The Viral Ki-ras Gene must be Expressed in the G₂ Phase if ts Kirsten Sarcoma Virus-Infected NRK Cells are to Proliferate in Serum-Free Medium

Abstract

NRK cells infected with a temperature-sensitive Kirsten sarcoma virus (ts371 KSV) are transformed at 36°C, but are untransformed at 41°C which inactivates the abnormally thermolabile oncogenic p21^Ki product of the viral Ki-ras gene. At 41°C, tsKSV-infected NRK cells were arrested in G₀/G₁ when incubated in serum-free medium, but could then be stimulated to transit G₁, replicate DNA, and divide by adding serum at 41°C or dropping the temperature to a p21-activating 36°C without adding serum. When quiescent cells at 41°C were stimulated to transit G₁ in serum-free medium by activating p21 at 36°C and then shifted back to the p21-inactivating 41°C in the mid-S phase, they continued replicating DNA but could not transit G₂. Reactivating p21 in the G₂-arrested cells by once again lowering the temperature to 36°C stimulated a rapid entry into mitosis. By contrast, while serum-stimulated quiescent G0 cells at 41°C replicate DNA and divide, serum did not induce G₂-arrested cells to enter mitosis, indicating that serum growth factors may trigger events in the G₁ phase that ultimately determine G₂ transit. These observations made with the viral ras product suggest that cellular ras proto-oncogene products have a role in G₂ transit of normal cells.