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Creation of human alkyltransferases resistant to O6-benzylguanine
F C Christians1, B J Dawson, M M Coates
1The Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle 98195-7705, USA.
Abstract:
O6-benzylguanine (BG), an inhibitor of O6-alkylguanine-DNA alkyltransferase, is being tested clinically for its ability to chemosensitize tumors to alkylating agents. Although this drug may increase the killing of tumors that express high levels of alkyltransferase, it would also be expected to reduce the already low alkyltransferase levels of hematopoietic stem cells and, thus, exacerbate the dose-limiting side effect of myelosuppression. One way to overcome this problem would be to transduce hematopoietic stem cells with a gene encoding a BG-resistant alkyltransferase prior to BG/alkylation treatment. We used the technique of random mutagenesis followed by positive genetic selection to create such a mutant gene. A pool of 6.5 x 10(6) human alkyltransferases that were randomly mutated at six amino acids near the alkyl-accepting cysteine was transformed into alkyltransferase-deficient Escherichia coli. Five mutants were selected based on their ability to provide the bacteria with resistance to both N-methyl-N'-nitro-N-nitrosoguanidine and BG. One mutant, V139F/P140R/L142M, not only had the highest BG resistance (50% inhibitory concentration, >500 microM) but also offered E. coli the best protection from N-methyl-N'-nitro-N-nitrosoguanidine and, thus, is a promising gene therapy candidate.
Insights
Researchers developed a modified O6-alkylguanine-DNA alkyltransferase gene to protect hematopoietic stem cells from chemotherapy side effects. This gene therapy candidate enhances resistance to O6-benzylguanine and alkylating agents, potentially improving cancer treatment safety.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Therapeutics
Background:
- O6-benzylguanine (BG) inhibits O6-alkylguanine-DNA alkyltransferase, enhancing alkylating agent chemotherapy.
- However, BG depletes alkyltransferase in hematopoietic stem cells, causing dose-limiting myelosuppression.
- Gene therapy offers a potential solution by engineering resistant stem cells.
Purpose of the Study:
- To create a mutant O6-alkylguanine-DNA alkyltransferase gene conferring resistance to BG.
- To identify a gene therapy candidate for protecting hematopoietic stem cells during BG/alkylating agent treatment.
Main Methods:
- Random mutagenesis of human O6-alkylguanine-DNA alkyltransferase was performed.
- Mutated genes were transformed into alkyltransferase-deficient Escherichia coli.
- Mutants were selected for resistance to N-methyl-N'-nitro-N-nitrosoguanidine and BG.
Main Results:
- Five BG-resistant O6-alkylguanine-DNA alkyltransferase mutants were identified.
- One mutant (V139F/P140R/L142M) exhibited high BG resistance (>500 microM IC50).
- This mutant also provided significant protection against N-methyl-N'-nitro-N-nitrosoguanidine in E. coli.
Conclusions:
- The V139F/P140R/L142M mutant is a promising candidate for gene therapy.
- This engineered enzyme can protect hematopoietic stem cells from BG and alkylating agents.
- This approach may mitigate myelosuppression and improve cancer therapy outcomes.