Cell proliferation as a requirement for development of the contact effect in Chinese hamster V79 spheroids

Chinese hamster V79 cells grown for several hours in suspension culture form spheroids which are more resistant to killing by ionizing radiation than cells grown on petri dishes, a phenomenon known as the "contact effect." Previous results using the alkali-unwinding assay as a measure of DNA damage have implicated differences in DNA conformation as contributing to this effect; spheroid DNA denatures more slowly in dilute alkali than monolayer DNA, perhaps due to the presence of constraints to DNA unwinding. In this paper, the rate of development of radiation resistance is shown to be similar when either cell survival or DNA unwinding is used as an end point. At the midpoint for development of resistance, approximately 10 h, the unwinding kinetics indicate that either half of the cells contain constraints to DNA unwinding, or half of the DNA in all of the cells contains constraints. The latter explanation appears more likely since all cells seem to develop these constraints at the same rate, regardless of position in the cell cycle or the degree of contact with other cells. Results using the microelectrophoresis assay to measure damage to individual nuclei confirm the fact that 10-h cultures show a homogeneous radiation response intermediate between that of monolayers and spheroids. Incubation of cells at room temperature or in the presence of drugs which inhibit cell cycle progression prevents full development of the contact effect. Conversely, incubation of cells in medium containing inhibitors of polyamine synthesis, adenylcyclase, glutathione synthesis, poly(ADP-ribose)polymerase, topoisomerase II, or cell-cell communication does not inhibit development of the contact effect as measured by DNA-unwinding kinetics.     

Recovery of thioguanine-resistant cells from Chinese hamster V79 spheroids

Multicell spheroids may prove useful in evaluting the interactions of mutagens with cells exposed in a tissue-like environment. However, direct comparisons among populations of Chinese hamster V79 spheroids of different sizes or with monolayers are complicated by the observation that as spheroids enlarge, the fraction of mutant cells resistant to 6-thioguanine (TG^r) gradually decreases from about 5 in 10⁵ to less than 1 in 10⁵. There appear to be at least 2 explanations for these observations. First, TG^r cells grow less well as spheroids than do 6-thioguanine-sensitive (TG^s) cells. Second, the clonal nature of spheroid growth means that small samples fo spheroids are likely to contain fewer pre-existing TG^r cells.     

Further studies on tetracycline-induced mutation in V79 Chinese hamster cells

The interaction between ultraviolet light and tetracycline in producing cell killing and mutation has been studied in V79 Chinese hamster cells. It has been established that these agents act independently of each other. Cycloheximide altered the response to tetracycline in the fractionation experiment: when cycloheximide was not present, fractionation of TC treatment resulted in a higher mutation yield but no change in survival level; in the presence of cycloheximide, however, mutation was greatly reduced but survival increased. The results were taken to indicate that for tetracycline action to take place, de novo protein synthesis during tetracycline treatment was necessary. Caffeine had no influence on tetracycline-induced lethality or mutagenicity. This observation was considered to suggest that tetracycline did not affect cellular repair processes.     

Comutagenesis of sodium arsenite with ultraviolet radiation in Chinese hamster V79 cells

Summary Solar ultraviolet radiation has been associated with the induction of skin cancer. Recent studies have indicated that near-ultraviolet, especially UVB, is mutagenic. Exposure to trivalent inorganic arsenic compounds has also been associated with increased skin cancer prevalence. Trivalent arsenic compounds are not mutagenicper se, but are comutagenic with a number of cancer agents. Here, we test the hypothesis that arsenite enhances skin cancer via its comutagenic action with solar ultraviolet radiation. Irradiation of Chinese hamster V79 cells with UVA (360 nm), UVB (310 nm) and UVC (254 nm) caused a fluence-dependent increase in mutations at thehprt locus. On an energy basis, UVC was the most mutagenic and UVA the least. However, when expressed as a function of toxicity, UVB was more mutagenic than UVC. Nontoxic concentrations of arsenite increased the toxicity of UVA, UVB and UVC. Arsenite acted as a comutagen at the three wavelengths; however, higher concentrations of arsenite were required to produce a significant (P < 0.05) comutagenic response with UVB. The increased mutagenicity of UVB and UVA by arsenite may play a role in arsenite-related skin cancers.     

