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1.
UV damage to CHO cell DNA, measured by formation of thymine-containing dimers, increases from mitosis to early S phase. Computer simulation of UV absorption by the DNA of an idealized CHO cell at different stages in the cell cycle resembles the cycle dependence of UV damage. Incision at UV damage sites, measured by the accumulation of breaks in preexisting DNA during 30 minutes' post-irradiation incubation with the DNA synthesis inhibitors 1-β-D arabinofuranosylcytosine and hydroxyurea, increases from mitosis to interphase. Analysis of the dose dependence of DNA break accumulation indicates that both the affinity of the endonuclease for dimer sites and the maximum enzyme activity at saturating levels of dimers are significantly lower in mitosis than in interphase. The killing of CHO cells by UV is enhanced if repair is temporarily inhibited by ara C. The DNA gyrase inhibitor novobiocin prevents UV-induced incision. 相似文献
2.
The lethal effect of UV radiation of HeLa cells is least in mitosis and greatest in late G1-early S. Photochemical damage to HeLa DNA, as measured by thymine-containing dimer formation and by alkaline sucrose sedimentation, also increases from mitosis towards early S phase. Computer simulations of UV absorption by an idealized HeLa cell at different stages of the cell cycle indicate that changes in damage could be due solely to changes in chromatin geometry. But survival is not exclusively a function of damage. 相似文献
3.
Hiwasa T Arase Y Kikuno K Hasegawa R Sugaya S Kita K Saido T Yamamori H Maki M Suzuki N 《Cell death and differentiation》2000,7(6):531-537
Human RSa cells are highly sensitive to apoptotic-like cell death by ultraviolet irradiation (UV) while UVr-1 cells are their variant with an increased resistance to UV. Three days after UV at 10 J/m2, the viability of RSa cells was approximately 17% while that of UVr-1 cells was 65%. This different survival might reflect apoptotic cell death since apoptosis-specific DNA ladder was more clearly observed in RSa cells than in UVr-1 cells after UV. Addition of ALLN/calpain inhibitor I to the culture medium after UV resulted in similar survival (14 - 18%) between RSa and UVr-1 cells. Immunoblot analysis showed down-regulation of protein kinase CTheta, Src, Bax and mu-calpain after UV was more prominent in UVr-1 than in RSa cells. Activated mu-calpain appeared within 1 h post-UV only in UVr-1 cells. The expression of calpastatin, a specific endogenous inhibitor of calpain, was higher in RSa than in UVr-1 cells. To further examine the role of calpain in UV-induced cell death, cDNA of human calpastatin was transfected into UVr-1 cells. The results showed that overexpression of calpastatin suppressed down-regulation of Src, mu-calpain and Bax. Concomitantly, colony survival after UV was reduced in calpastatin-transfected cells as compared to vector control cells. Our results suggest that activation of calpain might account for, at least in part, the lower susceptibility to UV-induced cell death in UVr-1 cells. 相似文献
4.
Lack of RB protein correlates with increased sensitivity to UV-radiation-induced apoptosis in human breast cancer cells 总被引:4,自引:0,他引:4
The underlying causes for different apoptotic responses in neoplastic cells are still not fully understood. We demonstrate here that a human breast cancer cell line, MDA-MB-468, which lacks the retinoblastoma protein (RB), is particularly sensitive to low doses of ultraviolet (UV) radiation. These cells are 15-20-fold more sensitive to UV radiation than RB-positive cell lines, as measured by both apoptosis and clonogenic assays. In addition, a prostate cancer cell line that lacks functional RB, DU-145, was found to have a similar apoptotic response to low doses of UV radiation. Based on these data, we hypothesized that the lack of RB is responsible for the extreme sensitivity of these cells to UV-radiation-induced apoptosis. To further examine the role of RB in apoptosis, cells of RB-positive human breast cancer and normal cell lines were infected with the human papilloma virus type 16 (HPV-16) E7 and assessed for UV-radiation sensitivity. The HPV-16 E7 protein is known to decrease levels of free RB in cells. Infection of RB-positive human breast cancer or normal cells with E7 resulted in a 4-5-fold increase in sensitivity to UV radiation compared to controls. The above data suggest a role for the RB protein in protecting cells from undergoing apoptosis in response to UV radiation. 相似文献
5.
