Melanoma Transition Is Frequently Accompanied by a Loss of Cytoglobin Expression in Melanocytes: A Novel Expression Site of Cytoglobin

<div><p>The tissue distribution and function of hemoglobin or myoglobin are well known; however, a newly found cytoglobin (CYGB), which also belongs to the globin family, remains to be characterized. To assess its expression in human malignancies, we sought to screen a number of cell lines originated from many tissues using northern blotting and real time PCR techniques. Unexpectedly, we found that several, but not all, melanoma cell lines expressed CYGB mRNA and protein at much higher levels than cells of other origins. Melanocytes, the primary origin of melanoma, also expressed CYGB at a high level. To verify these observations, immunostaining and immunoblotting using anti-CYGB antibody were also performed. Bisulfite-modified genomic sequencing revealed that several melanoma cell lines that abrogated CYGB expression were found to be epigenetically regulated by hypermethylation in the promoter region of <i>CYGB</i> gene. The RNA interference-mediated knockdown of the CYGB transcript in CYGB expression-positive melanoma cell lines resulted in increased proliferation <i>in vitro</i> and <i>in vivo</i>. Flow cytometric analysis using 2′-, 7′-dichlorofluorescein diacetate (DCFH-DA), an indicator of reactive oxygen species (ROS), revealed that the cellular ROS level may be involved in the proliferative effect of CYGB. Thus, CYGB appears to play a tumor suppressive role as a ROS regulator, and its epigenetic silencing, as observed in CYGB expression-negative melanoma cell lines, might function as an alternative pathway in the melanocyte-to-melanoma transition.</p></div

Sequencing histograms for the CpG island of the CYGB promoter region.

<p>Of the 24 CpG sites known to be methylated in the CYGB promoter region, 9 sites analyzed for methylation are shown. Cytosines methylated in A375 <b>(B)</b> are underlined. The corresponding cytosines are entirely unmethylated in melanocytes <b>(A)</b> and G361 <b>(C)</b>, resulting in the sequence “TpG” after bisulfite treatment.</p