Differential stability of the DNA-activated protein kinase catalytic subunit mRNA in human glioma cells. (1/2255)

DNA-dependent protein kinase (DNA-PK) functions in double-strand break repair and immunoglobulin [V(D)J] recombination. We previously established a radiation-sensitive human cell line, M059J, derived from a malignant glioma, which lacks the catalytic subunit (DNA-PKcs) of the DNA-PK multiprotein complex. Although previous Northern blot analysis failed to detect the DNA-PKcs transcript in these cells, we show here through quantitative studies that the transcript is present, albeit at greatly reduced (approximately 20x) levels. Sequencing revealed no genetic alteration in either the promoter region, the kinase domain, or the 3' untranslated region of the DNA-PKcs gene to account for the reduced transcript levels. Nuclear run-on transcription assays indicated that the rate of DNA-PKcs transcription in M059J and DNA-PKcs proficient cell lines was similar, but the stability of the DNA-PKcs message in the M059J cell line was drastically (approximately 20x) reduced. Furthermore, M059J cells lack an alternately spliced DNA-PKcs transcript that accounts for a minor (5-20%) proportion of the DNA-PKcs message in all other cell lines tested. Thus, alterations in DNA-PKcs mRNA stability and/or the lack of the alternate mRNA may result in the loss of DNA-PKcs activity. This finding has important implications as DNA-PKcs activity is essential to cells repairing damage induced by radiation or radiomimetric agents.  (+info)

The virulence plasmid-encoded impCAB operon enhances survival and induced mutagenesis in Shigella flexneri after exposure to UV radiation. (2/2255)

Upon exposure to UV radiation, Shigella flexneri SA100 displayed survival and mutation frequencies comparable to those of Escherichia coli AB1157, which contains a functional UmuDC error-prone DNA repair system. Survival of SA100 after UV irradiation was associated with the presence of the 220-kb virulence plasmid, pVP. This plasmid encodes homologues of ImpA and ImpB, which comprise an error-prone DNA repair system encoded on plasmid TP110 that was initially identified in Salmonella typhimurium, and ImpC, encoded upstream of ImpA and ImpB. Although the impB gene was present in representatives of all four species of Shigella, not all isolates tested contained the gene. Shigella isolates that lacked impB were more sensitive to UV radiation than isolates that contained impB. The nucleotide sequence of a 2.4-kb DNA fragment containing the imp operon from S. flexneri SA100 pVP was 96% identical to the imp operon from the plasmid TP110. An SA100 derivative with a mutation in the impB gene had reduced survival following UV irradiation and less UV-induced mutagenesis relative to the parental strain. We also found that S. flexneri contained a chromosomally encoded umuDC operon; however, the umuDC promoter was not induced by exposure to UV radiation. This suggests that the imp operon but not the umuDC operon contributes to survival and induced mutagenesis in S. flexneri following exposure to UV radiation.  (+info)

The inducible expression of dominant-negative epidermal growth factor receptor-CD533 results in radiosensitization of human mammary carcinoma cells. (3/2255)

Ionizing radiation activates the epidermal growth factor receptor (EGFR) and downstream signaling involving the cytoprotective mitogen-activated protein kinase (MAPK) pathway. In our effort to investigate the role of EGFR in cellular responses to radiation, we generated mammary carcinoma cell clones, MCF-TR5-EGFR-CD533 and MDA-TR15-EGFR-CD533, that inducibly express EGFR-CD533, a truncated EGFR mutant lacking mitogenic and transformation activity. EGFR-CD533 expression inhibits radiation- and EGF-induced EGFR autophosphorylation and MAPK activation and, therefore, functions as a dominant-negative mutant without blocking the expression of EGFR or erbB-2, another member of the erbB receptor Tyr kinase family. Expression of EGFR-CD533 only minimally inhibited cell growth and did not alter radiosensitivity to single radiation exposures. However, repeated 2 Gy radiation exposures of cells, under conditions of EGFR-CD533 expression, essentially abolished their ability for subsequent cell growth. These results identify the inhibition of EGFR function through genetic manipulation as a potential therapeutic maneuver. The concept of such an intervention would be the radiosensitization of cells by counteracting a radiation-induced cytoprotective proliferation response.  (+info)

