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1.
The poly(ADP-ribosyl)ation inhibitor 3 aminobenzamide (3AB) is used extensively to probe the involvement of post-translational modifications of proteins in the control of DNA repair and cell cycle progression. However, 3AB appears to lack specificity for the synthetase, and the use of excessive concentrations of the inhibitor may adversely affect the potential responsiveness of cells to DNA-damaging agents. Here we address the concentration dependency of the cellular impact of 3AB alone by using flow cytometry to analyze the cell cycle phase-dependent, anti-proliferative effects of 3AB on mouse L1210 cells together with fluctuations in RNA (predominantly ribosomal) levels. We report that 3AB, at cytostatic concentrations, does not block cells in G2 committed to mitosis but imposes an immediate G1 and S phase arrest. Eventually cells arrested in G1 and S phase can reenter cycle but become irreversibly blocked in G2 and are incapable either of progression to mitosis or of the reinitiation of DNA synthesis when cytokinesis is blocked by colcemid exposure. 3AB exposure rapidly reduced RNA levels in all phases of the cell cycle with recovery from depletion apparent only at nontoxic concentrations (5 mM). The responses of a 3AB-resistant subline, capable of sustained culture growth in a normally cytostatic concentration of inhibitor (25 mM), suggest a close association between the sensitivity to RNA depletion and cell cycle arrest.  相似文献   

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Abstract. The initiation of DNA synthesis and further cell cycle progression in cells during and following exposure to extremely hypoxic conditions in either G1 or G2+M has been studied in human NHIK 3025 cells. Populations of cells, synchronized by mitotic selection, were rendered extremely hypoxic (< 4 p.p.m. O2) for up to 24n h. Cell cycle progression was studied from flow cytometric DNA recordings. No accumulation of DNA was found to take place during extreme hypoxia. Cells initially in G1 at the onset of treatment did not enter S during up to 24 h exposure to extreme hypoxia, but started DNA synthesis in a highly synchronous manner within 1.5 to 2.25 h after reoxygenation. The duration of S phase was only slightly affected (increased by ≅10%) by the hypoxic treatment. This suggests that the DNA synthesizing machinery either remains intact during hypoxia or is rapidly restored after reoxygenation. Cells initially in G2 at the onset of hypoxia were able to complete mitosis, but further cell cycle progression was blocked in the subsequent G^ Following reoxygenation, these cells progressed into S phase, but the initiation of DNA synthesis was delayed for a period corresponding to at least the duration of normal G1 and did not appear in a synchronous manner. In fact, cell cycle variability was found to be increased rather than decreased as a result of exposure to hypoxia starting in G2. We interpret these findings as an indication that important steps in the preparation for initiation of DNA synthesis take place before mitosis. Furthermore, the change in cell cycle duration induced by hypoxia commencing in G1 is of a nature other than that induced by hypoxia commencing in other parts of the cell cycle.  相似文献   

4.
3-Aminobenzamide and benzamide, two potent inhibitors of poly-(ADP-ribose)-polymerase increase the frequencies of SCEs in Chinese hamster ovary cells in a dose-dependent manner. SCEs were studied in cells in which the inhibitors were present either during the first cell cycle or the second cell cycle or both. Most of the induced SCEs were found to be formed during the second cell cycle in which BU-containing DNA was used as template for DNA synthesis. In cells which were pregrown for 4 cell cycles in the presence of BrdUrd, in order to obtain both sister chromatids bifiliarly substituted with BU in their DNA, it was found that the presence of inhibitor even in the first cell cycle increased the frequencies of SCEs. It is concluded that the incorporated BrdUrd plays an important role in the origin of spontaneous and induced SCEs. 3-Aminobenzamide alone or benzamide in the presence of BrdUrd during culture, did not increase the frequencies of mutations to HGPRT? in these cells.  相似文献   

