RESUMEN
The objective of this study was to evaluate the time-course effects of UV-B exposure on expression of genes involved in the DNA repair system of zebrafish (Danio rerio) hepatocytes, a highly competent species in terms of damage repair induced by UV radiation. For gene expression analysis (RT-PCR), cells were exposed to 23.3 mJ cm(-2) UV-B, which was the dose that affected viable cell number (reduction of 30% when compared with the control group) and produced no visual alteration on cell morphology. The early response observed (6 h) showed induction in the expression of the CDKI gene (cyclin-dependent kinase inhibitor) and genes related to DNA damage repair (mainly XPC and DDB2), while the late response observed (24 h) was more related to up-regulation of p53 and genes involved in cell cycle arrest (gadd45a, cyclinG1). In all times analyzed, the anti-apoptotic gene Bcl-2 was down-regulated. Another interesting result observed was the up-regulation of the Apex-1 gene after UV-B exposure, which could indicate the induction of oxidative lesions in the DNA molecule. In conclusion, these results demonstrate an activation of the DNA repair system in hepatocytes of zebrafish exposed to UV-B radiation, mainly involving the participation of p53.
Asunto(s)
Reparación del ADN/genética , Regulación de la Expresión Génica/efectos de la radiación , Hepatocitos/metabolismo , Hepatocitos/efectos de la radiación , Rayos Ultravioleta , Pez Cebra/genética , Pez Cebra/metabolismo , Animales , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Línea Celular , Ciclina G , Ciclinas/genética , Ciclinas/metabolismo , Daño del ADN/genética , Cinética , Factores de TiempoRESUMEN
Leukemia inhibitory factor (LIF) and oncostatin M (OSM) induce DNA synthesis in Swiss 3T3 cells through common signaling mechanism(s), whereas other related cytokines such as interleukin-6 and ciliary neurotrophic factor do not cause this response. Induction of DNA replication by LIF or prostaglandin F2alpha (PGF2alpha) occurs, in part, through different signaling events. LIF and OSM specifically trigger STAT1 cytoplasmic to nuclear translocation, whereas PGF2alpha fails to do so. However, LIF and PGF2alpha can trigger increases in ERK1/2 activity, which are required for their mitogenic responses because U0126, a MEK1/2 inhibitor, prevents both ERK1/2 activation and induction of DNA synthesis by LIF or PGF2alpha treatment. PGF2alpha induces cyclin D expression and full phosphorylation of retinoblastoma protein. In contrast, LIF fails to promote increases in cyclin D mRNA/protein levels; consequently, LIF induces DNA synthesis without promoting full phosphorylation of retinoblastoma protein (Rb). However, both LIF and PGF2alpha increase cyclin E expression. Furthermore, LIF mitogenic action does not involve protein kinase C (PKC) activation, because a PKC inhibitor does not block this effect. In contrast, PKC activity is required for PGF2alpha mitogenic action. More importantly, the synergistic effect between LIF and PGF2alpha to promote S phase entry is independent of PKC activation. These results show fundamental differences between LIF- and PGF2alpha-dependent mechanism(s) that induce cellular entry into S phase. These findings are critical in understanding how LIF and other related cytokine-regulated events participate in normal cell cycle control and may also provide clues to unravel crucial processes underlying cancerous cell division.
Asunto(s)
Ciclina D1/biosíntesis , Replicación del ADN/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Interleucina-6/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Animales , Ciclina D1/genética , Ciclina G , Ciclina G1 , Ciclinas/biosíntesis , Ciclinas/genética , Citocinas/fisiología , Dinoprost/fisiología , Activación Enzimática/genética , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Cinética , Factor Inhibidor de Leucemia , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Oncostatina M , Fosforilación , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/fisiología , Proteína de Retinoblastoma/metabolismo , Fase S/fisiología , Factores de Transcripción STAT/fisiología , Transducción de Señal/fisiología , Células 3T3 SwissRESUMEN
The purpose of this study was to evaluate the overexpression of cyclin G in colorectal neoplasia, which may be a more frequent event than cyclin D1 during the cell cycle and thus may have a more enhanced therapeutic potential in treating colorectal cancer. Ninety formalin-fixed, paraffin-embedded human colon and rectal specimens were obtained from the Pathology Department of Norris Cancer Center/University of Southern California. The tissues had been obtained after surgical resection between 1995 and 2001, and had been processed by routine clinical histopathologic methods. Ninety-one percent of colorectal tumors had cyclin G overexpression. These cyclin-positive patients were evenly distributed between men and women, and between tumor locations, that is, 36% rectal tumors and 34% right-sided tumors. Thirty-two percent were well differentiated, and 66% were moderately differentiated. Thirty patients (38%) had stage I disease, 16 (20%) had stage II disease, 25 (32%) had stage III, and seven (9%) had stage IV disease. Eight patients (10%) in this group had recurrent disease during follow-up. There was no correlation between cyclin G overexpression and clinical and pathologic characteristics. Cyclin D1 overexpression was found to be present in only 42% of colorectal adenocarcinomas. There was no correlation between cyclin D1 overexpression and clinical and pathologic characteristics. The present study demonstrates that cyclin G overexpression is a frequent event in colorectal cancer. This frequent event in colorectal carcinogenesis may facilitate new therapeutic approaches acting as a target for gene therapy, possibly directed at downregulating cyclin G in colorectal cancer.
Asunto(s)
Adenocarcinoma/genética , Ciclo Celular/genética , Neoplasias del Colon/genética , Ciclinas/genética , Neoplasias del Recto/genética , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias del Colon/patología , Ciclina D , Ciclina G , Ciclina G1 , Femenino , Expresión Génica , Terapia Genética/métodos , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias del Recto/patologíaRESUMEN
PURPOSE: In a previous study, it was reported that docosahexaenoic acid (DHA) is essential to postpone apoptosis and to promote differentiation of rat retina photoreceptors in vitro. In the current study, the protective effects of GDNF on photoreceptor cells during development in vitro and its action when combined with DHA were investigated. METHODS: Rat retina neuronal cultures were incubated in a chemically defined medium, either without photoreceptor survival factors or supplemented with GDNF, DHA, or GDNF plus DHA. Evolution of survival, apoptosis, opsin expression, mitochondrial functioning, and cell proliferation were investigated at different times of development in vitro. RESULTS: Incubation with GDNF selectively increased the number of surviving photoreceptors, reduced their apoptosis, and augmented opsin expression. Proliferative cell nuclei antigen (PCNA) determination and addition of [(3)H]-thymidine or bromodeoxyuridine showed that GDNF promoted neuroblast proliferation during the first hours of development in vitro. The combined addition of GDNF and DHA enhanced opsin expression and photoreceptor survival in an additive manner. The advance of photoreceptor apoptosis in cultures without trophic factors correlated with an increased impairment in mitochondrial functionality. Addition of GDNF and DHA significantly diminished the loss of mitochondrial activity. CONCLUSIONS: These results show that GDNF stimulated the cell cycle progression, leading to neuroblast proliferation at early stages of development, and delayed the onset of apoptosis later on, improving differentiation and acting as a trophic factor for photoreceptors. The combination of GDNF with DHA had an additive effect both on photoreceptor survival and on opsin expression. Preservation of mitochondrial function may be involved in the antiapoptotic effect of both factors.