The contribution of DNA ploidy to radiation sensitivity in human tumour cell lines. (4/2255)

The contribution of DNA ploidy to radiation sensitivity was investigated in a group of eight human tumour cell lines. As previous studies suggest, while more aneuploid tumours tend to be more radioresistant, there is no significant relationship between ploidy and radiation sensitivity (SF2). The failure to observe a significant effect of ploidy on radiation sensitivity is due to the complex and multifactorial basis of radiation sensitivity. When we determined the relationship between survival and radiation-induced chromosome aberration frequency, a measure independent of most other modifiers of sensitivity, we observed a direct relationship between ploidy and mean lethal aberration frequency. The mean lethal frequency of aberrations increased from about 1 for diploid cells to about 2 for tetraploid cells. The mean lethal frequency of aberrations was independent of DNA repair variations. These observations demonstrate that changes in DNA ploidy are an important contributor to radiation sensitivity variations in human tumour cell lines. Therefore, any battery of predictive assays should include DNA ploidy measurements.  (+info)

Modification of non-conservative double-strand break (DSB) rejoining activity after the induction of cisplatin resistance in human tumour cells. (5/2255)

The induction of collateral radioresistance after the development of cisplatin resistance is a well-documented phenomenon; however, the exact processes that are responsible for the cisplatin-induced radioresistance remain to be elucidated. There was no obvious difference in the level of radiation-induced DNA double strand breaks (DSBs), in DSB rejoining rates, or the level of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) in the cisplatin- and radiation-sensitive 2780/WT and cisplatin-resistant 2780/CP cell lines. However, there was a significantly (P < 0.01) lower level of DSB misrejoining activity within nuclear protein extracts derived from the cisplatin- and radiation-sensitive 2780/WT and OAW42/WT tumour cell lines than in similar extracts from their cisplatin- (and radiation-) resistant 2780/CP and OAW42/CP counterparts. All of the DSB misrejoining events involved deletions of between 134 and 444 bp that arose through illegitimate recombination at short repetitive sequences, such as those that arise through non-homologous repair (NHR). These data further support the notion that the radiosensitivity of DSB repair proficient human tumour cell lines may be partly determined by the predisposition of these cell lines to activate non-conservative DSB rejoining pathways. Furthermore, our data suggest that the induction of acquired cisplatin resistance is associated with a two- to threefold decrease in the activity of a non-conservative DSB rejoining mechanism that appears to be a manifestation of NHR.  (+info)

Cyclin D1 overexpression enhances radiation-induced apoptosis and radiosensitivity in a breast tumor cell line. (6/2255)

Overexpression of cyclin D1, a G1 cell cycle regulator, is often found in many different tumor types, such as breast carcinoma and squamous cell carcinoma of the head and neck. The overexpression of this protein is, in several cases, associated with a poor prognosis. In this study, the effect of cyclin D1 on radiosensitivity was investigated in a breast tumor cell line, MCF7, containing a cyclin D1 gene construct under the control of a tetracycline-sensitive regulator. MCF7 cells cultured without tetracycline resulted in a 6-fold increase in the cyclin D1 protein. Cyclin D1-overexpressing MCF7 cells were more sensitive to ionizing radiation than the nonoverexpressing counterparts. The cyclin D1-overexpressing cells also exhibited a higher induction of apoptosis. Treatment with a dose of 5 Gy resulted in a rapid increase of p53 and p21 in the cyclin D1-overexpressing cells. Nonoverexpressing cells showed a more transient expression of these proteins after ionizing radiation. A pronounced G2-M block was observed in both cell lines. The cyclin D1-overexpressing cells were, however, released earlier from the block than the control cells. These data suggest that overexpression of cyclin D1 alters sensitivity toward ionizing radiation by modulating gamma-radiation-induced G2-M transition.  (+info)

Monoclonal antibody to HER-2/neureceptor modulates repair of radiation-induced DNA damage and enhances radiosensitivity of human breast cancer cells overexpressing this oncogene. (7/2255)

