Abstract
Purpose: There are several reports suggesting that the target of certain carcinogens would be tissue stem cells. To develop a model system assessing carcinogenic activity of chemical compounds in stem cells, we determined chemical-induced DNA damage in murine embryonic stem (mES) cells as well as the expression of xenobiotics-metabolizing enzymes (Cyp1a1 and Cyp1b1) and undifferentiation markers (Sox2 and alkaline phosphatase ALP). These patterns were compared with murine embryonic fibroblast (MEF), a representative differentiated cell to understand stem cell-specific responses.
Methods: 1) Cell lines: ES-D3 cells, and MEF prepared from fetuses. 2) Test chemicals: 7,12-dimethylbenz(a)anthracene (DMBA, 50-500 nM) or 3-methylcholanthrene (3-MC, 50-500 nM). 3) DNA adduct measurement: 32P-Postlabeling/PAGE analysis. 4) Gene expression: RT-PCR and real time RT-PCR for Cyps, and Sox2 expressions. 5) ALP assay: Cytochemical staining using ALP specific substrate.
Results and discussion: DNA adducts were detected in DMBA-treated cells. The level was almost two times higher in MEF as compared with mES cells, indicating a good correlation with the expression magnitude of Cyp1b1, not Cyp1a1. In mES cells, DMBA treatment suppressed the expression of Sox2, a known undifferentiation marker and decreased ALP-positive cells. 3-MC-treatment in mES cells induced Cyps expression with no detectable DNA adduct formation and no down regulation of undifferentiation markers, indicating that DNA damage induced by DMBA would be responsible for disruption of stemness by which damaged stem cells are being eliminated.
