摘要:

AbstractGrowth factor‐stimulated mitogenicity in mouse embryo‐derived AKR‐2B cells was inhibited in a dose‐dependent fashion by a mixture of alpha and beta mouse interferons (IFN). A 60% decrease in epidermal growth factor (EGF) and insulin‐stimulated DNA synthesis was observed with 10 kU/ml IFN, and half‐maximal inhibition was seen at 1 kU/ml. Likewise, the mitogenic effect of 5% fetal bovine serum (FBS) was inhibited by 60% with 10 kU/ml IFN and by 38% with 1 kU/ml IFN. IFN inhibition of DNA synthesis was paralleled by a decrease in monolayer growth of AKR‐2B cells by 60% on the 3rd day of culture and by 40% on the 6th day of culture. Soft agar growth of two AKR‐2B derived lines, AKR‐MCA and AKR‐2B (clone 84A), was also inhibited significantly with the addition of 1–10 kU/ml of IFN. The effect of IFN on EGF receptors was also examined. Treatment of AKR‐2B cells with 10 kU/ml IFN resulted in a 35% decrease in EGF binding to cell surface receptors. The reduced binding of EGF to cells treated with IFN was due to a loss of EGF receptors as determined by Scatchard analysis. IFN treatment of AKR‐2B cells neither altered the affinity of the EGF receptor for its ligand nor affected receptor internalization. Nuclear transcription and actinomycin D decay analysis indicated that within 2 hr, IFN reduced c‐mycmessenger RNA levels at the level of transcription with

ISSN:0021-9541    

DOI:10.1002/jcp.1041410302

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

年代:1989

数据来源: WILEY

摘要:

AbstractEvidence in the literature suggests that cancer cell growth in vitro is generally not sensitive to external calcium. A human ovarian carcinoma cell line (SKOV3) retained 60% of its normal growth in Dulbecco modified Eagle's medium (DME) when the calcium concentration was reduced from 3 mM to 10 μM. Chinese hamster ovary cells (CHO) were growth‐arrested in media containing less than 500 μM calcium. In low‐calcium (10 μM) DME, 10 μM of a calmodulin antagonist W7 inhibited the growth of SKOV3 cells by more than 90%, while 100 μM of its inactive analog W5 was mildly inhibitory (20%). The growth inhibition by W7 was antagonized by increasing calcium concentrations in the culture media, while the inhibition by W5 was calcium‐independent. The phorbol ester TPA was also effective in antagonizing W7's growth inhibition in low‐calcium DME, suggesting that the W7 effect is mediated via protein kinase C inhibition. SKOV3 total cellular protein kinase C activity was 1.6 times higher than CHO cells when incubated in normal DME. When incubated in low‐calcium DME, a large drop in protein kinase C activity in the CHO cells was observed while the enzyme activity was unchanged in the SKOV3 cells. Our data suggest that these human ovarian tumor cells have altered cellular calcium regulatory processes associated with the defective down‐regulation of protein kinase C. This defect may confer these cells the ability to proliferate independently of the external calcium concentration. Targeting the cellular signal transduction components may be useful in can

ISSN:0021-9541    

DOI:10.1002/jcp.1041410303

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

年代:1989

数据来源: WILEY

摘要:

AbstractWe have shown previously that insulin stimulates fluid phase endocytosis in 3T3‐L1 adipocytes (Gibbs et al., 1986). Using [14C]sucrose as an endocytotic marker, we show here that phenylarsine oxide, a trivalent arsenical which binds neighboring dithiols, blocked not only insulin‐stimulated fluid phase endocytosis, but basal endocytosis as well. The K1for this process was 6 μM in the presence or absence of insulin and the time required for inhibition was less than 2.5 min, the limit of detection in our assay system. These results can be compared with the inhibitory effect of phenylarsine oxide on insulin‐stimulated glucose transport. Although the K1for insulin‐stimulated transport (7 μM) was similar to that for inhibition of endocytosis, basal glucose transport was not affected by the inhibitor. Further, when cells were prestimulated with insulin causing maximal stimulation of the glucose transport rate, phenylarsine oxide induced a time‐dependent reduction to the basal rate (t1/2of 10 min), despite the fact that endocytosis was blocked immediately. This observation suggests that if the transporter is recycled by an exocytotic/endocytotic mechanism, it is distinct from fluid‐phase endocytosis/exocytosis, which is a vesicle‐mediated process, and provides further evidence that the transporter may undergo intrinsic activation/inactivation which does not require v

