In
cancer, the extensive methylation found in the bulk of
chromatin is reduced, while the normally unmethylated CpG islands become hypermethylated. Regions of solid
tumors are transiently and/or chronically exposed to
ischemia (
hypoxia) and reperfusion, conditions known to contribute to
cancer progression. We hypothesized that hypoxic microenvironment may influence local epigenetic alterations, leading to inappropriate silencing and re-awakening of genes involved in
cancer. We cultured human colorectal and
melanoma cancer cell lines under severe hypoxic conditions, and examined their levels of global methylation using HPLC to quantify
5-methylcytosine (5-mC), and found that
hypoxia induced losses of global methylation. This was more extensive in normal human fibroblasts than
cancer cell lines. Cell lines from metastatic
colorectal carcinoma or
malignant melanoma were found to be markedly more hypomethylated than cell lines from their respective primary lesions, but they did not show further reduction of 5-mC levels under hypoxic conditions. To explore these epigenetic changes in vivo, we established xenografts of the same
cancer cells in immune deficient mice. We used Hypoxyprobe to assess the magnitude of tissue
hypoxia, and immunostaining for 5-mC to evaluate DNA methylation status in cells from different regions of
tumors. We found an inverse relationship between the presence of extensive tumor hypoxia and the incidence of methylation, and a reduction of 5-mC in xenografts compared to the levels seen in the same
cancer cell lines in vitro, verifying that methylation patterns are also modulated by
hypoxia in vivo. This suggests that epigenetic events in solid
tumors may be modulated by microenvironmental conditions such as
hypoxia.