A major goal of the current year has been the direct determination of whether abnormally banding chromosome regions in antifolate-resistant Chinese hamster lung cells with relatively low levels of dihydrofolate reductase (DHFR) overproduction and only small increases in DHFR gene copy number indeed contain amplified DHFR genes. The abnormal regions have distinctly abnormal band patterns, as revealed by trypsin-Giemsa banding and karyotype analysis, and are usually located on the long (q) arm of one homologue of a chromosome 2. In our large series of independently selected, antifolate-resistant sublines, nonbanding, homogenously staining regions (HSRs) are also usually located on a chromosome 2q. HSRs are found in sublines with high levels of DHFR overproduction and high DHFR gene copy numbers. The major significance of our studies to date is the finding that cells with even low levels of resistance to antifolates such as methotrexate may overproduce target enzyme DHFR via a gene amplification process rather than via a solely transcriptional or translational control mechanism. Further, even low levels of DHFR gene amplification may result in profound chromosomal alterations that are detectable in metaphase cells. These findings, in conjunction with preliminary information from other laboratories, suggest that gene amplification may play a role in development of resistance of tumor cells to methotrexate in patients treated with this drug. Our goals for the coming years are: (1) Quantitative comparisons between gene copy number, as determined from gene titration data obtained by Dr. Melera, and in situ hybridization data (number of silver grains) will be continued. (2) High-resolution banding techniques will be utilized to determine whether gene clusters, as evidenced by grain clusters, can be assigned to specific Giemsa positive or Giemsa negative bands. (3) Attempts will be made to localize the resident DHFR gene in drug-sensitive control Chinese hamster lung cells. This undertaking will be facilitated by the use of a recombinant plasmid containing a 1,300 bp insert representing the full-length coding sequence of the Chinese hamster DHFR gene. (4) The coding sequence probe will be used to determine the location of amplified DHFR genes in mouse cell lines MAZ/A/MQ30 and MAZ/AM/Q60 that have either HSRs or unusually large double-minute chromosomes. Northern blot experiments have shown that the new probe hybridizes specifically with (amplified) mouse DHFR genes. (K)
