RESUMO
Ultraviolet radiation is the main cause of DNA damage to melanocytes and development of melanoma, one of the most lethal human cancers, which leads to metastasis due to uncontrolled cell proliferation and migration. These phenotypes are mediated by RhoA, a GTPase overexpressed or overactivated in highly aggressive metastatic tumors that plays regulatory roles in cell cycle progression and cytoskeleton remodeling. This work explores whether the effects of UV on DNA damage, motility, proliferation, and survival of human metastatic melanoma cells are mediated by the RhoA pathway. Mutant cells expressing dominant-negative (MeWo-RhoA-N19) or constitutively active RhoA (MeWo-RhoA-V14) were generated and subjected to UV radiation. A slight reduction in migration and invasion was observed in MeWo and MeWo-RhoA-V14 cells but not in MeWo-RhoA-N19 cells, which presented inefficient motility and invasiveness associated with stress fibers fragmentation. Proliferation and survival of RhoA-deficient cells were drastically reduced by UV compared to cells displaying normal or high RhoA activity, suggesting increased sensitivity to UV. Loss of RhoA activity also caused less efficient DNA repair, with elevated levels of DNA lesions such as strand breaks and cyclobutane pyrimidine dimers (CPDs). Thus, RhoA mediates genomic stability and represents a potential target for sensitizing metastatic tumors to genotoxic agents.
Assuntos
Melanoma/patologia , Raios Ultravioleta , Proteína rhoA de Ligação ao GTP/metabolismo , Proteínas Reguladoras de Apoptose , Linhagem Celular Tumoral/efeitos da radiação , Movimento Celular , Proliferação de Células , Ensaio Cometa , Dano ao DNA , Reparo do DNA , Proteínas de Ligação ao GTP , Glutationa Transferase/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Melanoma/metabolismo , Microscopia de Fluorescência , Invasividade Neoplásica , Metástase Neoplásica , Dímeros de Pirimidina/química , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologiaRESUMO
Radiotherapy with γ-radiation is widely used in cancer treatment to induce DNA damage reducing cell proliferation and to kill tumor cells. Although RhoA GTPase overexpression/hyperactivation is observed in many malignancies, the effect of RhoA activity modulation on cancer radiosensitivity has not been previously investigated. Here, we generated stable HeLa cell clones expressing either the dominant negative RhoA-N19 or the constitutively active RhoA-V14 and compared the responses of these cell lines with those of parental HeLa cells, after treatment with low doses of γ-radiation. HeLa-RhoA-N19 and HeLa-RhoA-V14 clones displayed reduced proliferation and survival compared to parental cells after radiation and became arrested at cell cycle stages correlated with increased cellular senescence and apoptosis. Also, Chk1/Chk2 and histone H2A phosphorylation data, as well as comet assays, suggest that the levels of DNA damage and DNA repair activation and efficiency in HeLa cell lines are correlated with active RhoA. In agreement with these results, RhoA inhibition by C3 toxin expression drastically affected homologous recombination (HR) and nonhomologous end joining (NHEJ). These data suggest that modulation of RhoA GTPase activity impairs DNA damage repair, increasing HeLa cell radiosensitivity.
Assuntos
Reparo do DNA/efeitos da radiação , Raios gama , Proteínas de Neoplasias/metabolismo , Neoplasias do Colo do Útero/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , ADP Ribose Transferases/farmacologia , Toxinas Botulínicas/farmacologia , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Feminino , Células HeLa , Humanos , Mutação , Proteínas de Neoplasias/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Cell division control protein 42 (CDC42) homolog is a small Rho GTPase enzyme that participates in such processes as cell cycle progression, migration, polarity, adhesion, and transcription. Recent studies suggest that CDC42 is a potent tumor suppressor in different tissues and is related to aging processes. Although DNA damage is crucial in aging, a potential role for CDC42 in genotoxic stress remains to be explored. Migration, survival/proliferation and DNA damage/repair experiments were performed to demonstrate CDC42 involvement in the recovery of HeLa cells exposed to ultraviolet radiation-induced stress. Sub-lines of HeLa cells ectopically expressing the constitutively active CDC42-V12 mutant were generated to examine whether different CDC42-GTP backgrounds might reflect different sensitivities to UV radiation. Our results show that CDC42 constitutive activation does not interfere with HeLa cell migration after UV radiation. However, the minor DNA damage exhibited by the CDC42-V12 mutant exposed to UV radiation most likely results in cell cycle arrest at the G2/M checkpoint and reduced proliferation and survival. HeLa cells and Mock clones, which express endogenous wild-type CDC42 and show normal activity, are more resistant to UV radiation. None of these effects are altered by pharmacological CDC42 inhibition. Finally, the phosphorylation status of the DNA damage response proteins γ-H2AX and p-Chk1 was found to be delayed and attenuated, respectively, in CDC42-V12 clones. In conclusion, the sensitivity of HeLa cells to ultraviolet radiation increases with CDC42 over-activation due to inadequate DNA repair signaling, culminating in G2/M cell accumulation, which is translated into reduced cellular proliferation and survival.
Assuntos
Proliferação de Células/efeitos da radiação , Reparo do DNA , Raios Ultravioleta/efeitos adversos , Proteína cdc42 de Ligação ao GTP/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Movimento Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Células HeLa , Humanos , Mutação , Tolerância a Radiação , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
Rac1 GTPase controls essential cellular functions related to the cytoskeleton, such as motility and adhesion. Rac1 is overexpressed in many tumor cells, including breast cancers, where it is also involved in the proliferation and checkpoint control necessary for the cell's recovery after exposure to ionizing radiation. However, its role in DNA damage and repair remains obscure in other tumor cells and under different genotoxic conditions. Here, we compare HeLa cells with mutants exogenously expressing a dominant-negative Rac1 (HeLa-Rac1-N17) by their responses to DNA damage induced by gamma or UV radiation. In HeLa cells, these treatments led to increased levels of active Rac1 (Rac1-GTP) and of stress fibers, with a diminished ability to migrate compared to untreated cells. However, the reduction of Rac1-GTP in Rac1-N17-deficient clones resulted in much higher levels of polymerized stress fibers accompanied by a strong impairment of cell migration, even after both radiation treatments. With regard to proliferation and genomic stability, dominant-negative Rac1 cells were more sensitive to gamma and UV radiation, exhibiting reduced proliferation and survival consistent with increased DNA damage and delayed or reduced DNA repair observed in this Rac1-deficient clone. The DNA damage response, as indicated by pH2AX and pChk1 levels, was increased in HeLa cells but was not effectively triggered in the Rac1-N17 clone after radiation treatment, which is likely the main cause of DNA damage accumulation. These data suggest that Rac1 GTPase plays an important role in signaling and contributes to the sensitivity of cervical cancer cells under UV or gamma radiation treatments.