Selection of N-(phosphonacetyl)-L-aspartate resistant Chinese hamster mutants in the presence of the uridine uptake inhibitor dipyridamole.

In mammalian cells selected in culture for resistance to PALA the CAD gene is amplified and these cells are a widely used model system to study gene amplification. Selection of resistant mutants is routinely performed in medium supplemented with dialyzed serum, because the cytotoxic effect of PALA is reversed by uridine, which is contained in serum. We have shown that in Chinese hamster cells dipyridamole reduced uridine uptake to less than 5% with limited effect on cell survival. Moreover, in medium supplemented with complete serum and 10 microM dipyridamole the toxicity of PALA was similar to that obtained in medium containing dialyzed serum. We then used 10 microM dipyridamole to inhibit uridine uptake during selection of PALA resistant colonies and found that both the frequency and the type of mutants were as those obtained in the presence of dialyzed serum. In particular, in the five mutants tested, the mechanism of resistance to PALA was amplification of the CAD gene.

Telomeric and nontelomeric (TTAGGG)n sequences in gene amplification and chromosome stability.

We have isolated eight PALA-resistant mutants from CHO-PV cells and have shown that the CAD gene was amplified. We then localized the CAD genes with fluorescence in situ hybridization followed by G-banding and identified 10 different marker chromosomes carrying amplified DNA. TTAGGG repetitions, which normally map to the telomeres and centromeres, have also been localized on the 10 marker chromosomes. The organization of amplified genes and of TTAGGG sequences suggests that dicentrics were formed during amplification and that breakage-fusion-bridge cycles may have generated 7 marker chromosomes. One isochromosome was probably derived from abnormal centromere segregation at anaphase. The most striking observation was that TTAGGG sequences of centromeric origin surrounded the amplified regions and were always localized at the telomeres of the chromosome arms carrying amplified DNA. These results indicate that the recombination events that accompanied gene amplification frequently involved centromeric DNA. Moreover, breakage within centromeric TTAGGG repeats may produce telomere-like structures that stabilize the ends of rearranged chromosomes.

Variation of minisatellites in chemically induced mutagenesis and in gene amplification.

A mutation assay in cultured mammalian cells was developed based on direct analysis of minisatellite DNA. Chinese hamster cells (V79) were mutagenized with nitrosoguanidine and independent colonies were isolated and expanded. DNA fingerprints were then obtained after digestion with HinfI or HaeIII and hybridization with 33.15 and 33.6 probes (Jeffreys et al., 1985). 12 colonies from untreated cells were also analyzed. Digestion with HaeIII and hybridization with 33.15 probe detected the highest frequency of induced variants. The results suggest that minisatellite sequences are hypermutable sites that can be used to monitor the mutagenic effect of chemical agents. We have also analyzed the DNA fingerprints of 17 independent Chinese hamster (CHO) cell lines carrying amplification of the CAD gene. The DNA fingerprint analysis showed a variation in minisatellite regions in 3 lines while no variation was observed in independent colonies from the CHO parental cell line. The results suggest that these sequences may be hot spots for recombination during gene amplification.

Sensitivity to DNA-damaging agents and mutation induction by UV light in UV-sensitive CHO cells.

Three UV-sensitive (UVs) mutants isolated from a CHO cell line were analyzed for survival after exposure to H2O2, EMS, MMC, CCNU, X-rays and for mutation induction after UV-irradiation. The UVs mutants showed normal sensitivities to EMS and H2O2, whereas they were hypersensitive to the bifunctional alkylating agents MMC and CCNU and to hypoxic X-irradiation. Compared to parental cells, one of the UV-sensitive clones showed approximately 3- and 7-fold enhancement in the mutagenic response per unit UV dose for 6-thioguanine and ouabain resistance, respectively.