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1.
We report the continuous growth maintenance of untransformed and chemically transformed fibroblasts (AKR-2B, AKR-MCA cells) in low concentrations of serum (0.1% FBS). The cell lines established (AKR-0.1F, MCA-0.1F) proliferated at rates comparable to cells maintained under high serum conditions (10% FBS). Complete removal of serum from the cells did not induce quiescence. The MCA-0.1F cells were more similar to the untransformed AKR-2B fibroblasts in their morphology, saturation density, inability to form colonies under anchorage-independent conditions, steady-state level of c-myc expression, and kinetics of induction of c-myc in response to specific growth factors. This report demonstrates the utility of this cell line as a nonquiescent model system for investigating growth factor-specific effects in serum-free, cycling cells. Addition of transforming growth factor-beta (TGF-beta) (5 ng/ml) to proliferating MCA-0.1F cells, in the absence of any serum, induced a multilayered growth pattern at confluency, similar to that of AKR-MCA cells maintained in 10% FBS. Other growth factors tested did not elicit this effect. The induction of this growth pattern by TGF-beta was associated with a sustained induction of the c-myc proto-oncogene at confluency, but not with a restoration of anchorage-independent growth. The data suggest that TGF-beta may play a role in the up-regulation of c-myc at confluency previously described for AKR-MCA cells maintained in 10% serum.  相似文献   

2.
The objective of this study was to investigate whether G1 cells could enter S phase after premature chromosome condensation resulting from fusion with mitotic cells. HeLa cell synchronized in early G1, mid-G1, late G1, and G2 and human diploid fibroblasts synchronized in G0 and G1 phases were separately fused by use of UV-inactivated Sendai virus with mitotic HeLa cells. After cell fusion and premature chromosome condensation, the fused cells were incubated in culture medium containing Colcemid (0.05 micrograms/ml) and [3H]thymidine ([3H]ThdR) (0.5 microCi/ml; sp act, 6.7 Ci/mM). At 0, 2, 4, and 6 h after fusion, cell samples were taken to determine the initation of DNA synthesis in the prematurely condensed chromosomes (PCC) on the basis of their morphology and labeling index. The results of this study indicate that PCC from G0, G1, and G2 cells reach the maximum degree of compaction or condensation at 2 h after PCC induction. In addition, the G1-PCC from normal and transformed cells initiated DNA synthesis, as indicated by their "pulverized" appearance and incorporation of [3H]ThdR. Further, the initiation of DNA synthesis in G1-PCC occurred significantly earlier than in the mononucleate G1 cells. Neither pulverization nor incorporation of label was observed in the PCC of G0 and G2 cells. These findings suggest that chromosome decondensation, although not controlling the timing of a cell's entry into S phase, is an important step for the initiation of DNA synthesis. These data also suggest that the entry of a S phase may be regulated by cell cycle phase-specific changes in the permeability of the nuclear envelope to the inducers of DNA synthesis present in the cytoplasm.  相似文献   

3.
db—cAMP对转化细胞钙调素基因表达与细胞骨架的影响   总被引:5,自引:0,他引:5  
We have demonstrated that the distribution of microtubules (MT), microfilaments (MF) and fibronectin (FN) were diminished, while the gene expression of the calmodulin and c-fos enhanced in the transformed C3 H10 T1/2 cells. After treatment with 1 mM db-cAMP for 1 hr. and 2 hrs., there was an early and rapidly reduced in gene expression of calmodulin and c-fos respectively. After db-cAMP treatment for 4-5 days, the number of Capping cells of ConA binding decreased significantly and the cell surface microvilli decreased also. The growth of treated cells was inhibited markedly. By using 4F1 cDNA probe, which is preferentially expressed in G1 phase, we have found that the db-cAMP treated cells were accumulated at G1 phase. Of particular interest is the fact that the distribution of microtubules, microfilaments and fibronectin were recovered after treatment with 1 mM db-cAMP for 6 days. It is suggested that the inhibition of proliferation, alteration of phenotype and recovery of cytoskeleton in transformed cells after treatment with db-cAMP are related to the inhibition of gene expression of calmodulin.  相似文献   

