Mitf-M is readily detectable in the nuclei of normal HeMa-LP cells and A2058 metastatic

melanoma cells, but is decreased or lost from the nuclei of MelJuso, M14, G361 and Malme-3 M cells. These data suggest that Mitf-M is necessary for the regulation of genes required for the maintenance and differentiation of melanocytes, as absence of Mitf-M in the nucleus is seen only in melanoma lines. The Mitf gene is amplified in some melanomas, and it has been suggested that Mitf can function as a melanoma oncogene [43]. Mitf is down-regulated in B-raf transformed murine melanocytes and B-raf overexpressing human melanocytes, and exogenous reexpression of Mitf inhibited the proliferation of these cells [44]. These data suggest a tumor-suppressive or differentiation-promoting role for Mitf in melanocytes. This role is consistent with the function GDC-0068 of Mitf in regulating cell cycle arrest via activation of p21/WAF1 and p16Ink4a [45] and [46]. Since the melanoma line A2058 shows abundant expression of Mitf-M and other Mitf isoforms in the nucleus, this suggests that Mitf can support both oncogenic and tumor suppressor functions. Cumulatively, these find more data suggest that Rad6 may be a more reliable marker than Mitf for melanoma development.

Double labeling analysis of Rad6 and Melan-A, and Rad6 and β-catenin in normal adjacent and transformed areas of the same SSMM specimens shed further light on the significance of Rad6 as Acyl CoA dehydrogenase a potential

early marker for neoplastic conversion to melanoma. When melanocyte homeostasis is tightly regulated by keratinocytes, a process occurring in normal skin, Rad6 is undetectable. However, when homeostasis regulation is lost, as evidenced by increases in the number of Melan-A positive cells, Rad6 expression becomes noticeable. However, it is interesting to note that Rad6 expression is not initially localized in melanocytes, but rather expressed in the neighboring keratinocytes, prompting us to speculate that up-regulation of Rad6 in neighboring cells likely plays a role in the deregulation of melanocyte homeostasis and contributes to the risk of melanoma development. This supposition is supported not only by concurrent increases in the number of Melan-A positive cells, but also by increases in Melan-A/Rad6 double positive cells in tumor regions. Since the first detectable increase in Rad6 expression occurs in the neighboring keratinocytes that strongly express β-catenin prompts us to speculate that Rad6 gene expression may be induced by β-catenin, it’s transcriptional activator [25].