Genetic factors and suppression of metastatic ability of v-Ha-ras-transfected rat mammary cancer cells.

Abstract

It has been demonstrated that when nonmetastatic rat mammary cancer (RMC1) cells are transfected with the mutated v-Ha-ras oncogene, an occasional resultant transfectant develops high metastatic ability (9). There is, however, no simple dose-response relationship between the level of the mutated v-Ha-ras expression in these transfectants and the development of metastases. Cytogenetic analysis of the same RMC1 system has demonstrated that the frequency of chromosomal changes in the mutated v-Ha-ras transfectants is significantly higher than that in control transfectants (10). These studies also suggest that if the appropriate chromosomal changes occur, these v-Ha-ras RMC1 transfectants acquire high metastatic ability. If such cytogenetic changes involve gain in gene expression only, then all hybrids formed by fusing highly metastatic v-Ha-ras RMC1 transfectants with the parental nonmetastic RMC1 should be highly metastatic. If the loss of a metastatic suppressor gene(s) is also involved, then such hybrids should be nonmetastatic since chromosomes from the nonmetastatic parental cells should supply the suppressor function. To test this possibility, highly metastatic cloned v-Ha-ras RMC1 transfectant cells were fused with nonmetastatic parental RMC1 cells and the metastatic behavior of the hybrid clones was examined. MATERIALS AND METHODSAnimals. All animals used in these studies were 6-to 8-wk-old female athymic nude mice obtained from HarlanSprague-Dawley. Tumor cells (5 x 105) were injected s.c. into nude mice and the tumor volume-doubling time was determined as described (11).Cell Culture and Transfection of RMC1 Cells. A hormonally independent dimethylbenz[a]anthracene-induced rat mammary cancer (i.e., RMC1) was established in culture as described (9). This RMC1 cell line was maintained as a monolayer culture in RPMI 1640 medium/10%o fetal bovine serum (HyClone), containing streptomycin (100 ,g/ml), penicillin (100 units/ml), and dexamethasone (250 nM) (standard medium) at 370C in 5% C02/95% air. RMC1 cells were transfected either with plasmid pY3, which encodes the hygromycin B-resistance gene (12), or with plasmid prasZip-6, which encodes the v-Ha-ras oncogene plus the neomycin-resistance gene (13) (i.e., coding for resistance to the G-418 neomycin analog) by the calcium phosphate precipitation procedure as described (14). Five independent clones were obtained from RMC1 cells transfected with plasmid pY3 and nine independent clones were obtained from RMC1 cells transfected with plasmid prasZip-6 using hygromycin B (500 ,&g/ml) selection and G-418 (500 ;Lg/ml) selection, respectively, as described (14). One clone from RMC1 cells transfected with plasmid pY3 (RMC1-hygro) and one highly metastatic clone from RMC1 cells transfected with plasmid prasZip-6 (RMC1-Ras) were used to produce hybrid cells. These RMC1-hygro cells and RMC1-ras cells were maintained in standard medium with hygromycin B (500 ug/ml; GIBCO) and G-418 (500 ,ug/ml; GIBCO), respectively.Cell Hybridization. Nonmetastatic RMC1-hygro cells...

Nm23-H1 was involved in regulation of KAI1 expression in high-metastatic lung cancer cells L9981

“…The function of KAI1 (CD82) in cancer progression was analyzed using genetic screening in a prostate cancer cell line aim to identify metastasis suppress genes (5). Down-regulation of KAI1 expression was associated with advanced stages of many malignancies including prostate, colon, lung, pancreatic, breast, ovarian and other cancers (6,7).…”

Generation of metastatic variants by transfection of a rat non-metastatic epithelial cell line with genomic DNA from rat prostatic carcinoma cells

“…Chromosometransfer studies have confirmed human chromosome 17q12-22 to contain a novel tumour-suppressor gene in this region (Murakami et al, 1995). Hybridization of the non-metastatic Dunning AT2.1 cell line with highly metastatic prostate carcinoma AT3.1 cells (Ichikawa et al, 1991) led to the discovery of a small metastasissuppressor gene and its human counterpart, KAII located on human chromosome 1 1.2 (Dong et al, 1995). Conversely, after differential display analysis of cell lines with distinct behavioural phenotypes from within the Dunning rat prostatic carcinoma model, the protein thymosin j15 has been identified to be selectively elevated in the metastatic carcinoma cells (Bao et al, 1996) and its gene regarded as a possible 'metastasis gene'.…”

Development of monoclonal antibodies for the identification of novel invasion associated targets in human cancer

“…These pathways typically involve phosphorylation of focal adhesion kinase (FAK), a cytoplasmic protein kinase that is localised at structures called focal adhesions (Brooks et al, 2010). FAK has been shown to promote cell migration through direct modulation of key proteins involved in the remodelling of the actin cytoskeleton, including the Rho subfamily of small GTPases (Hsia et al, 2003), N-WASP (Wu et al, 2004), and the Arp2/3 complex (Ichikawa et al, 1991).…”