4.
The object of this study was to develop a map of G1 phase on the basis of the progressive changes taking place in the morphology of the prematurely condensed chromosomes as the cells traverse through G1 and then use this technique to determine the cell cycle location of normal and transformed cell populations in plateau phase. The morphology of the prematurely condensed chromosomes (PCC) of G1 cells in random populations was found to be highly variable. For a better understanding of the relationship between the morphology of the G1-PCC and their position within G1 phase, synchronized populations of Chinese hamster ovary (CHO) cells in early, mid-, and late G1 phase were fused with mitotic cells. Early G1 cells resulted in highly condensed G1-PCC, while late G1 cells gave very extended G1-PCC. Mid-G1 cells resulted in PCC of intermediate condensation. To test the validity of these criteria for mapping the position of a cell in the cell cycle, synchronous G1 cell populations were treated with a variety of metabolic inhibitors. Cycloheximide and actinomycin D were shown to block cell in early G1 phase, while excess thymidine and hydroxyurea blocked cells in early S phase. The results presented here indicate that, upon reaching plateau phase, normal cell populations (BALB-C mouse 3T3, human PA-2, and WI 38) stop in early G1, while most cells in transformed cell lines (CHO, HeLa, and mouse SV-3T3) accumulate in late G1.  相似文献   

5.
The objective of this study was to determine the points in the cell cycle at which normal and transformed cells become arrested as a result of polyamine deprivation. Treatment of normal (human fibroblast line PA2 and mouse 3T3) and transformed (CHO, HeLa and SV3T3) cells with methylglyoxal bis-(guanyl-hydrazone) resulted in a significant decrease in the levels of spermidine and spermine which was associated with an inhibition of growth. Examination of the prematurely condensed chromosomes (PCC) of the polyaminedepleted cells, revealed that normal fibroblasts were preferentially arrested in early G1 phase while a majority of cells in the transformed lines were blocked in S phase. A close examination of the PCC of the transformed cells indicated a significant decrease in the number of DNA replication sites suggesting that polyamines have an important role in DNA chain initiation.  相似文献   

6.
Premature chromosome condensation and cell cycle analysis.   总被引:3,自引:0,他引:3  
The application of the phenomenon of premature chromosome condensation for cell cycle analysis in HeLa and CHO cells has been examined. Random populations of HeLa and CHO cells pulse labelled with H3-TdR were separately fused with mitotic HeLa cells using U.V. inactivated Sendai virus. The resulting prematurely condensed chromosomes (PCC) were scored and classified into G1, S and G2-PCC on the basis of both morphological and autoradiographic data, The results of this study indicated that the G1, S and G2 phase cells are equally susceptible to virus-induced fusion with mitotic cells and subsequent induction into PCC. Hence the PCC method for cell cycle analysis is both practical and accurate. This study also revealed that the process of chromosome decondensation initiated during the telophase of mitosis continues throughout the G1 period reaching an ultimate state of decondensation by the end of G1, at which point the fusion of such cells with those in mitosis yield PCC with the most diffused morphology instead of the discrete single stranded structures characteristic of early G1-PCC. Thus, the decondensation of chromatin during G1 appears to be a prerequisite for the subsequent initiation of DNA synthesis.  相似文献   

7.
P el-Achkar  P Mandel  M Mersel 《FEBS letters》1988,239(2):276-280
Cultured astrocytes derived from neonatal rats (normal cells) displayed maximal ethanolamine base exchange enzymatic activity (EBEE) when cultures reached confluency and cells almost ceased to divide. At this stage, ethanolamine phosphotransferase (EPT) and choline base exchange enzyme (CBEE) activities reached a plateau. In spontaneously transformed glial cells, no differential activity variation either between EPT and CBEE, or between EPT and EBEE was observed. The EBEE activity was mainly localized in the microsomal fraction and was completely absent from plasma membranes. Dibutyryl cyclic AMP (db-cAMP) treatment of the transformed cells reversed the pattern of these activities to that of normal cells. Moreover, treatment of the transformed cells with medium conditioned by normal astroblasts markedly increased EBEE activity. This study demonstrates that (i) variation of EBEE activity during cell growth differs in normal and in transformed cultured glial cells. (ii) EBEE activity may be modulated via both db-cAMP and normal cell conditioned medium. Our findings suggest a possible implication of EBEE in the maturation and contact inhibition of cell growth.  相似文献   

