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The mouse epidermal JB6P cell line consists of tumor promotion sensitive (JB6P+) and tumor promotion resistant cells (JB6P-) that have been widely utilized to study tumor promotion. We transformed the JB6P+ cell line with prolonged low dose, UVBR (10mJ/cm2), H2O2 (10µM), and CdCl2 (1µg/ml). Cell transformation was confirmed by anchorage-independence. JB6P+ parent cells formed colonies in soft agar culture (13.7±3.5 per 20 high-power fields) which are increased by stimulation with the tumor promotor TPA (44.7±2.9). For the new transformants in the absence of TPA, colonies formed (104.0± 17.3 per 20 high-power fields, JB6P+/UV; 56.7±15.0, JB6P+/H2O2; 64.3±22, JB6P+/Cd cells; p<0.05 for each compared to JB6P+). We measured the relative intracellular levels of hydrogen peroxide (DCF) and superoxide anion (HE). The DCF and HE fluorescence in the JB6P+ cells was increased 9.0 and 2.2 -fold that of JB6P- cells. Induction of DCF and HE florescence was also evident in the transformed JB6P+/UV (3.4,1.3-fold), JB6P+/H2O2 (6.5, 1.3-fold), and JB6P+/Cd cells (3.6,1.4 fold).

Many nuclear transcription factors, for example Activating Protein-1(AP-1), are rapidly activated by elevated ROS stresses. Using Western Blot analysis of nuclear protein extractions, the distinct AP-1 composition patterns are summarized in the table below. We hypothesize that the UVBR, H2O2 and CdCl2 transformed the JB6P+ cells through different mechanisms.

Relative l densitometric laser measurements of AP-1 subunits

Each band was scanned and the densitometric measurements normalized to the JB6P+ results. ( C-Fos was normalized to JB6P+/UV results). * The zero value represents <0.01 of control and was determined by serial dilution of the protein.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA