摘要:

AbstractStable mutants of Chinese hamster V79 cells deficient in thymidylate synthetase (TS; E.C. 2.1.1.45) have been selected from cultures grown in medium supplemented with folinic acid, aminopterin, and thymidine (FAT). After chemical mutagenesis, the frequency of colonies resistant to the “FAT” medium increased more than 100‐fold over the spontaneous frequency. The optimal expression time of the mutant phenotype was 5–7 days after mutagen treatment. The recovery of FAT‐resistant colonies in the selective medium was not affected by the presence of wild‐type cells at a density below 9,000 cells per cm2. All 21 mutants tested exhibited thymidine auxotrophy; neither folinic acid nor deoxyuridine could support mutant cell growth. There was no detectable TS activity in all 11 mutants so far examined and only about 50% of wild‐type activity in three prototrophic revertants, as measured by whole‐cell and cell‐free enzyme assays. The apparent Michaelis‐Menten constant (Km) for deoxyuridine‐5′‐monophosphate and inhibition constant (Ki) for 5‐fluorodeoxyuridine‐5′‐monophosphate, measured by whole‐cell enzyme assay, appear to be similar for the wild‐type and revertant cell lines. Using 5‐fluoro‐[63H]‐2′‐deoxyuridine 5′‐monophosphate as active site titrant, the relative amounts of TS in crude cell extract from the parental, revertant, and mutant cells were shown to exist in a 1:0.5:0 ratio. Furthermore, the enzymes from two revertants were more heat labile than that of V79 cells. These properties, taken together, suggest that the FAT‐resistant, thymidine auxotrophic phenotype may be the result of a structural gene mutation at the TS locus. The availability of such a mutant facilitates studies on thymidylate stress in rel

ISSN:0021-9541    

DOI:10.1002/jcp.1041200202

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

年代:1984

数据来源: WILEY

摘要:

AbstractFibroblasts in vivo adhere to a collagenous extracellular matrix. We present here a combined morphological and biochemical analysis of the adhesion sites of fibroblast‐like cells cultured in vitro on gelatin‐coated plastic, for comparison with earlier model studies using serum (plasma‐fibronectin [pFn])‐coated plastic. Scanning electron microscopy shows that cell adhesion to the gelatin is quite similar to that on plastic, but with some morphological differences reminiscent of those caused by higher concentrations of fibronectin adsorbed to the substratum. Measurement using125I‐radiolabeled pFn shows the level of substratum‐bound pFn adsorbed from serum in the growth medium is, however, comparable on gelatin or plastic; thus, differences due to pFn must be attributed to the quality of the adsorbed protein, not its absolute quantity. Gel electrophoretic analysis of cellular adhesion sites formed on the two substrata shows their compositions to be qualitatively similar, suggesting again that the same fundamental adhesion processes are involved. However, three protein bands do change; notably, cellular fibronectin is increased on gelatin. These three proteins are also the most resistant to saline extraction, suggesting their intrinsic importance in the adhesion sites. The nature of the growth substratum thus appears to modulate a fundamentally unvarying morphology and adhesion site composition of the cells that a

ISSN:0021-9541    

DOI:10.1002/jcp.1041200203

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

年代:1984

数据来源: WILEY

摘要:

AbstractA serum‐free, hormone‐supplemented medium (SFHM) for maintaining neonatal rat heart cells in culture has been developed in this laboratory (Mohamed et al., 1983). Morphological assessment of heart cells grown in SFHM show it to be similar to commonly used serum‐supplemented media. To quantitatively compare cell behavior in SFHM with serum‐supplemented media, the activities of ten regulatory enzymes which represent four metabolic pathways were studied in heart cells cultured in SFHM. The enzyme activities which were measured included hexokinase, glucose‐6‐phosphate dehydrogenase, 6‐phosphogluconate dehydrogenase, phosphofructokinase, pyruvate kinase, NAD+‐linkedsn‐glycerol‐3‐phosphate dehydrogenase, malate dehydrogenase, NAD+‐linked isocitrate dehydrogenase, NADH‐cytochromecreductase, and succinic cytochromecreductase. Rat heart cells maintained in culture on SFHM are not only qualitatively and quantitatively similar to those maintained in serum‐supplemented medium but also provide a more suitable model system for metabolic studies of neonatal cardiac tissue for several reasons: (1) many enzyme activities that may represent dedifferentiation are elevated by serum; (2) NAD‐linked glycerol‐3‐phosphate dehydrogenase activity in cells maintained on SFHM is similar to the in vivo activity; (3) cells beat at or near the in vivo frequency and can be maintained 3 months on SFHM; (4) the SFHM is chemically defined and thus can be completely manipulated by the investigator. The effects of three concentrations of hydrocortisone (HC) (5,000 ng/ml, 50 μg/ml, 0 ng/ml) on heart cells cultured in SFHM supported our previous conclusion that function (beating) and growth (protein accumulation) are inversely related in

