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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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Methylation status was determined after exposure to DNMT inhibitors using Western blot analysis of DNMT1, 3A/3B. The cells were treated with DNMT inhibitors for 18 hours. A drastic depletion of DNMT1 and DNMT3A by psammaplin A, 5-aza-2'deoxycytidine, and zebularine in both A549 and U373MG cell lines was observed. However, there was no depletion of DNMT3B by the three DNMT inhibitors in either of the cell lines. Each blot is representative of two independent experiments, with actin used as a loading control.
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Figure 2: Methylation status was determined after exposure to DNMT inhibitors using Western blot analysis of DNMT1, 3A/3B. The cells were treated with DNMT inhibitors for 18 hours. A drastic depletion of DNMT1 and DNMT3A by psammaplin A, 5-aza-2'deoxycytidine, and zebularine in both A549 and U373MG cell lines was observed. However, there was no depletion of DNMT3B by the three DNMT inhibitors in either of the cell lines. Each blot is representative of two independent experiments, with actin used as a loading control.

Mentions: DNMT1, DNMT3A, and DNMT3B are the three main functional methyltransferases responsible for the establishment and maintenance of DNA methylation patterns in mammals. The effects of DNMT inhibitors on the levels of DNMT expression were analyzed via Western blotting using specific antibodies against DNMT1, DNMT3A, and DNMT3B. Western blot analysis revealed a drastic depletion of DNMT1 and DNMT3A by psammaplin A, 5-aza-2'-deoxycytidine, and zebularine in both A549 and U373MG cell lines. However, no depletion of DNMT3B by the three DNMT inhibitors was observed in either of the cell lines (Figure 2). These results indicate that DNMT inhibitors induce selective demethylation in each of the evaluated tumor cell lines.


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)

Methylation status was determined after exposure to DNMT inhibitors using Western blot analysis of DNMT1, 3A/3B. The cells were treated with DNMT inhibitors for 18 hours. A drastic depletion of DNMT1 and DNMT3A by psammaplin A, 5-aza-2'deoxycytidine, and zebularine in both A549 and U373MG cell lines was observed. However, there was no depletion of DNMT3B by the three DNMT inhibitors in either of the cell lines. Each blot is representative of two independent experiments, with actin used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Methylation status was determined after exposure to DNMT inhibitors using Western blot analysis of DNMT1, 3A/3B. The cells were treated with DNMT inhibitors for 18 hours. A drastic depletion of DNMT1 and DNMT3A by psammaplin A, 5-aza-2'deoxycytidine, and zebularine in both A549 and U373MG cell lines was observed. However, there was no depletion of DNMT3B by the three DNMT inhibitors in either of the cell lines. Each blot is representative of two independent experiments, with actin used as a loading control.
Mentions: DNMT1, DNMT3A, and DNMT3B are the three main functional methyltransferases responsible for the establishment and maintenance of DNA methylation patterns in mammals. The effects of DNMT inhibitors on the levels of DNMT expression were analyzed via Western blotting using specific antibodies against DNMT1, DNMT3A, and DNMT3B. Western blot analysis revealed a drastic depletion of DNMT1 and DNMT3A by psammaplin A, 5-aza-2'-deoxycytidine, and zebularine in both A549 and U373MG cell lines. However, no depletion of DNMT3B by the three DNMT inhibitors was observed in either of the cell lines (Figure 2). These results indicate that DNMT inhibitors induce selective demethylation in each of the evaluated tumor cell lines.

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