Loss of oncogenic ras expression does not correlate with loss of tumorigenicity in human cells.

Abstract

ras oncogenes are mutated in at variety of human tumors, which suggests that they play an important role in human carcinogenesis. To determine whether continued oncogenic ras expression is necessary to maintain the malignant phenotype, we studied the human fibrosarcoma cell line, HT1080, which contains one mutated and one wild-type N-ras allele. We isolated a variant of this cell line that no longer contained the mutated copy of the N-ras gene. Loss of mutant N-ras resulted in cells that displayed a less transformed phenotype characterized by a flat morphology, decreased growth rate, organized actin stress fibers, and loss of anchorage-independent growth. The transformed phenotype was restored following reintroduction of mutant N-ras. Although loss of the oncogenic N-ras drastically affected in vitro growth parameters, the variant remained tumorigenic in nude mice indicating that mutated N-ras expression is not necessary for maintenance of the tumorigenic phenotype. We confirmed this latter observation in colon carcinoma cell lines that have lost activated K-ras expression via targeted knockout of the mutant K-ras gene.

A dominant negative RAS-specific guanine nucleotide exchange factor reverses neoplastic phenotype in K-ras transformed mouse fibroblasts

“…The observed heterogeneity might be due to development of mutations that allow ras-independent tumor growth (see for instance Aftab et al, 1997;Thomas and Hall, 1997), as well as to loss or inactivation of the dominant-negative protein. It is worth noting that in HT1080 cells and colon carcinoma cell lines loss of the mutated N-ras or kras gene resulted in reversion of in vitro growth parameters, with the mutated cells remaining tumorigenic in nude mice (Plattner et al, 1996). The recent discovery that oncogenic ras activation, in cooperation with the catalytic domain of telomerase and disruption of the intracellular pathway(s) regulated by the large T antigen is su cient to create a human tumor cell (Hahn et al, 1999) and the report of an essential role for ras ± extending beyond the regulation of VEGF expression in vivo ± in the maintenance of solid tumors (Chin et al, 1999) are good indications that downregulation of ras signal transduction may become a valuable tool in tumor therapy.…”

Differential contribution of the ERK and JNK mitogen-activated protein kinase cascades to Ras transformation of HT1080 fibrosarcoma and DLD-1 colon carcinoma cells

“…In the present study, we utilized two human tumor cell systems, where the contribution of oncogenic Ras to transformation has been established, in order to evaluate the role of the ERK and JNK pathways in cellular transformation. Parental and variant populations of HT1080 human ®brosarcoma and DLD-1 human colon carcinoma cells that di er in their Ras mutation status were evaluated (Plattner et al, 1996;Shirasawa et al, 1993). Whereas the expression of mutated N-Ras(61K) correlated with constitutive upregulation of the Raf/MEK/ERK and JNK pathways in HT1080 cells, no such correlation was observed for the expression of mutated K-Ras(13D) in DLD-1 cells.…”

“…We have shown previously that loss of the mutated N-ras allele caused an appearance of well organized actin stress ®bers in MCH603c8 cells (Plattner et al, 1996). Therefore, we determined whether blocking ERK activation alone could similarly restore actin stress ®ber formation.…”

“…Loss of mutated ras caused a dramatic impairment in growth in vitro (e.g., colony formation in soft agar) and in tumor formation in nude mice. Second, we have shown that a variant of the HT1080 human ®brosarcoma cell line (designated MCH603c8), that had lost the short arm of chromosome one harboring the mutated N-ras allele (Anderson et al, 1994), corresponded to a signi®cant impairment in its ability to grow in soft agar and to form tumors in nude mice (Plattner et al, 1996). This deletion occurred serendipitously during the microcell transfer of a copy of chromosome 1 from HT1080 cells that contained the wild type allele of N-ras into recipient HT1080 cells, i.e.…”

“…A valid criticism of experimental studies in rodent cell systems is that overexpression of an exogenously introduced mutated ras gene is used to cause transformation (Hua et al, 1997). The mutated N-ras(61K) and K-ras(13D) alleles present in HT1080 and DLD-1 cells, respectively, arose during the natural development of these tumor cells (Plattner et al, 1996;Shirasawa et al, 1993). The availability of variants of HT1080 and DLD-1 cells, that have lost their mutated ras alleles, allows for a comparative analysis to determine the signaling pathways that are activated by oncogenic Ras to cause tumorigenic transformation of these cells.…”