ISSN:0021-9541    

DOI:10.1002/jcp.1041200204

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

年代:1984

数据来源: WILEY

摘要:

AbstractNRK cells infected with a temperature‐sensitive, transformation‐defective mutant of avian sarcoma virus (ASV),tsLA23, are transformed at 36°C, but at 40°C they behave as nontransformed cells because of the inactivation of the abnormally thermolabile pp60v‐srcproduct of the virus' transformingsrcgene. At 40°C, thesetsLA23‐NRK cells were arrested in G1/G0by severe serum deprivation. They were induced to enter G1, initiate DNA synthesis 7 or 10 hours later, and then divide as (1) nontransformed cells by adding serum or platelet‐derived growth factor (PDGF) at 40°C, or (2) transformed cells by lowering the temperature to a pp60v‐src‐activating 36°C without adding exogenous growth factor(s). The level of pp60v‐srckinase activity rose dramatically in these serum‐deprived cells within 30 minutes of lowering the temperature to the permissive 36°C, and it fell just as rapidly when the cells were returned to the restrictive 40°C. As little as a 2‐hour exposure to 36°C, with an attendant 2‐hour burst of pp60v‐srckinase activity, was enough to stimulate serum‐deprivedtsLA23‐NRK cells to transit G1and initiate DNA replication, but not to divide. Much more prolonged pp60v‐srcactivity was needed for these serum‐deprived cells to complete their cycle and divide. The prereplicative development of quiescenttsLA23‐NRK cells stimulated by serum or PDGF was accompanied by greatly increased protein synthesis and slightly decreased protein degradation, but the pp60v‐src‐stimulated cells progressed through G1and initiated DNA replication without appreciably affecting the protein synthetic machinery of the cell. The cells stimulated by the mitogenic action of pp60v‐src, like the cells stimulated by serum, needed to activate early prereplicative genes in order to initiate DNA replication. The needed RNA transcripts induced by serum and pp60v‐srcwere produced with comparable efficiency, although it took longer for pp60v‐src‐stimulated cells to translate these transcripts and to initiate DNA replication, probably beca

ISSN:0021-9541    

DOI:10.1002/jcp.1041200205

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

年代:1984

数据来源: WILEY

摘要:

AbstractThe purpose of this study was to determine whether cyclic AMP (cAMP) plays any direct or indirect role in the antiproliferative effect of mouse L‐cell interferon in Swiss 3T3 cells. Firstly, we found that interferon did not affect intracellular levels of cAMP in these cells in the absence or the presence of cAMP‐elevating agents. Secondly, we examined the effect of interferon on the stimulation of DNA synthesis of quiescent 3T3 cells by a range of cyclic AMP‐elevating agents, including cholera toxin, cAMP derivatives, and prostaglandin E, added in the presence of insulin or vasopressin. Interferon inhibited cyclic AMP‐stimulated DNA synthesis as measured by incorporation of radioactive thymidine into acid‐insoluble material and autoradiographic analysis of the fraction of labelled cells. Dose‐response curves and kinetics of inhibition were identical to those obtained in cultures stimulated by combinations of growth factors that do not increase the intracellular level of cAMP. The inhibition by interferon of cAMP‐stimulated DNA synthesis was also observed in secondary cultures of mouse embryo fibroblasts, where cAMP‐elevating agents provide a mitogenic signal in the absence of other added growth factors. These results show that the inhibitory effect of interferon on DNA synthesis in Swiss 3T3 cells is not mediate

ISSN:0021-9541    

DOI:10.1002/jcp.1041200206

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

年代:1984

数据来源: WILEY

摘要:

