摘要:

AbstractA new spectrofluorometric method for heme quantitation in cultured fibroblasts is described. The method includes: (1) heme extraction by methanol/sulfuric acid, (2) partial purification of heme by a microchromatographic method, and (3) treatment of the purified heme by oxalic acid followed by fluorometric quantitation. Using this method, heme concentration was determined in chick embryo fibroblasts cultured in a medium supplemented with either 7% fetal bovine serum (FBS) or 10% horse serum (HS). In the presence of FBS, cultured cells actively divided and cells contained 34–55 pmol heme/mg protein. In contrast, cultures maintained in HS proliferated at a slower rate and contained 23–25 pmol heme/mg protein. The addition of 40 μM FeSO4to cultures maintained in the presence of HS stimulated cell proliferation, and the cellular heme concentration increased to 37–51 pmol/ mg protein. These findings suggest that the cessation of growth in the presence of HS may be due to decreased heme content in the cells and that the stimulation of cell growth by iron is mediated by its stimulation of heme syn

ISSN:0021-9541    

DOI:10.1002/jcp.1041130202

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

年代:1982

数据来源: WILEY

摘要:

AbstractPrimary mass cultures and cloned strains of bovine aortic endothelial and smooth muscle cells were investigated with respect to their growth responses to glucocorticoid hormones. The growth of primary endothelial cells was not influenced by glucocorticoid treatment in the absence of fibroblast growth factor (FGF) but was inhibited by about 30% in the presence of FGF; with cloned endothelial cells, glucocorticoids were also growth inhibitory only in the presence of FGF. In contrast, smooth muscle cell growth was inhibited 30%–70% by glucocorticoid treatment in both primary cultures and in the cloned strains in the absence of FCF, and this inhibition was totally abolished by the addition of FGF for both cultures. The corticosteroid influences on cell growth were glucocorticoid specific, concentration dependent, and were observed to be independent of the serum concentration. The results indicate that glucocorticoid hormones have direct and pronounced growth inhibiting effects on aortic smooth muscle cells but only minimal effects on endothelial cells when these components of the vascular wall are analyzed under identical conditions in vitr

ISSN:0021-9541    

DOI:10.1002/jcp.1041130203

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

年代:1982

数据来源: WILEY

摘要:

AbstractThe growth kinetics and population doubling limits of chick embryonic fibroblasts, chondroblasts, and retinal pigment cells were compared. Chondroblasts were found to have a cumulative population doubling level (37 ± 3 PDL) similar (p = 0.05) to that of control fibroblasts (42 ± 2 PDL), in individual and pooled clones. While both cell types have similar doubling potential, the proportion of tritium‐labeled nuclei decreases, and differs significantly as doubling level increases. This age‐associated decline is due to an extension in the population doubling time. Direct cell‐cycle analysis shows this increase to occur in the G1phase. Furthermore, cartilage colonies maintain their phenotypic expression (metachromasia) throughout their lifespan under conditions of subcloning at sparse density.When fibroblasts derived from 15 day chick embryos are compared with fibroblasts from 10 day embryos (41 ± 2 PDL) there is no significant difference (p = 0.05) in cumulative PDL or percent labeled nuclei, indicating that fibroblasts of different embryonic age have similar potential. The addition of hydrocortisone and insulin to the medium significantly shortens (25 ± 2 PDL) the lifespan of 10 day chick fibroblasts. Kinetics of retinal pigment cells show a population doubling potential (29 ± 1 PDL) different from fibroblasts and chondroblasts, suggesting that different cell types may not have similar limits on doubling potential when first determined in embryogenesis. Hydrocortisone and insulin have no effect on the growth kinetics or lifespan of retinal pigment cells

ISSN:0021-9541    

DOI:10.1002/jcp.1041130204

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

年代:1982

数据来源: WILEY

摘要:

AbstractIn culture medium supplemented with 10% clotted blood serum, the saturation density of BALB/c‐3T3 cells is determined jointly by cell replication and cell loss. By prelabelling cellular DNA with3H‐thymidine and also by time lapse photography, we studied cell loss independently of replication. Cell loss was accelerated when BALB/c‐3T3 cells were transferred from serum‐supplemented medium, which contains the platelet derived growth factor (PDGF), to medium supplemented with platelet‐poor plasma which lacks it. Loss occurred via the disintegration of cell attached to the surface of the tissue culture dish. Cytolysis of individual cells occurred rapidly; less than 15 minutes transpired between the first indication of a perturbance (by phase contrast microscopy) and fragmentation of the cell cytoplasm. Kinetic analysis was consistent with random cell death rather than a fixed lifetime.The percentage of cells undergoing cytolysis was governed by the cell density; at high densities, such as are present in confluent cultures, a higher percentage of cell loss was noted than at low density. Cell death was antagonized by partially purified or electrophoretically homogenous preparations of‐PDGF. Pure PDGF stimulated cell survivial at ng/ml in a concentration dependent fashion. The process of cell replication was not necessary for survival because PDGF prevented cytolysis in the presence of methotrexate, an inhibitor of DNA synthesis. A brief (4 hour) treatment with PDGF prevented cell death; such PDGF treated cells displayed increased survival after being taken up with trypsin and planted onto a fresh surface in plasma supplemented medium. Pituitary fibroblast growth factor, a functional analogue of PDGF for induc of DNA synthesis in BALB/c‐3T3 cells, also functioned as an anticytolytic agent. By contrast, epidermal growth factor and insulin did not. Cytolysis of SV40‐transformed cells occurred at a constitutively low rate and was inse

ISSN:0021-9541    

DOI:10.1002/jcp.1041130205

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

年代:1982

数据来源: WILEY

摘要:

AbstractEarly studies on the duration of mitotic stages and on metaphase‐to‐prophase ratios in a number of normal and neoplastic cells indicated that the process of mitosis becomes altered during the course of oncogenesis. However, the nature of these changes and their effects on each of the mitotic stages are still unclear. With the use of time lapse cinemicrography, we have compared the durations of mitotic stages of SV40/Wl‐38 and SV40/Wl‐26 cells to those of their normal counterparts and to other nontransformed human fibroblasts. We also examined the relative frequencies of the individual mitotic stages in fixed preparations of Wl‐38 and SV40/Wl‐38 cells. The data show that metaphase durations are increased in the transformed cells and as much as 3‐4.7‐fold in SV40/Wl‐38 cells compared to Wl‐38 and other nontransformed cells. Other stages are also prolonged though to lesser degrees. These findings suggest that increased metaphase/prophase ratios observed in many tumors are due to increases in duration of metaphase rather than to shorter prophases, and that increased mitotic indices commonly observed in malignant tumors and sometimes used as an index of growth rate are at least in part due to the prolongatio

ISSN:0021-9541    

DOI:10.1002/jcp.1041130206

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

年代:1982

数据来源: WILEY

摘要:

AbstractNormal, primary explanted, bovine granulosa cells grow reproducibly in agar culture as anchorage‐independent clones. Epidermal growth factor (EGF) and rat erythrocytes are effective stimulators of colony formation, and when both are added to the culture medium at optimal concentrations, there is an enhancement of colony numbers and colony size, indicative of an independent, and operationally additive, mode of action for the two factors. The ability of cells propagated from agar clones to secrete progesterone, and to augment progesterone secretion 4‐fold in the presence of 1 mM dbcAMP is proof that colonies originate from and are composed of functional granulosa cells.Maximal colony numbers are present at day 10 of incubation, and colony forming cells undergo self‐renewal as assessed by the ability of cells from primary colonies to redone in agar. Absolute cloning efficiency, however, is dependent on a number of factors. Inherent variability exists in cloning efficiency of granulosa cells from individual follicles. Quantitative and qualitative clonal growth was improved at an osmolality of less than 300 mOsm when compared with higher osmolalities. Cl‐1 medium and the alpha modification of Eagle's medium were equally effective in supporting agar clonogenic growth, whereas both Ham's F12 and NCTC 135 media exhibited poor clonogenic growth supporting properties. The substitution of agarose for agar did not affect colony numbers but colonies grown in the presence of agarose tended to be smaller and more uniform

ISSN:0021-9541    

DOI:10.1002/jcp.1041130207

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

年代:1982

数据来源: WILEY

摘要:

AbstractCultured heart cells from 2–3 day old and 5–6 day old neonatal rats have been used as a model system for the characterization of carbohydrate metabolism in developing cardiac tissue. The rate of depletion of glucose from the growth medium was dependent on (1) the age of the animals from which the cultured cells were obtained, and (2) the presence and absence of serum and/or insulin in the growth medium. The glucose depletion rate in insulin and serumcontaining medium was 9.63 ± 0.96 nmol/min/mg protein for heart cell cultures from, 2 day old rats and 3.51 ± 0.68 nmol/min/mg protein in heart cell cultures from 5 day old rats. Appearance of lactate in the medium during these experiments occurred at the rates of 18.6 ± 7.9 nmol/min/mg and 6.4 ± 1.2 nmol/min/mg, respectively. In the absence of serum and insulin, the medium glucose depletion rates were 5.7 ± 1.6 and 2.2 ± 0.5 nmol/min/mg for cells derived from 2‐day‐old and 5‐day‐old rats, respectively. It is apparent from these data that immature cardiac cells depend upon glucose as a primary source of energy for muscle contraction and cellular growth, and that less‐efficient energy‐yielding metabolic pathways a