Effect of intercellular contact on DNA conformation, radiation-induced DNA damage, and mutation in Chinese hamster V79 cells

Chinese hamster V79 cells, when grown as small spheroids in suspension culture, are more resistant to killing by ionizing radiation than when grown as monolayers. We have attempted to determine whether this enhanced survival following irradiation is reflected in DNA damage and repair at the structural level (by measuring alkali-induced DNA unwinding rates from strand breaks) and at the functional level (by measuring resistance to forward mutation at the HGPRT locus). For a given dose of radiation, the unwinding of DNA in high salt/weak alkali was less complete for spheroid DNA than for monolayer DNA, and the rate of repair of radiation damage was faster in spheroid DNA. These differential responses were lost 8 hr after separation of spheroids into single cells, coinciding with loss of radioresistance measured by clonogenicity. In addition, spheroid cells showed fewer numbers of induced mutants per Gray, although, for a given level of survival, the mutation frequency for monolayers and spheroids was identical. These results suggest that conformational changes in DNA resulting from cell growth as spheroids might enhance repair of radiation-induced lesions.     

Development of tetraploidy in V79 spheroids

Summary Chinese hamster V-79-171 cells, when placed in suspension culture, spontaneously form multicell spheroids. As the spheroids enlarge the fraction of polyploid (predominantly tetraploid) cells increases and can approach 100% in very large spheroids. Spheroid size, rather than age, seems to be a major determinant for increased ploidy. When cell separation techniques were used to select enriched populations of diploid and tetraploid cells, the growth rate and plating efficiency of the diploid cells was always marginally higher, and they gradually became predominant in mixed monolayer cultures. Cloned tetraploid cells, however, generally remained quite stable, and no consistent ploidy dependent changes in radiosensitivity were observed relative to normal, diploid cell lines. This research was supported by grants CA 28793 and CA 23511 awarded by the National Cancer Institute, Bethesda, MD.     

Oxygen enhancement ratio in V79 spheroids

Chinese hamster V79 spheroids were stained with a nontoxic fluorescent stain, Hoechst 33342, which penetrates slowly into the spheroids. Single cells from these spheroids were then sorted by a fluorescence-activated cell sorter according to staining intensity (and therefore position in the spheroids). Flow cytometry characterization of the various cell subpopulations indicated that the innermost cells were more radiosensitive than expected on the basis of cell cycle position or cell thiol content. However, comparison of the radiosensitivities of cells sorted from equivalent depths from completely aerobic or anoxic V79 spheroids indicated that the oxygen enhancement ratio remained remarkably constant at 2.7 +/- 0.2 through the spheroid.     

Serum, trypsin, and cell shape but not cell-to-cell contact influence the X-ray sensitivity of Chinese hamster V79 cells in monolayers and in spheroids

Nutrient concentration in the growth medium and trypsin affect cellular radiosensitivity in a manner that is related to cell shape (Reddy, Stevenson, and Lange, Int. J. Radiat. Biol. 55, 105-117 (1989); Reddy and Lange, Radiat. Res. 119, 338-347 (1989]. Hence we hypothesized that the concentration of serum in the medium could influence the X-ray sensitivity of cells and that the spread cells in monolayers and round cells in spheroids may differ in their response to the radiosensitizing effect of trypsin. We compared the X-ray sensitivity of monolayer and spheroid cells grown for 19 +/- 1 h in MEM supplemented with 5 or 15% serum. Cells were trypsinized and plated either immediately before, or 2.5 +/- 0.5 h after, irradiation and incubation for repair in situ. Survival of cells in monolayers and in spheroids was higher in MEM with 5% serum than with 15% serum. Trypsin treatment affected the shape and radiosensitivity of cells in monolayers but not in spheroids. When all cells were grown in the same serum concentration and a 2.5-h postirradiation incubation was allowed prior to trypsinization, the X-ray sensitivity of cells in spheroids was greater than that of cells in monolayers. The survival of cells in spheroids became equal to that of monolayer cells when cells in spheroids were converted to monolayers by placing them in 25-cm2 flasks and allowing them 3 h to attach and spread. Cell cycle distributions were nearly the same in monolayers and spheroids cultured in MEM with 5 or 15% serum. We conclude that: (1) serum concentration in the growth medium and trypsin do appear to contribute to the differences in the radiosensitivity of spheroids and monolayer V79 cells; (2) these differences are associated with changes in cell morphology.     

