Caffeine-induced modulation of the lethal action of X rays on Chinese hamster V79 cells

Caffeine-mediated enhancement of the killing of V79 cells by 220-kV X rays at various times in the cell cycle was compared with that of HeLa cells by measuring (i) the dependence of cell survival on the duration of treatment with 5-10 mM caffeine, (ii) the effect of caffeine treatment on the X-ray dose-survival curve, and (iii) the loss of sensitivity to caffeine as a function of time after irradiation. The behavior of V79, while similar in many respects to that of HeLa (reported previously), differs in several ways. Caffeine treatment causes rapid killing immediately after irradiation irrespective of cell age, while HeLa is refractory in S phase and highly sensitive in G2. As with HeLa, the (multitarget) dose-survival curve parameters are reduced by caffeine treatment, but the age-dependent fluctuations in D0 are not eliminated as completely as with HeLa and the extrapolation number assumes values less than unity in the latter part of the cycle rather than in the early part. Loss of sensitivity to caffeine after irradiation early in the cycle appears to undergo a transient reversal in the middle of the cycle, a phenomenon not observed in HeLa.     

Mutagenesis in cultured mammalian cells. II. Induction of gene mutations in Chinese hamster cells

Mutations controlling the resistance to 6-mercaptopurine (6-M) and the ability to multiply in a medium with a low concentration of glucose (“glucose-independent” mutants) were induced in cultured Chinese hamster cells by N-nitrosomethylurea (NMU), 5-bromodeoxyuridine (BUdR), UV and X-rays. The chemical agents were found to be very active in induction of mutations to 6-M resistance (NMU and BUdR) and mutations of “glucose independence” (NMU). These agents increase the yield of mutations as compared to the spontaneous mutation rate by about two orders of magnitude. The induced rate of 6-M-resistant mutations by X-rays was 2.0 ? 10⁻⁷ per viable cell per roentgen. BUdR approximately equally increases the cell's sensitivity to both inactivating and mutagenic action of X-rays. The maximum induction of mutations to 6-M resistance by UV was observed at 100 erg/mm². This dose leads to 1 16-fold increase of the mutation frequency as compared to the spontaneous rate. Further increase of the UV dose up to 200 erg/mm² resulted in a lower yield of mutations per dose unit. The highest yield of mutations to 6-M resistance induced by NMU, BUdR and X-rays was observed if cells were plated in selective medium several generations after the mutagenic treatment. The maximum yield of mutations to 6-M resistance induced by UV and of glucose-independence induced by NMU was recorded if cells were transferred to selective media immediately after treatment. The kinetics of expression of mutations and the decline of their number observed after prolonged incubation of treated cells in nonselective conditions are discussed.     

Characterization of ultraviolet light-induced ouabain-resistant mutations in Chinese hamster cells.

Ouabain-resistant mutations in Chinese hamster cells have been quantitatively characterized. The mutation frequencies were found to be induced curvilinearly with treatments of increasing doses of ultraviolet light (UV). For the range of UV doses tested (5--20 J/m(2)), the observed frequency, Y, as a function of UV dose X, follows a curvilinear function, Y = (-28 + 13.37 X--1.52X(2) + 0.08X(3)) . 10(-6). The frequencies of UV-induced mutations were directly correlated with cell survival, indicating a similar causal relationship between cell killing and mutation induction. Under the same experimental conditions, X-rays induced 6--thioguanine-, but not ouabain-, resistant mutations. UV-induced ouabain-resistant (ouar) mutants exhibit a selection disadvantage. Their phenotypic expressions are modifiable by various agents. Wild type and 16 ouar mutants were compared with respect to their sensitivity to ouabain inhibition of 86Rb uptake by whole cells. All the ouar mutants assayed are less sensitive to the drug than are wild-type cells. In the absence of ouabain, the Na+--K+--ATPase activities can be significantly higher or lower than that of the wild-type cells.     

Effects of ultraviolet irradiation on the rate and sequence of DNA replication in synchronized Chinese hamster cells.

The effects of ultraviolet light (UV) irradiation on the rate of DNA replication in synchronized Chinese hamster ovary (CHO) cells were investigated. A technique for measuring semiconservative DNA replication was employed that involved growing the cells in medium containing 5-bromodeoxyuridine and subsequently determining the amount of DNA that acquired hybrid buoyant density in CsCl density gradients. One of the advantages of this technique was that it allowed a characterization of the extent of DNA replication as well as rate after irradiation. It was found that while there was a dose-dependent reduction in the rate of DNA replication following UV-irradiation, doses of up to 10 J/m2 (which produce many dimers per replication) did not prevent the ultimate replication of the entire genome. Hence, we conclude that dimers cannot be absolute blocks to DNA replication. In order to account for the total genome replication observed, a mechanism must exist that allows genome replication between dimers. The degree of reduction in the rate of replication by UV was the same whether the cells were irradiated at the G1-S boundary or 1 h into S-phase. Previous work had shown that cells in early S-phase are considerably more sensitive to UV than cells at the G1-S boundary. Experiments specifically designed to test for reiterative replication showed that UV does not induce a second round of DNA replication within the same S-phase.     

