Moesin is a biomarker for the assessment of genotoxic carcinogens in mouse lymphoma

Abstract

1,2-Dibromoethane and glycidol are well known genotoxic carcinogens, which have been widely used in industry. To identify a specific biomarker for these carcinogens in cells, the cellular proteome of L5178Y mouse lymphoma cells treated with these compounds was analyzed by 2-dimensional gel electrophoresis (2-DE) and MALDI-TOF mass spectrometry (MS). Of 50 protein spots showing a greater than 1.5-fold increase or decrease in intensity compared to control cells on a 2-D gel, we focused on the candidate biomarker moesin. Western analysis using monoclonal rabbit anti-moesin confirmed the identity of the protein and its increased level of expression upon exposure to the carcinogenic compounds. Moesin expression also increased in cells treated with six additional genotoxic carcinogens, verifying that moesin could serve as a biomarker to monitor phenotypic change upon exposure to genotoxic carcinogens in L5178Y mouse lymphoma cells.

Fig. 1.

The 2-D gel of proteins isolated from L5178Y mouse lymphoma cells treated with genotoxic carcinogens. (A) Sample gel image of solvent control-treated proteome profile. Proteins were separated on the basis of pI (x-axis) and molecular weight (y-axis). Spots were visualized by Coomassie blue staining. (B) Enlarged images of upregulated protein spots. (C) Enlarged images of downregulated protein spots. Selected spots were defined as altered and identified by MALDI-TOF MS analysis.

Fig. 2.

Validation of increased expression of moesin by western analysis in response to genotoxic carcinogens treatments. Con: solvent control (DMSO), 1 to 3; 1,2-dibromoethane, glycidol (genotoxic carcinogen), and methylcarbamate (nongenotoxic carcinogen), respectively. L5178Y mouse lymphoma cells were treated with each compound for 2 h. β-actin was used as a loading control. Moesin expression level was increased to 170% and 160% with treatment of 1,2-dibromoethane and glycidol, respectively, compared to solvent control. In contrast, methylcarbamate did only 120% increase.

Fig. 3.

Specific expression of moesin by genotoxic carcinogen treatments. Cells were treated with compounds for 2 h. The effective concentration of each compound is shown in Tables 2 and 3. (A, B) Treatment of test compounds to L5178Y mouse lymphoma cells. (C, D) Treatment of test compounds to NIH3T3 mouse fibroblasts. (A, C) Treatment of genotoxic carcinogens. Con: solvent control (DMSO), 1 to 8: 1,2-dibromoethane, glycidol, diethylstilbestrol, urethane, chlorambucil, dibenz(a,h)anthraxcene, methyl methanesulfonate, and N-nitroso-N-methylurea, respectively. Cells were treated with each compound for 2 h at the concentrations shown in Table 2. (B, D) Treatment of non-genotoxic carcinogens. Con: solvent control (DMSO), 1 to 8: methylcarbamate, O-nitrotoluene, 1,4-dioxane, tetrachloroethylene, TCDD, chloroprene, nitrobenzene, and nitromethane, respectively.