E S Manuilova 《Genetika》1977,13(1):37-45
Lethal and mutagenic effects of UV light were studied in two synchronized UV-sensitive Chinese hamster cell clones differing in the degree of sensitivity (CHS1, CHS2). It is shown that the phase of mitosis is most resistant to the lethal effect of UV. The sensitivity of both cell clones increases in the pre-synthetic phase and reaches its maximum during the phase of DNA synthesis. Positive correlation of cell sensitivity to mutagenic and lethal action of UV was observed when studying induced mutability in both cell clones during the phase of DNA synthesis. However, the study of the mutagenic effect of UV on different phases of the synthesis. However, the study of the mutagenic effect of UV on different phases of the cell cycle (M, G1, S) in the less UV-sensitive cell clone has revealed that the maximal mutation yield takes place when cells are irradiated at G1 (CHS1). The discrepancy observed may be due to different probability of the phenotypic detection of pre-mutational lesions, arising at different phases of the cell cycle. It is shown that only one cell generation is necessary for the expression of pre-mutational changes. These data allow to conclude that the increased mutation rate observed at G1 (as compared with S) reveals rather a probability of the expression but not of the occurrence of pre-mutational lesions. It is suggested that the fixation of mutations in the cells studied proceeds during the post-replication repair synthesis. 相似文献
6.
Colony formation is the classic method for measuring survival of yeast cells. This method measures mitotic viability and can underestimate the fraction of cells capable of carrying out other DNA processing events. Here, we report an alternative method, based on cell metabolism, to determine the fraction of surviving cells after ultraviolet (UV) irradiation. The reduction of 2,3,5-triphenyl tetrazolium chloride (or TTC) to formazan in mitochondria was compared with cell colony formation and DNA repair capacity in wt cells and two repair-deficient strains (rad1Delta and rad7Delta). Both TTC reduction and cell colony formation gave a linear response with different ratios of mitotically viable cells and heat-inactivated cells. However, monitoring the formation of formazan in non-dividing yeast cells that are partially (rad7Delta) or totally (wt) proficient at DNA repair is a more accurate measure of cell survival after UV irradiation. Before repair of UV photoproducts (cis-syn cyclobutane pyrimidine dimers or CPDs) is complete, these two assays give very different results, implying that many damaged cells are metabolically competent but cannot replicate. For example, only 25% of the rad7Delta cells are mitotically viable after a UV dose of 12 J/m(2)75% of these cells are metabolically competent and remove over 55% of the CPDs from their genomic DNA. Moreover, repair of CPDs in wt cells dramatically decreases after the first few hours of liquid holding (L.H.; incubation in water) and correlates with a substantial decrease in cell metabolism over the same time period. In contrast, cell colony formation may be the more accurate indicator of cell survival after UV irradiation of rad1Delta cells (i.e., cells with little DNA repair activity). These results indicate that the metabolic competence of UV-irradiated, non-dividing yeast cells is a much better indicator of cell survival than mitotic viability in partially (or totally) repair proficient yeast cultures. 相似文献
7.
C J Gomer N Rucker A L Murphree 《International journal of radiation biology and related studies in physics, chemistry, and medicine》1988,53(4):651-659
The transformation and mutagenic potential of porphyrin photodynamic therapy has been examined in mammalian cells. The mutagenic frequency in Chinese hamster cells at the Na+/K+ ATPase locus was measured by resistance to ouabain following treatment with either photodynamic therapy (PDT) or UV irradiation. The C3H 10T 1/2 mouse embryo cell system was used to document the transformation frequency following PDT, UV irradiation, gamma irradiation or exposure to 3-methylcholanthrene (MCA). Treatments with UV irradiation were effective in producing mutants resistant to ouabain, and treatments with UV irradiation, gamma irradiation and MCA generated transformants at frequencies comparable to those which are reported in the literature. However, PDT treatment conditions (which produced a full range of cytotoxicity) did not induce any mutagenic or transformation activity above background levels. 相似文献
8.
