Tumorigenic and metastatic properties of "normal" and ras-transfected NIH/3T3 cells.

Abstract

To investigate the role of oncogene activation in the pathogenesis of malignant tumors, we have studied the tumorigenic and metastatic properties of NIH/3T3 secondary transfectants (designated A51) containing an activated c-Haras-) gene derived from the human T24 bladder carcinoma cell line and compared them Tumors are classically defined as benign or malignant (1). Benign tumors are noninvasive growths that do not spread to distant organs. Unless located in a functionally vital site (e.g., brain), they pose little threat to the patient and usually can be removed surgically. In contrast, malignant neoplasms are readily invasive, metastasize to organs throughout the body, and eventually kill their host (1). The biochemical events that distinguish malignant from benign neoplasms remain unidentified. Work in several laboratories has implicated oncogene activation in the expression of tumorigenicity but the role of oncogenes in the pathogenesis of malignant versus benign tumors has received little attention. The NIH/3T3 cell line, although immortalized in vitro, is reportedly nontumorigenic and is widely used as a recipient cell line for detecting transforming gene sequences isolated from tumorigenic cell lines or tissues (2-6). For example, upon transfection with the activated ras gene, NIH/3T3 cells form foci in tissue culture, exhibit anchorage independence, and, in those experiments in which in vivo studies have been conducted, produce tumors in nude mice (6-10). However, the behavior of the tumors formed by transfected NIH/3T3 cells has not been rigorously evaluated and it remains unclear whether oncogene activation is an event associated strictly with the pathogenesis of benign neoplasms or whether activation is also an essential feature for expression of metastatic properties. We report that transfection with the activated c-Ha-ras-l gene accelerates the tumorigenicity and enhances the metastatic potential of NIH/3T3 Tumorigenicity and Metastasis. Tumorigenicity and spontaneous metastatic potential were assayed by inoculating mice with different cell doses in the footpad (i.m.) or the supraclavicular region (s.c.). Tumor size was monitored at the supraclavicular site every 2-3 days by caliper measurement. For studies on experimental metastasis, different numbers of cells were injected into the tail vein of nude mice. At autopsy the major organs of all animals were examined both grossly and histologically for evidence of metastases. Single sections were prepared from each organ except the lung, in which case multiple sections were examined.Detection of Activated c-Ha-ras Oncogene and Human Alu Sequences. For preparation of DNA (12), cell monolayers established from primary tumors or metastatic foci were dispersed into phosphate-buffered saline (Pi/NaCl), pelleted, rinsed, resuspended in 10 mM Tris HCl, pH 8.0/0.35 M NaCl/1 mM EDTA, lysed in 0.5% NaDodSO4, and treated for 4-12 hr with Pronase (0.1 mg/ml) at 37°C. DNA was extracted with phenol, ethanol precipitated, and dissolved in 10 mM Tris-HCl, pH ...

Cell uptake, distribution and response to aluminium chloro sulphonated phthalocyanine, a potential anti-tumour photosensitizer

“…NIH/3T3 cells, derived from a Balb/c mouse, generally are considered to be nontumorigenic (Aaronson & Todaro, 1968) though tumorigenic conversion may occur following prolonged cultivation in tissue culture (Greig et al, 1985). UV-2237 is an ultraviolet light-induced fibrosarcoma of C3H/He mice (Kripke et al, 1978).…”

Evidence for a role of Met-HGF/SF during Ras-mediated tumorigenesis/metastasis

“…Members of the Ras family of small GTPases (H-, K-and N-Ras) are frequently mutated (activated) in a number of human tumors (Bos, 1989), and in addition, oncogenic mutants of Ras have been shown to confer both tumorigenic and metastatic phenotypes to a variety of cell types in culture (Bondy et al, 1985;Greig et al, 1985;Muschel et al, 1985;Thorgeirsson et al, 1985). Although a number of downstream genetic events have been characterized in Ras-transformed cells, precisely which of these are important for the acquisition of the tumorigenic and metastatic phenotypes are poorly understood (Chambers and Tuck, 1993).…”

p53 increases experimental metastatic capacity of murine carcinoma cells.

“…Most gene transfer studies in this field have been performed with oncogenes of the ras family which were used to induce metastatic competence in tumorigenic but nonmetastatic cells mostly of fibroblastic origin (1,8,24). In two independent studies (26,27), activated ras genes were transfected together with a pSV2neoR plasmid into nonmetastatic murine mammary carcinoma cell lines.…”