DNA repair protein O 6 -methylguanine-DNA methyltransferase in testis and testicular tumors as determined by a novel nonradioactive assay Georg Nagel, a Walburgis Brenner, b Kai Johnsson, c and Bernd Kaina a, * a Division of Applied Toxicology, Institute of Toxicology, University of Mainz, D-55131 Mainz, Germany b Department of Urology, University of Mainz, Mainz, Germany c Institute of Molecular and Biological Chemistry, Swiss Federal Institute of Technology, CH-1015 Lausanne, Switzerland Received 19 February 2003 Abstract The DNA repair protein O 6 -methylguanine-DNA methyltransferase (MGMT, alkyltransferase) is an important suicide enzyme involved in defense against O 6 -alkylating endogenous metabolites and environmental carcinogens. It also plays a pivotal role in primary and acquired resistance of tumors to alkylating anticancer drugs targeting the O 6 -position of guanine (i.e., methylating and chloroethylating agents). MGMT can thus be considered a crucial biomarker for individual susceptibility to alkylating carcinogens and tumor drug resistance. This implies a need for a fast and convenient method for determination of MGMT. Routinely, MGMT is being quantified by radioactive assays which are relatively laborious. Here we report a nonradioactive MGMT enzyme-linked immunosorbent assay (ELISA) for quantification of MGMT in cell and tissue homogenates. We compared the MGMT-ELISA with the standard radioactive assay and found it to be as sensitive but less time consuming. Therefore, it represents an alternative for the quantification of MGMT in cell and tissue homogenates. We applied the assay for determining MGMT in normal and tumor tissue of testes. In both normal and tumor tissue MGMT was quite variable, ranging from zero to 1300 fmol/mg protein. In various tumor samples MGMT was lower than MGMT in the normal tissue from the same patient or was even not detectable. The MGMT-ELISA might become a useful tool for MGMT determination in clinical routine and health control. Ó 2003 Elsevier Inc. All rights reserved. Keywords: DNA repair; Alkyltransferase; Drug resistance; Tumor therapy A large number of environmental alkylating carcino- gens, endogenous alkylating species, and various tumor chemotherapeutic agents attack DNA at the O 6 position of guanine, forming O 6 -alkylguanine [1]. This lesion is considered to be a major cause of mutations and malig- nant transformation induced by O 6 -alkylating agents [2]. It also provokes genotoxicity and cell death by inducing apoptosis [3,4] and, therefore, represents the underlying reason for the antineoplastic effect of O 6 -alkylating agents. O 6 -alkylguanine is repaired by the DNA repair protein O 6 -methylguanine-DNA methyltransferase (al- kyltransferase, MGMT) 1 which transfers the alkyl group to its own active center [5]. Thereby guanine in DNA is restored and MGMT gets inactivated. As such, MGMT is unique among the repair proteins: it is a suicide enzyme acting only once. Consequently, the repair capacity of a cell is determined by the amount of preexisting molecules of MGMT and the rate of its resynthesis. MGMT has strong impact on cellular survival and proliferation ca- pacity upon treatment with O 6 -alkylating agents includ- ing antineoplastic drugs [6] and, therefore, can be considered a crucial marker of resistance to methylating and chloroethylating drugs [7]. MGMT is unique also from another point of view: It is highly variably expressed in normal and tumor tissues [8]. For instance, in human lymphocytes it ranges in Analytical Biochemistry 321 (2003) 38–43 www.elsevier.com/locate/yabio ANALYTICAL BIOCHEMISTRY * Corresponding author. Fax: +49-6131-393-3421. E-mail address: kaina@mail.uni-mainz.de (B. Kaina). 1 Abbreviations used: MGMT, O 6 -methylguanine-DNA methyltransferase; ELISA, enzyme-linked immunosorbent assay; DTT, dithiothreitol; BSA, bovine serum albumin; PBS, phosphate-buffered saline; ABTS, 2,2 0 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid). 0003-2697/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0003-2697(03)00432-9