Oncogene activation and loss of tumor suppressor genes are known to play a role in tumor initiation as well as its progression.The potential roles of these genes in perturbation of genome stability has become of major

Oncogene activation and loss of tumor suppressor genes are known to play a role in tumor initiation as well as its progression.The potential roles of these genes in perturbation of genome stability has become of major interest. To better understand the relationship between expression of an oncogene and genetic instability, we have studied a cell line expressing an activated human Ha-ras under the control of bacterial lactose operon regulatory elements for changes in methotrexate resistance and dihydro folate reductase (dhfr) gene amplification following mutant Ha-ms induc tion. In these cells mutant Ha-ms is directed by an inducible SV4O promoter containing a bacterial lac operator sequence which is repressed due to constitutive expression of bacterial lac repressor gene. The expres sion of this Ha-ms is specifically induced by the addition of isopropyl-l thio-@3-D-galactopyranoside (IPTG), a lactose analogue, to the culture medium. During single-step methotrexate selection, these cells showed an increased frequency of methotrexate resistance in the presence of IPTG. More than 60% of the methotrexate-resistant colonies showed a 2-6-fold amplification of the dhfr gene. One clone with rearranged dhfr had about 100-fold amplification ofthe gene. The increased capacity to amplify DNA in response to mutant Ha-ras Induction was not locus specific since cells also displayed an Increased frequency ofreslstance toN-(phosphonacetyl) L-aspartic acid in the presence of JTPG. Four of the methotrexate-resis tant clones with amplified dhfr gene were cultured fOrther in the presence or absence of IPTG and subsequently compared for their ability to grow in soft agar as a measure of transformation. In medium containing methotrexate but no IPTG, the clones were unable to grow in soft agar, indicating that methotrexate resistance due to gene amplification is sep arabIc from transformation.