5.
5-Bromodeoxyuridine (BrdU)-induced sister chromatid exchanges (SCEs) are mainly determined during replication on a BrdU-substituted template. The BrdU, once incorporated, is rapidly excised as uracil (U), and the gap is repaired with the incorporation of BrdU from the medium, which leads to further repair. During the second S period in BrdU medium, this process continues as the strand acts as template. Experiments suggest that 3-amino-benzamide (3AB) delays the ligation of the gaps formed after U excision, resulting in enhanced SCE levels during the second cycle of BrdU incorporation. When normal templates of G1 cells are treated before BrdU introduction with methyl methanesulphonate (MMS), 3AB in the first cycle doubles the MMS-induced SCEs but has no effect on them during the second cycle. When the BrdU-substituted template is treated with MMS in G1 of the second cycle, 3AB again doubles the SCEs due to MMS and also enhances the SCEs resulting from delays in ligation of the gaps following U excision in the BrdU-substituted template. The repair processes of MMS lesions that are sensitive to 3AB and lead to SCEs take place rapidly, while the repair process of late repairing lesions that lead to SCEs appear to be insensitive to 3AB. A model for SCE induction is proposed involving a single-strand break or gap as the initial requirement for SCE initiation at the replicating fork. Subsequent events represent natural stages in the repair process of a lesion, ensuring replication without loss of genetic information. G1 cells treated with methylnitrosourea (MNU) and grown immediately in BrdU medium rapidly lose the O6-methylguanine from their DNA and the rate of loss is BrdU-dose dependent. The rapid excision of the U lesions can explain the effect of BrdU concentration on SCE reduction following both MNU or MMS treatment.  相似文献   

6.
Culture of cells in high exogenous levels (>10–4 M) of bromodeoxyuridine (BrdUrd) or thymidine will increase the baseline sister chromatid exchange (SCE) frequency. The effect is thought to be related to the balance of the DNA precursors thymidine and deoxycytidine. Exogenous addition of deoxycytidine will reverse this effect. Single and twin SCEs were analysed in Colcemid-induced tetraploid Chinese hamster ovary cells exposed to different concentrations of BrdUrd to determine at what stage SCEs are induced by high levels of BrdUrd. In cells exposed to low concentrations of BrdUrd (10–5 M), equal numbers of SCEs were induced in each of the two cell cycles. With increasing concentrations of BrdUrd (10–4 to 2×10–4 M), SCE frequency increased in both cell cycles, but far more SCEs were induced in the second cell cycle. Deoxycytidine (2×10–4 M) reduced the frequency of SCEs primarily by reducing the frequency of SCEs induced in the second cell cycle. Treatment with 3-aminobenzamide (3AB), a potent inhibitor of poly(ADP-ribose) polymerase, produced effects similar to exposure to high levels of BrdUrd including inducing SCEs in the second replication cycle. This suggests a similar mechanism of action. Deoxycytidine had no effect on 3AB-induced SCEs, however, and there was no interaction between 3AB and high exogenous levels of BrdUrd in SCE induction. Thus these two agents probably act through different mechanisms.  相似文献   

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Hepatocytes, isolated from adult (250-350 g) rats, attached and survived well in primary culture on highly diluted (less than 1 microgram/cm2) collagen gel in a synthetic medium without serum or hormones. About 20% of the cells "spontaneously" entered S phase during the first 4 days of culturing, and mitoses were easily demonstrated at the near physiological concentration (1.25 mM) of Ca++ prevailing in the medium. Cultures given 9 nM epidermal growth factor (EGF) and 20 nM insulin 20 h after inoculation showed vigorous DNA synthesis and mitotic activity. Autoradiography of such cells exposed to [3H]thymidine allowed the determination of the following cell cycle parameters: Lag period from EGF/insulin stimulation till onset of increased DNA synthesis, 17 h; rate of entry into S phase (kG1/S), 0.028/h; duration of S phase, 8.4 h; duration of G2 phase, 2.7 h. The peak DNA synthesis (pulse labelling index, 24%) and peak mitotic activity (mitotic index, 1.7%) occurred 35 and 43 h, respectively, after the stimulation with EGF/insulin. These values are comparable to those reported during the in vivo compensatory hyperplasia following partial hepatectomy of adult rats. A marked variation of the intranuclear [3H]thymidine pulse labelling pattern was noted: During the first 1.5 h of the S phase, the labelling was extranucleolar and during the last 1.5 h chiefly nucleolar. The cells survived well in the absence of glucocorticoid, whose effect on cell cycle parameters therefore could be studied. Dexamethasone (25-250 nM) did not appreciably affect the durations of S phase and G2 phase or the pattern of preferential extranucleolar and nucleolar DNA synthesis within the S phase.  相似文献   

