UV-light-induced mutations in synchronous CHO cells

Asynchronous and synchronous CHO cells were irradiated with germicidal UV light to determine the fluence response curve for cell killing, and the induction of resistance to 6-thioguanine, ouabain, and diphtheria toxin. For asynchronous populations the data show a sigmoidal response for induced reproductive death, as has been seen by other, with a D₀ of 6 J/m² and an extrapolation number of 2.5. The induction of mutations appears to be a linear function for all three mutagenic markers up to a dose of 17 J/m².Reproductive death induced in the synchronous populations is a function of the time at which exposure occurs in the cell cycle, with late G₁ and early S being the sensitive stages. The induction of resistance to 6TG, ouabain, and diphtheria toxin (DT) all seem to depend on the time of exposure in the cell cycle. As in the case of UV-induced reproductive death, the more sensitive periods for mutation induction appear also to be the G₁ and early S period of the cell cycle, with the largest cyclic variation occurring for induced DT resistance.A comparison of the results reported here for the UV exposure with exposures of synchronous CHO cells to X-rays and ethylnitrosourea suggests that there are different age-specific responses to mutation induction for each agent, and that there are often different age responses for different mutagenic end- points with the same mutagen.     

Effect of calcium phosphate and alumina C γ gels on the mutagenicity and cytotoxicity of dimethylnitrosamine as studied in the CHO/HGPRT system

When CaCl₂ was added in increasing concentrations to a rat liver metabolic activation system (S9) buffered with sodium phosphate, the mutagenic activity and cytotoxicity of dimethylnitrosamine (DMN) in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system were greatly increased. This effect was not observed with an S9 mix buffered with N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES). The calcium phosphate gel precipitate of the S9 mix possessed approximately $\text{built1}\text{3}$ of the total activity of the mix, while the supernatant had only slight activity. However, when the calcium phosphate gel precipitate of a solution of S9 salts (without S9 protein) was added to the supernatant, the remaining $\text{2}\text{3}$ of the activity was recovered. Commercially obtained calcium phosphate, tricalcium phosphate, and alumina C γ gels could substitute for CaCl₂ in the S9 mix, but diethylaminoethyl cellulose (DEAE cellulose) could not. Alumina C γ gel can exert its effect in the absence of both CaCl₂ and phosphate in the S9 mix. Increasing the time of contact between the S9 protein and the S9 salts increased the efficacy with which the S9 mix activated DMN; this is indicative of an adsorptive process by calcium phosphate gel.     

Phenotypic expression time of mutagen-induced 6-thioguanine resistance in Chinese hamster ovary cells (CHO/HGPRT system).

Mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells (referred to as the CHO/HGPRT system) can be quantitated by selection for the phenotype of resistance to 6-thioguanine (TG) under stringently defined conditions. The phenotypic expression time, that is, the time interval after mutagen treatment which is necessary befor all mutant cells are able to express the TG-resistant phenotype, has been found to be 7–9 days in this CHO/HGPRT system when the cells are subcultured every 48 h. Subculture in medium with or without hypoxanthine (HX) utilizing trypsin, ethylenediaminetetraacetic acid (EDTA), or ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) for cell removal yields identical results. When subculture at intervals greater than 48 h is employed, a slight lengthening of the expression time is observed. An alternative method to regular subculture has also been achieved by maintaining the cells in a viable, non-dividing state in serum-free medium. This procedure yields a similar time course of phenotypic expression and thus shows that continued cell division is not essential to this expression process. In addition, this observation offers methodology which can significantly reduce the investment of time and money for mutation induction determinations in this mammalian cell gene mutation assay.     

Mutagenicity of an antitumor protein, neocarzinostatin, in Escherichia coli.

An assay is described for the measurement of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells utilizing resistance to 6-thioguanine (TG). Optimal selection conditions are defined for such parameters as phenotypic expression time prior to selection, and TG concentration and cell density which permits maximum mutant recovery. The nature of the TG-resistant mutants is characterized by several physiological and biochemical methods. The data demonstrate that more than 98% of the mutant clones isolated by this selection procedure contain altered HGPRTase activity. The CHO/HGPRT system thus shows the specificity necessary for a specific gene locus mutational assay.     

