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The role of base excision repair proteins in the cellular responses to the anticancer drug cisplatin

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dc.contributor.advisor John M. Essigmann. en_US Kartalou, Maria, 1972- en_US
dc.contributor.other Massachusetts Institute of Technology. Division of Bioengineering and Environmental Health. en_US Cisplatin is one of the most widely used anticancer drugs. Its success in killing tumor cells results from its ability to form DNA adducts and the cellular processes triggered by the presence of those adducts in DNA. The geometric isomer of cisplatin, trans-DDP can also form DNA adducts but fails to show any antitumor activity. Variations in tumor response may result from altered expression of cellular proteins that recognize cisplatin and trans-DDP adducts. The human 3-methyladenine DNA glycosylase (AAG) is a repair enzyme that removes a number of damaged bases from DNA including adducts formed by some chemotherapeutic agents. Using site-specifically modified oligonucleotides containing each of the cisplatin intrastrand crosslinks we found that AAG readily recognized cisplatin adducts. The apparent dissociation constants for the 1,2-d(GpG), the 1,2-d(ApG) and the 1,3-d(GpTpG) oligonucleotides were 115 nM, 71 nM, and 144 nM, respectively. For comparison, the apparent dissociation constant for an oligonucleotide containing a single 1,M-ethenoadenine (EA), which is repaired efficiently by AAG, was 26 nM. Despite the affinity of AAG for cisplatin adducts, AAG was not able to release any of these adducts from DNA. Furthermore, it was demonstrated that the presence of cisplatin adducts in the reactions inhibited the excision of cA by AAG. These data suggest a previously unexplored dimension to the toxicological response of cells to cisplatin. We suggest that cisplatin adducts could titrate AAG away from its natural substrates resulting in higher mutagenesis and/or cell death because of the persistence of AAG substrates in DNA. The mutM and mutY genes encode DNA glycosylases involved in base excision repair; these genes constitute part of the "GO" repair system that, heretofore, has been associated with cellular defenses against the oxidative lesion, 7,8-dihydro-8-oxoguanine (8-oxoG). The MutM protein removes ring-opened purines and 8-oxoG from DNA. The MutY protein removes adenine from A:G and A: 8-oxoG mismatches. Purified MutM protein recognized the intrastrand crosslinks formed by cisplatin but had no excision activity on these adducts. Furthermore, in vivo experiments with wild type and mutM cells demonstrated that the presence of the MutM protein sensitized cells to cisplatin, and this effect of MutM was essentially abolished in a nucleotide excision repair deficient background, suggesting that MutM somehow modulates the activity of nucleotide excision repair proteins on cisplatin adducts. Purified MutY was also able to recognize cisplatin adducts. Moreover, MutY could excise adenine incorporated opposite a platinated guanine and was able to excise the platinated adenine of the 1,2-d(ApG) adduct when paired with guanine, suggesting that MutY might be directly involved in the repair of cisplatin adducts in vivo. These interactions between MutY and cisplatin adducts appears to be very important since muff cells were significantly more sensitive to cisplatin than wild type cells. Moreover, expression of MutY or p26, but not D 13 8N p26, in muff cells rescued their sensitivity to cisplatin, indicating that the catalytic activity of MutY is important for survival after treatment with cisplatin. Interestingly, MutY was also required for cisplatin induced mutagenesis, whereas MutM did not appear to play a role. Finally, a mutM or mutY mutation did not affect survival to trans-DDP, and MutM and MutY proteins displayed higher affinity for cisplatin than trans-DDP adducts. en_US 2012-09-27T15:23:47Z 2012-09-27T15:23:47Z 2000 en_US 2000 en_US
dc.description Thesis (Ph.D.)--Massachusetts Institute of Technology, Division of Bioengineering and Environmental Health, 2000. en_US
dc.description Includes bibliographical references. en_US
dc.description.statementofresponsibility by Maria Kartalou. en_US
dc.format.extent 270 leaves en_US
dc.language.iso eng en_US
dc.publisher Massachusetts Institute of Technology en_US
dc.rights MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission. en_US
dc.rights.uri en_US
dc.subject Division of Bioengineering and Environmental Health. en_US
dc.title The role of base excision repair proteins in the cellular responses to the anticancer drug cisplatin en_US
dc.type Thesis en_US Ph.D. en_US
dc.contributor.department Massachusetts Institute of Technology. Division of Bioengineering and Environmental Health. en_US
dc.identifier.oclc 47854261 en_US

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