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Ataxia Telangiectasia Group D Associated gene (ATDC)

Our group's studies are focusing on the pancreatic cancer and the Ataxia Telangiectasia Group D Associated gene (ATDC). Pancreatic cancer is a most deadly disease characterized by late diagnosis, aggressive invasion of surrounding tissues, early metastasis. It has the worst prognosis of any major malignancy (3% 5 year survival) and is the fourth most common cause of cancer death. Recent advances in surgical and medical therapy have had little impact on the mortality rate of this disease. Pancreatic cancer is notoriously resistant to many types of cytotoxic chemotherapy and ionizing radiation. The molecular basis of pancreatic cancer is incompletely understood. ATDC was initially described in the hunt for the gene responsible for the genetic disorder ataxia telangiectasia (AT). ATDC was isolated based on its ability to suppress the radiation sensitivity of AT complementation group D fibroblasts in cell fusion experiments. The ATDC gene, located on chromosome 11q23, possesses multiple zinc finger motifs and an adjacent leucine zipper motif. ATDC, also known as TRIM29, is a member of the tripartite motif (TRIM) protein family. TRIM proteins have a series of conserved domains, which include a RING (R), a B-box type 1 (B1) and B-box type 2 (B2), followed by a coiled-coiled (CC) region. ATDC contains the B1-B2-CC domains but lacks the R domain. However, very little is known about the biological mechanisms regulated by ATDC.

By affymetrix gene profiling, we recently determined that pancreatic cancer cells specifically overexpress ATDC at a level at least 20 fold higher than normal pancreas and chronic pancreatitis. We further validated our gene profiling data. RT-PCR indicated that ATDC message only up-regulated in pancreatic cancer, not in chronic pancreatitis and normal pancreas. Immunohistochemistry with specific anti-ATDC antibody showed that ATDC was expressed specifically in the neoplastic epithelium, and also was up-regulated in pancreatic cancer precursor lesions (PanIN lesions). To explore its precise biological function and clinical relevant in pancreatic cancer, we did following studies.

We first explored the effect of ATDC on cellular growth in vitro by utilizing two cell models with altered levels of ATDC. Following transfection with a construct expressing ATDC in HEK 293 cells, which normally do not express ATDC, demonstrated a significant increase in cellular proliferation. Conversely, when ATDC was silenced by stable transfection with the siRNAs targeting distinct regions of ATDC in Panc-1 pancreatic cancer cells, which have high endogenous levels of ATDC, cellular proliferation was inhibited. In addition, to examine the effects of ATDC silencing on pancreatic tumor growth and metastasis in vivo by bioluminescent imaging, we injected luciferase-expressing lentivirus infected Panc1 cells (with or without ATDC expression) into pancreas tails in immuno-deficient (NOD/SCID) mice. All of the animals injected with Panc-1 cells expressing control shRNA demonstrated tumor formation 14 days post-injection while tumors were not detected in the animals injected with Panc-1 cells expressing ATDC shRNA (ATDCshRNA). At 60 days post-injection, the control shRNA animals tumors grew significantly larger, with evidence of metastatic spread, while only 25% (2/8) of the ATDCshRNA animals demonstrated evidence of macroscopic tumors. The mean tumor volume was significantly larger in mice injected with Panc-1 cells expressing control shRNA compared to mice injected with Panc-1 cells expressing ATDC shRNA. These data support the role of ATDC in promoting growth and metastasis of pancreatic cancer cells. Although, addition studies are needed to analyze the signaling cascades and specific downstream targets, which mediate the stimulatory effects of ATDC on cellular proliferation and pancreatic cancer progression.

To evaluate whether ATDC overexpression is responsible for the radio-resistance capacity of pancreatic cancer, we examined the effect of ATDC silencing on DNA damage responses after ionizing radiation exposure in pancreatic cancer cells. We found that 1 hour after exposure to 10 Gy, ATDC silencing in Panc-1 cells significantly increased radioresistant DNA synthesis. This suggests that silencing of ATDC prevents the activation of the S phase checkpoint, potentially leading to genomic instability by replication of an unrepaired DNA template. Phosphorylation of the histone variant H2AX (?H2AX) is a well-recognized indicator of DNA double strand breaks. ?H2AX foci are rapidly formed at DNA double strand break sites and are thought to play a role in the repair of these breaks. Our data showed that ionizing radiation induced a transient H2AX phosphorylation, with induction seen as early as 10 minutes, with return to basal levels after 1 hour in control Panc1 cells. In contrast, irradiated Panc-1cells with ATDC silencing showed enhanced phosphorylation of H2AX and a prolonged recovery time, suggesting that either DNA damage with ionizing radiation was worsened or DNA double strand break repair was abrogated in these cells. As a predominately cytoplasmic protein, how does ATDC involve in DNA repair process remaining to be elucidate. By treatment with a nuclear export inhibitor leptomycin B, we were able to show that even though ATDC staining normally appears predominantly cytoplasmic, ATDC does traffic to the nucleus. Using double immunofluorescence staining, we observed that in response to ionizing radiation, ATDC co-localized with ?H2AX, presumably at DNA repair foci. To further investigate the role of ATDC in the DNA damage response, we examined the effect of silencing ATDC on phosphorylation of two critical molecules required for cell cycle arrest in response to DNA damage, p53 and Chk2. Our data showed that both p53 and Chk-2 underwent phosphorylation in response to ionizing radiation in control shRNA Panc-1 cells, but this was significantly decreased in ATDC-silenced Panc-1 cells, indicating that ATDC participates in downstream cell cycle checkpoint activation. Based on previous findings that ATDC overexpression rescued the radiosensitivity of AT cells, we hypothesized that ATDC might also confer radioresistance in HEK 293 cells. Expression of ATDC in HEK 293 cells resulted in higher cell survival when exposed to increasing doses of ionizing radiation compared to wild type HEK 293 cells. Similarly, the ability of 10 Gy of ionizing radiation to induce apoptosis (measured by annexin V expression), was reduced by overexpression of ATDC, confirming that ATDC confers radiation resistance. In contrast, we found that siRNA silencing of ATDC in Panc-1 cells rendered them more sensitive to ionizing radiation, with a marked decrease in the surviving fraction following irradiation. We also found that silencing of ATDC increased susceptibility to apoptosis due to ionizing radiation.

Therefore, we get following conclusions: ATDC is a novel DNA damage response protein that is expressed in the majority of pancreatic cancers and has dual functions of promoting pancreatic cancer cell growth and regulating cellular responses to DNA damage. This work has important implications for better understanding pancreatic tumorigenesis, as well as for exploring the potential role of ATDC in growth regulatory mechanisms and DNA damage responses in both neoplastic and normal cells, a topic which has not been previously explored. Furthermore, targeting ATDC for inactivation should have therapeutic value by both reducing proliferation and increasing susceptibility to radiotherapy of pancreatic cancer cells.

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