ROLE OF CRYPTOCHROME IN THE CIRCADIAN CLOCK AND DNA DAMAGE RESPONSES. Cryptochrome (CRY) is a photosensory flavoprotein and a core component of the molecular clock. We wish to determine the mechanism of action of CRY and of the animal circadian clock using mice and Drosophila as model systems. In addition, we will investigate how the interfacing of the mammalian circadian clock with DNA damage responses may be targeted for cancer prevention and cancer treatment.
Aim I : Reaction Mechanism of CRY and the Biochemistry of the Animal Circadian Clock A) Mammalian Circadian Clock. We will do the following to define the reaction mechanism of the mammalian circadian clock. (1) Biochemical assays with clock proteins purified from eukaryotic expression vectors and from mouse liver nuclei. (2) ChIP and immunofluorescence assays with wild-type and mutated cell lines and ChIP with liver nuclei of mice of different circadian genotypes to analyze clock protein-chromatin dynamics. (3) Transcription assays to correlate clock protein-chromatin interaction dynamics with the core clock mechanism as well as the regulation of clock-controlled genes. B) Drosophila Circadian Clock. We will use (1) photochemical/photophysical, (2) biochemical, and (3) cell biological methods to define the circadian photosensory system in Drosophila as well as the molecular mechanism of the core circadian circuitry.
Aim II : Circadian Control of Carcinogenesis and Chemotherapy A) Clock Control of Excision Repair and Cancer Prevention and Treatment. Excision repair activity exhibits high amplitude circadian oscillation in mice, and the rhythmicity of repair in mouse skin correlates with time-of-the-day of UV light-induced carcinogenicity. We will determine whether this repair rhythmicity exists in humans, and accordingly, develop strategies for reducing skin cancer risk by behavioral modification. In addition, using repair rhythmicity as a guide, we will conduct mechanism-based chronotherapy experiments with human colorectal cancer xenografts to improve the efficacy of oxaliplatin treatment of colorectal cancers. B) Clock Disruption and Carcinogenesis/Cancer Treatment. We have discovered that clock disruption by Cry- mutation reduces cancer risk in p53 mutant mice by enhancing both intrinsic and extrinsic apoptosis. Upregulation of intrinsic apoptosis is caused by circadian clock- and DNA damage-dependent upregulation of the p73 gene. We will pursue the therapeutic aspects of this finding in humans. Human p53-null colorectal cancer xenografts will be used to determine the efficacy of oxaliplatin in preferentially inducing apoptosis in cancer cells.
We propose to define the role of cryptochrome in the animal circadian clock and determine the molecular mechanism of the animal circadian clock. Based on our finding that the circadian clock controls DNA damage responses we will develop strategies for cancer prevention and cancer chemotherapy.
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