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DNMT (DNA methyltransferase) inhibitors radiosensitize human cancer cells by suppressing DNA repair activity.

Kim HJ, Kim JH, Chie EK, Young PD, Kim IA, Kim IH - Radiat Oncol (2012)

Bottom Line: Unlike the histone deacetylase inhibitors, which are known to exert radiosensitizing effects, there have only been a few studies thus far concerning the role of DNA methyltransferase (DNMT) inhibitors as radiosensitizers.Pretreatment with psammaplin A, 5-aza-2'-deoxycytidine, and zebularine radiosensitized both A549 and U373MG cells.Psammaplin A, 5-aza-2'-deoxycytidine, and zebularine induce radiosensitivity in both A549 and U373MG cell lines, and suggest that this effect might be associated with the inhibition of DNA repair.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Radiation Oncology, Seoul National University College of Medicine, Seoul, Republic of Korea.

ABSTRACT

Background: Histone modifications and DNA methylation are two major factors in epigenetic phenomenon. Unlike the histone deacetylase inhibitors, which are known to exert radiosensitizing effects, there have only been a few studies thus far concerning the role of DNA methyltransferase (DNMT) inhibitors as radiosensitizers. The principal objective of this study was to evaluate the effects of DNMT inhibitors on the radiosensitivity of human cancer cell lines, and to elucidate the mechanisms relevant to that process.

Methods: A549 (lung cancer) and U373MG (glioblastoma) cells were exposed to radiation with or without six DNMT inhibitors (5-azacytidine, 5-aza-2'-deoxycytidine, zebularine, hydralazine, epigallocatechin gallate, and psammaplin A) for 18 hours prior to radiation, after which cell survival was evaluated via clonogenic assays. Cell cycle and apoptosis were analyzed via flow cytometry. Expressions of DNMT1, 3A/3B, and cleaved caspase-3 were detected via Western blotting. Expression of γH2AX, a marker of radiation-induced DNA double-strand break, was examined by immunocytochemistry.

Results: Pretreatment with psammaplin A, 5-aza-2'-deoxycytidine, and zebularine radiosensitized both A549 and U373MG cells. Pretreatment with psammaplin A increased the sub-G1 fraction of A549 cells, as compared to cells exposed to radiation alone. Prolongation of γH2AX expression was observed in the cells treated with DNMT inhibitors prior to radiation as compared with those treated by radiation alone.

Conclusions: Psammaplin A, 5-aza-2'-deoxycytidine, and zebularine induce radiosensitivity in both A549 and U373MG cell lines, and suggest that this effect might be associated with the inhibition of DNA repair.

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Influence of DNMT inhibitors on radiation-induced γH2AX foci. A549 and U373MG cells growing in chamber slides were exposed to DNMT inhibitors for 18 hours, irradiated, and fixed at specific times for immunocytochemical analyses of nuclear γH2AX foci. Open columns, data from cells receiving radiation alone; grey columns, data from cells that were exposed to psammaplin A and radiation; filled columns, data from cells that were exposed to 5-aza-2'-deoxycytidine and radiation; hatched columns, data from cells that were exposed to zebularine and radiation. Cells with more than five foci per nucleus were classified as positive for radiation-induced γH2AX. Foci were evaluated in 50 nuclei. Bars, SE. *p < 0.01 as determined by a logistic regression compared with radiation alone (6 Gy) group.
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Figure 7: Influence of DNMT inhibitors on radiation-induced γH2AX foci. A549 and U373MG cells growing in chamber slides were exposed to DNMT inhibitors for 18 hours, irradiated, and fixed at specific times for immunocytochemical analyses of nuclear γH2AX foci. Open columns, data from cells receiving radiation alone; grey columns, data from cells that were exposed to psammaplin A and radiation; filled columns, data from cells that were exposed to 5-aza-2'-deoxycytidine and radiation; hatched columns, data from cells that were exposed to zebularine and radiation. Cells with more than five foci per nucleus were classified as positive for radiation-induced γH2AX. Foci were evaluated in 50 nuclei. Bars, SE. *p < 0.01 as determined by a logistic regression compared with radiation alone (6 Gy) group.

Mentions: γH2AX has been identified as a marker of DNA double-strand break (DSB) [15]. Immunocytochemical analysis using the anti-γH2AX antibodies was conducted in order to determine the effects of DNMT inhibitors on DNA repair. As shown in the representative micrographs in Figure 6, γH2AX foci could be clearly distinguished after the 6 Gy of radiation. The γH2AX expression of cells treated with radiation alone was compared with those treated with a combination of DNMT inhibitors and radiation. Although γH2AX foci expression was shown to be reduced in cells treated with radiation alone over time, γH2AX foci levels in the cells exposed to DNMT inhibitors prior to radiation remained constant over a 12-hour time course in A549 cells and over a 24-hour time course in U373MG cells (Figure 7). These results implicate an inhibition of the DNA damage repair process as the possible mechanism underlying the effects of DNMT inhibitors on radiosensitization.


