Consistent with TOP/Flash reporter activity data, β-catenin was detected in the nuclei of all melanoma lines with strongest nuclear β-catenin immunoreactivity in M14 and A2058 melanoma cells. Intense Rad6 staining was detected in the cytoplasm that colocalized with β-catenin in the melanoma lines ( Figure 2D). To analyze the potential role of Rad6 in melanoma development, we evaluated expression of Rad6 and the melanocyte differentiation antigen Melan-A in a melanoma tissue microarray by dual immunofluorescence staining. The numbers of Rad6 positive and Melan-A positive cells were scored,

and Poisson regression analysis was applied to compare the percentage of cells costaining for Rad6 and Melan-A in nevi vs. primary cutaneous melanomas. The percent of IWR-1 order Rad6 and Melan-A dual 3-Methyladenine ic50 positive cells ranged from 0% to 43.5% in the nevi group, and from 51.4% to 98.2% in

the melanoma group. Limiting Rad6 expression analysis to Melan-A positive cells could lead to underestimation of the number of Rad6 positive cells in the tissue specimens as Melan-A is not uniformly expressed in all nevi and melanomas. However, comparison of Melan- A expression in nevi and melanoma samples have been shown to have similar sensitivity and specificity values (75% to 92%, nevi vs. 95% to 100%, melanoma) [40]. Our data demonstrated that although the number of cells positive for Melan-A was not significantly different between the nevi and primary cutaneous melanoma groups (P = .5696), histological diagnosis of melanoma was significantly associated with the occurrence of Rad6/Melan-A dual positivity (P = .0029) with the odd ratio of 1.98 (95% confidence interval 1.6-2.46) compared to the nevi group. Also, compared to the nevi where only a few cell populations, if any, showed Rad6 staining ( Figure 3A), Rad6 was abundantly expressed in malignant melanomas ( Figure 3B). Similar analysis of Rad6

and β-catenin in nevi and malignant melanomas by dual immunofluorescence staining showed Rad6 and β-catenin costaining predominantly in melanoma specimens. In malignant melanomas, β-catenin staining was intense and widespread and colocalized with Rad6 ( Figure 4). β-catenin was localized ioxilan on the cell membrane and cytoplasm of nevi and melanomas, but was not found to localize in the nucleus. In rare cases of nevi that showed Rad6 expression, Rad6 was present in the cytoplasm, whereas in Rad6-positive melanomas, Rad6 was localized both in the cytoplasm and nucleus ( Figure 4). These data suggest that up-regulation of Rad6 may play a role in the conversion of nevus to cutaneous melanoma. The positive relationship between Rad6 expression and melanoma development was further verified in superficial spreading malignant melanoma (SSMM) biopsies, the most common form of cutaneous melanoma accounting for ~ 70% of all diagnosed melanomas [41].