• Biomolecules & Therapeutics
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• 제3권2호
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• pp.122-131
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• 1995

Five human cancer cell lines (HeLa S3, Hep 3B, KATO III, Hs 683, HeLa MR) and one human normal cell line (WI-38) were examined cell viability, northern blot analysis, western blot analysis, and in situ hybridization for the expression $O_{6}$ -methylguanine-DNAmethyltransferase (MGMT), which can repair $O_{6}$ -methylguanine produced in DNA by alkylating agents. In cell viability test, the lethal sensitivities of each strain against anti-tumor drug N,N-bis(2-chloroethyl)- N-nitrosourea (BCNU) were counted, and both BCNU treated and untreated cell extracts were examined for their MGMT inducibility by RNA dot blot analysis. Cell lines did not show MGMT induction by BCNU pretreatment. Tlle MGMT activity was assayed by measuring the $^3$H radioactivity transferred from the substrate DNA containing [methyl-$^3$H)-O$_{6}$ -methylguanine to acceptor molecules in the cell extracts. Extracts from the majority of tumor strains and normal cells contained substantial MGMT activity of varying degree, while the known Mer$^{[-10]}$ cell (lacked or severely depleted in MGMT activity) Hela MR, and Hs 683 (proved to be Mer$^{[-10]}$ ) were much more sensitive to BCNU than the rest of tumor strains, as measured by cell viability test. Overall results above, KATO III showed the highest expression level of MGMT among the strains examined. Furthermore, with all the tumor and normal strains tested, a good correlation was observed between MGMT expression and cellular resistance to BCNU. The varying levels of expression of MGMT in human cancer cells found in this study should provide a molecular basis for MGMT expression among tumor strains from different tissue origin, the information of antitumor agents selection for chemotherapy of cancers.

• BMB Reports
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• 제30권1호
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• pp.41-45
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• 1997

The present study was performed to analyze the molecular mechanism which dictates the transcription regulation of the $O^6$-methylguanine-DNA methyltransferase (MGMT) gene in Saccharomyces cerevisiae. Previously we identified one possible upstream repressing sequence (URS) in MGMT promoter by promoter deletion and competition analysis. In this paper we report another regulatory element (UAS: upstream activating sequence. -213 to -136) which affects the transcription activity of MGMT promoter. Gel mobility shift assay and Southwestern blot analysis using UAS probe showed several specific proteins which were able to bind to this sequence.