Daughter DNA synthesis in UV-resistant and UV-sensitive Chinese hamster clones cells under UV irradiation

By means of ultracentrifugation in alkaline sucrose gradients it has been shown that the size of DNA fragments synthesized in Chinese hamster cells of UV-sensitive clone (CHS-1) after exposure to UV light was equal to the distance between pyrimidine dimers in the parental DNA determined using endonuclease of Micrococcus luteus. With the UV-resistant clone (V-79), the length of fragments of the newly synthesized DNA was much longer than that between pyrimidine dimers in the parental DNA. The data obtained support the model according which DNA synthesis on the UV-irradiated template gives rise to gaps opposite to pyrimidine dimers.     

Hypermutability of a UV-sensitive aphidicolin-resistant mutant of Chinese hamster fibroblasts

An ultraviolet light (UV)-sensitive thymidine auxotroph of Chinese hamster V79 cells that exhibits pleiotropic effects such as a high level of deoxycytidine triphosphate, slow growth, sensitivity to cytidine, and high frequencies of site-specific bromodeoxyuridine-dependent chromosomal aberrations was selected by its resistance to aphidicolin. The UV-induced mutability of this mutant and one of its revertants, which retains some of the phenotypes listed above, was studied in 3 mutation assay systems. The results showed that the mutant was hypermutable for ouabain and diphtheria-toxin-resistant mutations compared to wild-type V79 cells at the same UV dose or the same survival level. The mutant exhibits a delayed expression of maximal frequency of induced 6-thioguanine-resistant mutants. When maximal frequencies are compared at the same UV dose, the mutant also has higher mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase locus. The revertant was similar to the wild-type in UV sensitivity and mutability. Our results showed that UV sensitivity and hypermutability are correlated and controlled by a single gene. Thymidine auxotrophy or nucleotide pool imbalance apparently did not contribute to the UV sensitivity and mutability of the mutant.     

Defect in the caffeine-dependent postreplication DNA repair in UV-sensitive Chinese hamster clone cells

The postreplication repair of DNA in the presence of caffeine was investigated in the Chinese hamster clones cells of different UV-sensitivity. Caffeine (10(-2)M) inhibits the repair of daughter DNA (PRR of DNA) in the UV-light irradiated cells of UV-resistant clones CHO-K1, 14-2C-1 and V79, but does not influence the PRR of DNA in cells of UV-sensitive clones CHS1 and CHS2. Thus, deficiency of PRR of DNA in cells of UV-sensitive clones (the repair of daughter DNA is significantly retarded) is associated with the defect of the caffeine-dependent component of this repair process.     

Over-replication of DNA in S phase Chinese hamster ovary cells after DNA synthesis inhibition

Agents that inhibit DNA synthesis increase the frequency of methotrexate resistance and gene amplification in cultured mammalian cells. Chinese hamster ovary cells blocked with hydroxyurea rereplicated dihydrofolate reductase gene sequences within a single cell cycle upon release from the block (Mariani, B.D., and Schimke, R.T. (1984) J. Biol. Chem. 259, 1901-1910). Perturbation of DNA synthesis was postulated to result in misfiring of replicon initiation, subsequent over-replication of DNA sequences, and amplification of specific genes. To test this hypothesis, we have exposed Chinese hamster ovary cells pulsed with bromodeoxyuridine to three agents that inhibit DNA synthesis and enhance gene amplification: UV irradiation, hydroxyurea, and aphidicolin. After release from the block, the progression of cells throughout the cell cycle was analyzed by flow cytometry through simultaneous measurement of total cellular DNA content and bromodeoxyuridine-labeled DNA. Although the cell cycle effects varied depending on the agent used for the block, in all cases a subset of cells that were in S phase at the time of the block exhibited DNA histograms with greater than 4C DNA content at various times after release and prior to cell division. Cells with the excess DNA were approximately 10-fold more resistant to methotrexate compared to treated cells with normal DNA content or untreated cells. Therefore, cells in S phase at the time of the block produce excess DNA per cell prior to division, and this over-replicated DNA may be relevant to gene amplification and drug resistance.     

