Transfection of human mutated K-ras in mouse NIH-3T3 cells is associated with increased cloning efficiency and DNA aneuploidization.

Abstract

The aim of the study was to test the hypothesis that a human mutated K-ras protein induces abnormalities in mitosis and development of sub-clones characterized by changes in DNA ploidy and proliferation. For this purpose, we used control and NIH-3T3 mouse cells transfected with the human codon 12 G-C-mutated K-ras oncogene. We found that abnormal mitoses, mainly characterized by lagging chromosomes in prometaphase or anaphase, had a significantly higher frequency in transfected cells than in control cells. The generation of sub-clones was screened by limiting-dilution experiments followed by cell expansion. Cloning efficiency was much higher for the K-ras transfected cells with 858/2112 (41%) successful sub-clones than for control, which provided 564/2592 (22%) sub-clones. DNA flow cytometry of 4.6-diamidino-2-phenilindole-2-hydrochloride-stained nuclei from randomly selected sub-clones was performed in order to evaluate DNA index and S-phase fraction values. We found 9 out of 100 DNA aneuploid sub-clones generated by the K-ras-transfected cells vs. 1 out of 100 for the controls. Overall, our data indicate that high expression of the mutationally activated human K-ras product in NIH-3T3 cells was associated with abnormal mitoses, increase of cloning efficiency and DNA aneuploidization.