Comparison between the binding of [3H]misonidazole and AF-2 in Chinese hamster V79 spheroids

Binding of two hypoxia probes, [3H]misonidazole and AF-2 (2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide), was compared in Chinese hamster V79 spheroids incubated under different oxygen concentrations. Fluorescence-activated cell sorting based on Hoechst 33342 penetration was used to obtain populations of cells from different depths within the spheroid, and sorted cells were analyzed by cytofluorometry for AF-2 content and by liquid scintillation counting for [3H]misonidazole content. The patterns of AF-2 and misonidazole binding were very similar, with about 20-fold more localization of both drugs in anoxic compared to aerobic cells. Similar results were obtained when cells were sorted on the basis of AF-2 rather than Hoechst 33342 fluorescence. When mean cellular fluorescence of AF-2 was plotted versus cpm misonidazole/cell for different oxygen tensions, it appeared that oxygen was equally effective in inhibiting AF-2 and misonidazole binding. Internal cells of anoxic spheroids bound about twice as much AF-2 and misonidazole as external cells, apparently due to an increased rate of nitroreduction by chronically hypoxic cells. AF-2 was found to enhance the retention of misonidazole in oxic and hypoxic spheroids when both drugs were present.     

Induction of mutation in V79 Chinese hamster cells by tetrachlorohydroquinone, a metabolite of pentachlorophenol

Tetrachlorohydroquinone (TCHQ) and tetrachlorocatechol (TCC), two metabolites of the environmental mutagen and carcinogen pentachlorophenol, were tested without exogenous activation in V79 Chinese hamster cells for the induction of mutation at the hypoxanthine phosphoribosyl transferase (HPRT) locus to 6-thioguanine resistance (TGr) and at the Na/K-ATPase locus to ouabain resistance (OuaR). Treatment was for 24 h at 37 degrees C. TCHQ produced statistically significant increases in the frequency of TGr mutants. The lowest observed effective dose (LOED) was 20 microM, where the relative cloning efficiency was 63%. The relationship between the dose of TCHQ and the frequency of TGr mutants was approximately linear over the range of 0-60 microM with an estimated slope (+/- 95% confidence limits) of 1.1 +/- 0.3 mutants per 10(6) clonable cells per microM. At the highest tested dose of TCHQ, 60 microM, the relative cloning efficiency was reduced to 7%. In contrast to TCHQ, TCC was unable to induce TGr mutants at doses up to 120 microM. The relative cloning efficiency at this dose was 5%. Both TCHQ and TCC were unable to induce OuaR mutants. The results suggest that TCHQ is at least partly responsible for the genotoxic activity of pentachlorophenol. TCHQ can produce reactive oxygen species, which may cause large genetic damage such as deletions, resulting in mutation to TGr but not to OuaR.     

Mutagenicity of 4-hydroxynonenal in V79 Chinese hamster cells

4-Hydroxynonenal (HNE), a major product of the peroxidation of liver microsomal lipids, was examined for mutagenic activity at the hypoxanthine-guanine phosphoribosyltransferase locus in V79 Chinese hamster lung cells. At concentrations ranging from 10 to 45 microM, HNE induced a dose-dependent increase in the number of mutations to 6-thioguanine resistance, which reached the level of 4.7X baseline at the highest concentration tested.     

Mutagenicity of hydrogen peroxide in V79 Chinese hamster cells

Hydrogen peroxide (H2O2) was investigated for its potential to induce gene mutations in V79 Chinese hamster cells. Exposure of 2-3 X 10(6) cells/100-mm dish to 0.5-4.0 mM H2O2 for 1 h resulted in a concentration-dependent increase in the frequency of 6-thioguanine-resistant clones. At 4 mM H2O2 the mutation frequency was increased about 6-fold above that in controls and survival of the cells was reduced by 50%. Cytotoxicity was markedly increased at lower cell densities. When only 100-200 cells/100-mm dish were exposed to H2O2 for 1 h, 50% were killed at an H2O2 concentration as low as 60 microM. The results show that mutagenicity of H2O2 in mammalian cells in vitro has escaped attention previously because the concentrations tested were too low, presumably because the likely toxicity of H2O2 to V79 cells treated at high cell densities was overestimated.     