AbstractThe mechanism whereby 25‐hydroxycholesterol, an inhibitor of the synthesis of cholesterol, depresses DNA synthesis in cycling P815 mastocytoma cells was investigated. The uptake of45Ca into P815 cells treated with 1 μg/ml 25‐hydroxycholesterol began to rise above control levels by 6 hours after initiation of treatment and was increased tenfold by 15 hours. Kinetic data of calcium uptake indicated the presence of at least two components of calcium uptake, fast and slow. The fast phase of calcium exchange at the cell surface was changed little by treatment with 25‐hydroxycholesterol. The slow phase of calcium exchange with the intracellular compartment was markedly affected by treatment with the inhibitor, there being a large increase in the flux and half‐time of uptake, and a fall in the rate constant. This resulted in a large elevation of the intracellular compartment size. Incorporation of [3H]thymidine into DNA began to decline between 9 and 12 hours posttreatment in these cultures. Uptake of calcium and depression of DNA synthesis were shown to be directly reiated to the dose of 25‐hydroxycholesterol used. The changes in45Ca uptake and DNA synthesis due to 25‐hydroxycholesterol treatment were abolished by addition of exogenous cholesterol to the incubation medium. The results are consistent with the hypothesis that 25‐hydroxycholesterol, by inhibiting cholesterol production, depresses DNA synthesis via an elevation in the uptake of calcium into the cell to a level incompatible with continued D

ISSN:0021-9541    

DOI:10.1002/jcp.1041200207

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

年代:1984

数据来源: WILEY

摘要:

AbstractThe transport of proteins across continuous capillary endothelium is believed to be mediated by micropinocytic vesicles which shuttle molecules between the lumenal and abluminal plasma membrane. We have studied the ability of capillary endothelial cells isolated from rat epididymal fat to endocytose fluorescently labelled ovalbumin within micropinocytic vesicles. Net association of fluorescent ovalbumin with endothelial cells reaches an equilibrium after 40 minutes of incubation. This equilibrium is presumably due to a balance between endocytosis and subsequent exocytosis of this protein. Capillaries equilibrated with fluorescent ovalbumin exhibited rapid exocytosis of this protein when it was removed from the external medium. The rate of endocytosis was concentration dependent and obeyed the kinetics expected for adsorptive phase endocytosis. High concentrations of ovalbumin stimulated the ingestion of14C‐sucrose, a marker of fluid endocytosis, suggesting that protein can affect the movement of vesicles within the endothelial cytoplasm. These results imply that capillary endothelium isolated from rat epididymal fat exhibits the ability to endocytose and subsequently exocytose protein. This demonstrates that the two components of endothelial vesicular transport or transcytosis can be observed and studied in a system of isolated capillary endotheliu

ISSN:0021-9541    

DOI:10.1002/jcp.1041200208

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

年代:1984

数据来源: WILEY

摘要:

AbstractProstaglandin (PG) production was evaluated in the three cell types (endothelial, smooth muscle, and fibroblast) comprising the bovine pulmonary artery. Prostacyclin (PG12) was the predominant prostaglandin (PG) produced by endothelial, smooth muscle, and fibroblast cells as they exist in culture or in freshly excised tissue fragments. In addition to PGl2, measurable amounts of PGE2, PGF2a, and thromboxane A2(TXA2) were also produced by these cells. Endothelial cells were the most active producers of PGs. However, the type of PG produced was characteristic of the particular cell type, while the level of production was dependent on external factors. Prostaglandin production by cultured cells, both under basal conditions and in response to stimulatory agents, was quite similar to that of the respective freshly excised tissue fragments containing a given cell type. These cells in culture could be stimulated to produce PGl2by both angiotensin and bradykinin at very low (physiological) concentrations, a further indication of the retention of the physiological responsiveness of these cells in culture. Endothelial cells and fibroblasts were activated by bradykinin at concentrations as low as 10−12M but did not respond to angiotensin. Smooth muscle cells in primary and first passage cultures were activated by both bradykinin and angiotensin at 10−12M concentrations. Serial subcultivations of smooth muscle cells resulted in a progressive loss in their responsiveness to bradykinin stimulation. The state of cell growth proved to be an important determinant of PG production. Actively growing cells in culture synthesized less PG when compared to cells which had entered into a “quiescent” nongrowt