In vitro metabolic activation of ethidium bromide and other phenanthridinium compounds: mutagenic activity in Salmonella typhimurium.

The induction of mutation by a variety of mutagens has been measured utilizing the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells (CHO/HGPRT) system). These mutagens include physical agents such as UV light and X-rays, and chemicals such as alkylating agents, ICR-191, and metallic compounds. This system can also be modified for study of the mutagenicity of promutagens such as dimethylnitrosamine (DMN) which require biotransformation for mutagenic action, either through the addition of a rat liver microsomal activation preparation or through a host-mediated activation step using Balb/c athymic mice.     

Heterogeneous cell-cycle behavior in response to UVB irradiation by a population of single cancer cells visualized by time-lapse FUCCI imaging

The present study analyzed the heterogeneous cell-cycle dependence and fate of single cancer cells in a population treated with UVB using a fluorescence ubiquitination-based cell-cycle (FUCCI) imaging system. HeLa cells expressing FUCCI were irradiated by 100 or 200 J/m² UVB. Modulation of the cell-cycle and apoptosis were observed by time-lapse confocal microscopy imaging every 30 min for 72 h. Correlation between cell survival and factors including cell-cycle phase at the time of the irradiation of UVB, mitosis and the G₁/S transition were analyzed using the Kaplan–Meier method along with the log rank test. Time-lapse FUCCI imaging of HeLa cells demonstrated that UVB irradiation induced cell-cycle arrest in S/G₂/M phase in the majority of the cells. The cells irradiated by 100 or 200 J/m² UVB during G₀/G₁ phase had a higher survival rate than the cells irradiated during S/G₂/M phase. A minority of cells could escape S/G₂/M arrest and undergo mitosis which significantly correlated with decreased survival of the cells. In contrast, G₁/S transition significantly correlated with increased survival of the cells after UVB irradiation. UVB at 200 J/m² resulted in a greater number of apoptotic cells.     

UV-induced mutagenesis at the hypoxanthine—guanine phosphoribosyl transferase locus in two L5178Y mouse lymphoma cell strains with different UV sensitivities

Two strains of L5178Y mouse lymphoma cells, L5178Y-R (LY-R) and L5178Y-S (LY-S), differ markedly in their sensitivity to 254 nm UV radiation (D₀ = 0.7 and 5.5 J/m²; n = 6.0 and 2.0 for LY-R and LY-S cells, respctively). In this study, the frequency o hypoxanthine-guanine-phosporibosyl-transferase-deficient mutants was determined, using 6-thioguanine (TG) as a selective agent, in populations of LY-R and LY-S cells exposed to various fluences of UV radiation. The spontaneous mutation frequency for LY-R cells was (3.7 ± 0.6) × 10^?5 TG^r mutants per viable cell, and the UV induction rate was (2.2 ± 0.8) × 10^?4 TG^r mutants per viable cell, per J/m². Both spontaneous and induced mutantion frequencies were much lower for LY-S cells. The sopntaneous mutation frequency for these cells were too low to make its measurement practicable ( < 0.0013 × 10^?5 TG^r mutants per viable cell). Mutation induction rate was (4.2 ± 2.2) × 10^?7 TG^r mutants per viable cell, per J/m². These differences in mutability do not appear to be due to gene duplication in LY-S cells, or to selective growth disadvantage of LY-S-derived TG-resistant mutants. Possible mechanisms underlying the differences in mutability of LY-R and LY-S cells are considered.     