8.
转化的C_3H_(10)T_(1/2)细胞表现增殖速度加快、表面微绒毛增加,细胞变圆,叠层生长,ConA受体呈帽状分布,微管、微丝、纤粘蛋白分布明显减少。与增殖有关的癌基因c-fos表达增强,同时发现与细胞增殖、转化和细胞骨架调节有关的钙调素(CaM)基因表达加强。用1mmo/Ldb-cAMP处理转化细胞,观察到CaM基因和原癌基因c-fos的表达分别在处理后1小时和2小时急剧下降。处理后4—5天,转化细胞表型趋正常化,大部分细胞恢复单层生长。细胞表面微绒毛和泡状物减少,ConA受体帽状分布消失,恢复分散分布在细胞膜上的特点。细胞生长明显被抑制,用优先在G_1期表达的4F_1 cDNA为探针进行分子杂交,证实了经db-cAMP处理后的细胞被阻抑在G_1期。经db-cAMP处理6天的转化细胞中微管、微丝、纤粘蛋白基本恢复正常分布。实验表明CaM的表达增强与转化细胞表型变化和细胞骨架组装减弱密切相关,db-cAMP作用后CaM表达下降是抑制转化细胞增殖并使细胞表型和细胞骨架分布趋于正常的关键事件之一。  相似文献   

9.
The uptake of 2-deoxy-d-glucose (2-DG) into human glioma cells (138 MG) was related to cell growth. The uptake of 2-DG was high at confluency but low in both rapidly growing sparse cultures and in growth-inhibited dense cultures. Lineweaver-Burke plots of uptake at different cell densities showed changes in Vmax; Km, however, remained constant. Dibutyryl cyclic-AMP (db-cAMP) doubled the uptake of 2-DG into rapidly growing sparse cultures but lacked effect at higher cell density. Independent of their density, cells treated with db-cAMP attained the characteristic morphology of differentiated glial cells.  相似文献   

10.
An important factor governing developmental rates of somatic cloned embryos is the phase of the cell cycle of donor nuclei. The aim of this experiment was to investigate the distribution of cell cycle phases in bovine cumulus and fibroblast cells cultured using routine treatment, and under cell cycle-arresting treatments. The highest percentages of cumulus cells in the G0 + G1 stage were observed in uncultured, frozen/thawed cells originating from immature oocytes (79.8 +/- 2.2%), fresh and frozen/thawed cells from in vitro matured oocytes (84.1 +/- 6.2 and 77.8 +/- 5.7%, respectively), and in cycling cells (72.7 +/- 16.3 and 78.4 +/- 11.2%, respectively for cumulus cells from immature and in vitro matured oocytes). Serum starvation of cumulus cultures markedly decreased percentages of cells in G0 + G1, and prolonged starvation significantly increased (P < 0.05) percentages of cells in G2 + M phase. Culture of cumulus cells to confluency did not increase percentages of cells in G0 + G1. Contrary to findings in cumulus cells, significantly higher percentages of cells in G0 + G1 were apparent when fibroblast cells were cultured to confluency or serum starved, and significantly increased (P < 0.01) as the starvation period was prolonged. It is concluded that for particular cell types specific strategies should be used to attain improvements in the efficiency of cloning procedures.  相似文献   