Delayed inhibition of subchromosomal DNA synthesis in V-79 Chinese hamster cells after gamma irradiation

Existence of a substantial fraction of replicon initiation events refractory to the effects of X irradiation in Chinese hamster cells has been reported by several laboratories. The work reported here examined whether this apparently refractive fraction resulted from a delayed inhibition of initiation events. Data obtained from velocity sedimentation studies indicated that the extent of inhibition increased over the first hour after irradiation from 35% inhibition immediately following exposure to 3 kR to 75% inhibition of initiation 1 hr after irradiation. Analysis of subsequent recovery of initiation radiosensitivity was performed using DNA fiber autoradiograms prepared from cells incubated up to 4 hr between 2-kR exposures. The data from these experiments indicated that some recovery occurs within 1 hr of irradiation and thus separation of the inhibition and recovery processes in V-79 cells may not be feasible.     

Continued initiation of DNA synthesis in arginine-deprived Chinese hamster ovary cells

When exponentially growing CHO cells were deprived of arginine (Arg), cell multiplication ceased after 12 h, but initiation of DNA synthesis continued: after 48 h of starvation with continuous [3H]thymidine exposure, 85% of the population had incorporated label, as detected autoradiographically. Consideration of the distribution of exponential cells in the various cell cycle phases leads to a calculation that most cells in G1 at the time that Arg was removed, as well as those in S, engaged in some DNA synthesis during starvation. In contrast, isoleucine (Ile)-starved cells did not initiate DNA synthesis, as has been reported by others. Experiments with cells synchronized by mitotic selection confirmed this difference in Arg- and Ile- deprived behavior, but also showed that cells which underwent the mitosis leads to G1 transition during Arg starvation remained arrested in G1 (G0?). The results suggest that Arg-deprived cells continue to maintain some proliferative function(s) while Ile-deprived cells do not.     

DNA synthesis inEscherichia coli B/r after UV-irradiation

When studying the kinetics of DNA synthesis, growth and cell division inEscherichia coli B/r after irradiation with different doses of UV-radiation (254 nm) we could demonstrate, by means of pulse incorporation of³H-thymidine, a lag in DNA synthesis after the irradiation. The relative rate of the restored DNA synthesis (related to the number of viable cells) was higher than in the non-irradiated culture. After 3 h the rate of DNA synthesis settled at a constant value, which was identical with the control rate up to the “critical dose” of 20 J/m². The irradiated cell population is heterogenous and contains basically two categories of cells — surviving and non-surviving. Cells of both types contribute to DNA synthesis restored after the lag period to a different extent. During the first hour after the irradiation even the nonviable portion of the population,i.e. cells that do not form colonies but are still penicillin-sensitive, is involved in the DNA synthesis.     

Identification of a new seventh complementation group of UV-sensitive mutants in Chinese hamster cells

The UV-sensitive mutant V-B11, isolated from the V79 Chinese hamster cell line (Zdzienicka and Simons, 1987) was further characterized. V-B11 has a slightly increased cross-sensitivity to 3me4NQO, whereas no increased sensitivity towards 4NQO was observed. A slightly increased sensitivity towards EMS and MMS was also found. The mutant shows a defect in the ability to perform the incision step of nucleotide-excision repair after UV irradiation: 2 h after UV exposure, the accumulation of incision breaks in V-B11, in the presence of HU and araC, was about 30% of that found in wild-type V79 cells. V-B11 was crossed to a panel of 6 UV-sensitive Chinese hamster ovary (CHO) cells, which represents all the previously identified 6 complementation groups of UV-sensitive Chinese hamster mutants. Since in all crosses complementation has been observed, V-B11 appears to be the first mutant of a new, 7th, complementation group.     