Analyses of differential sensitivities of synchronized HeLa S3 cells to radiations and chemical carcinogens during the cell cycle: Part V. Radiation- and chemical carcinogen-induced mutagenesis

8-Azaguanine (8AG)-resistant mutations induced by X-rays, ultraviolet radiation (UV) and a chemical carcinogen, 4-hydroxyaminoquinoline 1-oxide (4-HAQO) were examined during the cell cycle of synchronized HeLa S3 cells. Mutants induced by 400 R of X-rays occurred in a higher frequency in the X-ray sensitive G₁-S boundary phase than in the X-ray-resistant G₂, S and early g₂ phases. 8AG-resistant mutants induced by treatment with 10^?5 M 4-HAQO for 20 min appeared in a higher frequency in the early to middle S phases than in the other phases. In the case of UV, however, we found no significant difference in the induced mutation frequencies the cell cyle, because the mutation frequencies induced by the UV doses (0–20 Jm²) used were too low for detection of the difference. These results suggest that there is a close correlation between the critical damage induced in DNA molecule(s) at the DNA-synthetic phase in the cell cycle and mutagenesis, because mitotic cells have a low mutability in spite of their high radio-sensitivity.     

CIRCADIAN RHYTHMS IN MOUSE EPIDERMAL BASAL CELL PROLIFERATION

Several kinetic parameters of basal cell proliferation in hairless mouse epidermis were studied, and all parameters clearly showed circadian fluctuations during two successive 24 hr periods. Mitotic indices and the mitotic rate were studied in histological sections; the proportions of cells with S and G₂ phase DNA content were measured by flow cytometry of isolated basal cells, and the [³H]TdR labelling indices and grain densities were determined by autoradiography in smears from basal cell suspensions. The influx and efflux of cells from each cell cycle phase were calculated from sinusoidal curves adapted to the cell kinetic findings and the phase durations were determined. A peak of cells in S phase was observed around midnight, and a cohort of partially synchronized cells passed from the S phase to the G₂ phase and traversed the G₂ phase and mitosis in the early morning. The fluctuations in the influx of cells into the S phase were small compared with the variations in efflux from the S phase and the flux through the subsequent cell cycle phases. The resulting delay in cell cycle traverse through S phase before midnight could well account for the accumulation of cells in S phase and, therefore, also the subsequent partial synchrony of cell cycle traverse through the G₂ phase and mitosis. Circadian variations in the duration of the S phase, the G₂ phase and mitosis were clearly demonstrated.     

Ultraviolet light-induced mutation of diploid human lymphoblasts

Ultraviolet irradiation (254 nm) of immortal diploid human lymphoblasts killed cells, caused mutation at three genetic loci studied, and transiently inhibited ³H-TdR uptake into DNA. A shoulder of about 6 J/m² and a D₀ of 6 J/m² was observed for survival. Mutation rose in a monotonic non-linear fashion through 6 J/m²; above 6 J/m², complex behavior approximating a plateau in induced mutation was observed. Irradiation at 4.4 J/m² caused a transient increase in the number of cells synthesizing DNA and a decrease in the rate of DNA synthesis relative to mock-irradiated controls. The parameter of rate of DNA synthesis per cell in DNA synthetic phase showed a rapid recovery toward control values between 2 and 4 h after irradiation and a slower recovery to control values by 22 h post-irradiation.Fractionated dose schedules were used to measure the effects of allowing a time interval between doses at nontoxic fluences (2.2 j/m²), moderately toxic fluences (8.8 J/m²) and toxic fluences (17.6 J/m²). These measurements indicate that in the non-toxic range of fluences common to human exposure, mutational response is mediated by a post-irradiation process which seems to show to shkow enchanced ability to protect against mutation induced by subsequent irradiation. However, at moderately toxic fluences there was little effect of dose fractionation, and at toxic fluences, a time-dependent increase in mutation fraction was observed at separation times greater than 7 h. We suggest that these latter observations arise primarily from cell cycle heterogeneity with regard to sensitivity to UV killing and mutation.     