11.
Flow cytometric cell cycle analysis of cultured porcine fetal fibroblast cells   总被引:14,自引:0,他引:14  
Normal development of nuclear transfer embryos is thought to be dependent on transferral of nuclei in G0 or G1 phases of the cell cycle. Therefore, we investigated the cell cycle characteristics of porcine fetal fibroblast cells cultured under a variety of cell cycle-arresting treatments. This was achieved by using flow cytometry to simultaneously measure cellular DNA and protein content, enabling the calculation of percentages of cells in G0, G1, S, and G2+M phases of the cell cycle. Cultures that were serum starved for 5 days contained higher (p < 0.05) percentages of G0+G1 (87.5 +/- 0. 7) and G0 cells alone (48.3 +/- 9.7) compared with rapidly cycling cultures (G0+G1: 74.1 +/- 3.0; G0: 2.8 +/- 1.2). Growth to confluency increased (p < 0.05) G0+G1 percentages (85.1 +/- 2.8) but did not increase G0 percentages (6.0 +/- 5.3) compared to those in cycling cultures. Separate assessment of small-, medium-, and large-sized cells showed that as the cell size decreased from large to small, percentages of cells in G0+G1 and G0 alone increased (p < 0.05). We found 95.2 +/- 0.3% and 72.2 +/- 12.0% of small serum-starved cells in G0+G1 and G0 alone, respectively. Cultures were also treated with cell cycle inhibitors. Treatment with dimethyl sulfoxide (1%) or colchicine (0.5 microM) increased percentages of cells in G0 (24.8 +/- 20.0) or G2+M (37.4 +/- 4.6), respectively. However, cells were only slightly responsive to mimosine treatment. A more complete understanding of the cell cycle of donor cells should lead to improvements in the efficiency of nuclear transfer procedures.  相似文献   

12.
Double minutes (dm) characteristically exhibit greater numerical heterogeneity among tumor cells than do chromosomes. The biological basis of this heterogeneity was studied in human carcinoma cell line S 18. Pulse labeling of asynchronous cells with [3H]dThd, continuous labeling of synchronized cells with BrdUrd and prematurely condensed chromosome (PCC) studies of G1 and G2 phase S 18 cells indicate that dm-DNA replicates only once during S phase of the cell cycle. No evidence was found for replication of dm-DNA at G1 phase, G2 phase or mitotis. Cells observed at anaphase show imprecise distribution of dm to daughter cells. These studies suggest numerical heterogeneity of dm results from anomalous mitotic segregation rather than anomalous replication of dmDNA.  相似文献   

13.
We investigated the influence of the cell cycle stage of the nuclear donor on prematurely condensed chromatin (PCC) and spindle morphology and on chromosome constitution in rabbit nuclear transplant embryos. The configuration of PCC following nuclear transplantation with G1, early S, and late S phase donor nuclei (G1, early S, and late S transplants, respectively) was characterized in whole mounts and chromosome spreads. In addition, the influence of the donor cell cycle stage on chromosome constitution in cleavage stage-manipulated embryos was determined. Within 2 h after fusion of the donor blastomere, the recipient oocyte cytoplasm was able to induce formation de novo of a metaphase plate associated with a spindle in G1, early S, and late S transplants. Metaphase chromosomes and spindle were intact in most cases of PCC in G1 transplants. However, these structures displayed minor abnormalities in early S transplants and gross abnormalities in late S transplants, such as incomplete or absent spindle formation and incomplete chromatin condensation. Normal chromosomes were present in G1 and early S transplants, whereas chromosome abnormalities were detected in late S transplants. The results indicate that morphology of prematurely condensed G1 and early S chromatin has a minor influence on chromosome constitution of manipulated embryos. That of late S chromatin, however, affects chromosome constitution in embryos and may account for reduced development of nuclear transplant embryos when late S phase donor nuclei are used.  相似文献   

