| Cutaneous malignant melanoma (CMM), unlike other cancers of the integumentary system, is a highly aggressive skin cancer that effectively resists current treatments. Intermittent and intense exposure to sunlight, particularly in children, is implicated in its etiology. Until recently, few animal models were available. We have derived a mouse model for melanoma in which a single neonatal dose of UV radiation induces cutaneous melanomas that remarkably parallel the genetics and molecular progression of human melanoma. Although solar energy or ultraviolet (UV) radiation is known to be the environmental carcinogen responsible for initiating CMM, specifics concerning UV-induced events within the cutaneous network remain relatively unknown. Therefore, the goal of this research study was to investigate the early responses that occur in skin of melanoma-prone neonatal HGF/SF transgenic mice following treatment with UV radiation. Using surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) as an experimental tool, our approach was to investigate the relative abundance of proteins in 24 hour post-treated skin from HGF/SF transgenic (Tg) and wild-type (Wt) mice that were either UV-irradiated or untreated at 3.5 days of age. The mass spectral results demonstrate that the relative abundance of a protein ion at mass to charge ratio m/z 4109 is upregulated in Tg, but not Wt mice in response to UV irradiation. Searches performed on the Swiss-Prot mouse protein database suggested that the UV-altered protein is murine beta defensin-1 or mBD1. To investigate this hypothesis, a number of experimental approaches were used to ascertain whether the peptide upregulated with UV in skin extracts of neonatal HGF/SF transgenic mice is mBD1 and whether this hypothesized candidate is detectable in HGF/SF (UV or no UV) skin extracts. These approaches included: (1) High Performance Liquid Chromatography (HPLC)-fractionation; (2) Hybrid quadrupole time-of-flight tandem mass spectrometry (QgTOF-MS); (3) Protein G bead-antibody capture; (4) Western blotting; and (5) high-resolution mass spectrometry (MS), consisting of nanoflow reversed phase liquid chromatography (RPLC) and Fourier Transform Mass Spectrometry (FT-MS). The results of these studies support the conclusion that mBD1 protein, under the sets of experimental conditions examined, is not detectable and as a whole, do not support the hypothesis that mBD1 is the analyte upregulated by UV irradiation in the skin of neonatal HGF/SF transgenic mice. These findings, although inconclusive with regard to identifying m/z 4109 and its role in UV-induced events in HGF/SF transgenic mouse skin, support future studies in this area of investigation. Identifying UV-altered proteins in skin will allow future research inquiries to focus on evaluating the roles that these proteins play in disease initiation and as potential signaling molecules. |
