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Among various 150-nM methotrexate-resistant (MTXr) V79 clones isolated, we found that two near-tetraploid clones as well as a near-diploid clone with amplification in the dihydrofolate reductase (dhfr) gene readily developed resistant to 40 000 nM MTX within 3 months during stepwise increased MTX selection, while two near-diploid clones without gene amplification could not acquire resistance beyond 5000 nM MTX. Then, we studied how the clones increased the resistance to MTX, and compared the propensity for gene amplification among three types of clones. Dot blot analysis showed that the acquisition of the high levels of resistance to MTX observed in two near-tetraploid clones and a near-diploid clone with gene amplification was associated with amplification in the dhfr gene. The amplified dhfr gene was overexpressed at mRNA and protein levels in the clones. Southern blot analysis of Hind III- and Eco RI-digested DNA in the clones at the time when they became resistant to 10 000 nM MTX indicated that they amplified the dhfr gene fragments which existed in low amounts in parental V79 cells, and that no gross rearrangement of the amplified dhfr gene was detected. Furthermore, fluorescence in situ hybridization analysis showed that the amplified dhfr gene was located on one chromosome as cluster(s). On the other hand, two near-diploid clones without gene amplification did not show any amplification of the dhfr gene even at 5000 nM-MTX resistant stage. These combined results suggest that the near-tetraploid clone as well as the near-diploid clone with the dhfr gene amplification have genomic instability with the propensity for gene amplification during stepwise MTX selection, and have a similar process for the development of the dhfr gene amplification.

A comparison of the propensity for gene amplification between near-tetraploid and near-diploid V79 clones resistant to 150 nM methotrexate