Transformation of normal human fibroblasts into immortalized cells with the mutant p53 gene and X‐rays

Abstract

In vitro cell transformation is a valuable approach for studying the mechanisms of multistep carcinogenesis of human cells. Since immortalization is an essential step for in vitro neoplastic transformation of human cells, this study addresses the question of whether mutant p53 contributes to the immortalization process of human cells. The mutant p53 gene (mp53: codon273 Arg-His ) was introduced into normal human fibroblasts (OUMS-24 line) and a G418-resistant clone, OUMS-24/P6 line, was obtained. This clone showed an extended life span and chromosome abnormalities, but senesced at the 79th population doubling level (PDL). When these cells were subjected to intermittent X-ray treatment, they became an immortalized cell line (OUMS-24/P6X). Although these immortalized cells showed chromosome abnormalities, they were not tumorigenic. On the other hand, normal OUMS-24 cells into which mp53 had not been introduced were not immortalized by the same X-ray treatment. These results indicate that introduction and expression of mp53 alone were not sufficient for immortalization of human cells, and that mutations of the remaining wild-type p53 or other genes may have been necessary for immortalization. In vitro cell transformation is a valuable model for studying the mechanisms of multistep carcinogenesis. The human cell system is useful for studying the various stages of in vitro transformation, because events in the transformation process, from normal to neoplastic phenotypes, are clearly defined by the successive stages of aging, immortalization, and neoplastic transformation (Namba et al., 1988). For normal human cells to undergo neoplastic transformation, they must first be immortalized. However, normal human cells have a finite capacity to replicate and eventually enter a state of irreversible growth arrest (for review, Vojta and Barrett, 1995). This feature has been termed cellular senescence or replicative senescence. Cellular senescence is thought to be a genetically programmed process rather than the result of a random accumulation of damage. In normal human cells, in contrast to rodent cells, cellular senescence is particularly stringent and spontaneous immortalization is an extremely rare event.The immortalization process of human cells is probably related to deregulation of cellular senescence. Serum-stimulated senescent and quiescent human fibroblasts express many of the same genes regulating the cell cycle such as c-myc, c-jun and c-Ha-ras (Rittling et al., 1986;Seshadri and Campisi, 1989). However, senescent cells have lost the ability to express c-fos and cdc2-cyclin A kinase activity and to phosphorylate Rb protein (Seshadri and Campisi, 1990;Stein et al., 1990Stein et al., , 1991. p53 regulates the cell-cycle machinery partially through the expression of p21, because p21 inhibits the action of multiple cyclin-dependent kinase (cdk) complexes and arrests growth (Noda et al., 1994).We obtained 3 human cell lines immortalized with either 4-nitroquinoline 1-oxide (4-NQO) or 60 Co gamma-rays (Namba et al., 1988...

CD90 Is a Diagnostic Marker to Differentiate Between Malignant Pleural Mesothelioma and Lung Carcinoma With Immunohistochemistry

“…12 CD90 is also known as one of the candidates for cancer stem cell markers, 13 and the present study showed that immortalized Met-5A cells of mesothelium origin were negative for CD90 expression. Our study, however, also showed that human immortalized fibroblasts, HFF 14 and OUMS-24, 15 were positive for CD90, and 3 of 5 reactive mesothelial hyperplasia specimens expressed CD90 with the same staining pattern as found in mesothelioma (data not shown). In contrast, 6 cases of normal mesothelium were negative for CD90 expression (data not shown).…”

Inactivation of p53 and life span extension of human diploid fibroblasts by mot‐2

“…The expression of the p53‐induced cell cycle inhibitor p21 SDI1/WAF1 also increases at senescence [11]. Expression of a dominant negative p53 mutant in normal human diploid fibroblasts permitted them to continue dividing for a limited number of population doublings (PDs) beyond the point at which their normal counterparts became senescent [12,13]. Evidence that this effect was due to loss of wt p53 function and not due to a p53 gain‐of‐function mutation was provided by studies demonstrating that spontaneous loss of p53 function in fibroblasts from an individual with Li–Fraumeni syndrome resulted in a substantial, but finite, increase in proliferative capacity [14].…”

The Human P53 Gene Mutated At Position 249 Per Se Is Not Sufficient to Immortalize Human Liver Cells

“…39 However, it seems that in addition to the expression of mutant p53 further genetic events (e.g., the activation of a proto-oncogene or the inactivation of another tumor suppressor gene) are needed to immortalize human liver cells such as the HACL-1 cells. This also applies to normal human fibroblasts: Fushimi et al 40 very recently showed that the transfection and expression of a p53 cDNA mutated at codon 273 in the human embryo fibroblast strain OUMS-24 expands their life-span but does not lead to their immortalization. …”