearing the sensitizing alloantigen. Delay in addition to primary cultures of as little as 4 hr after culture initiation leads to loss of suppressive activity, suggesting that this antigen-specific allosuppressor factor inhibits an early step in the sensitization of precursor cytotoxic T lymphocytes.  相似文献   

18.
The c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway is activated by numerous cellular stresses. Although it has been implicated in mediating apoptosis and growth factor signaling, its role in regulating cell growth is not yet clear. Here, the influence of JNK on basal (unstimulated) growth of human tumor glioblastoma T98G cells was investigated using highly specific JNK antisense oligonucleotides to inhibit JNK expression. Transient depletion of either JNK1 or JNK2 suppressed cell growth associated with an inhibition of DNA synthesis and cell cycle arrest in S phase. The growth-inhibitory potency of JNK2 antisense ((JNK)2 IC(50) = 0.14 micrometer) was greater than that of JNK1 antisense ((JNK)1 IC(50) = 0.37 micrometer), suggesting that JNK2 plays a dominant role in regulating growth of T98G cells. Indeed, JNK2 antisense-treated populations exhibited greater inhibition of DNA synthesis and accumulation of S-phase cells than did the JNK1 antisense-treated cultures, with a significant proportion of these cells detaching from the tissue culture plate. JNK2 (but not JNK1) antisense-treated cultures exhibited marked elevation in the expression of the cyclin-dependent kinase inhibitor p21(cip1/waf1) accompanied by inhibition of Cdk2/Cdc2 kinase activities. Taken together, these results indicate that JNK is required for growth of T98G cells in nonstress conditions and that p21(cip1/waf1) may contribute to the sustained growth arrest of JNK2-depleted T98G cultures.  相似文献   

19.
Queuine modulates growth of HeLa cells depending on oxygen availability   总被引:1,自引:0,他引:1  
HeLa cells can be grown in media supplemented with horse serum that is lacking the nutrient factor queuine. The addition of 1 X 10(-8) M queuine to aerobically grown cells caused a slight, but significant, inhibition of growth, whereas cell proliferation was stimulated increasingly when the concentration of queuine was raised from 3 X 10(-8) M to 3 X 10(-7) M. This was also observed when the cells were transiently starved of serum factors. When the cells were grown under hypoxic stress, but otherwise identical conditions, they responded to queuine in an opposite manner. Under conditions of mitogenic stimulation, characteristic new proteins were found in cytosolic, nuclear and mitochondrial fractions of aerobically grown cells. The effects of queuine on cell proliferation at low concentrations are assumed to be mediated by the free base, whereas the effects at higher concentrations possibly involve both, queuine and Q-tRNAs. The 'Q system' appears to mediate growth control in dependence on oxygen availability.  相似文献   

20.
The effects of compounds with tumor promoting activity (mezerein, teleocidin and palytoxin) on rat growth hormone (rGH) release was compared to that of TPA (12-O-tetradecanoyl phorbol-13-acetate). Mezerein and teleocidin both of which are activators of protein kinase C (TPA type tumor promoter), elicited rGH release about 3.5 to 4 fold above control values. The ED 50 was 16 nM for mezerein, 1.1 nM for teleocidin and 1.5 nM for TPA. In contrast to mezerein or teleocidin, a non-TPA type tumor promoter (palytoxin) which does not activate protein kinase C failed to stimulate rGH release. These observations suggest that the activation of protein kinase C is essential in the release of rGH induced by the tumor promoters.  相似文献   

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