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6th ESACP Congress, Heidelberg, April 7-11, 1999 |
A059
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.
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