Synopsis The relative thickness of intracellular membranes of epithelial cells in the ventral lobe of the rat prostrate was measured by a densitometric method. Glutaraldehyde perfusion followed by ruthenium tetroxide immersion fixation appeared to be the most suitable method for membrane thickness measurements. By thickness, the membranes could be roughly subdivided into three groups. The inner and outer membranes of the mitochondrion made up the thinnest membranes of the cell. The second group of membranes consisted of the membranes of the rough-surfaced endoplasmic reticulum and the Golgi apparatus, the different faces of the latter organelle, and the Golgi vesicles. The thickest group of membranes included those of the cell membrane, secretory granules, condensing vacuoles, lysosomes, autophagic vacuoles and multivesicular bodies. The differences in thickness of the membranes are probably due to the varying protein/lipid ratio, and the qualities and proportions of the different lipids in the membranes. 相似文献
9.
The duration and variability of cell cycles in epithelial and fibroblast-like mammalian sister cells with different types of intercellular contacts were estimated using time-lapse cinemicrographic technique. To study a possible interrelation between cell cycles of the sister cells, one cell in each pair of sister cells was inactivated by selective UV microbeam irradiation at the beginning of its cell cycle. It is shown that this action may delay the cycle of the intact cell as well. Such an interrelation of sister cells was found only at the G1 phase of the cell cycle and only in epithelial cells. 相似文献
10.
The distribution of cells through the phases of the cell cycle by DNA flow cytofluorimetry was analysed to investigate the effects of UV irradiation on cell cycle progression in asynchronous Chinese hamster cells with different UV-sensitivity: cell line V79 (UV-resistant cells), and UV-sensitive clones: B6, CHS1, CHS2 and XII. The UV-irradiated cultures show a large accumulation of cells in S phase, the effect increasing with UV dose increase, which may point to an inhibition of the DNA chain elongation. UV-sensitive clones show a larger and more prolongated increase in the proportion of cells in S phase after irradiation with smaller dose than UV-resistant cells. Besides, the UV-sensitive clone XII shows an inhibition of movement of irradiated cells from G1 into S phase, that may testify to an inhibition of replicon initiation. These results suggest that there is a correlation in UV-irradiated Chinese hamster cells between alteration in cell cycle progression and UV-sensitivity of cells. 相似文献
11.
M Zernik-Kobak M Pirsel J Doniger J A DiPaolo A S Levine K Dixon 《Mutation research》1990,242(1):57-65
We have constructed a series of polyomavirus-based shuttle vectors for analyzing mechanisms of mutagenesis in rodent cell systems. These vectors contain the supF suppressor tRNA gene which serves as the mutagenesis target; the pBR327 replication functions and ampr gene for replication and selection in bacteria; and the polyomavirus genome which permits replication in rodent cells. The polyoma genomes used in these vectors vary in their enhancer regions, causing varying efficiencies of replication in different types of rodent cells. One of the vectors (pPySLPT-2) which replicates particularly well in several different rodent cell types (i.e., Chinese hamster ovary, mouse hepatoma and mouse lymphoma) was used to compare mutation induction by UV radiation in UV repair-deficient mouse lymphoma L5178Y-R cells with mutagenesis in the related UV repair-proficient line, L5178Y-S. In both cell types, UV-induced mutants could be recovered at frequencies up to 50-fold higher than that of the spontaneous background. At a given UV fluence the L5178Y-R cells were more highly mutable than the L5178Y-S cells. Our results indicate that these new polyomavirus-based vectors should be useful for analysis of the molecular mechanisms of mutation induction in rodent cell systems, and in particular should allow detailed analysis of mutagenesis in the well characterized rodent somatic cell mutants. 相似文献
12.
13.