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10.
Proliferating cell nuclear antigen (PCNA/cyclin) is a nuclear protein that can stimulate purified DNA polymerase delta in vitro, and its synthesis correlates with the proliferation rate of cells. We have attempted to determine whether synthesis of PCNA/cyclin in Chinese hamster ovary cells is necessary to regulate entry into S phase. We have measured cellular PCNA/cyclin concentration of the mRNA or protein throughout the cell cycle. Cells were separated by centrifugal elutriation into populations enriched for G-1, S, and G-2/M phases. Quantitative Northern hybridization analysis was performed on RNA isolated from each cell population by using a cDNA clone of PCNA/cyclin as a probe. Results demonstrated that although intact PCNA/cyclin mRNA is present during all phases of the cell cycle, an induction of about 3-fold occurs during S phase. Two-parameter staining for PCNA/cyclin and DNA, and analysis by flow cytometry, confirmed that the quantity of PCNA/cyclin protein in the cells increases severalfold in G-1 or early S phase but generally is invariant in S and G-2/M phases. This cell cycle dependence of PCNA/cyclin expression suggests that the observed synthesis is a prerequisite for initiation of DNA replication. Introduction of an antisense oligonucleotide complementary to the PCNA/cyclin mRNA to inhibit PCNA/cyclin synthesis effectively prevented entry of G-1 phase cells into S phase. A complementary sense oligonucleotide used as a control did not have an inhibitory effect. This result suggests that a threshold concentration of PCNA/cyclin is necessary for entry into S phase.  相似文献   

11.
The cell killing and induction of sister-chromatid exchanges (SCEs) by X-rays and short-wave ultraviolet (UV) irradiation in combination with inhibitors of DNA repair, 3-aminobenzamide (3AB), cytosine arabinoside (ara-C) or aphidicolin (APC) were studied in wild-type CHO-K1 and two X-ray-sensitive mutants, xrs 5 and xrs 6 cells. The spontaneous frequency of SCEs was similar in the mutants and the wild-type CHO-K1 cells (8.4-10.3 SCEs/cell). Though X-rays are known to be poor inducers of SCEs, a dose-dependent increase in the frequency of SCEs in xrs 6 cells (doubling at 150 rad) was found in comparison to a small increase in xrs 5 and no increase in wild-type CHO-K1 cells. 3AB, an inhibitor of poly(ADP-ribose) synthetase increased the spontaneous frequency of SCEs in all the cell types. 3AB did not potentiate the X-ray-induced frequency of SCEs in any of the cell lines. Ara-C, an inhibitor of DNA polymerase alpha, increased the frequency of SCEs in all the cell lines. In combined treatment with X-rays, ara-C had no synergistic effect in xrs 5 and xrs 6 cells, but the frequency of SCEs increased in X-irradiated wild-type CHO-K1 cells post-treated with ara-C. For the induced frequency of SCEs, CHO-K1 cells treated with X-rays plus ara-C behaved like xrs 6 cells treated with X-rays alone, suggesting a possible defect in DNA base damage repair in xrs 6 cells, in addition to the known defective repair of DNA double-strand breaks (DSBs). Survival experiments revealed higher sensitivity of xrs 5 and xrs 6 mutant cells to the cell killing effect of X-rays in S-phase when compared to wild-type CHO-K1 cells. The mutants responded with lesser sensitivity to cell killing effect of ara-C and APC than CHO-K1 cells, the relative sensitivity to ara-C or APC being CHO-K1 greater than xrs 5 greater than xrs 6 cells. When X-irradiation was coupled with ara-C, the results obtained for survival were similar to those of the SCE test, i.e., unlike wild-type CHO-K1, no synergistic effect was observed in xrs 5 or xrs 6 cells. After UV-irradiation, the frequency of SCEs increased similarly in wild-type CHO-K1 and xrs 6 cells, but xrs 5 cells responded with lower frequency of SCEs.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

