Antisense oligonucleotide targeting p53 increased apoptosis of MCF-7 cells induced by ionizing radiation.
ABSTRACT To investigate the effect of antisense compounds (AS) targeting human p53 mRNA on radiosensitivity of MCF-7 cells.
Western blotting and RTPCR were used to analyze the protein content and mRNA level. Additionally, cell proliferation, cell cycle and cell apoptosis were all analyzed in irradiated or sham-irradiated cells.
Among the five antisense compounds (AS), AS3 was identified to efficiently inhibit p53 mRNA level and protein content. Interestingly, AS3 transfer has little effect on cell proliferation in DU-145 cells (mutant p53) after ionizing radiation (IR). In contrast, a marked increase of cell apoptosis and growth inhibition were observed in MCF-7 cells (wild-type p53), suggesting that AS3 can increase radiosensitivity of MCF-7 cells. Additionally, it was also observed that the transfection of AS3 decreased the fraction of G1 phase cells, and increased the proportion of S phase cells compared to untreated cells 24 h after IR in MCF-7 cell lines.
AS3 transfection increases MCF-7 cell apoptosis induced by 5 Gy-radiation, and this mechanism may be closely associated with abrogation of G1 phase arrest.
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ABSTRACT: In the present study, we investigated the radiosensitivity profiles of three established human ovarian carcinoma cell lines, PA-1, Caov-3, and SK-OV-3, using the adenosine triphosphate-cell viability assay (ATP-CVA). We have correlated radioresponsiveness with the p53 status and the p53 accumulation after irradiation as well as with the Bcl-2 expression and the growth rate of these cell lines. The p53 status was examined by immunocytochemistry and a functional assay (functional analysis of separated alleles in yeast, FASAY); the p53 accumulation was determined by immunocytochemistry and flow cytometry. Furthermore, the Bcl-2 expression before and after irradiation was examined by immunocytochemistry. PA-1, expressing wild-type p53, showed an unequivocal accumulation of p53 protein following exposure to irradiation. This cell line was found to be strongly sensitive to irradiation. The two p53 mutant cell lines Caov-3 and SK-OV-3 showed radioresistance at different degrees and irradiation did not result in p53 accumulation. None of the cell lines examined expressed Bcl-2 protein and no change was seen after irradiation. Furthermore, the most sensitive cell line to irradiation, PA-1, showed the highest proliferative activity, while Caov-3 and SK-OV-3, the more resistant cell lines, exhibited lower growth rates. Our findings indicate that the presence of p53 protein is a possible determinant for the cytotoxicity induced by irradiation in the investigated ovarian carcinoma cell lines. Bcl-2 expression does not seem to determine the response to irradiation in these cell lines. Additionally, an association between radioresponsiveness and the growth rate is suggested in PA-1, Caov-3, and SK-OV-3.Cancer Letters 04/2000; 150(2):191-9. · 4.26 Impact Factor
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ABSTRACT: The current understanding of cellular responses to ionizing radiation fuses traditional radiobiological concepts with recent insight into underlying molecular mechanims. Cell cycle arrest, induction of specific cascades of genes, and activation of DNA repair mechanisms have been reported after radiation exposure. Of particular importance is the mitogen-activated protein (MAP) kinase signaling pathway, in which the ras and raf oncogenes participate. Both of these genes have been implicated in the cellular resistance to killing by ionizing radiation. Furthermore, regulated NF-kB transcriptional activation may be important for the pleiotrophic responses of cells to ionizing radiation. Disruption of this signaling cascade may contribute to the radiation sensitivity syndrome seen in ataxia telangiectasia. In this review, these pathways are consolidated into ionizing radiation signal transduction pathways that lead from the cell membrane/cytosol to the nuclear DNA.Seminars in radiation oncology 11/1996; · 4.32 Impact Factor
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ABSTRACT: The molecular basis of tumor response to therapeutic radiation is poorly understood. Recent evidence suggests the p53 tumor suppressor gene may be involved in production of the G1 arrest seen following DNA damage by X-irradiation. It has further been proposed that tumor cells lacking the p53 checkpoint function are likely to be more sensitive to cell killing by X-irradiation because these cells enter S phase despite unrepaired DNA damage. We tested the hypothesis that tumor cells with p53 mutations are more radiosensitive by correlating the in vitro surviving fraction at 2 Gy with the mutational status of 24 head and neck squamous cell cancer cell lines. p53 mutations were present in 15 of 24 (63%) of tumors; all were homozygous changes occurring within exons 5-9. The surviving fraction at 2 Gy for the group with mutations was 0.568 compared to 0.507 for tumors without mutations (P = 0.28, Mann-Whitney test). Furthermore, no association between radiosensitivity and mutational type, codon location, or predicted amino acid alteration was noted. Our data do not support the hypothesis that p53 gene alteration predisposes tumor cells to increased cell killing via radiation.Cancer Research 09/1993; 53(16):3667-9. · 8.65 Impact Factor