C H Ockey 《European journal of cell biology》1982,27(1):25-33
DNA fibre autoradiography, after incorporation of high specific activity 3H-thymidine and 3H-deoxycytidine, has been used to investigate repair in DNA fibres from single cells following UV, or methyl-methane sulphonate (MMS) treatment. Asynchronously growing human fibroblasts, leucocytes, and HeLa cells at different phases of the cell cycle have been investigated. Isotope incorporation in repair could be differentiated from that involved in replication by the distribution and density of silver grains along the DNA fibres. Grain distribution due to repair was continuous over long stretches of the fibres and was at a low density, occasionally interspersed with short slightly denser segments. Replication labelling on the other hand, was dense and usually in short tandem segments. Repair labelling was of a similar overall density in fibres from a single cell, but differed in intensity from cell to cell. In mutagen treated Go (leucocytes) or G1 (HeLa cells), repair labelling was not increased by the presence of the DNA inhibitors, hydroxyurea (HU) or 5-fluorodeoxyuridine (FUdR). Repair was not detectable in S cells however, without the use of these inhibitors to reduce endogenous nucleoside production. FUdR enhanced the repair labelling in S cells only slightly, while HU increased it beyond that observed in UV irradiated, HU treated, G1 cells. The intensity of repair labelling in fibres from mutagen treated S cells appears to be proportional to the degree of reduction of DNA chain elongation in replicons. 相似文献
14.
DNA repair in human cells: in Cockayne syndrome cells rejoining of DNA strands is impaired 总被引:1,自引:0,他引:1
Fibroblasts from patients with Cockayne Syndrome (CS) are hypersensitive to UV light. DNA repair was analyzed in these cells by sedimentation behaviour of DNA nucleoids in sucrose gradients and compared to normal control cells. The initiation of repair, the incision of the DNA strand next to the UV lesion appeared to be normal. The rejoining of DNA stretches, however, is retarded in CS cells. DNA repair synthesis of UV damages was measured by autoradiography of [14C]thymidine incorporation into resting cells. Up to 4 h the DNA repair synthesis was comparable with normal cells. From 4 to 7 h the incorporation of radioactive precursors declined in CS cells. Besides a defective DNA polymerase this could be due to accelerated excorporation of radioactive nucleotides as a consequence of delayed ligation. In ligation the enzyme itself could be affected as well as its activation by ADP-ribosylation. Nicotine adenine dinucleotide (NAD+) is needed for the ADP ribosylation process. The cellular NAD+ content, however, was found to be the same in normal and in CS fibroblasts. Increase of the extracellular NAD+ supply accelerated the rejoining of UV damaged DNA in CS cells. 相似文献
15.
The temperature dependency for the growth of tetraploid Meth-A cells established from diploid cells was examined in comparison with the parent diploid cells. Proliferation of the tetraploid cells was markedly suppressed below 35 degrees C. At above 40 degrees C, both the diploid and tetraploid Meth-A cells ceased growing. Flow cytometry (FCM) analysis showed that the hyperploid cell fraction increased in the tetraploid Meth-A cell population at low temperatures. The fluidity of cell membranes at different temperatures was measured by means of electron spin resonance (ESR), and it was almost the same between the diploid and tetraploid Meth-A cells. It was suggested that the decreased proliferation below 35 degrees C of the tetraploid Meth-A cells might be due to the increased volume of the cells. 相似文献
16.
Matsumura Y Byrne SN Nghiem DX Miyahara Y Ullrich SE 《Journal of immunology (Baltimore, Md. : 1950)》2006,177(7):4810-4817
UV exposure suppresses the immune response to a variety of microbial, fungal, and viral Ags. In addition, UV radiation is a complete carcinogen and the immune suppression induced by UV radiation is a major risk factor for skin cancer induction. In this study, we examined the mechanisms underlying the induction of immune suppression and tolerance induction by UV radiation. Transferring lymph nodes cells from UV-irradiated, FITC-sensitized mice into normal recipients transferred immune tolerance. Contrary to expectations, the cell responsible was an FITC(+), IL-10-secreting, CD19(+), B220(+) B cell. Because the lipid mediator of inflammation, platelet-activating factor (PAF) is released by UV-irradiated keratinocytes and is essential for the induction of immune suppression, we determined its role in tolerance induction. When UV-irradiated mice were injected with PCA 4248, a selective PAF receptor (PAFR) antagonist, transfer of tolerance was suppressed. However, immune suppression was not transferred when FITC(+) cells from the draining lymph nodes of UV-irradiated, PAFR-deficient donor mice were injected into the recipients. Because PCA 4248 also blocks serotonin receptor binding, we measured the effect that blocking both serotonin and PAFR binding has on the transfer of immune suppression. Only when both PAF and serotonin binding were blocked could we inhibit tolerance induction. These data identify a novel function for PAF and serotonin in modulating immune function, the activation of immunoregulatory B cells. 相似文献
17.
