damage was clearly greater in FRA16D AT island MAR compared to the loop DNA of the �-globin segment. IR dose of 10Gy produced, respectively, 1.27 � 0.02 vs. 0.54 � 0.02 lesions/kbp. For comparison, cells were exposed to bizelesin, anAT-specific alkylating drug which preferentially targets AT islands. Bizelesin-induced markedly more lesions in FRA16D ATisland compared to the �-globin region (3.6 � 0.6 vs. 0.7 � 0.01 lesions/kbp/�M drug, respectively). The differential effectof this highly AT-specific drug was thus more pronounced than the difference observed for IR. Still, the preference of IR forFRA16D MAR is remarkable, given that IR is known to have no specificity at the DNA sequence level.

To extend the findings with the specific MAR domain in FRA16D, we used the same irradiation/post-incubation scheme toexamine damage to all cellular MARs versus all loop DNA. FIGE revealed marginal differences in size distribution of loopDNA fragments from control and irradiated cells, indicating that most of DSB in loop DNA were repaired during the 24hpost-incubation. In contrast, matrix-associated DNA fraction showed clearly dose-dependent reduction in fragment populationsizes, indicative of progressive persistent DSB.

Conclusions: Collectively, the results demonstrate that IR-induced persistent lesions are more frequent in cellular MARs thanin loop DNA, which may reflect either enhanced formation or impeded removal of IR damage in MAR domains. Persistentdamage in MAR domains of the critical sites of genomic instability, such as FRA16D, is likely to be highly relevant to thelethality of IR in cancer cells.

2003 Radiation Sensitization of Prostate Carcinoma Cells by Onc 01910, A Novel Protein Kinase Inhibitor andCell Cycle Modulator

A. K. Sharma,1 S. Mohan,1 A. Alfieri,1 M. Garg,1 B. Xi,1 S. Cosenza,2 M. Reddy,2 S. Bell,3 E. Reddy,2C. Guha1

1Radiation Oncology, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, NY, 2Fels Cancer Research,

Temple University, Philadelphia, PA, 3Onconova Therapeutics, Princeton, NJ

Purpose/Objective: The ONC 01910 is a benzyl styryl sulfone that inhibits CDK1 kinase activity and targets the G2/M cellcycle check-point in tumor cells. ONC 01910 demonstrates anti-cancer activity against a broad spectrum of cancer cell lines.In this study, we examined the effects of combination treatments of ONC 01910 and irradiation in prostate carcinoma cell lines,normal human fibroblasts and prostate epithelial cells.

Materials/Methods: Prostate carcinoma (PC) cell lines, murine RM-1 (p53 ) and human PC-3 (androgen-independent,p53-mutant), normal human fibroblast (HF-1) and human primary prostate epithelial cells (PE) were evaluated as exponentiallygrowing cells. Cells were incubated with 1910 (25nM-2.0 uM) for 5–18 hrs, followed by irradiation (0-6Gy). Alternatively,cells were chronically incubated with very low doses of ON 01910 (40–100 nM) and irradiated 24hr after exposure. The effectof drug plus irradiation was examined for cell survival (WST-1 assay), clonogenic survival, flow cytometric analysis bypropidium iodide and annexin-stained cells (cell cycle and apoptosis), and immunoblot analyses (cyclin B1, CDK1, cdc25c,bcl-2, PARP and active Caspase-3). Differences between treatment cohorts were determined using Student’s t test.

Results: Pulse incubation with ON 01910 (5–18 hours) was cytotoxic (IC50 50–200nM) with drug induced G2/M arrest andapoptosis in both RM-1 and PC-3 cells, which was further enhanced by irradiation. Seventy-two hours after drug exposure, theapoptotic index in PC-3 cells was 54 � 6%, 77 � 7% and �10% in cells treated with ON01910, ON01910�6Gy and 6 Gy,respectively. In contrast, normal fibroblasts and prostate epithelial cells were arrested in both G1 and G2 phases of the cell cycleand exhibited only minimal apoptosis (� 5%). Immunoblot analyses demonstrated a reduction in cyclin B1 and bcl-2 expressionfor both RM-1 and PC-3 cells, following ON01910 treatment. Furthermore, there was activation of Caspase 3, degradation ofPARP and DNA laddering in ON01910-treated PC-3 cells. Chronic exposures (40–50 nM) resulted in decreased clonogenicityand apoptotic values that were significantly greater than untreated or RT alone controls. Survival fraction at 2 Gy (SF2) was0.79 in controls and decreased to 0.32 and 0.23 with 40 and 50nM of ON 01910, respectively. The dose modification factorsat isodose (SF � 0.1) were 1.6 and 2.4, respectively for the two doses.

Conclusions: Pulse and chronic exposures to ON 01910 demonstrated significant cytotoxicity in human and murine PC celllines, at doses that exhibited minimal cytotoxicity for normal cells. This selective cytotoxicity, combined with enhancedradiosensitization at low doses of ON01910 (40–50 nM), makes it a very attractive drug for chemoradiation therapy of PC.

2004 Mechanisms of Radiation-Induced Hippocampal Neuron Cytotoxicity and Implications on Memory andLearning Disorders

E. Edwards,1 W. Whetsell,1 E. Shinohara,1 J. Tan,1 K. Osusky,1 D. Hallahan1

1Vanderbilt University, Nashville, TN

Purpose/Objective: To determine the mechanisms of memory and learning disorders in the irradiated brain. Children whoreceive cranial radiation therapy often have devastating long-term side effects, including mental retardation. Particularlysusceptible are neural precursor cells within the subgranular layer of the dentate gyrus of the hippocampus, which plays a crucialrole in learning and memory. Few strategies have been implemented to protect the normal brain against radiation. Studiessuggest that lithium chloride, an inhibitor of the pro-apoptotic signaling protein glycogen synthase kinase 3 � (GSK-3�), mayact as a neuroprotective agent against various cytotoxic insults. We therefore hypothesized that lithium could protect thehippocampus against radiation.

Materials/Methods: In vitro clonogenic survival analysis was performed using HT-22, a mouse hippocampal neuronal cell line.Survival analysis was also performed on Daoy medulloblastoma cells. Cells were treated with various doses of radiation withor without prior treatment with 3 mM lithium chloride for one week. After 10 days, the cells were fixed and stained, and the

S341Proceedings of the 46th Annual ASTRO Meeting