Messenger RNA synthesis in synchronized Chinese hamster ovary cells

Chinese hamster ovary cells were synchronized without inhibitors by mitotic selection and labelled in G1, S or G2 phase by incubation for 90 min with [3H]- OR [14C]uridine. Purified polyribosomes were extracted with phenol and the polyadenylated mRNA prepared by poly(U)-Sepharose chromatography. Poly-adenylated [3H]uridine-labelled mRNA from the G1 phase of the cell cycle was compared by exponential polyacrylamide gel electrophoresis in formamide with [14C] uridine-labelled polyadenylated nRNA from the S or G2 phase. The electrophoretic patterns obtained correspond to the size range expected for mRNA (7-28 S). No prominent differences were detected between mRNAs synthesized in different phases of the cell cycle. From these data we conclude that the major size classes of polyribosomal poly(A)-containing mRNA are synthesized in equal ratios throughout the cell cycle.     

The nature of ultraviolet light-induced mutations at the heterozygous aprt locus in Chinese hamster ovary cells

A system for studying mutational specificity at a heterozygous locus in Chinese hamster ovary (CHO) cells is described. The strategy employed is based on restriction fragment analysis and DNA sequencing of enzymatically amplified mutant adenine phosphoribosyltransferase (aprt) alleles. We have demonstrated the usefulness of this approach through the characterization of a collection of aprt- mutants with respect to the role played by loss of heterozygosity events in ultraviolet light (UV) induced mutagenesis. A similar strategy has also been applied to speculate on the identity of the premutational lesion responsible for a UV-induced mutational hotspot at the aprt locus.     

Antimutagenic action of cobaltous chloride on radiation-induced mutations in cultured Chinese hamster cells

The effects of cobaltous chloride on 8-azaguanine (8AG)-resistant mutations induced by gamma-rays or ultraviolet (UV) light in cultured Chinese hamster V79 cells were examined. Cobaltous chloride alone had no significant effects on survival and mutations of V79 cells at concentrations less than 1 x 10(-5) M. Cobaltous chloride at a concentration of 3 x 10(-6) M had a marked effect in reducing 8AG-resistant mutations induced by gamma-rays of 2-6 Gy, when cells were incubated for 6-7 days in the presence of cobaltous chloride after gamma-ray irradiation (posttreatment). The pretreatment of cells with cobaltous chloride for 6 days before gamma-ray irradiation reduced 8AG-resistant mutations induced by gamma-rays. Pre- or post-treatment with cobaltous chloride had no such effect on UV-induced mutations, however. The difference in responsiveness to cobaltous chloride between bacterial and mammalian cell systems is discussed.     

Effect of neon ions on synchronized Chinese hamster cells

The variation in radiosensitivity across the cell cycle after exposure to neon ions and 60Co gamma-rays is reported for cultured hamster cells. The cells were first synchronized by mitotic selection, then resynchronized in the region of the G1/S boundary by treatment with 10(-3)M hydroxyurea. Although the use of hydroxyurea improves the synchrony, it does sensitize cells at the G1/S boundary to some degree. The cells were exposed at the plateau and the distal peak position of a neon ion beam modified by a 10 cm wide ridge filter. The results indicate that the variation (ratio of maximum to minimum survival after fixed doses of radiation that are approximately matched to produce similar cell killing) was approximately 80 to 100-fold for 60Co gamma-rays and neon ions at the plateau, and 25-fold for distal peak neon ions. While the r.b.e. of distal peak neon ions decreased rapidly with increasing dose for cells in late S-phase, the r.b.e. is independent of dose for cells at the G1/S boundary.     

Tyrosyltubulin ligase and colchicine binding activity in synchronized Chinese hamster cells

Tyrosyltubulin ligase (TTL) was found to be present in CHO and V79 Chinese hamster cells grown in tissue culture. The enzyme is soluble and requires potassium, magnesium, and ATP for maximum activity and requires tubulin as a substrate. TTL was analyzed through the cell cycle of V79 and CHO Chinese hamster cells. The enzyme showed two peaks of activity in V79 cells at 4 h and 7 h after mitotic selection, corresponding to the early S and mid to late S phases of the cell cycle. In CHO cells the enzyme displayed a major peak of activity at mid S and a minor peak or plateau during early S. Tubulin, as measured by (3H)colchicine binding, was shown to increase through S phase and reach a maximum late in the cycle during G2 approx. 3 h after maximum TTL activity.     

