Projects and Grants per year
Grants and Contracts Details
Description
We seek to understand the molecular responses that occur when skin is exposed to UV radiation in order to
devise novel strategies to protect against damage and carcinogenesis. In this proposal we will examine two
major risk factors for skin cancer: IN radiation and pigment (melanin) phenotype. Our studies focus on the
melanocyte, and its cell surface receptor, the melanocortin-1 receptor (Mclr), which regulates melanin
synthesis. We hypothesize that Mclr protects melanocytes against UV-mediated mutagenesis and
transformation by modulating melanization and recovery from UV-mediated cellular injury. Loss-of-function
polymorphisms in Mclr correlate with fair skin and a high incidence of melanoma, while robust Mclr function
correlates with darker skin and UV resistance. We have developed a novel genetically-matched murine model
of human skin as well as a protocol for growing primary melanocytes from these mice. These isogenic
melanocytes span eumelanotic, pheomelanotic, and amelanotic melanin composition, will serve a unique and
potent tool to delineate the role of Mci r function in response to UV. Using our unique animal mode! and
primary melanocytes derived thereof, we propose the following aims: Aim 1. Characterize the mechanism of
McI r-mediated enhancement of nucleotide excision repair (NER); Aim 2. Determine whether Mclr signaling
protects against UV-mediated oxidative damage; and Aim 3. Determine the ability of pharmacologic Mci r
rescue to protect against UV damage. With respect to Aim `1, we will examine the role of Mclr in the repair of
UV-induced photolesions by southwestern and flow cytometric analysis and determine the influence of Mci r on
cellular levels of nucleotide excision repair enzymes by qRT-PCR and Western analysis. We will investigate a
molecular link between Mci r signaling and enhanced repair enzyme levels by examining expression of the
NER enzymes basally and in the setting of UV irradiation and by investigating the ability of cAMP-responsive
transcription factors downstream of Mci r signaling (namely Miff and CREB) to bind to and induce transcription
of NER enzyme promoters. In Aim 2 we will study whether pheomelanin, which is produced as a result of low
Mclr function, promotes oxidative damage in melanocytes by complementary measures of oxidative load
(DCF-mediated fluorescence flow cytometry, Southwestern blotting, hOGO 1-adapted Comet assay, TBARS
assay, and direct quantification of oxidative DNA adducts by GC/MS). Importantly, we will directly test whether
pheomelanin functions as a pro-carcinogen by an HPRT-based forward mutagenesis approach in primary
melanocytes. Finally, we will directly test the ability of forskolin, an activator of adenylyl cyclase, to bypass
defective Mclr function to enhance recovery from UV-mediated DNA damage and to rescue UV protection in
melanocytes and in whole skin. Together, these studies will lay the foundation for future translational studies
designed to develop novel small molecule-based approaches for UV and cancer protection to prevent many
cases of melanoma.
Status | Finished |
---|---|
Effective start/end date | 7/1/10 → 4/14/16 |
Funding
- National Cancer Institute: $1,625,377.00
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Projects
- 1 Finished
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Diversity Supplement for Amaro-Ortiz: The role of Mc1r in melanocytic UV-induced DNA damage and repair responses
D'Orazio, J. (PI)
7/1/10 → 4/30/15
Project: Research project