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Inactivation of human O(6)-alkylguanine-DNA alkyltransferase by modified oligodeoxyribonucleotides containing
A E Pegg1, K Goodtzova, N A Loktionova
1Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033, USA. aep1@psu.edu
Abstract:
Inactivation of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) enhances tumor cell killing by therapeutic alkylating agents. O(6)-Benzylguanine (b(6)G) can inactivate AGT and is currently in clinical trials to enhance therapy. Short oligodeoxyribonucleotides containing b(6)G are much more effective inactivators, but their use for therapeutic purposes is likely to be compromised by metabolic instability. We have therefore examined the ability to inactivate AGT of an 11-mer oligodeoxyribonucleotide containing b(6)G (11-mpBG) when modified with terminal methylphosphonate linkages to protect it from nucleases. This modification did not reduce the ability to serve as a substrate/inactivator for AGT, and 11-mpBG had an ED(50) value of 1.3 nM, more than 300-fold lower than that for b(6)G. A similar oligodeoxyribonucleotide containing O(6)-methylguanine (m(6)G) was also found to be a good substrate (ED(50) value of 10 nM), but the benzylated form was repaired more rapidly and preferentially. When added to HT29 cell cultures, 5 microM 11-mpBG was able to cause a prolonged inactivation of cellular AGT for at least 72 h and to greatly sensitize the cells to killing by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The 11-mpMG was ineffective at up to 20 microM, suggesting that the benzyl group allows better uptake into the cell. However, even with 11-mpBG, the 1000-fold decrease in potency toward AGT in HT29 cells compared to that toward the protein in vitro suggests that uptake may be a limiting factor. These results suggest that oligodeoxyribonucleotides such as 11-mpBG may prove to be useful drugs for potentiation of alkylating agent chemotherapy if uptake can be improved.
Insights
Modified oligodeoxyribonucleotides containing O(6)-Benzylguanine (b(6)G) effectively inactivate the DNA repair protein AGT. These compounds show promise for enhancing chemotherapy by sensitizing tumor cells to alkylating agents.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Therapeutics
Background:
- The DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) protects tumor cells from alkylating agents.
- Inactivating AGT can enhance the efficacy of chemotherapy.
- O(6)-Benzylguanine (b(6)G) is an AGT inhibitor in clinical trials, but short oligodeoxyribonucleotides are more effective but unstable.
Purpose of the Study:
- To evaluate the AGT-inactivating ability of a nuclease-resistant oligodeoxyribonucleotide containing b(6)G (11-mpBG).
- To compare the efficacy of 11-mpBG with a similar compound containing O(6)-methylguanine (m(6)G).
- To assess the potential of 11-mpBG to sensitize cancer cells to chemotherapy.
Main Methods:
- Synthesis of an 11-mer oligodeoxyribonucleotide with terminal methylphosphonate linkages containing b(6)G (11-mpBG).
- In vitro assessment of AGT inactivation by 11-mpBG and an analogous O(6)-methylguanine compound (11-mpMG).
- In vitro and cell culture studies using HT29 cells to evaluate AGT inactivation, cellular uptake, and sensitization to BCNU.
Main Results:
- 11-mpBG demonstrated potent AGT inactivation with an ED(50) of 1.3 nM, over 300-fold more effective than b(6)G.
- 11-mpBG caused prolonged AGT inactivation in HT29 cells for at least 72 hours.
- 5 microM 11-mpBG significantly sensitized HT29 cells to BCNU, while 11-mpMG was ineffective.
- Cellular uptake appeared to be a limiting factor for 11-mpBG efficacy.
Conclusions:
- Nuclease-resistant oligodeoxyribonucleotides like 11-mpBG are highly effective AGT inhibitors.
- 11-mpBG can prolong AGT inactivation and sensitize cancer cells to alkylating agents.
- Further improvements in cellular uptake are needed for 11-mpBG to be a viable therapeutic agent for potentiating chemotherapy.