Abstract
[Purpose] Nevoid basal cell carcinoma syndrome (NBCCS), also known as Gorlin syndrome or basal cell nevus syndrome, is an autosomal dominant disorder that predisposes to both developmental defects and cancer. Fibroblast cells derived from (NBCCS) patients show increased levels of DNA synthesis after X-ray irradiation. We recently reported that the induction of DNA synthesis after irradiation could be caused by the down-regulation of SUMO-2 gene expression in NBCCS cells. Post-translational modification marked by the covalent attachment of the ubiquitin-like protein SUMO-1 has been implicated in a wide variety of cellular stress responses. By contrast, the functions of SUMO-2 remain largely uncharacterized. At the last meeting, we reported that after X-ray irradiation, an increase in DNA synthesis activity was detected in HeLa cells with knockdown of SUMO-2. To investigate the molecules of DNA synthesis induction due to the down-regulation of SUMO-2, we searched for the proteins whose expression levels differ pre- and post-X-ray irradiation in HeLa cells with knockdown of SUMO-2.
[Methods] DNA synthesis activity was measured by a pulse-labeling method with 14C and 3H. When comparative proteomic analysis was performed between the siRNA for SUMO-2 and mimic control siRNA treated cells using two-dimensional (2D) electrophoresis and mass spectrometry, the NM23-H1 protein was identified as a candidate protein. Knockdowns of SUMO-2 and NM23-H1 were performed using their siRNAs. Immunoprecipitation assay was carried out to analyze of NM23-H1 modification by SUMO-2.
[Results and discussion] Eight hours after X-ray irradiation(2Gy), an increase in DNA synthesis activity was found in HeLa cells with knockdown of SUMO-2. Under these conditions, the expression levels of Nm23-H1, a metastatic suppressor gene, was altered. The decrease of NM23-H1 protein after X-ray irradiation was confirmed by western blot analysis. As expected, treatment of HeLa cells with the siRNA of NM23-H1 resulted in the enhancement of DNA synthesis. These findings suggest that the intimate relationship with the expression of SUMO-2 and the stability of NM23-H1 after X-ray irradiation, lead to the induction of DNA synthesis.
Nm23-H1 may be modified by SUMO-2 after X-ray irradiation. The reduction of Nm23-H1 seems to be casually related to the decrease in sumoylation, which in turn, is involved in the induction of DNA synthesis after X-ray irradiation.
