Misregulation of RAD50 in Melanoma Cells.
Richard A Owings, Nathan L Avaritt, Josie M Munkberg, Stephanie Byrum, Bruce R Smoller, Wang L Cheung. UAMS, Little Rock, AR
Background: Ultraviolet radiation has been linked to increased risk of skin cancer including melanoma. One hypothesis is the accumulation of damaged DNA by way of UV radiation, leading to DNA double stranded breaks (DSB). When this reaches an irreparable sum the cell activates the pathway of cell death or apoptosis. Previously, we determined that DNA DSBs, as detected by histone H2AX phosphorylation, are increased in human melanoma tissue. We decided to determine if the downstream effectors of DNA DSB, namely RAD50 expression are altered in human melanoma cells.
Design: Eleven melanoma, 7 severely and 11 mildly dysplastic melanocytic nevi were retrieved from our archives. Age, sex, and location were not significantly different between the melanocytic lesions (p > 0.5). Routine immunohistochemistry was performed with commercially available antibodies against RAD50 (Cell Signaling). The intensity of staining was scored from nil to high (0-3). The percentage of cell staining was scored as 0 (none), 1 (<25% staining), 2 (<50% staining), and 3 (>50% staining). The intensity was multiplied by the percentage and the product was categorized as follows: 0-1 is 0; 2-3 is 1+; 4-5 is 2+; 6-9 is 3+. Chi square analysis was used to determine statistical significance with a p < 0.05 considered significant.
Results: RAD50 staining is significantly increased in melanoma (82%) cases compared to cases of mildly (27%) and severely (29%) dysplastic melanocytic nevi. Severely and mildly dysplastic melanocytic nevi had average staining intensities of 1.25 and 1, respectively. The majority of the melanoma cases demonstrate intense 3+ staining with an average staining intensity of 2.45. Both staining intensity and percentage of positive cells were statistically significant (p<0.001). Interestingly, RAD50 localized to cytoplasm in 7 of 9 (78%) positive melanoma cases, whereas all (100%) of the RAD50 staining localized to the nucleus in severely and mildly dysplastic melanocytic nevi (p < 0.01).
Conclusions: This is the first study that demonstrates activation and misregulation of the DNA repair pathway in human melanoma cells. Previous studies have shown increased histone H2AX phosphorylation in melanoma, suggesting an increase in DNA DSB's. In this study, the staining features of RAD50, a component of an essential DNA DSB repair protein, are clearly increased in melanoma cells. Interestingly, the majority of RAD50 staining in melanoma cells is cytoplasmic indicating aberrant localization of at least one DNA DSB repair protein. Future studies will determine the mechanism of aberrant RAD50 localization in human melanoma cells.
Monday, February 28, 2011 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 67, Monday Morning