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In the present study, we report the development of a sensitive and selective assay based on LC (liquid chromatography)-MS/MS (tandem MS) to simultaneously measure N7-MeG (N7-methylguanine) and N7-EtG (N7-ethylguanine) in DNA hydrolysates. With the use of isotope internal standards (15N5-N7-MeG and 15N5-N7-EtG) and on-line SPE (solid-phase extraction), the detection limit of this method was estimated as 0.42 fmol and 0.17 fmol for N7-MeG and N7-EtG respectively. The high sensitivity achieved here makes this method applicable to small experimental animals. This method was applied to measure N7-alkylguanines in liver DNA from mosquito fish (Gambusia affinis) that were exposed to NDMA (N-nitrosodimethylamine) and NDEA (N-nitrosodiethylamine) alone or their combination over a wide range of concentrations (1-100 mg/l). Results showed that the background level of N7-MeG in liver of control fish was 7.89+/-1.38 mmol/mol of guanine, while N7-EtG was detectable in most of the control fish with a range of 0.05-0.19 mmol/mol of guanine. N7-MeG and N7-EtG were significantly induced by NDMA and NDEA respectively, at a concentration as low as 1 mg/l and increased in a dose-dependent manner. Taken together, this LC-MS/MS assay provides the sensitivity and high throughput required to evaluate the extent of alkylated DNA lesions in small animal models of cancer induced by alkylating agents.

Glycine-N-methyltransferase (GNMT) is a protein with multiple functions. It can affect genetic stability by (1) regulating the ratio of S-adenosylmethionine to S-adenosylhomocystine and (2) binding to folate. In addition to act as a tumor suppressor for liver cancer, GNMT participated in the detoxification pathway by interacting with environmental carcinogens [such as benzo(a)pyrene (BaP), aflatoxin B1 (AFB1)] and decreasing subsequent DNA adducts formation and cytotoxicity. GNMT was primarily expressed in cytoplasm and partially translocated to cell nuclei after BaP treatment. Despite the mechanism of GNMT nuclear translocation remains unclear, it has been shown that the phosphorylation modification is important for GNMT nuclear translocation. The results from LC-MS/MS (liquid chromatography-tandem mass spectrometry) analysis showed that serine 9 of GNMT was phosphorylated after BaP treatment. The GNMTS9A mutant did not undergo nuclear translocation after BaP treatment by using indirect immunofluorescence assay. GNMT serine 9 was identified within an Akt1 consensus site by Scansite program. The co-immunoprecipitaion assay demonstrated that GNMT interacts with Akt. Akt phosphorylated GNMT only on serine 9 in vitro. Furthermore, BaP induced GNMT nuclear translocation was blocked by LY294002 treatment. In conclusion, the results suggested that the Serine 9, can be phosphorylated by Akt1, is an important phosphorylation site responsible for GNMT nuclear translocation after BaP treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1335. doi:10.1158/1538-7445.AM2011-1335

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GNMT is a tumor suppressor for hepatocellular carcinoma (HCC). We found that DEPTOR, a novel mTOR binding protein, interacts with GNMT through its PDZ domain. DEPTOR expression is significantly increased in HCC, especially among patients with HCV infection. Loss of DEPTOR in HuH-7 cells activates S6K but reduces Akt activation and cell growth. DEPTOR overexpression activates Akt and extends survival of HCC cells. Overexpression of GNMT counteracts DEPTOR induced Akt activation and activates mTORC1 downstream signaling via disruption of the DEPTOR-mTOR interaction. Moreover, GNMT overexpression delays G2/M progression, which causes senescence and slows proliferation of HuH-7 cells, sensitizing them to rapamycin. We suggest that GNMT by interacting with DEPTOR regulates the cellular homeostasis of hepatocytes through modulation of Akt/mTORC1 signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1990. doi:10.1158/1538-7445.AM2011-1990