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Chair(s): Begg, Adrian; Li, Chuan-Yuan
(000) DNA-dependent protein kinase is a molecular target for the development of non-cytotoxic radiation sensitizing drugs.
Gene, L.1, Shinohara, E.1, Hallahan, Dennis*,1, 1 Vanderbilt Univ., Nashville, TN, United States
ABSTRACT- Purpose/Objective: IC87361 is a morpholine derivative that is 50-fold more selective for DNA-PK than for PI3K and other kinases. DNA-PK defective SCID cells were studied to determine whether radiosensitization by the specific inhibitor of DNA-PK, IC87361, is eliminated in the absence of functional DNA-PK. Materials/Methods: Clonogenic and apoptosis assays were performed in cancer, SCID and normal cells treated with IC87361. Tumors were implanted into SCID, nude and C57BL6 mice. The mean volume of the tumors in mice at the start of treatment (day 0) was approximately 100 mm3 in all groups. DNA-PK inhibitor was also studied in C57BL6 mice separated into control, irradiated, IC87361 alone and IC87361 given prior to irradiation. Mice treated with intraperitoneal injection of 75 ug IC87361. Radiation was administered as 3 Gy fractions per day given one hour after drug for 7 days. Tumor volumes were measured 3 days per week using skin calipers. Results: To determine whether the radiosensitizing effect of IC87361 is specific to DNA-PK in the mouse model, we studied the response of SCID blood vessels by use of the tumor vascular window. LLC cells were injected into the vascular window skin fold. This induced the formation of blood vessels which were treated with IC87361, 3 Gy alone or IC87361 + 3 Gy. C57BL6 mice treated with IC87361 + 3 Gy showed significant enhancement of radiation-induced tumor vascular destruction in wild type host with vascularity of 5% at 72 hours (P05). IC87361 or 3 Gy alone produced minimal reduction in tumor vascularity. The radiosensitizing effect of IC87361 was not observed in tumor vasculature in SCID mice which lack DNA-PK enzyme activity. To determine the specificity of DNA-PK inhibitors, clonogenic survival was studied in irradiated SCID and wild type endothelial cells following treatment with IC87361. DNA-PK defective SCID endothelial cells treated with IC87361 and radiation showed no reduction in clonogenic survival as compared to SCID cells treated with radiation alone. Wild type endothelial cells treated with IC87361 showed a significant increase in radiosensitivity (P=0.01) that was similar to the results in SCID mice. We next studied tumor growth delay following fractionated radiotherapy. Tumors were implanted into C57BL6 and SCID mice. Tumors were treated with daily fractionated radiotherapy using 3 Gy doses. B16F0 tumors in C57BL6 mice showed growth of 381 mm3/day. Radiation induced a significant increase in tumor growth delay in SCID mice, which grew 10 times slower at 38 mm3 /day (P01) as compared to either nude or C57BL6. There was also a four-fold greater radiation-induced growth delay in SCID mice implanted with B16F0 as compared to tumors in C57BL6. There was a 2-fold increase in growth delay resulting in tumors that were 5 times the original volume in SCID (requiring 7 days) as compared to C57BL6 mice (14 days) or nude mice (14 days). To determine whether inhibition of DNA-PK with IC87361 reproduces the radiosensitivity of tumors in SCID mice, C57BL6 mice were implanted with B16F0 and LLC tumors and treated with either 3 Gy fractions to 21 Gy, IC87361 alone, or IC87361 + each 3 Gy fraction. Tumor growth curves showed a reduction in growth of B16F0 treated with 3 Gy. Untreated tumor growth rate was 613 mm3 compared to tumors treated with radiation alone which showed tumor growth delay of 327 mm3 (P5). IC87361 alone had a growth of 687 mm3 as compared to 3 Gy + IC87361 growth of 193 mm3 (P01). Conclusions: DNA-PK inhibitors induced no cytotoxicity and no toxicity in mouse normal tissues. Mouse models deficient in enzyme activity are useful to assess the specificity of novel kinase inhibitors. DNA-PK is an important target for the development of novel radiation-sensitizing drugs that have little intrinsic cytotoxicity.
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