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.

Specific K-ras2 Mutations in Human Sporadic Colorectal Adenomas Are Associated with DNA Near-Diploid Aneuploidy and Inhibition of Proliferation

“…This finding, which appears in agreement with other literature data and hypotheses, 14,15,22,27 suggests that G3 C/T transversions disrupt chromosome stability. Although the exact mechanisms are not known, they may be related to mitotic checkpoints that maintain chromosome stability.…”

“…[7][8][9][10][11] Ras proteins were also reported to regulate the formation of stress fibers, focal cell adhesion, and cytokinesis. 12,13 Transfection of human mutated K-ras2 in mouse NIH-3T3 cells has been shown to induce destabilization of the chromosomes in mitosis 14,15 and generation of DNA aneuploid subclones as detected by flow cytometry (FCM). 15 Chromosome losses and chromatin textural changes by image cytometry were also shown to occur in H-ras-transformed human breast epithelial cells.…”

“…12,13 Transfection of human mutated K-ras2 in mouse NIH-3T3 cells has been shown to induce destabilization of the chromosomes in mitosis 14,15 and generation of DNA aneuploid subclones as detected by flow cytometry (FCM). 15 Chromosome losses and chromatin textural changes by image cytometry were also shown to occur in H-ras-transformed human breast epithelial cells. 16 Dependence of aneuploidy from H-ras mutations was also shown in chemically induced mouse skin papillomas from a very early stage.…”

Phylogenetic fate mapping

“…NIH 3T3 cells are diploid (23), and each marker displayed a distribution of intensities centered at either one or two maximal peaks (Fig. 2), corresponding to homozygosity or heterozygosity, respectively, for alleles of a particular autosomal marker.…”

Chromosome instability in human lung cancers: possible underlying mechanisms and potential consequences in the pathogenesis

“…Several lines of evidence also point to a role of mutational activation of the ras genes, which are known to be present in certain types of human cancers including lung adenocarcinomas. Abnormal mitotic figures, mainly characterized by lagging chromosomes in prometaphase, and the resulting various ranges of aneuploidy were frequently observed in NIH-3T3 expressing activated human K-ras (Hagag et al, 1990;Nigro et al, 1996). Increased karyotypic alterations were also observed in a chromosomally stable human colorectal cancer cell line, SW480, in proportion to the expression levels of ectopic human c-H-ras (de Vries et al, 1993).…”