The effects of ultraviolet light on cellular DNA replication were evaluated in an asynchronous Chinese hamster ovary cell population. BrdUrd incorporation was measured asa function of cell-cycle position, using an antibody against bromodeoxyuridine (BrdUrd) and dual parameter flow cytometric analysis. After exposure to UV light, there was an immediate reduction ( 50%) of BrdUrd incorporation in S phase cells, with most of the cells of the population being affected to a similar degree. At 5 h after UV, a population of cells with increased BrdUrd appeared as cells that were in G1 phase at the time of irradiation entered S phase with apparently increased rates of DNA synthesis. For 8 h after UV exposure, incorporation of BrdUrd by the original S phase cells remained constant, whereas a significant portion of original G1 cells possessed rates of BrdUrd incorporation surpassing even those of control cells. Maturation rates of DNA synthesized immediately before or after exposure by alkaline elution, were similar. Therefore, DNA synthesis measured in the short pulse by anti-BrdUrd fluorescence after exposure to UV light was representative of genomic replication. Anti-BrdUrd measurements after DNA damage provide quantitative and qualitative information of cellular rates of DNA synthesis especially in instances where perturbation of cell-cycle progression is a dominant feature of the damage. In this study, striking differences of subsequent DNA synthesis rates between cells in G1 or S phase at the time of exposure were revealed. 相似文献
18.
Thrombin-induced activation of RhoA in platelet shape change 总被引:4,自引:0,他引:4
Bodie SL Ford I Greaves M Nixon GF 《Biochemical and biophysical research communications》2001,284(1):71-76
19.
A Chinese boy was identified to be suffering from progeria (Hutchinson-Gilford syndrome), the first case of the disease ever reported in China. Cells originating from the patient had a reduced amount of unscheduled DNA synthesis after irradiation with ultraviolet light (UV). The fractions of the progeria cells surviving against UV irradiation measured by colony-forming ability, and the host-cell reactivation capacity of the progeria cells, measured by the plaque formation of UV-irradiated herpes simplex virus were lower than those measured in normal cells. The progeria cells appear to have a reduced capacity to repair UV excision damage. 相似文献
20.
We have studied the effect of UV irradiation on the cell cycle progression of synchronized Chinese hamster ovary cells. Synchronization of cells in S or G2 phase was accomplished by the development of a novel protocol using mimosine, which blocks cell cycle progression at the G1/S boundary. After removal of mimosine, cells proceed synchronously through the S and G2 phases, allowing manipulation of cells at specific points in either phase. Synchronization of cells in G1 was achieved by release of cells after a period of serum starvation. Cells synchronized by these methods were UV irradiated at defined points in G1, S, and G2, and their subsequent progression through the cell cycle was monitored. UV irradiation of G1-synchronized cells caused a dose-dependent delay in entry into S phase. Irradiation of S-phase-synchronized cells inhibited progression through S phase and then resulted in accumulation of cells for a prolonged interval in G2. Apoptosis of a subpopulation of cells during this extended period was noted. UV irradiation of G2-synchronized cells caused a shorter G2 arrest. The arrest itself and its duration were dependent upon the timing (within G2 phase) of the irradiation and the UV dose, respectively. We have thus defined a previously undescribed (in mammalian cells) UV-responsive checkpoint in G2 phase. The implications of these findings with respect to DNA metabolism are discussed. 相似文献