Coordinate modulation of Sp1, NF-kappa B, and p53 in confluent human malignant melanoma cells after ionizing radiation

Regulation of transcriptional responses in growth-arrested human cells unde r conditions that promote potentially lethal damage repair after ionizing r adiation (TR) is poorly understood. Sp1/retinoblastoma control protein (RCP ) DNA binding increased within 30 min and peaked at 2-4 h after IR (450-600 cGy) in confluent radioresistant human malignant melanoma (U1-Mel) cells. Increased phosphorylation of Spl directly corresponded to Sp1/RCP binding a nd immediate-early gene induction, whereas pRb remained hypophosphorylated. Transfection of U1-Mel cells with the human papillomavirus E7 gene abrogat ed Sp1/RCP induction and G(0)/G(1) cell cycle checkpoint arrest responses, increased apoptosis and radiosensitivity, and augmented genetic instability (i.e., increased polyploidy cells) after IR. Increased NF-KB DNA binding i n U1-Mel cells after LR treatment lasted much longer (i.e., >20 h). U1-Mel cells overexpressing dominant-negative I kappa R alpha S32/36A mutant prote in were significantly more resistant to IR exposure and retained both G(2)/ M and G(0)/G(1) cell cycle checkpoint responses without significant genetic instability (i.e., polyploid cell populations were not observed). Nuclear p53 protein levels and DNA binding activity increased only after high doses of IR (>1200 cGy). Disruption of p53 responses in U1-Mel cells by E6 trans fection also abrogated G(0)/G(1) cell cycle checkpoint arrest responses and increased polyploidy after IR, but did not alter radiosensitivity. These d ata suggest that abrogation of individual components of this coordinate IR- activated transcription factor response may lead to divergent alterations i n cell cycle checkpoints, genomic instability, apoptosis, and survival. Suc h coordinate transcription factor activation in human cancer cells is remin iscent of prokaryotic SOS responses, and further elucidation of these event s should shed light on the initial molecular events in the chromosome insta bility phenotype.