Gemcitabine is a chemotherapeutic with multiple proposed mechanisms of action, including depletion of deoxynucleotide triphosphates that are necessary for DNA synthesis and completion of DNA repair39. Interestingly, gemcitabine has been shown to specifically inhibit GADD45A mediated gene activation via DNA demethylation and DNA repair40. To investigate the role of DNA repair in expression of AREG and EREG, cells were treated with 10 μM gemcitabine for 72 hours. As demonstrated in Fig. 5A, AREG and EREG expression dramatically decreased in cells with high integrin α6β4 in response to treatment, thus indicating that DNA repair is required to maintain expression. As summarized in Fig. 5B, GADD45A mediated active DNA demethylation is achieved through BER. Therefore, we next investigated the role of TET1, GADD45A, TDG, and PARP1 in the regulation of AREG and EREG as key regulators of DNA repair-mediated DNA demethylation.Figure 5GADD45A is both required for and the rate-limiting step in activation of AREG and EREG expression. (A) Cells were treated with 10 μM Gemcitabine for 72 hours and expression of AREG and EREG measured by QPCR. (B) Summary of current literature for how GADD45A mediated DNA demethylation is achieved. Transient knockdown of GADD45A was achieved using electroporation and specific siRNA (E). Adenovirus was used to overexpress GADD45A in Panc1-2G6 and Panc1-3D7 cells (H). Changes in AREG and EREG expression were measured by QPCR in Panc1-2G6 (C,F) and Panc1-3D7 (D,G). Data depicted here are representative of at least three different experiments and represent the mean +/− standard deviation. Statistical significance was calculated using a one-tailed t-test in which * denotes P < 0.05 as compared to controls.