Enzymatic elimination of O6-ethylguanine and stability of O4-ethylthymine in the DNA of malignant neural cell lines exposed to N-ethyl-N-nitrosourea in culture

Abstract

The capacity of cells for enzymatic repair of DNA structurally altered by DNA-reactive agents is of particular interest in relation to carcinogenesis and with regard to the sensitivity/resistance of cancer cells towards chemotherapeutic drugs. The developing rat brain is characterized both by a pronounced susceptibility to the tumorigenic effect of N-ethyl-N-nitrosourea (EtNU), and by its incapacity, relative to other rat tissues, for enzymatic removal of O6-ethylguanine from DNA. We have investigated whether the latter property is preserved or altered in a panel of nine tumorigenic neural cell lines derived from pre-natal BDIX-rat brain exposed to EtNU in vivo. Using a competitive radioimmunoassay (RIA) in conjunction with a monoclonal antibody (Mab ER-6) specific for O6-ethyl-2'-deoxyguanosine (O6-EtdGuo), the kinetics of O6-EtdGuo elimination from cellular DNA were determined after a 20-min exposure to EtNU in vitro (100 micrograms/ml; resulting average 'input' O6-EtdGuo/2'-deoxyguanosine molar ratio in DNA, 0.74 X 10(-5)). All of the cell lines showed rapid removal of O6-EtdGuo from DNA. During a 280-min period of culture at 37 degrees C following exposure to EtNU, 36-88% of the 'input' amount of O6-EtdGuo was eliminated. Several of the cell lines removed O6-EtdGuo at least as efficiently as fetal BDIX-rat liver (the most O6-EtdGuo elimination-proficient rat tissue thus far documented). In agreement with published data on postnatal rat tissues, O4-ethyl-2'-deoxythymidine (O4-EtdThd) was not eliminated from DNA to any significant degree in a cell line chosen on the basis of its high O6-EtdGuo removal capacity, as assayed by RIA using a Mab (Mab ER-01) specific for O4-EtdThd. Seven subclones derived from one of the cell lines exhibited considerable variation of O6-EtdGuo removal capacity (elimination of 43-93% of the 'input' O6-EtdGuo within 280 min). The relevance of the observed O6-alkylguanine repair-proficiency of malignant neural rat cell lines in relation to carcinogenesis by EtNU in rat brain is not yet clear; however, the apparent instability and intracell population heterogeneity of O6-alkylguanine repair capacity in malignant cells deserve attention with respect to chemotherapy with, e.g. chloroethylnitrosoureas.