Fluctuation analyses of spontaneous mutations to 6-thioguanine resistance in Chinese hamster ovary cells in culture

Fluctuation analyses of the spontaneous appearance of 6-thioguanine (TG)-resistant mutants in cultured Chinese hamster ovary (CHO) cells were performed to investigate (1) whether the resistance is induced by the selective agent or is the result of a mutation which occurs prior to the TG selection and (2) to estimate the spontaneous mutation rate at the hypoxanthine—guanine phosphoribosyl transferase (hgprt) locus. The potential problem of phenotypic delay was minimized by allowing an adequate expression time through maintenance of the cultures in a division-arrested, viable state. The results demonstrate that the TG-resistant (TG^r) cells arise randomly in the cultures, independently of the selective agent, which is consistent with spontaneous mutations. The average values for mutation rate ± standard deviation, based on 4 independent determinations and 2 methods of calculation, are 3.4 ± 1.2 × 10^?7 (median method) and 5.1 ± 1.8 × 10^?7 (mean method) mutants/cell/generation.     

Fission Yeast Ste9, a Homolog of Hct1/Cdh1 and Fizzy-related, Is a Novel Negative Regulator of Cell Cycle Progression during G1-Phase

When proliferating fission yeast cells are exposed to nitrogen starvation, they initiate conjugation and differentiate into ascospores. Cell cycle arrest in the G₁-phase is one of the prerequisites for cell differentiation, because conjugation occurs only in the pre-Start G₁-phase. The role of ste9⁺ in the cell cycle progression was investigated. Ste9 is a WD-repeat protein that is highly homologous to Hct1/Cdh1 and Fizzy-related. The ste9 mutants were sterile because they were defective in cell cycle arrest in the G₁-phase upon starvation. Sterility was partially suppressed by the mutation in cig2 that encoded the major G₁/S cyclin. Although cells lacking Ste9 function grow normally, the ste9 mutation was synthetically lethal with the wee1 mutation. In the double mutants of ste9 cdc10^ts, cells arrested in G₁-phase at the restrictive temperature, but the level of mitotic cyclin (Cdc13) did not decrease. In these cells, abortive mitosis occurred from the pre-Start G₁-phase. Overexpression of Ste9 decreased the Cdc13 protein level and the H1-histone kinase activity. In these cells, mitosis was inhibited and an extra round of DNA replication occurred. Ste9 regulates G₁ progression possibly by controlling the amount of the mitotic cyclin in the G₁-phase.     

Mitochondrial protein synthesis in chinese hamster cells (line CHO) synchronized by isoleucine deprivation

Mitochondrial protein synthesis was measured in line CHO cells after phases of the cell cycle were synchronized by isoleucine deprivation or mitotic selection. Maximum incorporation of [³H] leucine into mitochondrial polypeptides occurred within 2 hours after isoleucine was added to initiate G₁ traverse. In cells synchronized in G₁ by mitotic selection, the rate of mitochondrial protein synthesis was fairly constant throughout the cell cycle. SDS-polyacrylamide gel electrophoretic profiles of labeled mitochondrial polypeptides were similar in cells synchronized by either isoleucine deprivation or mitotic selection. Obvious changes in the distribution of polypeptides were not detected during various phases of the cell cycle. The increased rate of incorporation of [³H] leucine into mitochondrial polypeptides after reversal of G₁-arrest may indicate that mitochondrial protein synthesis and possibly mitochondrial biogenesis are synchronized in CHO cells deprived of isoleucine.     

Autoradiographic studies on the incorporation of3H-amino acids into chromosomes during the mitotic cell cycle ofHaplopappus gracilis

The synthesis of chromosomal proteins and the incorporation of labelled proteins into chromosomes in the mitotic cell cycle ofHaplopappus gracilis, 2n=4, were traced autoradiographically with³H-arginine,³H-lysine, and³H-tryptophane. The duration of the mitotic cell cycle in the root tip cells was determined by³H-thymidine autoradiography and was measured to be 13.0 hr (G₁ 1.3 hr, S 6.5 hr, G₂ 3.8 hr and M 1.4 hr).³H-arginine labelled proteins which were synthesized at S and G₂ were found to be incorporated into chromosomes to a greater extent than proteins which were synthesized either at G₁, at the transition phase from late S to early G₂, or at the mitotic phase. Such varied incorporation was also found in³H-lysine labelled proteins, but not in³H-tryptophane labelled proteins. These findings indicate that the chromosomal proteins are synthesized mainly at S and G₂. Some of the³H-arginine labelled proteins which were synthesized during the first mitotic cell cycle, were found to be incorporated into the chromosomes of the second mitotic cell cycle. The incorporation of the proteins synthesized at one stage of the mitotic cell cycle was found to occur locally in some regions of the chromosomes, while the pattern of incorporation was observed to be similar between euchromatic and heterochromatic regions.     