Post-treatment with caffeine and the induction of gene mutations by ultraviolet irradiation and ethyl methanesulphonate in V-79 Chinese hamster cells in culture.

The effect of caffeine on V-79 Chinese hamster cells after ultraviolet irradiation or treated with ethyl methanesulphonate was investigated. Caffeine strongly potentiated the killing of both agents, but it had no effect on the induction of mutations at the hypoxanthine-guanine phosphoribosyl transferase locus. The results are consistent with the notion that caffeine slows down an error-prone post-replicative repair mechanism without changing the mutation frequency.     

Kinetics of micronucleus expression in synchronized irradiated Chinese hamster ovary cells

The kinetics of expression of radiation-induced micronuclei (MN) in synchronized Chinese hamster cells (CHO) was examined. The purpose of the study was to determine if the cell cycle distribution of a population significantly influences the levels of radiation induced MN, thereby obscuring the exact quantification of the radiation effect. Cells were synchronized by centrifugal elutriation, irradiated, and then different phases of the cell cycle were examined for: cell cycle progression, division probability, and temporal expression of MN. The results demonstrate that the time interval for maximal MN expression is long enough that the position of cells in the cell cycle and radiation induced division delays do not prevent the majority of cells from completing their first post-irradiation mitosis, therefore, expressing MN. By following the progression of synchronized cell populations by flow cytometry and also examining the time of division of individual cells for 24 hr after irradiation, we observed that the maximum number of cells from all phases of the cell cycle are in their first post-irradiation interphase at that time, thus explaining the MN results.     

X-ray sensitivity of synchronized Chinese hamster cells irradiated during hypoxia

Synchronized Chinese hamster cells were irradiated in air and in nitrogen at various points in the cell cycle. The irradiations were carried out after flushing with air or nitrogen with the medium removed from the mono-layer of cells. Under these conditions the dose-modifying factor, or oxygen enhancement ratio, was between 2.0 and 2.3 for survival in asynchronous cells. The variation in x-ray sensitivity evident as the cell progresses through its cycle was not differentially affected by its state of oxygenation at the time of irradiation. The x-ray age-response curves for irradiation in air and in nitrogen were similar at each point, except for the dose-modifying factor. This was true not only for the cells of a normal short generation time (10 hours) subline of the V79 line but also for a longer generation time (with longer GC period) subline derived from a "small colony". The variation in radiosensitivity as the cell progresses through its cycle must therefore be due to factors other than change in oxygen tension within the cell. The fact that the same variation in x-ray sensitivity with age exists for hypoxic cells as for well-oxygenated cells has a bearing on the radiotherapy of tumors which contain cells at low oxygen tensions.     

Induction of mutations resistant to 6-thioguanine by fast neutrons in cultured Chinese hamster cells

A study was made of induction of mutations, resistant to 6-thioguanine (TGr), and reproductive death of Chinese hamster cells after irradiation by fission-spectrum fast neutrons (mean energy of 0.75 MeV) with doses of 10-130 cGy. A high relative biological effectiveness (RBE) of fast neutrons was shown. The maximum RBE values (13-16) were within the dose range inducing minimum mutagenic and lethal effects. RBE decreased with the dose increase. Inspite of high mutagenic effectiveness of neutrons, estimated according to TGr mutation frequency per cell per dose unit, their relative mutagenic effectiveness, estimated per cell per one lethal event, did not substantially differ from that of X-radiation.     

Novel point mutations in mitochondrial 16S rRNA gene of Chinese hamster cells

To know the nature and mechanisms of spontaneous mutations in mitochondrial DNA (mtDNA), we determined, by direct cycle sequencing, the nucleotide sequence of the 3' terminal region of the mitochondrial 16S rRNA gene from chloramphenicol-resistant (CAP-R) mutants isolated in Chinese hamster V79 cells. Four different base substitutions were identified in common for the six CAP-R mutants. All mutations were heteroplasmic. One A to G transition was mapped at a site within the putative peptidyl transferase domain, the target region for chloramphenicol, and one G to A transition and two T to G transversions were located within the two different segments which form the stems of the hairpin loop structures attached to this key domain in the predicted secondary structure of 16S rRNA. The mutations detected in this study do not map to the same sites where CAP-R mutations were found previously in mammalian cells. Allele specific-PCR analyses revealed that all four mutations occurred on a single mutant-DNA molecule, but not on several ones independently. Together with the other previous reports, our data suggest that spontaneous mtDNA mutations may not be caused exclusively by oxidative DNA damage at least in 16S rRNA gene.