Gene-specific and strand-specific DNA repair in the G1 and G2 phases of the cell cycle.

We have analyzed the fine structure of DNA repair in Chinese hamster ovary (CHO) cells within the G1 and G2 phases of the cell cycle. Repair of inactive regions of the genome has been suggested to increase in the G2 phase of the cell cycle compared with other phases. However, detailed studies of DNA repair in the G2 phase of the cell cycle have been hampered by technical limitations. We have used a novel synchronization protocol (D. K. Orren, L. N. Petersen, and V. A. Bohr, Mol. Cell. Biol. 15:3722-3730, 1995) which permitted detailed studies of the fine structure of DNA repair in G2. CHO cells were synchronized and UV irradiated in G1 or early G2. The rate and extent of removal of cyclobutane pyrimidine dimers from an inactive region of the genome and from both strands of the actively transcribed dihydrofolate reductase (DHFR) gene were examined within each phase. The repair of the transcribed strand of the DHFR gene was efficient in both G1 and G2, with no major differences between the two cell cycle phases. Neither the nontranscribed strand of the DHFR gene nor an inactive region of the genome was repaired in G1 or G2. CHO cells irradiated early in G2 were more resistant to UV irradiation than cells irradiated in late G1. Since we found no major difference in repair rates in G1 and G2, we suggest that G2 resistance can be attributed to the increased time (G2 and G1) available for repair before cells commit to DNA synthesis.     

Synthesis of poly(adenosine diphosphate ribose) in synchronized Chinese hamster cells.

Chinese hamster ovary cells were synchronized by mitotic selection and used to study the relation of poly(adenosine diphosphate ribose) synthesis to DNA synthesis and the different phases of the cell cycle. DNA synthesis was measured in cells rendered permeable to exogenously supplied nucleotides. Poly(ADPR) synthesis was also measured in permeable cells in the presence of both minimum and maximum DNA damage. The maximum DNA damage was produced by treating the cells with saturating concentrations of DNase. As anticipated, the DNA synthesis complex showed its maximum activity during S phase and showed 4–5-fold less activity during the other phases of the cell cycle. The basal level of poly(ADPR) synthesis was elevated during G1, fell to its lowest level during S phase, then increased during G2 and rose to its highest level during G1. The DNase responsive activity of poly(ADPR) synthesis was relatively constant thru the cell cycle but showed a peak at the end of S phase; then the activity decreased during the subsequent G2-M period.     

RNA dependence in the cell cycle of V79 cells

The cell cycle of V79 Chinese hamster lung cells synchronized by hydroxyurea was investigated by flow cytometry. The metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA of V79 cells. Green and red fluorescence from individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry. Periodic changes of cellular DNA and RNA contents were observed over nine cell cycles. The duration of G1, S, and G2 + M phases of synchronized V79 cells whose RNA content was close to that of the cells in balanced growth was 3, 4.5, and 1.5 hours, respectively. The duration of G1 and S phases of cells containing RNA above a certain threshold was inversely proportional to the RNA content. The RNA content of cells containing RNA above the normal level regressed to normal after a few generations. Coefficients of variation for RNA content were significantly larger than those for DNA. An explanation for the decay of synchrony in a synchronized cell population is proposed.     

Enhanced repair of DNA interstrand crosslinks in S phase

Hela cells synchronized in G1 and S phases of the cell cycle were transfected with pEGFP crosslinked with trioxsalen. Twelve hours later the number of fluorescent cells was determined by fluorescent microscopy. Cells in S phase have repaired 0.2-0.3 ICL/kb over the 12h period, while cells in G1 phase repaired interstrand crosslinks much more poorly. The crosslinked plasmids were efficiently recruited to the nuclear matrix both in G1 phase and S-phase, which showed that the poor repair of G1 cells was a result of a lack of DNA replication rather than of a lack of matrix attachment.     

DNA repair endonuclease activity during synchronous growth of diploid human fibroblasts.

