RESUMO
Fibroblast growth factor 2 (FGF2) is considered to be a bona fide oncogenic factor, although results from our group and others call this into question. Here, we report that exogenous recombinant FGF2 irreversibly inhibits proliferation by inducing senescence in Ras-dependent malignant mouse cells, but not in immortalized nontumorigenic cell lines. We report the following findings in K-Ras-dependent malignant Y1 adrenocortical cells and H-Ras V12-transformed BALB-3T3 fibroblasts: (a) FGF2 inhibits clonal growth and tumor onset in nude and immunocompetent BALB/c mice, (b) FGF2 irreversibly blocks the cell cycle, and (c) FGF2 induces the senescence-associated beta-galactosidase with no accompanying signs of apoptosis or necrosis. The tyrosine kinase inhibitor PD173074 completely protected malignant cells from FGF2. In Y1 adrenal cells, reducing the constitutively high levels of K-Ras-GTP using the dominant-negative RasN17 mutant made cells resistant to FGF2 cytotoxicity. In addition, transfection of the dominant-negative RhoA-N19 into either Y1 or 3T3-B61 malignant cell lines yielded stable clonal transfectants that were unable to activate RhoA and were resistant to the FGF2 stress response. We conclude that in Ras-dependent malignant cells, FGF2 interacts with its cognate receptors to trigger a senescence-like process involving RhoA-GTP. Surprisingly, attempts to select FGF2-resistant cells from the Y1 and 3T3-B61 cell lines yielded only rare clones that (a) had lost the overexpressed ras oncogene, (b) were dependent on FGF2 for proliferation, and (c) were poorly tumorigenic. Thus, FGF2 exerted a strong negative selection that Ras-dependent malignant cells could rarely overcome.
Assuntos
Proliferação de Células , Senescência Celular/fisiologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Proteína Oncogênica p21(ras)/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Células 3T3 , Animais , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Nuclear phenotypes of clonal and polyclonal T24 H-ras-transformed NIH 3T3 cells differing in p21 expression and experimental metastatic ability were studied in Feulgen-stained preparations by image analysis. The objective was to determine if these cells varied in their degree of chromatin condensation, as previously reported with cell transformation, or in any other chromatin texture property highlighted by image analysis parameters. The majority of nuclei in all of these ras-transformed cells exhibited increased levels of chromatin condensation, independent of ras levels or metastatic properties. This chromatin texture characteristic was assumed to be related to the transformed phenotype. No significant changes in chromatin supraorganization that could be correlated directly with ras levels or metastatic ability were found, with the exception of an increased frequency of a relatively rare phenotype in highly metastatic cells. This phenotype was characterized by an extreme contrast in packing state between condensed and noncondensed chromatin. It is suggested that ras transformation results in alterations in chromatin structure but that there is not a simple relationship between ras p21 expression levels or ras-induced metastatic ability and the degree of these changes.