6th ESACP Congress, Heidelberg, April 7-11, 1999

A059
IN VITRO AND IN VIVO ROLE OF K-RAS2 MUTATIONS TO INDUCE DEREGULATION OF PROLIFERATION-APOPTOSIS AND CHROMOSOME STABILITY
Giaretti W, Rapallo A, Di Vinci A, Infusini E, Sciutto A, Orecchia R, Geido E

Laboratory of Biophysics-Cytometry, National Cancer Institute, Genoa, Italy

Methods used to analyze proliferation-apoptosis in K-ras2 transfected 3T3 cells included pulse and continuous labelling of Bromodeoxyuridine and "TdT-mediated dUTP-biotin nick end labelling". DNA Index and S-phase fraction of human pathological colorectal specimens were, instead, analyzed by DAPI multiparameter flow cytometry, while K-ras2 spectrum analysis was done by Sequence Specific Oligonucleotide hybridization. K-ras2 transfected versus control cells had a significant time elongation of the G1 cell cycle phase of about 40%, a reduction of the G2M transit time of 25%, and decrease of the cell loss factor of 40%. In addition, they showed a twenty-fold higher frequency of abnormal mitoses and DNA aneuploid subclones. Sulindac sulfide apoptosis was induced in a dose-time dependent way (100-300µM; 24-72h) up to 70% in control but not higher than 20% in K-ras2 transfected 3T3 cells. Colorectal adenomas showed K-ras2 mutations and DNA aneuploidy in 29/116 (25%) cases. DNA aneuploidy (mainly near-diploid) was strongly associated with G-C/T transvertions while low S-phase correlated with G-A transitions. Multivariate logistic regression accounted for the effects of size, dysplasia, site, type, age and sex. Among adenocarcinomas, K-ras2 mutations showed mainly (87.5%) intratumor homogeneous distribution, were more frequently associated with near-diploid aneuploidy, and, in particular, G-T transvertions were associated to increased S-phase and higher risk of recurrence and death (Rascal study, JNCI 1998, 90:675-684; and extentions of AJP 1996, 149:237-245 and AJP 1998, 153:1201-1209). In conclusion, our data suggest that K-ras2 mutations in vitro and in the human colorectal adenoma-carcinoma sequence model is accompanied by destabilization of the genome and deregulation of proliferation-apoptosis and that apoptotic response to sulindac sulfide (antioxidants are under study) is higher in absence of K-ras2 mutations.