Glycoprotein synthesis in a temperature-sensitive Chinese hamster cell cycle mutant

A temperature-sensitive mutant of Chinese hamster cells is described which has two interesting properties: (1) it is a cell cycle mutant and (2) glycoprotein synthesis appears to be affected at the at the non-permissive temerature (40degreesC). Synchronized cells shifed to 40degreesC in the beginning of their G1 phase do not incorporate [3H]-thymidine into DNA during the expected S-phase, but once DNA synthesis has been initiated ( approximately 10 hours after termination of serum starvation) a shift to 40 degrees C no longer leads to an arrest of DNA synthesis. Flow microfluorimetric analysis of DNA content/cell supports this conclusion and indicates that a majority of cells become arrested in the G1 phase of the cell cycle when a non-synchronized population of cells is transferred to 40degreesC. Apparently at all times in the cell cycle there is a drastic reduction if incorporation of labeled sugars (particularly fucose) into glycoproteins. The uptake of fucose and its conversion to GDP-fucose appears to be normal at 40degreesC. Chromatographic analysis indicates that all classes of glycoproteins are affected, and we do not find any evidence for partially completed oligosaccharides at 40 degrees C. Overall protein synthesis is not reduced at he nonpermissive temperature during the time interval under consideration and the number of polysomes attached to membranes (RER) is also normal at 40degreesC. This suggests that the defect is at an early step in the synthesis or regulation of synthesis of glycoproteins. The mutation is a recessive mutation in hybrid cells and mutagen induced revertants can be obtained which grow normally at 40degreesC and in which glycoprotein synthesis at 40 degrees C is restored to normal, wild type levels.     

Distribution of UV-induced unscheduled DNA synthesis in metaphase chromosomes of Chinese hamster cells

Unscheduled DNA synthesis (USD) occurred in metaphase chromosomes of cultured Chinese hamster cells after ultraviolet light (UV) irradiation. When the chromosomes were labeled by UV-induced USD in metaphase, the number of grains was in proportion to the amount of chromosomal DNA and the grain densities were approximately equal all over the segments of chromosomes.     

Cellular and genetic studies in three UV-sensitive Chinese hamster mutants

Three UV sensitive (UV^s) mutants (CHO43RO, CHO423PV, CHO30PV), characterized by different levels of reduction in their ability to perform unscheduled DNA synthesis (UDS), were analysed for spontaneous and UV-induced frequency of chromosomal aberrations and for sensitivity to alkylating agents. The baseline frequency of chromosomal aberrations was in the normal range, whereas after UV irradiation a positive correlation between the degree of UV sensitivity and the rate of chromosomal breakage was observed. Survival experiments after mutagen exposure indicated that the UV^s clones are characterized by different levels of hypersensitivity to bifunctional alkylating agents whereas the sensitivity to monofunctional alkylating agents is in the normal range. Genetic analysis performed by measuring the survival after UV in hybrids produced by fusing UV^s cells with wild-type or UV^s cells belonging to the six Chinese hamster complementation groups, indicated that the three clones carry recessive mutations and belong to c.g. 2. These findings suggest that defects in the same gene may result in different degrees of phenotypic alterations.Abbreviations CG complementation group - EMS ethyl methane sulfonate - MMS methyl methane sulfonate - MMC mitomycin C - UV ultraviolet - UDS unscheduled DNA synthesis     

Studies on temperature-sensitive mutants of Chinese hamster ovary cells affected in DNA synthesis

DNA synthesis in two mutants of Chinese hamster overy cells, ts 13A and ts 15C, which were temperature sensitive for growth, was found to be shut off rapidly at the nonpermissive temperature. The mutants did not complement each other and the ts lesion was not located on the X chromosome. Both isolates were found to be considerably more sensitive to the alkylating agents, ethylmethanesulfonate (EMS) and methylmethanesulfonate (MMS), as compared to the parental cells, but showed normal sensitivity to UV irradiation. The mutants also showed interesting differences in their response to EMS-induced mutation frequencies at the ouabain-resistant and thioguanine-resistant loci. At high survival (50%) the frequencies of mutations at these genetic loci were markedly low in the ts mutants as compared to the parental cells. In ts+ revertants isolated from the mutants, the ts phenotype and the increased sensitivity to EMS and MMS were affected simultaneously, indicating that both these characteristics resulted from a single genetic lesion.     

