Expression of H-ras correlates with metastatic potential: evidence for direct regulation of the metastatic phenotype in 10T1/2 and NIH 3T3 cells.

Abstract

Using three independent approaches, we studied the effects of H-ras on metastasis formation. Analysis of five in vitro-ras-transfected 1OT1/2 clones with either flat or refractile morphologies revealed a relationship between metastatic potential, H-ras expression, and anchorage-independent growth. Four metastatic variants derived from a poorly metastatic, low-H-ras-expressing line all expressed high levels of H-ras RNA and grew efficiently in soft agar. Activation of H-ras expression in the metastatic tumors had occurred through amplification and rearrangement of H-ras sequences. In addition, preinduction of p21 synthesis in NIH 3T3 line 433, which contains v-H-ras under transcriptional control of the glucocorticoid-sensitive mouse mammary tumor virus long terminal repeat, significantly increased metastatic efficiency. Glucocorticoid treatment of normal or pEJ-transformed NIH 3T3 cells did not affect metastatic potential. These data reveal a direct relationship between ras expression and metastasis formation and suggest that metastatic and transformed phenotypes may be coregulated in ras-transformed 10TI/2 and NIH 3T3 cells.Tumor progression is the tendency of tumors to become more aggressive with time. The widely accepted model is that described originally by Foulds (11), in which progression was characterized by emergence of new variants with a selective advantage for growth in the host. Metastatic spread is the most important form of tumor progression because it is the most life-threatening aspect of the disease. Metastasis is a complex process involving invasion through host barriers into the vasculature and survival against circulating host immune defenses, followed by implantation, extravasation, and growth at sites distant to the primary neoplasm (24,25,27,34).Much effort has been directed at understanding the metastatic cascade, yet little is known about the mechanisms involved. On the other hand, the critical events in cell immortalization and transformation have been partially elucidated and attributed to mutation or disregulation of a group of genes collectively known as oncogenes. These genes are normally responsible for maintenance of control over diverse cellular functions including proliferation, differentiation, morphological regulation, communication, and motility (3,20,22,33,40). Consequently, they are good candidates for study of the metastatic process, which also requires alterations of many of these functions (24,27,34).Recent studies showed that primary and established rodent fibroblasts transformed by activated ras sequences can form metastases (12,18,23,27,36). Exclusive selection and analysis of in vitro-transformed foci, however, raises the possibility that ras transformation may be a permissive event for expression of the metastatic phenotype but may not be directly involved. To resolve this question we analyzed the relationship between ras expression and metastatic poten-* Corresponding author.tial. ras-mediated transformation may be the initial event which allows expression of second...

Amplification of Ki-ras and elevation of MAP kinase activity during mammary tumor progression in C3(1)/SV40 Tag transgenic mice

“…The relationship of cell growth and metastases to enhanced expression of normal Ras proteins has been demonstrated in both cell lines and in human breast cancers (Czerniak et al, 1989;Egan et al, 1987;Shekhar and Miller, 1995). For example, a quantitative correlation between Ras expression and experimental metastases has been shown in Ras transfected 10T1/2 cells (Egan et al, 1987).…”

“…The relationship of cell growth and metastases to enhanced expression of normal Ras proteins has been demonstrated in both cell lines and in human breast cancers (Czerniak et al, 1989;Egan et al, 1987;Shekhar and Miller, 1995). For example, a quantitative correlation between Ras expression and experimental metastases has been shown in Ras transfected 10T1/2 cells (Egan et al, 1987). Studies of human breast cancers show that Ras protein levels are signi®cantly higher in cancer cells than in epithelial cells of control specimens and that higher levels of Ras p21 are associated with axillary lymph node metastases (Czerniak et al, 1989).…”

Fibroblasts transformed by combinations of ras, myc and mutant p53 exhibit increased phosphorylation of histone H1 that is independent of metastatic potential

“…10T1A cells are nontumorigenic [19,23]. b Metastatic potential was determined after intravenous injection of lxl05 cells into the tail veins of immunocompetent C3IM HeN syngeneic mice (five mice/clone) as described previously [19,23].…”

A link between ferritin gene expression and ribonucleotide reductase R2 protein, as demonstrated by retroviral vector mediated stable expression

“…The retroviral vector package lines ¥2, PA317, and their derivatives were maintained in Dulbecco's modified Eagle's medium (Gibco) containing 10% calf serum and 0.45% glucose. Determinations of cell division times, and sensitivities to hydroxyurea cytotoxicity by estimating relative colony forming efliciencies, were carried out as described previously [10,11].…”

“…Electrophoresis, 32p-labelling of eDNA probes [5,6] and blotting procedures have been described [5,6,10]. Radioactivity on filters was detected by a phophorimager (Molecular Dynamics Inc., Sunnyvale, CA), and quantitation was performed using Image Quant software (Molecular Dynamics, Inc.).…”