The management of human breast cancer frequently includes radiation therapy as an important intervention, and improvement in the clinical efficacy of radiation is desirable. Overexpression of the HER-2 growth factor receptor occurs in 25-30% of human breast cancers and correlates with poor clinical outcome, including earlier local relapse following conservative surgery accompanied by radiation therapy. In breast cancer cells with overexpression of HER-2 receptor, recombinant humanized monoclonal antibodies (rhuMAbs) to HER-2 receptors (rhuMAb HER-2) decrease cell proliferation in vitro and reduce tumor formation in nude mice. Therapy with rhuMAb HER-2 enhances tumor sensitivity to radiation at doses of 1-5 Gy, exceeding remission rates obtained with radiation alone. This benefit is specific to cells with HER-2 overexpression and does not occur in cells without overexpression. Treatment of cells with radiation (2-4 Gy) alone provokes a marked increase in unscheduled DNA synthesis, a measure of DNA repair, but HER-2-overexpressing cells treated with a combination of rhuMAb HER-2 and radiation demonstrate a decrease of unscheduled DNA synthesis to 25-44% of controls. Using an alternate test of DNA repair, i.e., radiation-damaged or undamaged reporter DNA, we introduced a cytomegalovirus-driven beta3-galactosidase into HER-2-overexpressing breast cancer cells that had been treated with rhuMAb HER-2 or control. At 24 h posttransfection, the extent of repair assayed by measuring reporter DNA expression was high after exposure to radiation alone but significantly lower in cells treated with combined radiation and rhuMAb HER-2 therapy. To further characterize effects of rhuMAb HER-2 and the combination of antibody and radiation on cell growth, analyses of cell cycle phase distribution were performed. Antibody reduces the fraction of HER-2-overexpressing breast cancer cells in S phase at 24 and 48 h. Radiation treatment is also known to promote cell cycle arrest, predominantly at G1, with low S-phase fraction at 24 and 48 h. In the presence of rhuMAb HER-2, radiation elicits a similar reduction in S phase at 24 h, but a significant reversal of this arrest appears to begin 48 h postradiation exposure. The level of S-phase fraction at 48 h is significantly greater than that found at 24 h with the combined antibody-radiation therapy, suggesting that early escape from cell cycle arrest in the presence of antireceptor antibody may not allow sufficient time for completion of DNA repair in HER-2-overexpressing cells. Because it is well known that failure of adequate p21WAF1 induction after DNA damage is associated with failure of cell cycle arrest, we also assessed the activity of this critical mediator of the cellular response to DNA damage. The results show induction of p21WAF1 transcripts and protein product at 6, 12, and 24 h after radiation treatment; however, increased levels of p21WAF1 transcript and protein are not sustained in HER-2-overexpressing cells exposed to radiation in the presence of rhuMAb HER-2. Although transcript and protein levels increase at 6-12 h, they are both diminished by 24 h. Levels of p21WAF1 transcript and protein at 24 h are significantly lower than in cells treated by radiation without antibody. A reduction in the basal level of p21WAF1 transcript also occurred after 12-24 h exposure to antibody alone. The effect of HER-2 antibody may be related to tyrosine phosphorylation of p21WAF1 protein. Tyrosine phosphorylation of p21WAF1 is increased after treatment with radiation alone, but phosphorylation is blocked by combined treatment with antireceptor antibody and radiation. This dysregulation of p21WAF1 in HER-2-overexpressing breast cells after treatment with rhuMAb HER-2 and radiation appears to be independent of p53 expression levels but does correlate with reduced levels of mdm2 protein. (ABSTRACT TRUNCATED)  (+info)

Fas (APO-1/CD95) signaling pathway is intact in radioresistant human glioma cells. (8/2255)

Radiation-induced apoptosis can be mediated through pathways initiated by either DNA damage or ceramide-induced Fas signaling. Glioblastoma multiforme is a primary brain tumor that is highly resistant to irradiation, and U-87 MG, SF126, and T98G are glioblastoma-derived cell lines that mimic this characteristic. We found that these radioresistant glioma cells are susceptible to Fas-mediated cell death induced by treatment with either anti-Fas antibody or exogenous ceramide. Fas-mediated cell death in these cell lines is p53-independent. These data demonstrate that apoptosis can be induced by ceramide and mediated through the Fas pathway in glioma cells, although high-dose ionizing radiation fails to trigger this pathway.  (+info)