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

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. © 1996 Wiley‐Liss, Inc.