Rate and time of DNA synthesis of individual Chinese hamster cells.

The duration of DNA synthesis of a diploid cell line of Chinese hamster fibroblasts was determined in a comparative study by the FLM technique, and also by a new technique for measuring the rate of DNA synthesis of individual cells. These methods produced comparable results when applied during exponential growth of the cells. The rate of DNA synthesis was measured by means of quantitative autoradiography following a short-term incubation of the cells with 5 X 10(-6) M FUdR and 10(-5) M 14C-TdR. The choice of the medium for this purpose did not seem to be critical. The autoradiographic silver grains over cells and 14C-standard sources are counted by microphotometry using incident light bright-field. The direct measurements of DNA synthesis rate are 'compartment' statistics which have been converted into 'flux' parameters for comparison with the FLM method and applicability in cell-kinetic calculations. Frequency distributions of the rate of DNA synthesis of individual cells thus obtained may resemble normal distributions quite closely. They result from several factors: differences in the rate of synthesis in different parts of the S-phase, the density distribution of cells within the S-phase, the variation in the time of DNA synthesis among individual cells, and the experimental error. In the case of a pronounced partial synchronization as probably has been present in one experiment performed in the lag phase, an incorrect time of DNA synthesis may result from the rate values. Due to the variation in DNA synthesis rate in different parts of the S-phase it is not possible to determine the duration of DNA synthesis of an individual cell. However, the mean values of DNA synthesis time are reliable. The new method will be preferentially applied for determining the duration of DNA synthesis of human cells in as far as difficulties are encountered with the classical methods. In addition, it may be used to advantage for studying cells which make up low percentages in mixed populations. It finally permits a safer morphological classification of the cells under study than is possible with the classical methods.     

Recovery from effects of heat on DNA synthesis in Chinese hamster ovary cells

The hyperthermic inhibition of cellular DNA synthesis, i.e., reduction in replicon initiation and delay in DNA chain elongation, was previously postulated to be involved in the induction of chromosomal aberrations believed to be largely responsible for killing S-phase cells. Utilizing asynchronous Chinese hamster ovary cells heated for 15 min at 45.5 degrees C, an increase in single-stranded regions in replicating DNA (as measured by BND-cellulose chromatography) persisted in heated cells for as long as replicon initiation was affected. Alkaline sucrose gradient analyses of cells pulse-labeled immediately after heating with [3H]thymidine and subsequently chased at 37 degrees C revealed that these S-phase cells can eventually complete elongation of the replicons in operation at the time of heating, but required about six times as long relative to control cells which completed replicon elongation within 4 h. DNA chain elongation into multicluster-sized molecules was prevented for up to 18 h in these heated cells, resulting in a buildup of cluster-sized molecules (approximately 120-160 S) mainly because of the long-term heat damage to the replicon initiation process. Utilizing bromodeoxyuridine (BrdU)-propidium iodide bivariate analysis on a flow cytometer to measure cell progression, control cells pulsed with BrdU and chased in unlabeled medium progressed through S and G2M with cell division starting after 2 h of chase time. In contrast, the majority of the heated S-phase cells progressed slowly and remained blocked in S phase for about 18 h before cell division was observed after 24 h postheat. Our findings suggest that possible sites for where the chromosomal aberrations may be occurring in heated S-phase cells are either (1) at the persistent single-stranded DNA regions or (2) at the regions between clusters of replicons, because this long-term heat damage to the DNA replication process might lead to many opportunities for abnormal DNA and/or protein exchanges to occur at these two sites.     