DNA-repair endonuclease activity in response to UV-induced DNA damage was quantified in diploid human fibroblasts after synchronizing cell cultures to selected stages of the cell cycle. Incubation of irradiated cells with aphidicolin, an inhibitor of DNA polymerases alpha and delta, delayed the sealing of repair patches and allowed estimation of rates of strand incision by the repair endonuclease. The apparent Vmax for endonucleolytic incision and Km for substrate utilization were determined by Lineweaver-Burk and Eadie-Hofstee analyses. For cells passing through G1, S or G2, Vmax for reparative incision was, respectively, 7.6, 8.4 and 8.4 breaks/10(10) Da per min, suggesting that there was little variation in incision activity during these cell-cycle phases. The Km values of 2.4-3.1 J/m2 for these cells indicate that the nucleotidyl DNA excision-repair pathway operates with maximal effectiveness after low fluences of UV that are in the shoulder region of survival curves. Fibroblasts in mitosis demonstrated a severe attenuation of reparative incision. Rates of incision were 11% of those seen in G2 cells. Disruption of nuclear structure during mitosis may reduce the effective concentration of endonuclease in the vicinity of damaged chromatin. The extreme condensation of chromatin during mitosis also may restrict the accessibility of reparative endonuclease to sites of DNA damage. Confluence-arrested fibroblasts in G0 expressed endonuclease activity with Vmax of 5.5 breaks/10(10) Da per min and a Km of 5.5 J/m2. The greater condensation of chromatin in quiescent cells may restrict the accessibility of endonuclease to dimers and so explain the elevated Km. When fibroblasts were synchronized by serum-deprivation, little variation in reparative endonuclease activity was discerned as released cells transited from early G1 through late G1 and early S. Proliferating fibroblasts in G1 were shown to express comparatively high numbers of reparative incision events in the absence of aphidicolin which was normally used to inhibit DNA polymerases and hold repair patches open. It was calculated that in G0, S and G2 phase cells, single-strand breaks at sites of repair remained open for 30, 19 and 14 sec, respectively. In G1 phase cells, repair sites remained open for 126 sec. Addition of deoxyribonucleosides to G1 cells reduced this time to 42 sec suggesting that the slower rate of synthesis and ligation of repair patches in G1 was due to a relative deficiency of deoxyribonucleotidyl precursors for DNA polymerase.(ABSTRACT TRUNCATED AT 400 WORDS)     

The simulation of thymidine kinase enzyme kinetics in cultured cells using the computer language cellsim

Thymidine kinase is an enzyme that occurs in cells actively synthesizing DNA. In studies of synchronized cell populations, it has been shown that the enzyme activity disappears during the G1 phase of the cell cycle and reappears during the S and G2 phases. Its reappearance is consistent with the synthesis of the mRNA for this enzyme during the S and G2 phases and its immediate translation into active enzyme by the protein synthesis machinery within the cell. The disappearance of the enzyme is consistent with the cessation of mRNA synthesis by mitotic cells. We have now tested this concept by computer simulation of a growing cell population in which a specific mRNA is generated while cells are in the S and G2 phases of the cell cycle. The computer simulation was done using the simulation language Cellsim designed for modeling populations of cells. The Cellsim program which we developed allowed each cell to make about 1 mRNA molecule per min during the S and G2 phases. Every 3 min each mRNA molecule generated a protein enzyme molecule. The mRNA had a half-life of about 9 min, and the enzyme had a half-life of about 150 min. When these molecular parameters were coupled to the cell cycle parameters for Chinese hamster fibroblasts, the resulting curve of enzyme production with time closely matched the observed kinetics of enzyme activity seen in synchronized cells. The only part of the curve that did not fit was the rapid drop in enzyme activity which was seen as the population of mitotic cells was permitted to enter G1. This drop in activity was not seen in mitotic cells blocked with Colcemid where mRNA synthesis must be lacking. Earlier studies have shown that the Gl cells do not contain any inhibitor of enzyme activity. It therefore appears that the enzyme molecule is more unstable during the G1 phase than in any of the other phases of the cell cycle.     

