A melanocyte malignant transformation model was developed in our laboratory, in which different melanoma cell lines were obtained after submitting the non-tumorigenic melanocyte lineage melan-a to sequential cycles of anchorage impediment.,Our group has already showed that increased superoxide level leads to global DNA hypermemethylation as well increased Dnmt1 expression few hours after melanocyte anchorage blockade.,Here, we showed that Ras/Rac1/ERK signaling pathway is activated in melanocytes submitted to anchorage impediment, regulating superoxide levels, global DNA methylation, and Dnmt1 expression.,Interestingly, Ras and Rac1 activation is not related to codon mutations, but instead regulated by superoxide.,Moreover, the malignant transformation was drastically compromised when melan-a melanocytes were submitted to sequential cycles of anchorage blockage in the presence of a superoxide scavenger.,This aberrant signaling pathway associated with a sustained stressful condition, which might be similar to conditions such as UV radiation and inflammation, seems to be an early step in malignant transformation and to contribute to an epigenetic reprogramming and the melanoma development.
Down-regulation of the immune system facilitates tumor progression at different stages of cutaneous melanoma.,Sentinel nodes, the first lymph nodes on lymphatics draining directly from a primary melanoma are immune down-regulated by tumor-generated immune-suppressive cytokines including interleukin-10.,To better understand the kinetics of sentinel node suppression, we investigated interleukin-10 expression by melanoma cells and tumor-associated macrophages and lymphocytes at different stages of primary melanoma evolution.,We used reverse transcriptase in situ polymerase chain reaction to identify cellular sources of interleukin-10 mRNA in 39 melanomas.,Interleukin-10 mRNA was identified in tumor cells of 2/6 melanomas in situ (33%), 17/21 invasive melanomas (81%) and 11/12 metastatic melanomas (92%).,Higher interleukin-10 expression correlates with tumor progression, with differences between melanoma in situ, invasive melanoma and metastatic melanoma.,In primary melanomas, the interleukin-10 mRNA content of tumor cells correlates with Clark level.,There was significantly more interleukin-10 mRNA in vertical growth phase melanoma cells than in radial growth phase cells.,In a logistic regression model, moderate to high interleukin-10 mRNA expression by tumor cells was significantly associated with vertical growth phase melanoma.,Interleukin-10 mRNA was detected in melanoma-associated macrophages and lymphocytes.,In invasive melanomas, the interleukin-10 mRNA reactivity of macrophages declined as Clark level increased.,Alterations of immunity by interleukin-10 derived from melanoma cells and melanoma-associated macrophages and lymphocytes potentially facilitate evolution of the primary melanoma and render regional lymph nodes susceptible to metastases.