Increased apoptosis and increased clonogenic survival of 12V-H-ras transformed rat fibroblasts in response to cisplatin

Mutationally activated Ras is involved in tumor progression and likely also in drug resistance. Using survival, viability and apoptosis assays, we hav e here compared the cisplatin sensitivities of FR3T3 rat fibroblasts and a 12V-H-ras transformed subline (Ras2:3). Around 24 h after cisplatin treatme nt Ras2:3 cells showed higher apoptosis levels and lower viability than FR3 T3. This increased sensitivity correlated with weaker cisplatin-induced act ivation of Jun N-terminal kinase (JNK). In contrast to apoptosis assays, co lony formation assays showed that Ras2:3 were more resistant to cisplatin t han were FR3T3. This was partly due to the increased cisplatin sensitivity of FR3T3 seeded at low densities, as required in colony formation assays. I n addition, Ras2:3 cisplatin survivors had a higher relative proliferative capacity. Cell cycle analyses showed that FR3T3 cells initially responded w ith a dose-dependent G2 arrest, while Ras2:3 accumulated in S-phase. Experi ments with an anti-apoptotic mutant of MEKK1 suggested that the apoptotic r esponse of Ras2:3 cells is not specific to the S-phase fraction. In summary , the cisplatin response of ras-transformed fibroblasts is distinct from th at of parental cells, in that they show increased apoptosis, a different ce ll cycle response and increased post-treatment proliferative capacity. The results illustrate the need to carefully consider methods and protocols for in vitro studies on chemotherapy sensitivity.