t-Darpp antagonizes ceramide-induced apoptosis: up-regulation of Bcl2 through active CREB/ATF-1

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Our laboratory identified Darpp-32 (Dopamine and cAMP-regulated phosphoprotein of Mr 32,000) as the critical target at the 17q12 amplicon in gastric cancer. We have also cloned a novel truncated isoform that we named t-Darpp. We showed overexpression of Darpp-32 and t-Darpp in a number of common adenocarcinomas such as colon, breast, prostate, and stomach. Herein, we report the biological function of t-Darpp. We performed a dual TUNEL assay and immunofluorescence on transiently transfected RKO cells with t-Darpp expressing pcDNA3.1 vector and following treatment with 5 μM camptothecin. The results showed a significant reduction in apoptosis in t-Darpp expressing cells. This strong anti-apoptotic potential for t-Darpp was independently confirmed by Annexin V assay and FACS analysis following treatment with CPT, ceramide, and butyrate treatment. Luciferase assays using the p53 and p21 reporter plasmids and western blot with antibodies specific for p53 transcriptional targets, such as Hdm2 and p21, indicated that t-Darpp does not interfere with p53 function. Alternatively, we demonstrate that t-Darpp confers antiapoptotic property to cancer cells through transcriptional up-regulation of Bcl2 and protection of mitochondrial transmembrane potential. The mitochondrial release of cytochrome c and activation of caspase 9 were abolished by t-Darpp expression in ceramide-treated cells. The use of siRNA-specific oligo for t-Darpp confirmed that protein expression of Bcl2 was regulated by t-Darpp. In addition, using luciferase, EMSA, and quantitative real-time RT-PCR assays, indicated that t-Darpp transcriptionally upregulates Bcl2 levels. The t-Darpp mediated upregulation of Bcl2 correlated with increased expression and phosphorylation of CREB and ATF-1 transcription factors. The use of siRNA-specific oligos for CREB and ATF led to a dramatic suppression of Bcl2 protein levels in t-Darpp expressing cells. These results confirm that the t-Darpp-mediated up-regulation of Bcl2 requires active CREB and ATF-1 transcription factors. Further studies are ongoing to fully characterize the oncogenic pathways of t-Darpp.