14.
The objective of this study was to determine whether transformed cells have greater DNA synthesis-inducing ability (DSIA) than normal cells when fused with G1 phase cells. HeLa cells synchronized in G1 phase, prelabeled with large latex beads, were fused separately with (a) quiescent human diploid fibroblasts (HDF), (b) HDF partially synchronized in late G1, and random populations of (c) HeLa, (d) WI-38, (e) SV-40 transformed WI-38, (f) CHO, (g) chemically transformed mouse cells (AKR-MCA), and (h) T98G human glioblastoma cells (all prelabeled with small latex beads) using UV-inactivated Sendai virus. The fusion mixture was incubated with [3H] thymidine, sampled at regular intervals, and processed for radioautography. Among the heterodikaryons, the frequency of those with a labeled and an unlabeled nuclei (L/U) were scored as a function of time after fusion. The faster the induction of DNA synthesis in HeLa G1, the steeper the drop in the L/U class and hence the higher DSIA in the S phase cells. The DSIA, which is indicative of the intracellular levels of the inducers of DNA synthesis, was the highest in HeLa and virally transformed WI-38 cells and the lowest in normal human diploid fibroblasts (HDF) while those of chemically and spontaneously transformed cells are intermediate between these two extremes. Higher level of DNA synthesis inducers appears to be one of the pleotropic effects of transformation by DNA tumor viruses. These studies also revealed that initiation of DNA synthesis per se is regulated by the presence of inducers and not by inhibitors.  相似文献   

15.
N6,O2′-dibutyryl adenosine 3′,5′-cyclic-phosphate (db-cAMP) has been shown to convert Chinese hamster cells of ovarian origin (CHO-K1) from compact, randomly oriented cells growing in multilayers to elongated fibroblast-like cells which grow in monolayers. This compound also has been reported to have a variety of effects on the cell cycle. Most such studies have employed synchronized cells to determine cell cycle effects, and consequently have been limited to the short-term effects of the compound. We have looked for chronic effects on the cell cycle in cultures exposed continuously to db-cAMP from the initiation of the cultures until they had reached or approached the plateau phase. This was done by combined autoradiography and Feulgen microspectrophotometry plus measurements of the protein content of mitotic cells to detect any influence on cell size. The overall results were that continuous exposure to db-cAMP had at most only minor effects on the cell cycle and cell size when the culture medium was renewed daily. Somewhat greater effects were found on plateau-phase cells in cultures in which the medium was not renewed. In this case fewer cells appeared to remain in the cell cycle in the cultures with db-cAMP. Comparison with our earlier results with Chinese hamster V79 cells led to the conclusions that cell cycle parameters and cell size at mitosis were less altered during culture growth in CHO cells, but that CHO cells seemed to be less able to maintain cells in the cell cycle in crowded cultures.  相似文献   

16.
Functional and optimal activities of the (Na+-K+)ATPase, as determined by ouabain-sensitive K+ influx in intact cells and ATP hydrolysis in cell homogenates respectively, have been measured during the cell cycle of neuroblastoma (clone Neuro-2A) cells. The cells were synchronized by selective detachment of mitotic cells. The ouabain-sensitive K+ influx decreased more than fourfold from 1.62 +/- 0.11 nmoles/min/10(6) cells to 0.36 +/- 0.25 nmoles/min/10(6) cells on passing from mitosis to early G1 phase. On entry into S phase a transient sixfold increase to 2.07 +/- 0.30 nmoles/min/10(6) cells was observed, followed by a rapid decline, after which the active K+ influx rose again steadily from 1.03 +/- 0.25 nmoles/min/10(6) cells in early S phase to 2.10 +/- 0.92 nmoles/min/10(6) cells just prior to the next mitosis. The ouabain-insensitive component rose linearly through the cycle in the same manner as the protein content/cell. Combining total K+ influx values with efflux data obtained previously showed that net loss of K+ occurred with transition from mitosis to G1 phase while net accumulation occurred with entry into S. Throughout mid-S phase net K+ flux was virtually zero, but a large net influx occurred again just before the next mitosis. The (Na+-K+)ATPase activity measured in cell homogenates decreased rapidly from mitosis to G1 phase and increased steadily throughout S phase, but the transient activation on entry into S phase was not observed. Complete inhibition of the (Na+-K+)ATPase mediated K+ influx by ouabain (5 mM) prevents the cells from entering S phase, while partial inhibition by lower concentrations of ouabain (0.2 and 0.5 mM; km = 0.17 mM) causes partial blockage in G1 and, to a lesser extent, a reduced rate of progression through the rest of the cell cycle. We conclude that the transient increase in (Na+-K+)ATPase mediated K+ influx at the G1/S transition is a prerequisite for entry into S phase, while maintenance of adequate levels of K+ influx is necessary for normal rate of progression through the rest of the cell cycle.  相似文献   