Phase-Specific Polypeptides and Poly(A) RNAs during the Cell Cycle in Synchronous Cultures of Catharanthus roseus Cells

This study shows an overall analysis of gene expression during the cell cycle in synchronous suspension cultures of Catharanthus roseus cells. First, the cellular cytoplasmic proteins were fractionated by two-dimensional gel electrophoresis and visualized by staining with silver. Seventeen polypeptides showed qualitative or quantitative changes during the cell cycle. Second, the rates of synthesis of cytoplasmic proteins were also investigated by autoradiography by labeling cells with [³⁵S]methionine at each phase of the cell cycle. The rates of synthesis of 13 polypeptides were found to vary during the cell cycle. The silverstained electrophoretic pattern of proteins in the G₂ phase in particular showed characteristic changes in levels of polypeptides, while the rates of synthesis of polypeptides synthesized during the G₂ phase did not show such phase-specific changes. This result suggests that posttranslational processing of polypeptides occurs during or prior to the G₂ phase. In the G₁ and S phases and during cytokinesis, several other polypeptides were specifically synthesized. Finally, the variation of mRNAs was analyzed from the autoradiograms of in vitro translation products of poly(A)⁺ RNA isolated at each phase. Three poly(A)⁺ RNAs increased in amount from the G₁ to the S phase and one poly (A)⁺ RNA increased preferentially from the G₂ phase to cytokinesis.     

Reactivation of UV and γ-inactivated herpes virus in BHK cells

The DNA-repair capabilities of baby hamster kidney (BHK) cells were investigated by comparing the reactivation of irradiated herpes simplex virus type I (HSV1) in BHK cells with its reactivation in mouse fibroblasts and in normal and repairdeficient human diploid fibroblasts. BHK cells were found to have an intermediate ability to reactive UV-irradiated HSV1 (the viral D_o was 14 J/m²) relative to normal human fibroblasts (viral D_o = 19 J/m²) and xeroderma pigmentosum (XP) group A cells (viral D_o = 4.5 J/m²). With mouse L929 cells as the host, the response of the UV-irradiated virus was biphasic with D_os of 4.6 and 30 J/m² for the low- and high-dose components respectively. In contrast to the response following UV radiation, γ-irradiated HSV1 was similarly reactivated by BHK and normal human cells (the D_os for the irradiated virus in BHK and CRl 1106 were 55 and 51 krad, respectively, whereas xeroderma pigmentosum cells were slightly less efficient in the repair of γ-irradiated virus (D_o = 45 krad). UV irradiation of BHK host cells 0–48 h prior to infection enhanced the reactivation of UV-irradiated HSV.     

Relationship between excision repair and the cytotoxic and mutagenic effect of the ‘anti’ 7,8-diol-9,10-epoxide of benzo[a]pyrene in human cells

The cytotoxic and mutagenic effect of (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti BPDE) in normally excision diploid human cells treated just prior to onset of S was compared with that of cells allowed ～ 16 h for excision repair before onset of S and with that observed in excision-deficient serodema pigmentosum (SP12BE) cells. The cells were synchronized by release from density inhibition of cell replication. DNA synthesis began ～ 22 h after the cells were plated at lower density (i.e., 1.4 × 10⁴ cells/cm²). The frequency of thioguanine-resistant mutants induced in normal cells treated just prior to onset of S was ～ 12- to 16-fold higher than that observed in cells treated in early G₁ or treated in G₀ (confluence) and then plated at lower density. The frequency approximated that expected for XP12BE cells from extrapolation of data obtained at lower doses. The frequency of mutants measured in normal cells treated in exponential growth was also much higher than that in the cells treated in early G₁ or in G₀, No such difference could be seen in XP12BE cells treated in exponential growth or in G₀. In contrast to the mutagenicity data in the normal cells, there was no significant difference in the slope of the survival curve of normal cells treated at various times prior to S phase at low densities. However, normal cells treated even at the onset of S exhibited survival equal to XP12BE cells give a 4- to 5-fold lower dose. The data support the hypothesis that DNA synthesis is the cellular event which converts unexcised DNA lesions into mutations. However, they indicate that S is not the event primarily responsible for translating DNA damage into cell death. Accompanying studies on the rate of excision of anti BPDE adducts from the normal cells during the period priot to S support the conclusions.     

