Skin encounters major challenges in maintaining genomic fidelity as its cells divide in the presence of environmental DNA damaging agents such as UVB and chemicals. When DNA is damaged or DNA synthesis is otherwise delayed, a cell requires additional time to ensure complete DNA replication. The replication checkpoint is the mechanism by which a cell ensures that DNA replication is complete prior to initiating chromatin condensation, an initial stage of mitosis. We have shown that the protein kinase ATR and its downstream target, Chk-1, are required for the replication checkpoint and for survival after DNA damage. Critical to this proposal is that pre-malignant and cancer cells are deficient in p53 and other early cell cycle checkpoints and are thus highly sensitive to inhibition of ATR, leading to cell death. Complete loss of function of the replication checkpoint by genetic deletion of either ATR or Chk-1 is rapidly lethal to a developing embryo or to dividing cells in culture. In contrast, partial inhibition of the replication checkpoint by small molecule inhibitors or by expression of dominant negative ATR or Chk-1 is tolerated for many cell divisions in unstressed cells. The precise consequences of partially inhibiting the replication checkpoint are not known for normal or DNA damaged cells in vivo. The broad, long-term objectives of this proposal are: i) To further elucidate the molecular mechanism of the replication checkpoint in maintaining genomic fidelity, ii) To define the in vivo role of the replication checkpoint using skin as a model organ system, iii) To determine whether inhibiting the replication checkpoint can be used to eliminate DNA-damaged cells at risk of malignant progression. We will achieve these objectives with a variety of approaches including in vitro studies of chromosomal fragile sites, transgenic mice with diminished replication checkpoint function in skin, and topical application of small molecule inhibitors of the replication checkpoint to UVB-damaged mouse skin. This proposal will define the molecular mechanisms of the replication checkpoint in maintaining genomic integrity in a model organ system (mouse skin) and investigate its inhibition as a selective means of eliminating DNA-damaged pre-malignant cells in vivo.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR049832-05
Application #
7117633
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Baker, Carl
Project Start
2003-09-01
Project End
2008-08-31
Budget Start
2006-09-01
Budget End
2007-08-31
Support Year
5
Fiscal Year
2006
Total Cost
$326,427
Indirect Cost
Name
University of Washington
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
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Conney, Allan H; Lu, Yao-Ping; Lou, You-Rong et al. (2013) Mechanisms of Caffeine-Induced Inhibition of UVB Carcinogenesis. Front Oncol 3:144
Conney, Allan H; Lou, You-Rong; Nghiem, Paul et al. (2013) Inhibition of UVB-induced nonmelanoma skin cancer: a path from tea to caffeine to exercise to decreased tissue fat. Top Curr Chem 329:61-72
Lu, Yao-Ping; Lou, You-Rong; Peng, Qing-Yun et al. (2011) Caffeine decreases phospho-Chk1 (Ser317) and increases mitotic cells with cyclin B1 and caspase 3 in tumors from UVB-treated mice. Cancer Prev Res (Phila) 4:1118-25
Kawasumi, Masaoki; Lemos, Bianca; Bradner, James E et al. (2011) Protection from UV-induced skin carcinogenesis by genetic inhibition of the ataxia telangiectasia and Rad3-related (ATR) kinase. Proc Natl Acad Sci U S A 108:13716-21
Huryn, Donna M; Brodsky, Jeffrey L; Brummond, Kay M et al. (2011) Chemical methodology as a source of small-molecule checkpoint inhibitors and heat shock protein 70 (Hsp70) modulators. Proc Natl Acad Sci U S A 108:6757-62
Heffernan, Timothy P; Kawasumi, Masaoki; Blasina, Alessandra et al. (2009) ATR-Chk1 pathway inhibition promotes apoptosis after UV treatment in primary human keratinocytes: potential basis for the UV protective effects of caffeine. J Invest Dermatol 129:1805-15
Lu, Yao-Ping; Lou, You-Rong; Peng, Qing-Yun et al. (2008) Effect of caffeine on the ATR/Chk1 pathway in the epidermis of UVB-irradiated mice. Cancer Res 68:2523-9
Kawasumi, Masaoki; Nghiem, Paul (2007) Chemical genetics: elucidating biological systems with small-molecule compounds. J Invest Dermatol 127:1577-84

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