ISSN:0021-9541    

DOI:10.1002/jcp.1041410304

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

年代:1989

数据来源: WILEY

摘要:

AbstractPrimary cultures of myogenic cells from progressively older embryonic and adult chickens were incubated in medium containing Merocyanine 540 (MC540) and were exposed to white light during the incubation period. After exposure, the cultures were followed to determine cell survival and differentiation. MC540 attached to the surface membranes of all cells. In cultures from 10‐day embryos (E10 cells), concentrations of MC540 ≥ 60 μg/ml resulted in death of nearly all myogenic cells upon exposure to light, but non‐myogenic cells survived and replicated. Below 60 μg/ml, there was a dose‐dependent reduction in muscle differentiation. At concentrations<40 μg/ml, there was no effect on myogenesis. Cultures of cells from 18‐day (E18) embryos (myogenic stem cells) and from adult muscle (satellite cells) were resistant to doses of MC540 that killed E10 cells. E14 myogenic cell populations contained both resistant and sensitive sub‐populations. Terminally differentiated muscle cells were more sensitive to MC540 than precursor cells from any age embryo. Progeny of E18 cells acquired sensitivity to MC540 as differentiation proceeded. In clonal cultures, cells that normally give rise to small muscle clones (committed cells) were selectively destroyed by exposure to the dye. These observations demonstrate that an MC540‐resistant myogenic population is present in low numbers in 10‐day embryonic pectoral muscle. As development proceeds, this population increases such that, by 18 days of gestation, most of the myogenic cells are resistant to MC540. The results also suggest that embryonic chick myogenic stem cells and adult satellite cells have surface membrane properties which differ from those of their

ISSN:0021-9541    

DOI:10.1002/jcp.1041410305

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

年代:1989

数据来源: WILEY

摘要:

AbstractTwo clones (LD3 and LC3) were isolated from the established renal cell line LLC‐PK1. They differed with respect to the development of the Na+‐dependent α‐methyl‐D‐glucoside (AMG) uptake. The higher uptake capacity in LD3 as compared to LC3 was owing to the expression of a higher number of carrier molecules as was shown by the specific phlorizin binding. The intracellular cyclic AMP level, the D‐glucose concentration in the growth medium and the cell density could be excluded as the causes of the difference between both clones. We found that the faster development of the Na+‐dependent hexose carrier in LD3 was parallelled by a faster expression of trehalase in this clone. This suggests that the expression of both apical proteins is correlated. From the growth curves it was concluded that renewal of the undifferentiated population was roughly the same in both clones. The recruitment of cells from the undifferentiated to the growth arrested state seems also very similar as was deduced from the development of tight junctions and from the down‐regulation of the α‐methylaminoisobutyric acid (meAIB) uptake. The start of differentiation was identical as was shown by the similar rate of expression found for γ‐glutamyl transferase. The difference between both clones is most likely situated at the traverse to a fully differentiated cell. This process takes more time in LC3 than in LD3. Also the fully differentiated state seemed to be different in both clones. We conclude that the pattern of differentiated characteristics found in both clones is different and a model incorporating these di

ISSN:0021-9541    

DOI:10.1002/jcp.1041410306

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

年代:1989

数据来源: WILEY

摘要:

AbstractContact‐inhibited Kaposi's sarcoma‐derived cells (KS cells) were transfected with Simian Virus 40 (SV40) DNA. Transformed cells (SV‐KSC) were selected for their capacity to form foci on monolayers of the low‐malignant KS cells. Isolated SV‐KSC foci were found to contain integrated SV40 DNA sequences and to express SV40 large T‐antigen. Several differentiation properties of KS cells are retained in the SV40 transformants, e.g., expression of vimentin and the endothelial cell marker BMA 120. In contrast to the maternal KS cells, SV‐KSC are capable of growing in platelet‐derived growth factor (PDGF)‐depleted plateletpoor‐plasma serum (PPPS) and in soft agar. However, they are not tumorigenic in nude mice. Expression of the oncogenes c‐myc, c‐N‐ras, c‐Ha‐ras, and p53 is significantly elevated in SV‐KSC, whereas c‐fos and c‐erb B expression is comparable to that of KS cells and fibroblasts. Conditioned medium from SV‐KSC can substitute for PDGF when PDGF‐dependent, nontransformed KS cells are grown in PPPS. Biochemical analysis of the SV‐KSC supernatant and PDGF A and B mRNA expression analysis provide evidence that the mitogenic activity is not due to a PDGF‐like growth factor. On the other hand, there is evidence to indicate that the SV‐KSC mitogen is a member of the fibroblast growth factor family. SV‐KSC represent an interesting model system for the study of different degrees of malignancy of cultured mesenchymal cells and especially provide an important source for the isolation of a potent growth factor fo

ISSN:0021-9541    

DOI:10.1002/jcp.1041410307

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

年代:1989

数据来源: WILEY

摘要:

AbstractNorepinephrine (NE), acting through the α1‐adrenergic receptor, modulates the response of rat hepatocytes in primary culture to transforming growth factor type β1(TGFβ) by increasing the amount of TGFβ required for a given degree of inhibition of epidermal growth factor (EGF)‐induced DNA synthesis (Houck et al., J. Cell. Physiol.135:551–555, 1988). This effect was also found in hepatocytes isolated from regenerating livers but was greatly magnified in cells isolated between 12 and 18 hr after two‐thirds partial hepatectomy (PHX). During this period of enhanced sensitivity, NE was equally potent in terms of dose but more efficacious in the regenerating hepatocytes. As it did in control hepatocytes (Cruise et al., Science227:749–751, 1985), the α1‐adrenergic receptor mediated the activity of NE in regenerating hepatocytes. Vasopressin (VP) and angiotensin‐II (AG) also antagonized the effect of TGFβ and showed increased activity in regenerating hepatocytes but at only 50% or less of the maximal effect reached by NE. Regenerating hepatocytes isolated 24–72 hr after PHX exhibited decreased sensitivity to inhibition by TGFβ, with a nadir in 48‐hr‐regenerating cells. These findings suggest that NE may be involved in triggering the early phase of DNA synthesis during liver regeneration, with the subsequent acquisition of innate resistance to TGFβ responsible for continued proliferation at a time when TGFβ mRNA is known to be increasing in the liver (Braun et al., Proc. Natl. Acad. Sci. USA85:1539–1543, 1988). EGF induced increased DNA and protein synthesis in cultures of control hepatocytes; TGFβ inhibited the EGF‐induced DNA synthesis but had no effect on protein synthesis. This may be relevant to the latter stages of liver regeneration, when high levels of TGFβ mRNA are detected in liver and cellular hypertr

ISSN:0021-9541    

DOI:10.1002/jcp.1041410308

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

年代:1989

数据来源: WILEY

摘要:

