Regulation of RASSF1A in nasopharyngeal cells and its response to UV irradiation.

RASSF1A, which is frequently found inactivated in human cancers, is revealed as a tumor suppressor gene in nasopharyngeal carcinoma (NPC). Using RASSF1A-expressing (NP69 and HK-1) and non-RASSF1A-expressing (C666-1) cell models, the transcriptional regulation of RASSF1A was studied. By deletion analysis of 3.1 kb of 5' flanking region, the core promoter of RASSF1A was identified in the region between -431 and -1 upstream of the translation start site. Sequence analysis of this core promoter revealed several putative transcription factor binding sties. Using NP69 cells and by block replacement mutagenesis, the presence of three functional GC-boxes were identified, to which by competitive and supershift electrophoretic mobility shift assays (EMSA), the in vitro bindings of Sp1 and Sp3 were suggested. The in vivo functions of Sp-proteins in regulating RASSF1A gene were then investigated by overexpression studies; among the tested Sp-proteins, Sp1 or Sp3, but not Sp4, was able to augment promoter activities. More interestingly, co-expression of Sp1 and Sp3 could synergistically enhance RASSF1A promoter function. UV irradiation induces oxidation stresses and hence is routinely used to investigate expressions of oncogenes and tumor suppressors. In this report, upon UV irradiation, the RASSF1A promoter activity and endogenous transcript levels were found to be reduced. By chromatin immunoprecipitation (ChIP) and EMSA, we demonstrated that the binding of Sp1 and Sp3 onto -431 to -202 were significantly reduced after UV irradiation. This UV-mediated effect on RASSF1A promoter, as shown by specific inhibitors that interrupt cellular pathways, is MEK1-, but not JNK-dependent. In summary, our data provided a simple model to explain the potential development of NPC, via silencing of the tumor suppressor RASSF1A by reduced bindings of activators Sp1 and Sp3 onto the GC-boxes in the core promoter of the gene.

Significance of scheduling on the cytotoxicity of radiation and cisplatin combination treatment in nasopharyngeal carcinoma cells.

The use of cisplatin as a potential radiosensitizer in nasopharyngeal carcinoma (NPC) has produced encouraging results in clinical trials. In order to provide information on improving the design of clinical treatments, we investigated the effect of cisplatin dose, and the time interval and sequence between administration of cisplatin and radiation on cell survival of two NPC cell lines, CNE1 and SUNE1. When cisplatin was applied first, an exposure time of 24 h resulted in up to 2.6-fold increase in cell death and 7-fold increase in radiation effect (cell survival after cisplatin/cell survival after cisplatin plus radiation) in the cisplatin-radiation combination treatment compared to the cells treated with cisplatin for 4 h. When radiation was applied first, a shorter interval time of 4 h followed by cisplatin treatment resulted in up to 3-fold increase in cell death and a 3-fold enhanced radiation effect over longer time intervals of 24 h. By changing the order of radiation and cisplatin treatment alone, a 2-fold difference in radiation effect was observed. The differential cytotoxicity was partially explained by the alterations in cell cycle distribution. Our results indicate the importance of scheduling the radiation and cisplatin combination regimens on the survival of NPC cells.