ISSN:0021-9541    

DOI:10.1002/jcp.1041200209

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

年代:1984

数据来源: WILEY

摘要:

AbstractWe have investigated the responsiveness of established rat hepatocyte cell cultures to inducers of cytochrome P‐450. One Reuber hepatoma‐derived line (Fu5‐C8), which under normal culture conditions produces no detectable cytochrome P‐450(MC) or cytochrome P‐450(PB)—the major cytochrome P‐450 isozymes induced by 3‐methylcholanthrene and phenobarbital, respectively‐ was tested for the ability to accumulate either cytochrome P‐450 isozyme in response to treatment with various xenobiotics. By immune‐precipitation from [35S]‐methionine‐labeled cell extracts, using monospecific anticyto‐chrome P‐450(MC) antybody or monoclonal anticytochrome P‐450(PB) antibody, it was demonstrated that these cells possess the capability to synthesize cytochrome P‐450(MC) in response to 3‐methylcholanthrene treatment, while none of the drug treatments caused the synthesis of detectable quantities of cytochrome P‐450(PB). RNA extracted from Fu5‐C8 cells directed the in vitro synthesis of immune‐precipitable cytochrome P‐450(MC) only after treatment of the cells with 3‐methylcholanthrene Kinetic analysis of the response of these cells to 3‐methylcholanthrene induction revealed detectable levels of immune‐precipitable cytochrome P‐450(MC) 2 h after drug treatment with maximal induction occurring between 12 and 16 h of exposure. Another cell line (HF 1.5), obtained originally by hybridization of Fao X H5 variants of a Reuber H35 hepatoma, produces cytochrome P‐450(MC) and also cytochrome P‐450(PB) constitutively, as determined by specific immune‐precipitation from labeled cell extracts. Exposure of confluent monolayers to either phenobarbital or 3‐methylcholanthrene resulted in an induction of cytochrome P‐450(PB) or cytochrome F‐450(MC), respectively. Double‐labeling immunofluorescence studies indicate that all cells in the culture produce albumin and most of the cells produce cytochrome P‐450(MC), but only a subset of cells synthesize cytochrome P‐450(PB). Our results demonstrate that some continuously dividing hepatocyte cell cultures retain the capacity to respond to xenobiotics, including phenobarbital, a response which is typically exhibited by fully differentiated liver cells. Such established hepatocyte cell cultures should prove useful

ISSN:0021-9541    

DOI:10.1002/jcp.1041200210

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

年代:1984

数据来源: WILEY

摘要:

AbstractTo elucidate conditions which affect the lag time for resting cells to enter S phase after serum stimulation, we used a wild‐type 3Y1 rat fibroblast line and four temperature‐sensitive mutants of 3Y1 (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203). Among these five lines, in only tsG125 cells was there an obviously prolonged lag time with increase in time in resting state at 33.8°C. The resting wild‐type 3Y1 cells, preexposed to 39.8°C, also showed a prolongation of lag time. The prolongation in tsG125 had a certain limit. Preexposure to 39.8°C before serum stimulation accelerated such prolongation in tsG125 to its limit, but did not change the limit, per se. Resting tsG125 cells stimulated by serum at 39.8°C, did not enter S phase, yet they did advance toward S phase. When they were kept at 39.8°C, they retreated toward a deeper resting state (“GO”) with time. These retreats correlated with the decrease in stimulating activity in the culture media. About 20% of the resting tsG125 cells stimulated by serum at 39.8°C were committed to enter S phase, when the extent of commitment was examined at 33.8°C. Most of the tsG125 cells committed at 33.8°C did not enter S phase, when the extent of commitment was examined at 39.8°C. More cells were committed after stimulation at 33.8°C than at 39.8°C, when the test was done at 33.8°C. We suggest that resting cells may be reversibly changed within range of resting states, in either direction, that is, advance toward S phase or retreat toward deeper “GO”. These changes may be determined by alterations in the balance between synthesis and decay of the preparedness for the initiation of DNA synthesis caused by cellular response to environmental changes (e.g., medium activity, temperature, etc.). The ts defect in tsG125 may affect the cell cycle progression, both before and

ISSN:0021-9541    

DOI:10.1002/jcp.1041200211

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

年代:1984

数据来源: WILEY