AbstractAn overall increase of 40% in nuclear‐associated protein has been shown to be one of the sequellae of exposure of eukaryotic cells to elevated temperatures. Several investigators have shown that the increased protein/DNA ratios correlated well with the degree of cytotoxicity. In previous investigations, we have shown that cycloheximide, which protects the cell from the killing effects of heat, produces a dramatic reduction of the bulk nuclear‐associated proteins after heating. In this investigation, we studied a previously unobserved efflux of a 26 kDa protein after heat shock and the preferential accumulation of the 70 kDa protein. The 26 kDa protein was shown not to be a member of previously described heat shock protein families. Preferential reduction of a 26 kDa protein and accumulation of a 70 kDa protein was observed in nuclei isolated from Chinese hamster ovary cells after heating at 43°C. After heat treatment, the 26 kDa protein in the nucleus was decreased to a level 0.1–0.3 times the original amount in unheated cells, and the 70 kDa protein in the nucleus increased by a factor of 1.6–1.8. The normal levels of these two proteins were restored when cells were incubated at 37°C following heat shock. Cells treated with heat protectors, cycloheximide and histidinol, demonstrated approximately the same redistribution in nuclear 26 and 70 kDa proteins immediately after heating as those not exposed to these drugs. On the other hand, restoration to control levels was much faster in the protector‐treated cells, suggesting that “repair” of heat‐induced damage is an important factor in the cells ability to survive this insult. Return to normal protein levels did not require new

ISSN:0021-9541    

DOI:10.1002/jcp.1041410309

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

年代:1989

数据来源: WILEY

摘要:

AbstractNormal FBAE AG 7680 cells and chemically transformed FBAE GM 7373 cells were compared for their capacity to produce and to respond to bFGF. Normal FBAE cells showed higher levels of bFGF protein and of poly(A)+bFGF mRNA than transformed GM 7373 cells, indicating that chemical transformation in FBAE cells is paralleled by a decrease of bFGF gene expression. Basic FGF induced cell proliferation in both normal and transformed FBAE cells. However, bFGF appeared to be much more potent in transformed than in normal cells. No differences in bFGF membrane receptors were observed between normal and transformed FBAE cells in terms of apparent molecular weight, number per cell, dissociation constant, and kinetic of downregulation. In respect to normal cells, however, transformed GM 7373 cells showed higher basal levels of PKC activity. This kinase is activated by bFGF and is involved in mediating the mitogenic activity of bFGF, as shown by the capacity of the PKC inhibitor H‐7 to abolish the mitogenic activity of bFGF both in normal and transformed FBAE cells. Like bFGF, the PKC activators DAG and TPA exerted a stronger mitogenic activity in transformed than in normal FBAE cells. Thus, the different susceptibility of normal and transformed FBAE cells to bFGF appears to depend on differences in the post‐receptor signal transduction mediated by PKC rather than on differences in bFGF receptors.The results indicate that chemical transformation causes significant modifications of bFGF physiology in FBAE cells. The relevance of these modifications to the genesis of tumors of vascular origin deserves further investigat

ISSN:0021-9541    

DOI:10.1002/jcp.1041410310

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

年代:1989

数据来源: WILEY

摘要:

AbstractWe have studied the effect of insulin concentration on the kinetics of insulin internalization and efflux in isolated rat adipocytes. To determine internalization rates adipocytes were incubated with125l‐insulin at 37°C; and at frequent, early time points surface‐bound and intracellular insulin were quantitated. Surface‐bound and intracellular insulin were discriminated by the sensitivity of the former to rapid dissociation by a pH 3.0 buffer at 4°C. From this data the endocytotic (internalization) rate constant (ke) was calculated for six insulin concentrations ranging from 0.3 to 100 ng/ml. Ke was found to decrease in an insulin concentration‐dependent manner (P<.001). Thus, values for ke were 0.121 ± 0.006 min−1versus 0.074 ± 0.011 min−1at 0.3 ng/ml and 100 ng/ml, respectively. The decrease in ke did not parallel insulin concentration‐dependent changes in insulin receptor affinity indicating it was not the result of an inability of low affinity receptors to be internalized. The kinetics of insulin efflux were determined by loading various concentrations of125l‐insulin into the adipocyte interior, washing away surface‐bound and extracellular insulin, and then monitoring the subsequent efflux of pre‐loaded insulin into medium that contained the same concentration of insulin used in the loading step. The overall rate of efflux was independent of insulin concentration. In summary, these results show that at high insulin concentrations the efficiency of insulin internalization is impaired. In contrast, the rate of insul

ISSN:0021-9541    

DOI:10.1002/jcp.1041410311

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

年代:1989

数据来源: WILEY