Anti-proliferative mechanisms induced by FGF2 and phorbol ester in murine cell lines transformed by Ras

Author(s)

Tatiana Guimarães de Freitas Matos

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2007-09-17

Examining board members

Hugo Aguirre Armelin; Marcelo Ribeiro da Silva Briones; Alícia Juliana Kowaltowski; Eduardo Moraes Rego Reis; Sara Teresinha Ollala Saad

• Advisor: Hugo Aguirre Armelin

Abstract

Amplification and gain of function mutations in ras proto-oncogenes are frequent genetic lesions in human cancers of bad prognostic. This thesis aimed to investigate novel anti-proliferative mechanisms induced by two mitogens, FGF2 (\"Fibroblast Growth Factor 2\") and PMA (\"Phorbol-12-Myristate-13-Acetate\", a phorbol diester), in murine cell lines transformed by ras and resistant to apoptosis. To this end, we took two different mouse malignant cell lines: Y1, a cell line derived from an adrenal tumor, naturally transformed by K-ras amplification and another one, 3T3-B61, obtained by transformation of Balb-3T3 fibroblasts with the H-rasV12 oncogene. To elucidate FGF2 mechanisms of action, we selected, isolated and characterized clonal sublines resistant to FGF2 from both Y1 and 3T3-B61 parental lines. FGF2-resistant clones are rare normal-like revertant sublines that no longer display Ras over expression, dependent on FGF2 for growth, do not grow in suspension cultures and exhibit low tumorigenicity in Nude mice. These results show that FGF2 exerts a strong selective pressure against ras-transformed cells, inducing senescence and irreversibly blocking proliferation. Differently from FGF2 , PMA citotoxic effect is completely dependent on PKC activity. In addition, PMA is highly toxic to K-Ras transformed Y1 cells, poorly toxic to H-Ras-transformed 3T3-B61 cells and not toxic to immortalized non tumorigenic cell lines. Attempts to select PMA-resistant cells fropm Y1 parental line have yielded very rare, highly clonal sublines, dependent on FGF2 for proliferation. In conclusion, two mitogens, FGF2 and PMA, can selectively inhibit Ras-driven proliferation, a phenomenon of great interest for biology and therapy of tumors dependent on ras oncogenes. (AU)