RESUMEN
A CAN1/can1Delta heterozygous allele that determines loss of heterozygosity (LOH) was used to study recombination in Saccharomyces cerevisiae cells exposed to ultraviolet (UV) light at different points in the cell cycle. With this allele, recombination events can be detected as canavanine-resistant mutations after exposure of cells to UV radiation, since a significant fraction of LOH events appear to arise from recombination between homologous chromosomes. The radiation caused a higher level of LOH in cells that were in the S phase of the cell cycle relative to either cells at other points in the cell cycle or unsynchronized cells. In contrast, the inactivation of nucleotide excision repair abolished the cell cycle-specific induction by UV of LOH. We hypothesize that DNA lesions, if not repaired, were converted into double-strand breaks during stalled replication and these breaks could be repaired through recombination using a non-sister chromatid and probably also the sister chromatid. We argue that LOH may be an outcome used by yeast cells to recover from stalled replication at a lesion.
Asunto(s)
Pérdida de Heterocigocidad , Fase S , Saccharomyces cerevisiae/genética , Rayos Ultravioleta , Ciclo Celular/efectos de la radiación , ADN/metabolismo , ADN/efectos de la radiación , Reparación del ADN , Modelos Genéticos , Fase S/genética , Fase S/efectos de la radiación , Saccharomyces cerevisiae/efectos de la radiación , Factores de TiempoRESUMEN
Plasminogen activator inhibitor (PAI)-1 is predictive of poor outcome in several types of cancer. The present study investigated the biological role for PAI-1 in ovarian cancer and potential of targeted pharmacotherapeutics. In patients with ovarian cancer, PAI-1 mRNA expression in tumor tissues was positively correlated with poor prognosis. To determine the role of PAI-1 in cell proliferation in ovarian cancer, the effects of PAI-1 inhibition were examined in PAI-1-expressing ovarian cancer cells. PAI-1 knockdown by small interfering RNA resulted in significant suppression of cell growth accompanied with G2/M cell cycle arrest and intrinsic apoptosis. Similarly, treatment with the small molecule PAI-1 inhibitor TM5275 effectively blocked cell proliferation of ovarian cancer cells that highly express PAI-1. Together these results suggest that PAI-1 promotes cell growth in ovarian cancer. Interestingly, expression of PAI-1 was increased in ovarian clear cell carcinoma compared with that in serous tumors. Our results suggest that PAI-1 inhibition promotes cell cycle arrest and apoptosis in ovarian cancer and that PAI-1 inhibitors potentially represent a novel class of anti-tumor agents.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Ováricas/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Inactivadores Plasminogénicos/farmacología , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Concentración 50 Inhibidora , Terapia Molecular Dirigida , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/patología , Piperazinas/farmacología , Inhibidor 1 de Activador Plasminogénico/genética , Pronóstico , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Regulación hacia Arriba , para-Aminobenzoatos/farmacologíaRESUMEN
After DNA damage, p53 is accumulated in the nucleus and transactivates downstream genes and induces apoptosis. There are two pathways in p53-dependent apoptosis, the transactivation-dependent and -independent pathway. In this study, we constructed p53-inducible glioblastoma cell lines and analyzed them for the induction of apoptosis and transactivation of p53-downstream genes after the nuclear or cytoplasmic expression of p53. To sequester p53 in the cytoplasm, we used p53 mutant with arginine to glycine substitution at residue 306 (R306G). Wild-type p53 retained the ability to arrest the cell cycle, and a p53 mutant with serine to phenylalanine substitution at residue 121 (S121F), which has a strong ability to induce apoptosis, retained this ability even when both the wild-type and p53 and S121F mutant were exclusively sequestered from the nucleus into the cytoplasm. Notably, cytoplasmically sequestered wild-type p53 and S121F mutant transactivated the downstream genes with distinct expression profiles, and the strong apoptotic ability of S121F was not associated with its transactivation activity. These results underscore the existence of transactivation-independent apoptosis and cytoplasmic function of p53.
RESUMEN
Second-site suppressor (SSS) mutations in p53 found by random mutagenesis have shown to restore the inactivated function of some tumor-derived p53. To screen novel SSS mutations against common mutant p53s, intragenic second-site (SS) mutations were introduced into mutant p53 cDNA in a comprehensive manner by using a p53 missense mutation library. The resulting mutant p53s with background and SS mutations were assayed for their ability to restore the p53 transactivation function in both yeast and human cell systems. We identified 12 novel SSS mutations including H178Y against a common mutation G245S. Surprisingly, the G245S phenotype is rescued when coexpressed with p53 bearing the H178Y mutation. This result indicated that there is a possibility that intragenic suppressor mutations might restore the protein function in an intermolecular manner. The intermolecular mechanism may lead to novel strategies for restoring inactivated p53 function and tumor suppression in cancer treatment.