17.
Cloudman S91 mouse melanoma cells lose their ability to demonstrate an MSH-induced increase in tyrosinase activity as cell density increases. This loss in hormone responsiveness occurs before confluency is reached and cannot be reversed by exposure of cells to increasing concentrations of MSH. The failure of high-density cultures to respond to MSH is apparently not the result of an inability of MSH to stimulate cAMP production, since either low- or high-density cultures exposed to MSH demonstrate equivalent increases in intracellular levels of cAMP. Further, neither theophylline (1mM), dibutyryl cyclic AMP (10(-4)M), or prostaglandin E1 (10(-6)M) is effective in stimulating tyrosinase activity in melanoma cells cultured at densities exceeding 6 X 10(4) cells/cm2. This finding suggests that the decay of hormone responsiveness occurs at a cellular site distal to cAMP production. The decrease in tyrosinase stimulation by MSH as cell density increases is also apparently not the result of an increase in activity of any soluble inhibitor of the enzyme, for cytosol preparations from high-density cultures (10(5) cells/cm2) fail to inhibit tyrosinase activity in cell homogenates from low-density cultures treated with MSH.  相似文献   

18.
A density dependent stimulation of glutamine synthetase (GS) activity has been observed in cultures of mouse teratoma cells. GS specific activity increased as cultures approached confluency to a level greater than 2-fold over the basal level found in sparse cultures. After confluency the GS specific activity returned to the basal level found in sparse cultures. The enzyme increase could not be attributed to age of cultures, medium or glutamine depletion, cell leakage of GS, or change in the amount of cellular protein. Dibutyryl cyclic AMP (db-cAMP) plus theophylline lowered GS specific activity both in cultured teratoma and in teratoma obtained from ascites grown tumors. The enzyme increase observed in cultured teratoma cells could be prevented by cycloheximide, and enhanced by hydrocortisone or actinomycin D.  相似文献   

19.
Treatment of the transformed mouse embryo fibroblast cell line AKR-MCA with 1% N,N-dimethylformamide (DMF) resulted in the restoration of a nontransformed phenotype in these cells. In order to determine if an increase in growth inhibitory peptides might be responsible for these changes in growth properties of the DMF-treated AKR-MCA cells we examined the serum-free conditioned medium for its ability to inhibit the anchorage-independent growth of a human colon carcinoma cell line. The extracellular levels of inhibitory activity were two-fold higher in conditioned medium derived from AKR-MCA cells than in AKR-MCA cells grown in 1% DMF (AKR-MCA/DMF). Fractionation of the crude conditioned medium indicated the presence of an Mr 20,000 inhibitory fraction in AKR-MCA/DMF conditioned medium which was reduced in AKR-MCA cells. This Mr 20,000 inhibitory activity was acid and heat stable and sensitive to dithiothreitol and trypsin. In addition to inhibiting the growth of a human colon carcinoma cell line this protein induced colony formation in AKR-2B cells and competed for binding to the transforming growth factor beta (TGF-beta) receptor. Therefore, this Mr 20,000 inhibitory polypeptide induced by DMF is probably TGF-beta. TGF-beta was also shown to inhibit the growth of AKR-MCA cells in monolayer culture.  相似文献   

20.
Synchronization of the human promyelocytic cell line HL 60 by thymidine   总被引:2,自引:0,他引:2  
Cultures of the promyelocytic cell line HL 60 were synchronized with thymidine. A concentration of 0.05 mM thymidine and an exposure time of 24 hr was found optimal for blocking about 90% of the cells in S phase. Following release from the thymidine block the cell cultures were followed intermittently over 40 hr for fluctuation in cell numbers, labelling with radioactive thymidine and nuclear DNA distributions. Mathematical evaluation of the results revealed a cycling time of 18.6 hr and a duration of specific cell phases of 8.6 hr, 7.1 hr and 2.9 hr for G1, S and G2 + M, respectively. The doubling time was 26 hr and the growth fraction was estimated as 1.  相似文献   

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