Dataset Information

MicroRNA-29a regulates the benzo[a]pyrene dihydrodiol epoxide-induced DNA damage response through Cdc7 kinase in lung cancer cells.

ABSTRACT: Cdc7 kinase is a key regulator of DNA replication and has an important role in the cellular DNA damage response by controlling checkpoint signaling and cell survival. Yet, how the activity of Cdc7 kinase is regulated is poorly understood. In silico analysis identified microRNA-29 (miR-29)-binding sites in the 3'-untranslated region (UTR) of both Cdc7 and its activating subunit Dbf4. We show that miR-29a binds to Cdc7 and Dbf4 3'-UTRs and regulates kinase levels. We find that in response to DNA damage, upregulation of Cdc7 kinase correlates with a downregulation in miR-29a. Enforced miR-29a expression prevents the accumulation of Cdc7 in response to the environmental genotoxin, benzo[a]pyrene dihydrodiol epoxide (BPDE) present in cigarette smoke, resulting in aberrant checkpoint signaling and increased cell lethality. As BPDE sensitivity was rescued by overexpression of miRNA-resistant Cdc7/Dbf4, we propose that Cdc7 kinase is an important target of miR-29a in determining cell survival from genotoxic stress caused by this environmental toxin.

SUBMITTER: Barkley LR

PROVIDER: S-EPMC3740286 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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MicroRNA-29a regulates the benzo[a]pyrene dihydrodiol epoxide-induced DNA damage response through Cdc7 kinase in lung cancer cells.

Barkley L R LR Santocanale C C

Oncogenesis 20130722

Cdc7 kinase is a key regulator of DNA replication and has an important role in the cellular DNA damage response by controlling checkpoint signaling and cell survival. Yet, how the activity of Cdc7 kinase is regulated is poorly understood. In silico analysis identified microRNA-29 (miR-29)-binding sites in the 3'-untranslated region (UTR) of both Cdc7 and its activating subunit Dbf4. We show that miR-29a binds to Cdc7 and Dbf4 3'-UTRs and regulates kinase levels. We find that in response to DNA d ...[more]

PMID: 23877787

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Project description:This study demonstrates that benzo[g]chrysene-11,12-dihydrodiol (B[g]C-11,12-dihydrodiol) derived from the fjord-region parent hydrocarbon B[g]C is oxidized by rat AKR1C9 with a k c a t/ K m 100 times greater than that observed with the commonly studied bay-region benzo[ a]pyrene-7,8-dihydrodiol (B[a]P-7,8-dihydrodiol). Conversely, despite its strikingly similar structure to B[ g]C-11,12-dihydrodiol, benzo[ c]phenanthrene-3,4-dihydrodiol (B[ c]Ph-3,4-dihydrodiol) is consumed by AKR1C9 at sluggish rates comparable to those observed with B[ a]P-7,8-dihydrodiol. CD spectroscopy revealed that only the (+)-B[ g]C-11,12-dihydrodiol stereoisomer was oxidized, while AKR1C9 oxidized both stereoisomers of B[a]P-7,8-dihydrodiol and B[ c]Ph-3,4-dihydrodiol. The (+)- S, S- and (-)- R, R-stereoisomers of B[g]C-11,12-dihydrodiol were purified by chiral RP-HPLC. The 11 S,12 S-stereoisomer was oxidized at the same rate as the racemate. The 11 R,12 R-stereoisomer did not act as an inhibitor to AKR1C9, indicating that the (-)- R, R-stereoisomer was excluded from the active site. To understand the basis of stereochemical preference, we screened alanine-scanning mutants of active site residues of AKR1C9. These studies revealed that in comparison to the wild type, F129A, W227A, and Y310A enabled the oxidation of both the B[g]C-11 S,12 S-dihydrodiol and the B[g]C-11 R,12 R-dihydrodiol. Molecular modeling revealed that unlike B[a]P-7,8-dihydrodiol and B[ c]Ph-3,4-dihydrodiol, B[g]C-11,12-dihydrodiol enantiomers are significantly bent out of plane. As a consequence, the (-)- R, R-stereoisomer was prevented from binding to the active site because of unfavorable interactions with F129, W227, or Y310. Additionally, LC/MS validated that the product of the reaction of B[g]C-11,12-dihydrodiol oxidation catalyzed by AKR1C9 was B[g]C-11,12-dione, which was trapped in vitro with the nucleophile 2-mercaptoethanol. The similarity between rates of trans-dihydrodiol oxidation by the rat and human liver specific AKRs (AKR1C9 and AKR1C4) implicate these enzymes in hepatocarcinogenesis in rats observed with the fjord-region PAH.

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Dataset Information

MicroRNA-29a regulates the benzo[a]pyrene dihydrodiol epoxide-induced DNA damage response through Cdc7 kinase in lung cancer cells.

Publications

MicroRNA-29a regulates the benzo[a]pyrene dihydrodiol epoxide-induced DNA damage response through Cdc7 kinase in lung cancer cells.

Similar Datasets

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