Cell cycle analysis of synchronized chinese hamster cells using bromodeoxyuridine labeling and flow cytometry

Summary Chinese hamster ovary cells were synchronized into purified populations of viable G1-, S-, G2-, and M-phase cells by a combination of methods, including growth arrest, aphidicolin block, cell cycle progression, mitotic shake-off, and centrifugal elutriation. The DNA content and bromodeoxyuridine (BrdUrd) labeling index were measured in each purified fraction by dual-parameter flow cytometry. The cell cycle distributions determined from the DNA measurements alone (single parameter) were compared with those calculated from both DNA and BrdUrd data (dual parameter). The results show that highly purified cells can be obtained using these methods, but the assessed purity depends on the method of cell cycle analysis. Using the single versus dual parameter measurement to determine cell cycle distributions gave similar results for most phases of the cell cycle, except for cells near the transition from G1- to S-phase and S- to G2-phase. There the BrdUrd labeling index determined by flow cytometry was more sensitive for detecting small amounts of DNA synthesis. As an alternative to flow cytometry, a simple method of measuring BrdUrd labeling index on cell smears was used and gave the same result as flow cytometry. Measuring both DNA content and DNA synthesis improves characterization of synchronized cell populations, especially at the transitions in and out of S-phase, when cells are undergoing dramatic shifts in biochemical activity.     

Arrest of irradiated G1, S, or G2 cells at mitosis using nocodazole promotes repair of potentially lethal damage

The ability of synchronized Ehrlich ascites tumor cells, irradiated in G1, S, and G2 phases, to repair potentially lethal damage when arrested at mitosis by using 0.4 microgram/ml nocodazole, a specific inhibitor of microtubule polymerization, has been studied. Cells irradiated in these phases were found to repair potentially lethal damage at mitosis. The extent of this repair was similar to that observed for cells irradiated at the same stages in the cell cycle but allowed to repair potentially lethal damage by incubating in balanced salt solution for 6 hr after X irradiation.     

Increase of uv-induced DNA repair synthesis during blast transformation of human lymphocytes.

UV-induced DNA repair synthesis, measured as unscheduled DNA synthesis, was studied in human peripheral lymphocytes in various phases of the cell cycle. Mitogen transformation of the lymphocytes was effected with phytohemagglutinin (PHA), and the stage in the cell cycle was determined by measuring the Feulgen DNA content and the dry mass in individual cells by cytophotometry. The initial rate of repair was determined by autoradiography after UV-light irradiation (19.2 J/m²) and incubation of the cells for 30 min with [³H]thymidine. When the cells progressed from the G0 to the G1 phase there was a 3-fold increase in the grain count. The correlation between the grain count and the dry mass indicated an increase in the initial rate of repair during the progression of cells from G0 to G2 phase. G2 cells were more heavily labelled than those in G1, but there did not seem to be any difference between these two phases as regards the relationship between grain count and DNA content. The results indicate that the initial rate of UV-induced DNA repair may differ in various phases of the lymphocyte cell cycle.     

Base excision repair of ionizing radiation-induced DNA damage in G1 and G2 cell cycle phases

Background

Major genomic surveillance mechanisms regulated in response to DNA damage exist at the G₁/S and G₂/M checkpoints. It is presumed that these delays provide time for the repair of damaged DNA. Cells have developed multiple DNA repair pathways to protect themselves from different types of DNA damage. Oxidative DNA damage is processed by the base excision repair (BER) pathway. Little is known about the BER of ionizing radiation-induced DNA damage and putative heterogeneity of BER in the cell cycle context. We measured the activities of three BER enzymes throughout the cell cycle to investigate the cell cycle-specific repair of ionizing radiation-induced DNA damage. We further examined BER activities in G2 arrested human cells after exposure to ionizing radiation.

Results

Using an in vitro incision assay involving radiolabeled oligonucleotides with specific DNA lesions, we examined the activities of several BER enzymes in the whole cell extracts prepared from synchronized human HeLa cells irradiated in G1 and G2 phase of the cell cycle. The activities of human endonuclease III (hNTH1), a glycosylase/lyase that removes several damaged bases from DNA including dihydrouracil (DHU), 8-oxoguanine-DNA glycosylase (hOGG1) that recognizes 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) lesion and apurinic/apyrimidinic endonuclease (hAPE1) that acts on abasic sites including synthetic analog furan were examined.

Conclusion

Overall the repair activities of hNTH1 and hAPE1 were higher in the G1 compared to G2 phase of the cell cycle. The percent cleavages of oligonucleotide substrate with furan were greater than substrate with DHU in both G1 and G2 phases. The irradiation of cells enhanced the cleavage of substrates with furan and DHU only in G1 phase. The activity of hOGG1 was much lower and did not vary within the cell cycle. These results demonstrate the cell cycle phase dependence on the BER of ionizing radiation-induced DNA damage. Interestingly no evidence of enhanced BER activities was found in irradiated cells arrested in G2 phase.     