12.
Rat kangaroo(Potorous tridactylus) cells efficiently repair 254 nm ultraviolet light (UV) induced cyclobutane pyrlmidine dimers (CPDs) through photoreactivation, leading to an enhancement of survival when cells are exposed to photoreactivation light (PRL) immediately after UV-irradiation. This work presents evidence that at least part of the UV-irradiated cells die through apoptosis, as demonstrated by DNA fragmentation and chromatin condensation. The induction of this kind of cell death can be reversed through photoreactivation immediately after irradiation, indicating that CPDs are essential signals for the initiation of apoptosis by UV-irradiation. Exposure to PRL 24 h after UV-irradiation does not reverse the induction of apoptosis, implying that the cells are committed to die at this time after irradiation. Inhibition of DNA synthesis during this period of time following UV-irradiation, and before exposure to PRL, does not avoid apoptosis. Since similar results were obtained in Go confluent and G1/S synchronized cells, the signals for the UV-induced apoptosis do not seem to be related to a specific phase of cell cycle. Nevertheless, by adding 3-aminobenzamide (3AB—an inhibitor of poly(ADP-ribose) polymerase) in the cell medium after UV-irradiation, apoptosis endpoints were partially reversed if cells are exposed to PRL 24 h later. This result strongly indicates that poly(ADP-ribose) is an intermediary signal for UV-induced apoptosis in mammalian cells.  相似文献   

13.
The nuclear enzyme DNA topoisomerase II catalyzes the breakage and resealing of duplex DNA and plays an important role in several genetic processes. It also mediates the DNA cleavage activity and cytotoxicity of clinically important anticancer agents such as etoposide. We have examined the activity of topoisomerase II during the first cell cycle of quiescent BALB/c 3T3 cells following serum stimulation. Etoposide-mediated DNA break frequency in vivo was used as a parameter of topoisomerase II activity, and enzyme content was assayed by immunoblotting. Density-arrested A31 cells exhibited a much lower sensitivity to the effects of etoposide than did actively proliferating cells. Upon serum stimulation of the quiescent cells, however, there was a marked increase in drug sensitivity which began during S phase and reached its peak just before mitosis. Maximal drug sensitivity during this period was 2.5 times greater than that of log-phase cells. This increase in drug sensitivity was associated with an increase in intracellular topoisomerase II content as determined by immunoblotting. The induction of topoisomerase II-mediated drug sensitivity was aborted within 1 h of exposure of cells to the protein synthesis inhibitor cycloheximide, but the DNA synthesis inhibitor aphidicolin had no effect. In contrast to the sensitivity of cells to drug-induced DNA cleavage, maximal cytotoxicity occurred during S phase. A 3-h exposure to cycloheximide before etoposide treatment resulted in nearly complete loss of cytotoxicity. Our findings indicate that topoisomerase II activity fluctuates with cell cycle progression, with peak activity occurring during the G2 phase. This increase in topoisomerase II is protein synthesis dependent and may reflect a high rate of enzyme turnover. The dissociation between maximal drug-induced DNA cleavage and cytotoxicity indicates that the topoisomerase-mediated DNA breaks may be necessary but are not sufficient for cytotoxicity and that the other factors which are particularly expressed during S phase may be important as well.  相似文献   

14.
Exposure of suspension-cultured HeLa cells to a 45° thermal shock resulted in cell inactivation and inhibition of both protein and DNA synthesis. DNA synthesis was inhibited in a biphasic manner with a more sensitive (D0 = 7 min) and a less sensitive (D0 = 20 min) phase. The less sensitive process was demonstrated to be DNA chain elongation. Transport of thymidine into intracellular pools was significantly less sensitive to thermal shock (D0 in excess of 200 min). When HeLa cells were heated at 45° for 15 min there was an 80% inhibition of incorporation of precursors into both DNA and protein with little effect on precursor transport into cellular pools. While the rate of synthesis of whole cell and histone protein (H2a, H2b, H3, and H4) and DNA chain elongation recovered by 6 h after cell heating, total precursor incorporation into DNA was only 0.4 of control levels. The long-term depression of the DNA synthetic rate could not be explained by a cell cycle redistribution, a depression in the total fraction of S phase cells synthesizing DNA, or by a depression in the rate of DNA chain elongation. We conclude that thermal shock results in a long-term depression in the fraction of cell replicons involved in DNA replication.  相似文献   