CELLULAR AND NUCLEAR VOLUME DURING THE CELL CYCLE OF NHIK 3025 CELLS

The distribution of cellular and nuclear volume in synchronous populations of NHIK 3025 cells, which derive from a cervix carcinoma, have been measured by electronic sizing during the first cell cycle after mitotic selection. Cells given an X-ray dose of 580 rad in G₁, were also studied. During the entire cell cycle the volume distribution of both cells and nuclei is an approximately Gaussian peak with a relative width at half maximum of about 30%. About half of this width is due to imperfect synchrony whereas the rest is associated with various time invariant factors. During S the mean volume of the cells grows exponentially whereas the nuclear volume increases faster than for exponential kinetics. Hence, although cellular and nuclear volumes are closely correlated, their ratio does not remain constant during the cell cycle. Volume growth during the first half of G₁ is negligible especially for nuclei where the growth appears to be closely associated with DNA-synthesis. For unirradiated cells the growth of cellular and nuclear volume is negligible also during G₂+ M. In contrast, the X-irradiated cells continue to grow during the 6 hr mitotic delay with a rate that is constant and about half of that observed in late S. Hence, radiation induced mitotic delay does not appear merely as a lengthening of an otherwise normal G₂. During G₁ and S the irradiated cells were identical to unirradiated ones with respect to all the parameters measured.     

Mitochondrial Fragmentation Due to Inhibition of Fusion Increases Cyclin B through Mitochondrial Superoxide Radicals

During the cell cycle, mitochondria undergo regulated changes in morphology. Two particularly interesting events are first, mitochondrial hyperfusion during the G₁-S transition and second, fragmentation during entry into mitosis. The mitochondria remain fragmented between late G₂- and mitotic exit. This mitotic mitochondrial fragmentation constitutes a checkpoint in some cell types, of which little is known. We bypass the ‘mitotic mitochondrial fragmentation’ checkpoint by inducing fragmented mitochondrial morphology and then measure the effect on cell cycle progression. Using Drosophila larval hemocytes, Drosophila S2R⁺ cell and cells in the pouch region of wing imaginal disc of Drosophila larvae we show that inhibiting mitochondrial fusion, thereby increasing fragmentation, causes cellular hyperproliferation and an increase in mitotic index. However, mitochondrial fragmentation due to over-expression of the mitochondrial fission machinery does not cause these changes. Our experiments suggest that the inhibition of mitochondrial fusion increases superoxide radical content and leads to the upregulation of cyclin B that culminates in the observed changes in the cell cycle. We provide evidence for the importance of mitochondrial superoxide in this process. Our results provide an insight into the need for mitofusin-degradation during mitosis and also help in understanding the mechanism by which mitofusins may function as tumor suppressors.     

Analysis of differential sensitivities of synchronized HeLa S3 cells to radiations and chemical carcinogen during the cell cycle. II. Ultraviolet light

UV-induction of thymine dimers in cellular DNA and their excision during different phases of the cell cycle of HeLa S3 cells were studied. Induction of thymine dimers was higher in the mitotic phase and the middle of the S phase than in the G₁ phase and from the late S phase to the early G₂ phase which are rather insensitive to UV. However, there is no significant difference in excision rate of UV-induced thymine dimers from the irradiated cells through the cell cycle. These findings indicate that the cyclic variation of UV-survivals during the cell cycle may be due to differences in the amount of thymine dimers in cellular DNA induced by UV-irradiation.