The relation between length of the cell cycle duration and RNA content in HeLa S3 cells

The cell cycle of HeLa S3 cells synchronized by hydroxyurea was investigated by flow cytometry. Metachromatic fluorochrome acridine orange was used to strain the DNA and RNA of the cells differentially. Periodic changes in the cellular DNA and RNA contents were observed through five cell cycles. The G1 and S phases of synchronized HeLa S3 cells that contained large amounts of RNA became shorter than those of cells that contained smaller amounts of RNA.     

Differential response of human fibroblasts to the cytotoxic and mutagenic effects of UV radiation in different phases of the cell cycle

The effects of DNA repair on UV-induced mutagenesis and cell killing in human diploid skin fibroblasts in different phases of the cell cycle were studied. The cells were synchronized in G1 by culturing at 30 degrees C. Using this synchronization method, it could be demonstrated that cells irradiated at 30 degrees C and allowed to carry out excision repair for various lengths of time, show a much lower mutation frequency than cells irradiated in the exponentially growing state. Irradiation in early G1 gives rise to less mutations than irradiation in S. However, the surviving fraction is not decreased when cells are irradiated in S in comparison with irradiation in G1. Moreover, there is no recovery from UV-induced lethal effects when irradiated cells are kept stationary at 30 degrees C for various periods of time. This is in contrast with the results obtained with density-inhibited fibroblasts held at 37 degrees C, which show a recovery from the UV-induced lethal effects.     

Chemical oncogenesis in cultured mouse embryo cells in relation to the cell cycle.

Using the C3H/10T 1/2 CL8 line of mouse embryo fibroblasts and three different methods of obtaining cell cycle synchrony, namely arginine or isoleucine deficiency and release from postconfluence inhibition of growth, a sensitive phase for oncogenic transformation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been found. This sensitive phase is located somewhere between the G1/S boundary and a point 4 hr prior to this marker. Methylation of cellular macromolecules by tritiated MNNG is not cycle-dependent in cells synchronized by arginine deficiency. The capacity of cells to repair DNA single strand breaks produced by MNNG was examined by alkaline sucrose sedimentation analysis in cells synchronized by arginine deficiency and treated with MNNG during phases of the cell cycle sensitive and insensitive to oncogenic transformation. Whereas DNA repair was found to be equally rapid in cells treated just before S phase (I), or just after commencement of DNA synthesis (III), transformation was maximal in I. By contrast, cells treated when blocked by arginine deficiency (II) repaired DNA slowly and were not sensitive to malignant transformation. Cells in I and II, which repaired DNA at very different rates, were equally sensitive to MNNG-induced lethality, while cells in III, which repaired DNA at the same rate as cells in I, suffered greater lethality. Thus, in this system it was concluded that there was no direct correlation between DNA repair, as measured by alkaline sucrose sedimentation analysis of prelabeled DNA, and malignant transformation or lethality produced by MNNG. In preliminary experiments malignant transformation induced by cytosine arabinoside (1-beta-D-arabinofuranosylcytosine, ara-C) has been found to occur mainly in S phase, indicating that diverse chemical oncogens may have different sites of action, or that activation of chemical oncogens is cell cycle-specific for some agents.     

Cell sorting analysis of cell cycle-dependent X-ray sensitivity in end joining-deficient human cells

Non-homologous end joining (NHEJ) plays a major role in the repair of ionizing radiation-induced DNA double-strand breaks (DSBs), especially during the G1-phase of the cell cycle. Using a flow cytometric cell sorter, we fractionated G1- and S/G2-phase cells based on size to assess the DSB-repair activity in NHEJ factor-deficient DT40 and Nalm-6 cell lines. Colony formation assays revealed that the X-ray sensitivities of the G1-enriched populations correctly reflected the DSB-repair activities of both the DT40 and Nalm-6 cell lines. Furthermore, as assessed by γ-H2AX foci formation, the sorted cells exhibited less DNA damage than chemically synchronized cells. Given that it does not use fluorescent labeling or chemical agents, this method of cell sorting is simpler and less toxic than other methods, making it applicable to a variety of cell lines, including those that cannot be synchronized by standard chemical treatments.     