ISSN:0021-9541    

DOI:10.1002/jcp.1041130208

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

年代:1982

数据来源: WILEY

摘要:

AbstractAmino acid transport in Madin‐Darby canine kidney (MDCK) cells, grown in a defined medium, was investigated as a function of cell density, exposure to specific growth factors, and transformation. MDCK cells were found to transport neutral amino acids by systems similar to the A, ASC, L, and N systems which have been characterized using other cell lines. Experimental conditions were developed for MDCK cells which allowed independent measurement of A, ASC, and L transport activities. The activity of the L system was measured as Na+‐independent leucine or methionine uptake at pH 7.4. The activity of the A system was measured as Na+‐dependent α(methylamino)isobutyric acid (mAIB) uptake at pH 7.4, the activity of the ASC system was measured as Na+‐dependent alanine uptake in the presence of 0.1 mM mAIB at pH 6.0, and the activity of system N was observed by measuring Na+‐dependent glutamine uptake at pH 7.4 in the presence of high concentrations of A and ASC system substrates. The L transport system responded minimally to changes in growth state, but Na+‐dependent amino add transport responded to regulation by growth factors, cell density, and transformation. The activities of the A and ASC systems both decreased at high cell density, but these activities responded dissimilarly under other conditions. The activity of the A system was stimulated by insulin, was inhibited by PGE1, and was elevated 3–7 fold in the transformed cell line, MDCK‐T1. The activity of the ASC system was slightly stimulated by insulin and by PGE1, but was unchanged after chemical transformation. Changes in cellular growth were monitored and were found to correlate best with the activity of the A system. These results suggested that MDCK cell growth may be more closely related to the activity of the A than o

ISSN:0021-9541    

DOI:10.1002/jcp.1041130209

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

年代:1982

数据来源: WILEY

摘要:

AbstractAmiloride is known to inhibit both influx of sodium ions and activation of quiescent cells by growth factors. The coincidence of these effects has been cited to support the proposal that influx of sodium ions acts as a mitogenic signal. Although it was noted that amiloride inhibited protein synthesis, this was attributed to an action on transport of amino acids, particularly those coupled to sodium fluxes. We find, however, that amiloride directly inhibits polypeptide synthesis in a reticulocyte lysate. In Swiss 3T3 cells, concentrations of amiloride and of cycloheximide that are nearly matched in their degree of inhibition of protein synthesis, produce about the same degree of inhibition of transit of cells from G0to S. Inhibition of protein synthesis is sufficient to explain the effect of amiloride on mitogenesis; the drug, therefore, is not suitable for testing the hypothesis that sodium influx is a mitogenic signal.

ISSN:0021-9541    

DOI:10.1002/jcp.1041130210

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

年代:1982

数据来源: WILEY

摘要:

AbstractTransglutaminase and ornithine decarboxylase activities have been assayed at intervals after partial hepatectomy in regenerating liver cells fractionated to obtain nuclear, cytoplasmic‐particulate, and cytoplasmic‐soluble fractions. Ornithine decarboxylase activity, localized entirely in the cytoplasmic fractions, undergoes a dramatic induction during the first 4 h after partial hepatectomy and remains elevated. This induction is very sensitive to inhibition by cycloheximide and actinomycin D, as previously reported. Transglutaminase activity is localized in both the cytoplasm and the nucleus with the highest specific activity in the nucleus. Nuclear transglutaminase activity approximately doubles in the first 2 h of liver regeneration, apparently as a result of a translocation of enzyme from the cytoplasm to the nucleus. Inhibitor studies indicate that the translocation is not dependent upon protein or RNA synthesis. In the first 2 h, actinomycin D slightly activates transglutaminase activity in the cytoplasmic‐particulate and nuclear fractions. Only at 4 h after the onset of regeneration do actinomycin D and cycloheximide show some inhibition of transglutaminase activity indicating de novo synthesis at this time. A broad increase of transglutaminase activity occurs from hours 12–16 to hour 32 after partial hepatectomy in the nuclear and cytoplasmic‐particulate fraction. These data suggest the existence of a function for transglutaminase in the nucleus of rat li

ISSN:0021-9541    

DOI:10.1002/jcp.1041130211

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

年代:1982

数据来源: WILEY