15.
A cut was made on the ear conch of mouse and an extract containing epidermal chalone was injected subcutaneously 2 days later. The time changes after the chalone administration in the number of cells labeled with 3H-thymidine, in the number of grains on labeled cells and in the number of mitoses within the regenerating epidermis surrounding the wound were investigated by means of autoradiography (ARG). Grain counts decreased temporarily in early phase (0–2 h) after chalone injection. This decrease in grain count resulted in a decrease in the number of labeled cells on the ARG of a short exposure but not in that on the ARG of a long exposure. A decrease in the number of labeled cells on the ARG of a long exposure was evident at 6 h when the grain counts reverted to a level similar to the control without chalone. The number of mitoses reached a minimum at 2 h and then recovered quickly, indicating a rapid disappearance of the inhibition of cells in G 2 from entering M phase. Mitoses decreased again thereafter, presumably as a result caused by inhibition of cells in the preceding S phase from completing DNA synthesis. The extract made similarly from liver or kidney affected neither the mitotic nor the DNA synthetic activities.These results indicate that the epidermal chalone or chalones inhibit the epidermal cell proliferation in, at least, 3 different processes of the cell cycle; the DNA synthesis in S phase, the transition from G 1 to S phase and the transition from G 2 to M phase.  相似文献   

16.
Suspension-cultured mouse plasmacytoma cells (MPC-11) were accumulated in the late G1 phase by exposure to isoleucine-deficient medium for 20- 24 h. The arrested culture was fed with complete medium enabling the cells to continue the cell cycle synchronously, undergo mitosis, and enter a second cycle of growth. This method of synchronization left the protein-synthesizing ability intact as judged by the polysome profile and the capacity of the cells to incorporate labeled amino acids into protein after the restoration of isoleucine. After incubation in isoleucine-deficient medium and the addition of isoleucine to the culture, cells entered the S phase after a short lag, as judged by [3H]thymidine incorporation into nucleic acid and by spectrophotometric measurement of nuclear DNA. The cells were in mitosis between 12 and 18 h as judged by the increase in cell count and analysis of cell populations on albumin gradients. Synthesis and secretion of light- chain immunoglobulin were maximal in the late G1/early S phase of the first cycle. During late S phase, G2 phase, and mitosis, both synthesis and secretion were observed to be at a low level; however, immediately after motosis the cells which then entered the G1 phase apparently commenced synthesis of light chain immunoglobulin straight away, although secretion of labeled material remained at a low level.  相似文献   

17.
ts20 is a temperature-sensitive mutant cell line derived from BALB/3T3 cells. DNA synthesis in the mutant decreased progressively after an initial increase during the first 3 h at the restrictive temperature. RNA and protein synthesis increased for 20 h and remained at a high level for 40 h. Cells were arrested in S phase as determined by flow microfluorimetry, and DNA chain elongation was retarded as measured by fiber autoradiography. Infection with polyomavirus did not bypass the defect in cell DNA synthesis, and the mutant did not support virus DNA replication at the restrictive temperature. After shift down to the permissive temperature, cell DNA synthesis was restored whereas virus DNA synthesis was not. Analysis of virus DNA synthesized at the restrictive temperature showed that the synthesis of form I and replicative intermediate DNA decreased concurrently and that the rate of completion of virus DNA molecules remained constant with increasing time at the restrictive temperature. These studies indicated that the mutation inhibited ongoing DNA synthesis at a step early in elongation of nascent chains. The defect in virus and cell DNA synthesis was expressed in vitro. [3H]dTTP incorporation was reduced, consistent with the in vivo data. The addition of a high-salt extract prepared from wild-type 3T3 cells preferentially stimulated the incorporation of [3H]dTTP into the DNA of mutant cells at the restrictive temperature. A similar extract prepared from mutant cells was less effective and was more heat labile as incubation of it at the restrictive temperature for 1 h destroyed its ability to stimulate DNA synthesis in vitro, whereas wild-type extract was not inactivated until incubated at that temperature for 3 h.  相似文献   