Fluctuation of trypsin-like proteinase activity in the cell cycle of synchronized and growing HeLa cells, and the effect of GMCHA-OPhBut

HeLa cells synchronized by double-thymidine block were grown in Eagle's minimum essential medium supplemented with 10% calf serum, and the fluctuation of trypsin-like protease activity in the cell cycle was examined. Seven distinct activity peaks were observed in one cell cycle at a cell density of 2%: two peaks in S phase, one peak at the S/G2 boundary, one peak in early M phase and one at the M/G1 boundary, and two peaks in G1 phase. HeLa cells synchronized by a mitotic detachment technique also showed similar results at cell density of 4.8%. The appearance of trypsin-like proteinase activity in the cell cycle was markedly affected by cell density, and no definite peak was observed above 8%. trans-Guanidinomethylcyclohexanecarboxylic and 4-tert-butylphenyl ester (GMCHA-OPhBut), a specific inhibitor for trypsin and a strong inhibitor of HeLa cell growth, had no effect on the various events in the first S, G2 and M phases, such as the incorporation of [methyl-3H]thymidine into DNA, the increase in the cell concentration, and the appearance of trypsin-like proteinase activity, whereas it retarded the onset of the second S phase and the various events in the second S, G2 and M phases for 3 h. In particular, it induced the appearance of a new proteinase peak at the G1/S boundary.     

Cell proliferation and cell cycle dependent changes in the methylthioadenosine phosphorylase activity in mammalian cells

The objective of this study is to investigate the activity of methylthioadenosine phosphorylase (MTA-Pase) in mammalian cells stimulated by serum to proliferate and during their cell cycle. A direct correlation between growth rate and MTA-Pase activity in chinese hamster ovary (CHO) cells was observed. High MTA-Pase activity was observed during the exponential growth phase followed by a low enzyme activity during plateau phase of growth. To understand whether the fluctuations in the enzyme activity was cell cycle dependent, initially the activity of MTA-Pase was studied in plateau phase (G0) CHO cells as they synchronously go into S phase upon plating in fresh medium. The MTA-Pase activity in G0 cells before initiation of growth was 10.3 n.mol/mg protein/30'. A peak activity of 16.0 n.mol/mg/30 min was found at 12 hr after stimulation of proliferation by serum. These results indicate a peak MTA-Pase activity between 10-12 hr after stimulation of proliferation coinciding with the initiation of DNA synthesis. The activity of the enzyme slowly decreased as the cells completed their DNA synthesis. To understand whether these fluctuations are cell cycle specific, HeLa cells were synchronized in different phases and MTA-Pase activity was studied. The specific activities of the enzyme were 2.76, 2.99, 3.97, 3.28 and 3.65 n.moles/mg/30 min. in mitosis, early G1, late G1, S and G2 phases of the cell cycle respectively. These results indicate that MTA-Pase activity peaks in late G1 phase before the initiation of DNA synthesis, similar to the polyamine biosynthetic enzymes and might play a role in the initiation of DNA synthesis by salvage of adenine into nucleotide pools.     

Possible Involvement of Glutathione S-Transferases in the Cell Growth of C6 Astroglioma Cells

The changes of glutathione S-transferase activity were investigated using rat brain astroglioma C6 cells that were synchronized at different phases of the cell cycle. The enzyme showed two significant activity peaks at G2 and G1 phases. Furthermore, when C6 glioma cells were exposed to a culture medium supplemented with specific glutathione S-transferase inhibitors, ethacrynic acid and caffeic acid, cell growth was remarkably suppressed. These results suggest that glutathione S-transferases may be closely related to the mechanism of cell proliferation.     

Cell-cycle phase-dependence of drug-induced cycle progression delay.

The effect of adriamycin on cell cycle phase progression of CHO cells synchronized into the various phases of the cell cycle by elutriation was investigated by high resolution pulse cytophotometry. Cells treated in all phases of the cell cycle showed delay in their subsequent progression. In addition to the wellknown block of cells in the G2-phase, a delay in passage of cells from G1 to S and a decreased rate of transit through the S-phase were observed. A broadening of the DNA distributions of the treated cells was observed after cell division indicating induction of chromosomal abnormalities.