DNMT (DNA methyltransferase) inhibitors radiosensitize human cancer cells by suppressing DNA repair activity.

Kim HJ, Kim JH, Chie EK, Young PD, Kim IA, Kim IH - Radiat Oncol (2012)

Influence of DNMT inhibitors on radiation-induced γH2AX foci. A549 and U373MG cells growing in chamber slides were exposed to DNMT inhibitors for 18 hours, irradiated, and fixed at specific times for immunocytochemical analyses of nuclear γH2AX foci. Open columns, data from cells receiving radiation alone; grey columns, data from cells that were exposed to psammaplin A and radiation; filled columns, data from cells that were exposed to 5-aza-2'-deoxycytidine and radiation; hatched columns, data from cells that were exposed to zebularine and radiation. Cells with more than five foci per nucleus were classified as positive for radiation-induced γH2AX. Foci were evaluated in 50 nuclei. Bars, SE. *p < 0.01 as determined by a logistic regression compared with radiation alone (6 Gy) group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3375186&req=5

Figure 7: Influence of DNMT inhibitors on radiation-induced γH2AX foci. A549 and U373MG cells growing in chamber slides were exposed to DNMT inhibitors for 18 hours, irradiated, and fixed at specific times for immunocytochemical analyses of nuclear γH2AX foci. Open columns, data from cells receiving radiation alone; grey columns, data from cells that were exposed to psammaplin A and radiation; filled columns, data from cells that were exposed to 5-aza-2'-deoxycytidine and radiation; hatched columns, data from cells that were exposed to zebularine and radiation. Cells with more than five foci per nucleus were classified as positive for radiation-induced γH2AX. Foci were evaluated in 50 nuclei. Bars, SE. *p < 0.01 as determined by a logistic regression compared with radiation alone (6 Gy) group.
Mentions: γH2AX has been identified as a marker of DNA double-strand break (DSB) [15]. Immunocytochemical analysis using the anti-γH2AX antibodies was conducted in order to determine the effects of DNMT inhibitors on DNA repair. As shown in the representative micrographs in Figure 6, γH2AX foci could be clearly distinguished after the 6 Gy of radiation. The γH2AX expression of cells treated with radiation alone was compared with those treated with a combination of DNMT inhibitors and radiation. Although γH2AX foci expression was shown to be reduced in cells treated with radiation alone over time, γH2AX foci levels in the cells exposed to DNMT inhibitors prior to radiation remained constant over a 12-hour time course in A549 cells and over a 24-hour time course in U373MG cells (Figure 7). These results implicate an inhibition of the DNA damage repair process as the possible mechanism underlying the effects of DNMT inhibitors on radiosensitization.

Bottom Line: Unlike the histone deacetylase inhibitors, which are known to exert radiosensitizing effects, there have only been a few studies thus far concerning the role of DNA methyltransferase (DNMT) inhibitors as radiosensitizers.Pretreatment with psammaplin A, 5-aza-2'-deoxycytidine, and zebularine radiosensitized both A549 and U373MG cells.Psammaplin A, 5-aza-2'-deoxycytidine, and zebularine induce radiosensitivity in both A549 and U373MG cell lines, and suggest that this effect might be associated with the inhibition of DNA repair.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Radiation Oncology, Seoul National University College of Medicine, Seoul, Republic of Korea.

ABSTRACT

Background: Histone modifications and DNA methylation are two major factors in epigenetic phenomenon. Unlike the histone deacetylase inhibitors, which are known to exert radiosensitizing effects, there have only been a few studies thus far concerning the role of DNA methyltransferase (DNMT) inhibitors as radiosensitizers. The principal objective of this study was to evaluate the effects of DNMT inhibitors on the radiosensitivity of human cancer cell lines, and to elucidate the mechanisms relevant to that process.

Methods: A549 (lung cancer) and U373MG (glioblastoma) cells were exposed to radiation with or without six DNMT inhibitors (5-azacytidine, 5-aza-2'-deoxycytidine, zebularine, hydralazine, epigallocatechin gallate, and psammaplin A) for 18 hours prior to radiation, after which cell survival was evaluated via clonogenic assays. Cell cycle and apoptosis were analyzed via flow cytometry. Expressions of DNMT1, 3A/3B, and cleaved caspase-3 were detected via Western blotting. Expression of γH2AX, a marker of radiation-induced DNA double-strand break, was examined by immunocytochemistry.

Results: Pretreatment with psammaplin A, 5-aza-2'-deoxycytidine, and zebularine radiosensitized both A549 and U373MG cells. Pretreatment with psammaplin A increased the sub-G1 fraction of A549 cells, as compared to cells exposed to radiation alone. Prolongation of γH2AX expression was observed in the cells treated with DNMT inhibitors prior to radiation as compared with those treated by radiation alone.

Conclusions: Psammaplin A, 5-aza-2'-deoxycytidine, and zebularine induce radiosensitivity in both A549 and U373MG cell lines, and suggest that this effect might be associated with the inhibition of DNA repair.

Show MeSH
Related in: MedlinePlus