“This paper adds substantial fuel to the fire of concern about using cultured cells to study phenotypes associated with cancer in vivo (such as drug resistance),” Michael Gottesman, chief of the Lab of Cell Biology at the National Cancer Institute’s Center for Cancer Research, told The Scientist in an e-mail. “Obviously, these studies are done using mouse embryo fibroblasts and not human cancer cells, but the changes in 5hmC levels are so dramatic and so rapid that they cannot be ignored.”
5hmC is the result of Tet enzymes adding a hydroxyl group to 5-methylcytosine, or 5mC, which is a cytosine with a methyl group attached.
Following up on their observation that 5hmC levels were low to undetectable in various human cancer cell lines, Richard Meehan at the Medical Research Council’s Human Genetics Unit in Edinburgh and his colleagues decided to figure out what might contribute to this phenomenon. “Obviously, these have been in culture for a long time,” said Meehan. “What happens when you try to establish cells?”
So the team cultured mouse embryonic fibroblasts and measured the levels of 5hmC over time. “To our surprise, they disappeared almost overnight,” Meehan told The Scientist. “We thought it was going to take a bit longer than that.” The researchers tried again with murine CD4+ T cells and found the same thing—the marks vanished. 5mC levels, however, appeared to remain intact.
“What we would like to know is, does the same phenomenon occur in human cancer cells taken out of tumors and put in culture, and whether there is anything that can obviate this effect,” said Gottesman.
The loss of 5hmC may in part have to do with a loss of Tet protein. Meehan’s group found that Tet gene expression declined as cells spent more time in culture. Yun Nancy Huang, an epigenetics researcher at Texas A&M Health Science Center who was not involved in the study, said that Tet enzymes are sensitive to environmental changes; any differences between in vivo and in vitro conditions—such as nutrient availability—might also impact their activity.
In fact, the researchers found that adding Tet cofactor vitamin C to the culture rescued some of the loss of 5hmC. “This result suggests that the observed loss of 5hmC results from both a reduction of Tet1 levels and general loss of Tet activity due to limiting cofactors,” the authors wrote in their report.
Meehan also proposed that the loss of 5hmC may have to do with the disruption of a de-methylation cycle in the cell, in which 5mC marks are normally converted into 5hmC marks that are then removed to return the cytosine to an unmethylated state. In a disruptive environment, 5mC would remain, according to a hypothesis supported by his findings.
Meehan added that his team’s results indicate a more dynamic epigenetic profile than is traditionally appreciated. “We need ways of following this process,” he said. “We need real-time trackers.”
The study points to how researchers might further improve culture conditions, Meehan said. “One possibility is, if the pattern we see in the tissue is the real pattern, essentially we should be adjusting our culture conditions so cells in the dish have this pattern.”
C.E. Nestor et al., “Rapid reprogramming of epigenetic and transcriptional profiles in mammalian culture systems,” Genome Biology, doi:10.1186/s13059-014-0576-y, 2015.