RESUMO
Nonmelanoma skin cancer (NMSC) is the most frequently diagnosed cancer in the United States. Deregulation of bcl-2 and ras family members is commonly observed in NMSC. It has been previously demonstrated that simultaneous bcl-2 and Ha-ras gene expression in keratinocytes results in disordered differentiation and resistance to cell death induced by ultraviolet (UV) radiation. It was, therefore, interest to assess the extent of cooperation between bcl-2 and Ha-ras during multistep skin carcinogenesis. The keratin 1 promoter was used to generate HK1.ras and HK1.bcl-2 transgenic mice, which were subsequently crossed to generate HK1.ras/bcl-2 double transgenic mice. The apoptotic index (AI) following UV-irradiation was significantly lower in HK1.bcl-2 and HKI.ras/bcl-2 epidermis compared to control littermates. Interestingly, the AI of HK1.ras/bcl-2 mice was significantly lower than even HK1.bcl-2 mice following UV-irradiation. To investigate the interaction of these oncogenes in skin tumorigenesis, a two-stage chemical carcinogenesis protocol was used to induce tumors. The individual contributions of Ha-ras and bcl-2 to papilloma latency, incidence, and growth rate in HK1.ras/bcl-2 double transgenic mice was marginally additive. Papillomas arising in HK1.ras transgenic mice exhibited the highest rate of apoptosis whereas papillomas arising in the HK1.ras/bcl-2 double transgenic mice exhibited rates of apoptosis that were significantly lower than papillomas arising in either control littermate or HK1.ras mice. Constitutive expression of either Ha-ras or bcl-2 exhibited similar rates of malignant tumor progression and they were not significantly different than control littermates. Importantly, when these two oncoproteins were coexpressed, a significant, and synergistic, increase in malignant transformation was observed.
Assuntos
Apoptose , Transformação Celular Neoplásica , Genes ras/fisiologia , Papiloma/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Neoplasias Cutâneas/metabolismo , Animais , Animais Recém-Nascidos , Células Epidérmicas , Epiderme/metabolismo , Feminino , Queratinócitos/citologia , Queratinócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Papiloma/patologia , Regiões Promotoras Genéticas , Pele/citologia , Pele/metabolismo , Neoplasias Cutâneas/patologia , Raios UltravioletaRESUMO
Chk1 (checkpoint kinase 1) is an evolutionarily conserved serine/threonine kinase involved in DNA damage responses. Originally identified as a kinase regulating the G2/M transition checkpoint, its role has broadened to include the S-phase checkpoint response and essential functions in early embryonic development. In this manuscript we investigated the potential of chemo-sensitization via ablation of Chk1 in cells treated with anti-metabolite cancer drugs, hydroxyurea (HU) and cytosine arabinoside (ara-C). Exposure to these replication interfering drugs in cells carrying Chk1 targeted siRNA provoked markedly increased rates of apoptosis. Although cell death was accompanied by an increase in p53 and activation of Chk2, the increased susceptibility to apoptosis was not dependent on p53 or Chk2. Additionally, we found that cells with reduced Chk1 expression displayed increased gamma-H2A.X expression, a marker for damaged DNA, and phosphorylated 32kDa subunit of replication protein A (RPA). Thus, Chk1 may play an essential role in maintaining DNA integrity during the replication block. Significantly, normal cells such as WS1 did not exhibit increased DNA damage or subsequent increases in apoptosis following replication stress, in the absence of Chk1. Thus, the essential role Chk1 plays in maintaining viability during the replication block in cancer cell lines can be exploited to sensitize cancer cells when abrogation of Chk1 is combined with DNA anti-metabolite chemotherapeutic drugs. Taken together, these data suggest that inhibition of Chk1 in combination with DNA anti-metabolite chemotherapy is a viable therapeutic strategy.
Assuntos
Sobrevivência Celular , Replicação do DNA , Proteínas Quinases/fisiologia , Antineoplásicos/farmacologia , Apoptose/genética , Apoptose/fisiologia , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Citarabina/farmacologia , Dano ao DNA/genética , Dano ao DNA/fisiologia , Replicação do DNA/efeitos dos fármacos , DNA de Neoplasias/genética , Histonas/genética , Histonas/fisiologia , Humanos , Hidroxiureia/farmacologia , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , RNA Interferente Pequeno/efeitos dos fármacos , RNA Interferente Pequeno/fisiologia , Proteína de Replicação A/genética , Proteína de Replicação A/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologiaRESUMO
The utility of dominant acting proapoptotic molecules to induce cell death in cancer cells is being evaluated in preclinical studies and clinical trials. We recently developed a binary adenoviral expression system to enable the efficient gene transfer of Bax and other proapoptotic molecules. Using this system, overexpression of Bax protein in four non-small-cell lung cancer (NSCLC) cell lines, H1299, A549, H226 and H322, was evaluated. The H322 line exhibited significant resistance to Bax-induced cell death compared to the other cell lines. H322 cells had the highest level of glutathione (GSH). GSH levels were significantly decreased following buthionine sulfoximine treatment and this coincided with enhanced apoptosis induction by Ad-Bax in H322 cells. GSH depletion enhanced Bax protein translocation to mitochondrial membranes. These findings suggest that the redox status may be a determinant of Bax-mediated cell death and that manipulation of intracellular thiols may sensitize cells to apoptosis by facilitating Bax insertion into mitochondrial membranes.
