Modulation of metastatic potential of v-src transformed fibroblasts as a result of exogenous DAP-kinase activity

E Zueva 1, E Tchevkina 1, A Kimchi 2, A Tatosyan 1

1 Cancer Research Centre, Moscow, Russia 2 Weizmann Institute, Israel

Aim: In order to identify cellular genes and proteins involved in the metastatic process of transformed cells a panel of cell lines transformed by variants of v-src oncogene with different metastatic potential in vivo were used. Recently discovered DAP-kinase gene (a novel type of calmodulin-dependent serine-threonine kinase with antitumorigenic and antimetastatic effects) was introduced into cells with different metastatic capacity. The activity of selected signal transduction proteins and biological properties of new isolated cells were examined. Methods: transfection ?f v-src-transformed fibroblasts having different metastatic potential by DAP-kinase. Analysis of experimental (EMA i.v. injection of the cells) and spontaneous (SMA s.c. injection of the cells) metastatic characteristics of the transfectants in vivo. Comparative immunoblot analysis of different signal transduction proteins supposed to be involved in metastatic processes. Results: DAP-kinase significantly suppressed EMA and SMA of transformed cell lines. Parental and control (carrying empty vector) lines induced from 50 to 300 metastatic nodules in the lungs of all i.v. injected animals. DAP-kinase expressing cells never induced more than 20 nodules. SMA test also shows multiple reductions of metastatic activities of DAP-kinase expressing cells: 40% of these cells were not able to produce metastasis at all, 60% produced less than 50 nodules in the lungs. The differences in metastatic potential of analyzed cells in vivo were also confirmed in vitro by collagen degradation test: cells producing DAP partially lost collagenase activity i.e. this parameter correlates with suppression of metastatic potential of transformed cells. It was found that production and activity of selected members of signaling pathways (MAP-k, FAK, Shc, etc.) were changed in cells carrying different phenotype after introduction of DAP-kinase gene. Production and enzymatic function of v-src protein in transfected lines was not changed. It was shown, that wildtype p53 protein is more stable in DAP-kinase-expressing cells than in parental cells. Conclusion The described collection of new cells (8 lines) with modulated metastatic properties is unique model system for identification of specific genetic and molecular factors responsible for invasive and metastatic behavior of tumour cells.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Metastasis.