UV mutagenesis and the CHO cell cycle

This dissertation presents several studies of cytotoxicity, mutagenesis, lesion induction and repair, and cellular recovery in Chinese hamster ovary (CHO) cells following ultraviolet light (UV) irradiation. These phenomena were investigated primarily as a function of position in the cell cycle at the time of UV exposure. Synchronous cell populations were obtained with or else cells a mitotic harvest procedure, CHO were synchronized utilizing a serum starvation/refeeding protocol, cells were demonstrated to become increasingly more sensitive to the cytotoxic effects of UV as they traversed the cell cycle from G₁ through early-mid S, when sensitivity was maximal. This trend then reversed with the cells becoming significantly more resistant as they progressed into G₂ and M. During cell cycle traverse, D_q values remained approxmately constant, while those for D_O varied by more than a factor of two.

Mutation induction at the hypoxanthine-guanine phosphoribosyl transferase for resistance to (HGPRT) locus was monitored by selecting the purine analogue, 6-thioguanine (TG⁶). Cells irradiated either during a period in mid-late G₁, or 3-4 h later during early-mid S, were discovered to be particularly mutable to TG^R . Optimum phenotypic and was expression of TG ^R was achieved 7-9 days after UV irradiation, essentially independent of both UV fluence and cell cycle position at the time of exposure. Similar mutability patterns were obtainable in cells synchronized by serum starvation and refeeding, though results were somewhat more variable, particularly during the Gg/G-] phases. TG ^R mutants induced by irradiation at different times during the cell cycle were cloned and tested for their HGPRT activity.

Virtually all clones exhibited similar low levels of residual enzyme activity, regardless of the cell cycle time of their induction. It was also demonstrated that cell-cycle-dependent changes in cellular morphology are not a factor in determining the mutability response. Induction to three other variant phenotypes was also investigated: ouabain resistance (OUA ^R), 2,6-diaminopurine resistance (DAP ^R), and loss of glucose-6phosphate dehydrogenase activity (G6PD ^-). All demonstrated a cell cycle dependence, with the precise temporal pattern apparently dependent upon the particular marker and cell clone investigated.

The possible importance of the DNA replication and/or chromosome condensation cycles was further suggested by several other observations. Treatment of early G ₁, cultures with hydroxyurea, an inhibitor of semiconservative DNA replication, had virtually no effect on the time-course of the mid-late G ₁, period of enhanced mutability, but the early-mid S period was inhibited in a manner similar to DNA replication. It was confirmed that hypertonic treatment induces chromatin condensation in CHO cells, and while such treatment had little effect on the survival or mutability of asynchronous cultures, it did appear to alter the cell cycle mutability pattern of synchronous cells. Furthermore, co-induction of mutation at the closely linked HGPRT and G6PD loci was 9 times higher during mid-late G₁, and 50 times higher during early-mid S ₁, than expected if the induction at each locus were an independent event. Thus, enhanced mutability may represent a condition localized both in time during the cell cycle, and in space along the genome (e.g., gene replication or transcription).

The role of DNA repair and cellular recovery mechanisms in mammalian cell mutagenesis remain unclear. CHO-K ₁ -BH ₄ cells were demonstrated capable of excising ~ 50% of the induced thymine dimers within 8 h after a Uv exposure of 10J/M² Cell Cycyle traverse and DNA replication are inhibited to varying degrees by Uv, depending upon cell cycle position at the time of irradiation. However, the data indicates that essentially all the genome can be replicated, even though many dimers remain. Time course and quantitation studies suggest that perhaps lesions other than dimers may largely be responsible for inhibition of DNA replication, and possibly mutagenesis as well. Variation in dimer induction during the cell cycle afyer irradiation with 200 or 500 J/m² of Uv was not clearly demonstrated. Cellular recovery between fractionated UV exposures was found to occur during all of S, and only during S. Capacity for repair replication did not correlate well with mutability, and during early-mid S and late S/G₂/M was approximately 20 and 80%, respectively, of that seen during G₁. Caffeine, an inhibitor of post replication repair, was found to have a complex effect on the cell cycle mutability pattern.

In an adjunct study, conditions necessary for optimally and quantitatively measuring induction of OUA^R in CHO cells were investigated, and characterization of the OUA^R phenotype was pursued. Among the major findings were that although induction of OUA ^R in tetraploid cells was consistent with its being a co-dominant genetic trait, other data indicate that parameters such as phenotypic expression time and variant phenotype stability may vary radically according to the particular mutagen used.