Hopkins researchers have found that 5-hydroxymethylcytosine (5hmC) content closely correlates with the differentiation state of cells. Terminally differentiated cells contain the highest levels of 5hmC, while less differentiated stem/progenitor cell compartments exhibit very low levels. In addition, 5hmC levels are significantly reduced in prostate, breast and colon carcinomas as compared to normal tissues, a finding which sheds some light on the characteristics that enable cancer cells to survive.
DNA methylation at the 5-position of cytosine, one of the four main bases found in DNA and RNA, is an epigenetic modification that plays a role in cellular differentiation. Such epigenetic modifications have been implicated in several diseases, including cancer.
According to recent studies, 5-methylcytosine can be enzymatically converted to 5hmC using proteins from the ten-eleven translocated family in mammalian genomes. While 5hmC modifications have been known to exist in embryonic stem cell DNA and in the brain, the precise distribution of 5hmC and the role of this DNA modification in epigenetic regulation has not been extensively studied until recently.
This study, published this month online in Oncotarget, explored the different levels of 5hmC in a large set of human and mouse tissues. Researchers obtained embryonic mouse tissue from 17-day-old C57BL embryos, while adult mouse tissue came from 11-week-old FVB mice – a strain of inbred homozygous mice. They applied several methods to test 5hmC levels in these cells.
"We optimized a protocol to robustly and specifically detect 5hmC with a commercially available antibody using immunohistochemistry and immunofluorescence methods," Srinivasan Yegnasubramanian, assistant professor at the Hopkins School of Medicine and one of the study's authors, said. "These methods allow visualizing 5hmC in situ and can help us to understand the cell-to-cell variability in tissues. With these methods, we noticed that more differentiated cells contained much higher levels of 5hmC than less differentiated cells in tissue stem/progenitor cell layers."
The group first determined 5hmC patterns in mouse embryonic cells by staining with 5hmC-specific antibodies. Using the techniques described by Yegnasubramanian, they found significant levels of 5hmC in the mouse cerebral cortex and cerebellum. They also managed to detect 5hmC across the majority of tissues in the mouse embryo.
The interesting aspect of these results was that the 5hmC content was strongly associated with the state of cellular differentiation in hierarchically organized cells. Micrographs showed that 5hmC staining in basal cell compartments of 17-day-old mouse embryo tissues was lower than that in more differentiated luminal/apical epithelial cells. In human colon tissue, apical epithelial cells exhibited strong 5hmC staining, while basal epithelial cells showed much lower staining intensities. "The results suggest that 5hmC accumulation may be generally important in the differentiation of stem and progenitor cells to more specialized cells," Yegnasubramanian said.
A particularly intriguing finding was the reduction of 5hmC in human cancers. Tumors are known to lose certain features of differentiation while gaining functions similar to those of less differentiated cells, such as self-renewal. The researchers set about to determine whether these tumor cells have 5hmC levels closer to terminally differentiated cells or to tissue stem cell compartments from the original tissue. They tested a total of 78 carcinoma and 28 normal tissue samples from breast, prostate and colon.
In the normal tissues, terminally differentiated luminal cells showed much greater 5hmC staining than basal cells, which were likely to contain the tissue stem/progenitor cells. These results were similar to those found in mouse tissues. However, a great reduction of 5hmC levels was observed in cancers arising from the same tissues for all three cancer types. Even small lesions showed a reduction in 5hmC, suggesting that the global loss of 5hmC could occur early on in carcinogenesis.
The study provides evidence for cancer-associated loss of 5hmC and suggests that this loss can aid the survival and renewal of cancer cells. However, Yegnasubramanian believes further research into this area is required.
"Cancer cells appear to adopt 5hmC levels that are closer to the stem/progenitor cell compartments than to the more differentiated cells," he said. "It is possible that the reduced 5hmC in cancer cells allows these cancer cells to acquire properties such as self-renewal that are characteristic of normal tissue stem cells. These hypotheses would need to be tested more mechanistically in future work."