Normal DNA ligase activity in a gamma-ray-sensitive Chinese hamster mutant

A Chinese hamster cell mutant (XR-1) was previously described that is extremely deficient in the repair of double-strand DNA breaks produced by gamma-irradiation during the sensitive G1--early-S period and somewhat deficient in repair of gamma-ray-induced single-strand DNA breaks. To determine whether a deficiency in DNA ligase activity might underlie the biochemical defect, protein extracts from mutant and parental cells were examined for their ability to ligate single- and double-strand breaks in DNA. The kinetics of ligation of single 5'-phosphate-3'-hydroxyl breaks in double-stranded DNA were the same in protein extracts from both cells. After separation of protein extracts by gel-filtration chromatography, the percentage of activity in the large and small molecular forms of DNA ligase was also similar in the two cells. Finally, protein extracts prepared from exponentially growing or G1-synchronized mutant and parental cells were equal in their ability to ligate blunt-end DNA substrates. These data suggest that a deficiency in DNA ligase is not the cause of the repair defect in the XR-1 mutant cell.     

DNA repair in a UV-sensitive mutant of of mouse cell line

A UV-sensitive mutant, Q31, isolated from mouse-lymphoma L5178Y cells, was studied for excision and post-replication rerpairs. A nearly equal number of UV endonuclease-sensitive sites was induced by UV in L5178Y, Q31, and human Raji cells. L5178Y cells irradiated with 10 J/m² removed 18% of sensitive sites from DNA during incubation for 24 h, and Q31 cells removed 3% of the sites, a fraction less than the limit of detection, whereas Raji cells eliminated about 60% of the sites. These results indicate that mouse-lymphoma cells are capable of excision repair to a limited extend as compared with human cells and that mutant Q31 cells are essentially devoid of dimer excision. The newly synthesized DNA was of smaller size in UV-irradiated and unirradiated Q31 cells than that in the corresponding L5178Y cells, but the DNAs in both strains increased to comparable sizes after a 2-h chase.     

Reproductive death of Chinese hamster V79 cells after exposure to chemical inhibitors of DNA synthesis.

1. The results of this study have contributed to the definition of three categories of chemical inhibitors of DNA replication in mammalian cells. 2. Inhibitors of replicon cluster initiation [4-nitroquinoline-N-oxide (4-NQO), etoposide (VP-16), teniposide (VM-26), amsacrine (m-AMSA), N-methyl-N'-nitro-N-nitrozoguanidine (MNNG), cis-Pt(II)diammine dichloride (cis-PDD)], which needed similar doses to produce a slow and persistent (up to 4 hr) inhibition of DNA synthesis, followed by significant cell killing. 3. Inhibitors of DNA replication by indirect action [3-aminobenzamide [correction of 3-aminobezamide] (3-AB), cycloheximide (CHX), puromycin (PRC), bisbenzimide Hoechst No. 33258 (H-33258]), that showed reduced cytotoxic effects, and caused a slow (60 min) and reversible inhibition of DNA synthesis. 4. Inhibitors of formation and/or polymerization of deoxyribonucleotides [5-aminouracil (5-AU), bisbenzimide Hoechst No. 33342 (H-33342)], which induced a fast (20 min) and reversible suppression of DNA replication, associated with limited cell killing.     

Subchromosomal DNA synthesis in synchronous V-79 chinese hamster cells after X irradiation

A substantial fraction of replicon initiation events in Chinese hamster V-79 cells have been shown to be refractory to the effects of X irradiation immediately after exposure. This study examines the possibility that the initiation radiorefractive portion is the result of changes in replicon radiosensitivity as a function of position in S phase. The data obtained from DNA fiber autoradiograms and kinetic incorporation of radiolabeled thymidine from cells irradiated at various positions in S phase showed only slight changes in the proportion of replicons refractive to X irradiation immediately after exposure. These results indicate that initiation radiorefractive replicons may be an intrinsic property of V-79 cells and that cell-cycle-specific heterogeneity in radiation response cannot fully account for this phenomenon. The results also indicate that delayed inhibition of initiation events may play a larger role in the observed radiorefractive fraction than previously thought.