We hypothesize that ionizing radiation stimulates the activation of a nuclear tyrosine or serine/threonine kinase (e.g., c-abl or the double- strand DNA-dependent Ku kinase) that phosphorylates the nuclear Spl transcription factor (TF), which is dormant via its complexation with Sp1- I, its inhibitor. Phosphorylation of Spl de-stabilizes the Sp1-Sp1-I inhibitory complex and results in retinoblastoma (pRB)-mediated dissociation of Sp1 from Sp1-I. Activated Spl then associates with a group of undefined retinoblastoma control proteins (RCPs). The resulting Sp1/RCP TF complexes bind in various combinations to unique consensus sequences [i.e., Sp1 sites and retinoblastoma control elements (RCEs) within certain genes [e.g., immediate-early growth response genes, thymidine kinase (TK), tissue-type plasminogen activator (t-PA), gadd45, and other X-ray-inducible transcripts (xips), etc.] to induce their transcription in confluence-arrested, G0/GI human cells. These genes then act to regulate cell cycle arrest checkpoints and stimulate further DNA repair processes that may ultimately be involved in radioresistance. Therefore, we hypothesize that induction of Sp1-responsive genes in arrested human cells are necessary for, and are coupled to, DNA repair. The efficacy of Sp1-mediated transcriptional repression or induction of certain genes after radiation is, therefore, regulated by (coupled to) DNA damage, Ku kinase (repair) and Sp1 activation, pRB, and RCPs. The goal of this grant is to elucidate the biochemical mechanisms and interactions between these factors and Sp1/RCE elements within specific genes in human cells that control their X-ray-inducibility.
The Specific Aims of this proposal are to: #1 - Determine the promoter elements required for transcriptional induction of two known X-ray-responsive genes, thymidine kinase (TK) and tissue-type plasminogen activator (t-PA), by promoter deletion and CAT expression studies in confluence-arrested human cells (Years 0-2). #2. Examine the composition, time course, and dose-responsive activation of TF complexes which bind to key X-ray-responsive TK or t-PA promoter regions (from Specific Aim #1) using DNA band shift, Shift-Western, and primer extension-DNase l footprinting analyses (Years 2-3). #3. Investigate the intracellular signaling mechanism(s) by which Ku kinase, Sp1, pRB, and retinoblastoma control proteins (RCPs) mediate increased TF binding to RCE and Sp1 sites within the TK and t-PA promoters to elicit transcription following ionizing radiation (Years 0-3). #4. Purify Sp1, RCPs, and other as yet undefined associated proteins using Spl consensus site-DNA affinity column chromatography and SDS-PAGE analyses from X-irradiated compared to control human Ul-Mel cells to characterize TF component additions/deletions and posttranslational alterations that influence transcription following ionizing radiation (Years 1-4). The overall goal of this grant is to elucidate a novel biochemical mechanism whereby human cells respond to DNA damage by coupling DNA repair to SP1-responsive transcription of genes which regulate radioresistance and cell cycle checkpoints allowing increased survival after a genetic insult. Elucidating these mechanisms will allow us to understand how human cells respond to damage creating by ionizing radiation.
| Yang, C R; Wilson-Van Patten, C; Planchon, S M et al. (2000) Coordinate modulation of Sp1, NF-kappa B, and p53 in confluent human malignant melanoma cells after ionizing radiation. FASEB J 14:379-90 |
| Limoli, C L; Hartmann, A; Shephard, L et al. (1998) Apoptosis, reproductive failure, and oxidative stress in Chinese hamster ovary cells with compromised genomic integrity. Cancer Res 58:3712-8 |
| Boothman, D A; Burrows, H L; Yang, C R et al. (1997) Damage-sensing mechanisms in human cells after ionizing radiation. Stem Cells 15 Suppl 2:27-42 |
