DNA Repair-Mediated Alkylating Agent Resistance in Human Gliomas

Background:

Alkylating agent-based chemotherapy when used as an adjuvant to surgery and radiation modestly increases response rates and survival times for adult malignant gliomas. The chloroethylating agents 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, carmustine) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU, lomustine), and the methylating agents procarbazine and temozolomide, used alone or in combination, are the most commonly employed drugs for malignant glioma. However, alkylating agents provide no benefit to ~50% of patients, seldom produce long term remission, and the modest benefit that they confer depends greatly on patient and clinical parameters, including age, neurological status and extent of resection. Intrinsic and acquired resistance to alkylators reduces cell killing in gliomas and are, therefore, major factors affecting outcome. Intensive investigation during the last three decades has not yielded new chemotherapy that is superior to alkylating agent-based regimens. This, together with the dismal prognosis for malignant glioma, emphasizes the need to develop and evaluate strategies to overcome resistance to agents of known clinical efficacy.

Our long-term objective is to define the contribution of DNA repair to the resistance of human gliomas to alkylating agent-based chemotherapy, and to identify strategies to combat resistance.

Cytotoxic DNA damage produced by alkylator used to treat human adult gliomas

Research Question:

Methylating (procarbazine, temozolomide) and chloroethylating agents (BCNU, CCNU) readily transfer their alkyl moieties to ~15 nucleophilic sites on DNA purines and pyrimidines. The majority (55% to 70%) of alkylation occurs at the ring nitrogens in purines (e.g., N7-methylguanine, N7-hydroxyethylguanine) and are the precursors of cytotoxic abasic sites. O6-methyguanine and O6-chloroethylguanine, the precursor of inter-strand cross-links, are also known lethal adducts.

MGMT and Ape1/Ref-1 are promising targets for anti-resistance therapies in gliomas. MGMT is a good candidate because it is the sole mechanism that removes O6-methylguanine and O6-chloroethylguanine. In addition, there is a specific inhibitor of MGMT, O6-benzylguanine (O6-BG), which enables quantitation of the contribution of repair of O6-alkylguanine adducts to alkylator resistance. Ape1/Ref-1 is also a good anti-resistance target. Ape1/Ref-1 is the major activity that initiates repair of cytotoxic abasic sites, the most abundant lesion produced by alkylating agents. Ape1/Ref-1 catalyzes > 90% of the abasic site incising activity in human cells. Ape1/Ref-1 is also a reduction-oxidation protein (ref-1) that participates in critical cellular functions, including response to oxidative stress and the induction of apoptosis. Notably, Ape1/Ref-1 protein and activity is elevated in response to oxidative stress, suggesting a possible mechanism of acquired resistance to alkylators.

Research Highlights:

I. MGMT contributes to glioma cell line alkylator resistance

II. Apurinic endonuclease (Ape1/Ref-1) contributes to glioma cell line alkylator resistance

III. Oxidative stress elevates Ape1/Ref-1 level and increases alkylator resistance

IV. Elevated MGMT and apurinic endonuclease activity accompanies glial tumorigenesis

V. Gliomas lacking detectable MGMT activity (i.e., Mer- status) are not more responsive to alkylator therapy

Research Methods:

We utilize the following experimental techniques to analyze the contribution of specific DNA repair activities and the repair of specific DNA adducts to human glioma resistance to alkylating agents and radiation

• Biochemical assay of DNA repair activities
• Western analysis of DNA repair protein abundance
• Immunocytochemical analysis of intra-tumoral heterogeneity and intra-cellular localization of DNA repair activities
• Antisense oligonucleotides to suppress DNA repair activities in human glioma cell lines
• Quantitation of abasic sites using an aldehyde reactive probe
• Immunocytochemical analysis of intra-tumoral heterogeneity and intra-cellular localization of alkylation- and oxidation-induced DNA damage

Laboratory Members and Collaborators:

Selected Publications:

1. Silber, J.R., Bobola, M.S., Blank, A., Schoeler, K.D., Haroldson, P.D. and Kolstoe, D.D. (2002) The apurinic/apyrimidinic endonuclease activity of Ape1/Ref-1 contributes to human glioma cell resistance to alkylating agents and is elevated by elevated stress. Clin Cancer Res. 8: 3008-3018..

2. Poot, M., Silber, J.R. and Rabinovitch, P.S. (2002) A novel flow cytometric technique for drug cytotoxicity that is comparable to colony-forming assays. Cytometry 48: 1-5.

3. Poot, M. Gollahon, K.A. Emond, M.J., Silber, J.R. and Rabinovitch, P.S. (2002) Werner Syndrome diploid fibroblasts are sensitive to 8-methoxypsoralen and 4-nitroquinoline-N-oxide. FASEB J. 16:757-758.

4. Silber, J.R., Bobola, M.S., Blank, A., Berger, M.S., and Stevens, B.A. (2001) Apurinic/apyrimidinic Endonuclease Activity is Elevated in Human. Clin Cancer Res. 7: 3510-3518.

5. Bobola, M.S., Berger, M.S., Ellenbogen, R.G., Roberts, T.S., Geyer, J.R. and Silber, J,R. (2001) O6-Methylguanine-DNA methyltransferase in pediatric primary brain tumors: relation to patient and tumor characteristics. Clin Cancer Res. 7: 613-619.

6. Bethune, C., Blum, A., Geyer, J.R., Silber, J.R. and Ho, R.J.Y. (1999) Lipid association increases the potency against primary medulloblastoma cells and systemic exposure of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in rats. Pharm. Res. 16: 896-903.

7. Tseng, S.-H.,Bobola, M.S., Berger, M.S. and Silber, J.R. (1999) Characterization of Taxol Sensitivity in Human Glioma and Medulloblastoma-derived Cell Lines. Neuro-Oncology 1: 101-108.

8. Silber, J.R., Bobola, Blank, A., Bobola, M.S., Ghatan, S., M.S., Kolstoe, D.D., and Berger, M.S. (1999) O6-methylguanine-DNA methyltransferase activity-deficient phenotype in human gliomas: Frequency and time to tumor progression after alkylating agent-based chemotherapy. Clin. Cancer Res. 5: 807-814

9. Lundin, D.A., Berger, M.S., Blank, A. and Silber., J.R. (1998) Deficiency of O6-methylguanine-DNA methyltransferase is not associated with microsatellite instability in adult gliomas. Oncol. Res. 10: 421-428.

10. Silber, J.R., Bobola, M.S., Ghatan, S., Kolstoe, D.D., Blank, A. and Berger, M.S. (1998) O6-methylguanine-DNA methyltransferase activity in adult gliomas: Relation to patient and tumor characteristics. Cancer Res. 58: 1068-1073.

11. Silber, J.R., Blank, A., Bobola, M.S., Mueller, B.A., Kolstoe, D.D., Ojemann, G.A. and Berger, M.S. (1996) Lack of the DNA repair protein O6-methylguanine-DNA methyltransferase in histologically normal brain adjacent to primary brain tumors. Proc. Natl. Acad. Sci. USA 93: 6941-6946.

12. Bobola, M.S., Tseng, S.H., Blank, A., Berger, M.S. and Silber, J.R. (1996) Role of O6-methylguanine-DNA methyltransferase in resistance of human brain tumor cell lines to the clinically relevant methylating agents temozolomide and streptozotocin. Clin. Cancer Res. 2: 735-741.