18.
本文观察了FL细胞中ADP-核糖基转移酶(ADPRT)底物NAD含量的细胞周期性变化及其与DNA复制之间的关系。FL细胞NAD含最在G_1期最高,而在S期DNA合成高峰后0—3小时(S/G_2期)达到最低点。ADPRT抑制剂3 AB能够抑制NAD含量的细胞周期性变化,而且S期DNA合成亦受到抑制,并呈现S期延长,提示ADP-核糖基化作用可能参与DNA复制过程。本文还观察了三种DNA损伤剂MNNG、MMS及4NQO对处于细胞周期不同时相的FL细胞NAD含量的影响,以及ADPRT抑制剂3 AB及尼克酰胺对此影响的作用。证明ADPRT抑制剂可以特异地抑制DNA损伤性NAD含量下降而对正常FL细胞NAD含量及代谢抑制剂2,4-DNP所致的NAD含量下降没有影响。从而有可能建立一个以测量细胞内NAD含量为指标的简便、快速、特异的检测DNA损伤因子的方法。  相似文献   

19.
Takaji Ikushima 《Chromosoma》1990,99(5):360-364
The cell cycle dependence of sister chromatid exchanges (SCEs) induced by luminol, a new potent inhibitor of poly(ADP-ribose) synthetase, was studied in Chinese hamster V79 cells. Continuous treatment with luminol during two whole cell cycles in the presence of 5-bromo-2-deoxyuridine (BrdUrd), or in the first or second cycle induced SCEs very efficiently in a linear dosedependent manner. However, no enhancement of SCE levels was observed after luminol treatment in a cycle preceding BrdUrd treatment, in contrast to results found with other strong SCE inducers such ascis-diammine-dichloroplatinum (II) (CDDP) and mitomycin C (MMC). Luminol was about ten times as effective in inducing SCEs as 3-aminobenzamide (3AB), an inhibitor of the NAD+ site of poly(ADP-ribose) synthetase. The induction of SCEs by luminol was restricted to the Sphase of the cell cycle with peaks at an early and a late stage, corresponding to the biphasic replication of DNA. The mechanism of SCE appears to be the same at the early and late stages of S-phase for luminol-induced SCE formation.  相似文献   

20.
The staurosporine analogues, K-252a and RK-286C, were found to cause DNA re-replication in rat diploid fibroblasts (3Y1) without an intervening mitosis, producing tetraploid cells. Analysis of cells synchronized in early S phase in the presence of K-252a revealed that initiation of the second S phase required a lag period of 8 h after completion of the previous S phase. Reinitiation of DNA synthesis was inhibited by cycloheximide, actinomycin D, and serum deprivation, but not by Colcemid, suggesting that a functional G1 phase dependent on de novo synthesis of protein and RNA is essential for entry into the next S phase. In a src-transformed 3Y1 cell line, as well as other cell lines, giant cells containing polyploid nuclei with DNA contents of 16C to 32C were produced by continuous treatment with K-252a, indicating that the agent induced several rounds of the incomplete cell cycle without mitosis. Although the effective concentration of K-252a did not cause significant inhibition of affinity-purified p34cdc2 protein kinase activity in vitro, in vivo the full activation of p34cdc2 kinase during the G2/M was blocked by K-252a. On the other hand, the cyclic fluctuation of partially activated p34cdc2 kinase activity peaking in S phase still continued. These results suggest that a putative protein kinase(s) sensitive to K-252a plays an important role in the mechanism for preventing over-replication after completion of previous DNA synthesis. They also suggest that a periodic activation of p34cdc2 is required for S phases in the cell cycle without mitosis.  相似文献   

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