Genotoxicity of 1-nitronaphthalene in Chinese hamster V79 cells

1-Nitronaphthalene (1-NN) has been identified in the U.S. National Toxicology Program as a non-carcinogen showing some evidence of in vitro genotoxicity. We tested this compound in Chinese hamster V79 cells at 20-80 micrograms/ml with two endpoints: sister-chromatid exchange (SCE) and thioguanine resistance (TGR), with 5 repeat experiments. The SCE values in the presence of rat or hamster hepatocytes were consistently above the 95% and usually the 99% upper confidence limits for the corresponding control. Without hepatocyte activation, the control upper confidence limits were not exceeded except in one experiment in which the control SCE value was unusually low. TGR was scored both as proportion of plates with mutant colonies and as number of mutant colonies per plate. In 2 of 5 experiments, these values exceeded control 95% or 99% upper confidence limits; on the other hand, these values were substantially lower than those of the positive controls, dimethylbenz[a]anthracene (2.6 micrograms/ml) with activation and ethyl methanesulfonate (155 microgram/ml), which is direct-acting. For TGR, activation of 1-NN by either rat or hamster hepatocytes produced inconsistent results. Overall we would consider this compound to be a weak genotoxin, to which a cancer bioassay would be expected to be relatively insensitive.     

Genotoxic effects of paracetamol in V79 Chinese hamster cells

Paracetamol was studied for possible genotoxic effects in V79 Chinese hamster cells. Paracetamol (0.5 mM for 30 min) reduced the rate of DNA synthesis in exponentially growing V79 cells to about 50% of control. A further decrease in the DNA synthesis was seen during the first 30 min after termination of paracetamol exposure. Paracetamol (3 and 10 mM for 2 h) caused a small increase in DNA single-strand breaks, as measured by the alkaline elution technique. After 16 h elution, the amount of DNA retained on the filters was 79 and 70% of controls in cells treated with 3 and 10 mM paracetamol respectively. No indication of DNA damage was seen in measuring the effect of paracetamol (0.25-10 mM for 2 h) on unscheduled DNA synthesis in growth-arrested cultures of V79 cells. At the highest concentrations (3 and 10 mM paracetamol), decreased unscheduled DNA synthesis was observed. Also UV-induced DNA-repair synthesis was inhibited by 3 and 10 mM paracetamol. DNA-repair synthesis was, however, inhibited at a much higher concentration than that inhibiting replicative DNA synthesis. The number of sister-chromatid exchanges (SCE) increased in a dose-dependent manner on 2 h exposure to paracetamol from 1 mM to 10 mM. At the highest dose tested (10 mM), the number of SCE increased to 3 times the control value. Co-culturing the V79 cells with freshly isolated mouse hepatocytes had no further effect on the paracetamol induced sister-chromatid exchanges. The present study indicates that paracetamol may cause DNA damage in V79 cells without any external metabolic activation system added.     

Repair, redistribution and repopulation in V79 spheroids during multifraction irradiation

Abstract. Development of predictive assays for measuring tumour radiosensitivity has generated much recent interest, particularly with the recognition that tumour cell survival at doses of about 2 Gy may correlate well with tumour curability. Clinical data, however, suggest that overall treatment time may be of considerable significance in radioresponsive tumours, especially for rapidly growing tumours capable of accelerated repopulation. Because neither factor can be repeatedly assessed in human tumours, we used cells growing as multicell spheroids to determine whether the initial radiation response would be predictive for multifraction exposures, or whether other factors including repopulation rate should be considered. Potential problems of hypoxia and reoxygenation were avoided by using small spheroids which had not yet developed radiobiologically hypoxic regions. Repair and redistribution dominated the responses in the first two or three exposures, with repopulation playing a minor role. As the fractionation schedule was extended, however, repopulation between fractions largely determined the number of viable cells per spheroid. We conclude that the radiation response of cells from untreated spheroids provides a general indication of net sensitivity, but that repair and redistribution produces considerable variation in radiosensitivity throughout a fractionation protocol. Ultimately, repopulation effects may dominate the multifraction response.