Ki-ras activation in vitro affects GI and G2M cell-cycle transit times andapoptosis

Mutant ras genes occur frequently in human neoplasia and, in particular, in pancreatic, colorectal, and lung adenocarcinomas, Recent evidence suggests that G-->T and G-->C transversions of the Ki-ras gene in codon 12 may lead to biological effects in vitro and ill vivo that may be associated with an abnormal cell cycle and increased tumour aggressiveness. The role of Ki-ra s activation (a G-->C transversion in codon 12, arginine for glycine) in th e cell cycle and apoptosis was investigated using control and permanently t ransfected NIH3T3 mouse fibroblasts. Flow cytometry was used to evaluate th e G1-, S- and G2M-phase transit times, the potential doubling time, the gro wth fraction, and the cell loss factor during asynchronous exponential grow th. Apoptosis was induced in both cell lines by absence of growth factors f or an extended period of time (72 h) and quantitatively evaluated using the TUNEL method coupled with flow cytometry, It was found that codon 12 G-C K i-ras transfected cells compared with controls, had a significant prolongat ion of G1 by about 50%, a reduction of the G2M transit time by 30%, and a d ecrease of the cell loss factor by about 90%. Apoptotic cells were about 10 % in control and less than 0.5% in Ki-ras transfected cells after 72 h star vation-confluency. These data suggest that codon 12 G-->C Ki-ras activation in mouse NIH3T3 fibroblasts is associated with deregulation of checkpoint controls in the GI and G2M phases of the cell cycle and inhibition of apopt osis. It appears plausible that these cell mechanisms are related to a prol iferative advantage and that they may also be important in the progression of human tumours characterized by specific Ki-ras mutations. Copyright (C) 2000 John Wiley & Sons, Ltd.