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...

Oncogenes encoding serine/threonine or tyrosine kinases were introduced into the established rodent fibroblast cell line NIH 3T3 and tested for tumorigenic and metastatic behavior in T cell-deficient nude mice. Transforming oncogenes of the ras family were capable of converting fibroblast cell lines to fully metastatic tumors. Cell lines transformed by the kinase oncogenes mos, raf, src, fes, and fms formed experimental metastases and (in some cases) these genes were more efficient at metastatic conversion than a mutant ras gene. In contrast, cells transformed by either of two nuclear oncogenes, myc or p53, were tumorigenic when injected subcutaneously but were virtually nonmetastatic after intravenous injection. These data demonstrate that, in addition to ras, a structurally divergent group of kinase oncogenes can induce the metastatic phenotype.

Apoptosis and necrosis need to be differentiated in order to distinguish drug-induced cell death from spontaneous cell death due to hypoxia. The ability to differentiate between these two modes of cell death, especially at an early stage in the process, could have a significant impact on accessing the outcome of anticancer drug therapy in the clinic. Nuclear magnetic resonance spectroscopy was used to distinguish apoptosis from necrosis in human cervical carcinoma (HeLa) cells. Apoptosis was induced by treatment with the topoisomerase II inhibitor etoposide, whereas necrosis was induced by the use of ethacrynic acid or cytochalasin B. We found that the intensity of the methylene resonance increases significantly as early as 6 h after the onset of apoptosis, but that no such changes occur during necrosis. The spectral intensity ratio of the methylene to methyl resonances also shows a high correlation with the percentage of apoptotic cells in the sample (r 2 =0.965, P50.003). Cell Death and Differentiation (2001) 8, 219 ± 224.

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