Alteration of apoptotic signaling molecules as a function of time after radiation in human neuroblastoma cells

Ascertaining the time-dependent regulation of induced apoptosis and radioresistance is important to understand the relationship between the level of spontaneous apoptosis in cells and their radiosensitivity. Accordingly, we investigated the time-dependent expression of apoptosis related genes and radioresistance in neuroblastoma cells. Serum-starved human SK-N-MC cells were exposed to low linear energy transfer (LET) radiation (2 Gy) and incubated for 15, 30, 45 min, and 48 h. Radioresistance was investigated by examining the NFκB DNA-binding activity, cellular toxicity, DNA fragmentation, and expression of apoptotic signal transduction molecules. NFκB DNA binding activity was analyzed using electrophoretic mobility shift assay (EMSA). Cellular toxicity was measured using MTT assay. DNA fragmentation was quantified by labeling with fluorescein-conjugated deoxynucleotides. Microarray analysis was performed using cDNA microarray and relative gene expression was measured as % GAPDH and, subsequently validated using Q-PCR. Induction of NFκB analyzed using EMSA showed an increased DNA-binding activity at all time points investigated. Induced DNA fragmentation was observed after 15, 30, and 45 min post-radiation. Relatively, induced fragmentation was reduced after 48 h. Compared to the untreated controls cellular toxicity was induced with low LET radiation after 15, 30, and 45 min. Conversely, cytotoxicity was relatively less at 48 h after low LET radiation. Microarray analysis after low LET radiation revealed time-dependent modulation of apoptosis-related genes that are involved in radio-adaptation, spontaneous apoptosis-related early-responsive genes and late response genes. These results suggest that the time-dependent regulation of apoptotic response may determine the relationship between the level of spontaneous apoptosis in cells and their radiosensitivity.