Down but Not Out
Normal cells do not grow and divide forever. Even before they get old and die, many cells in the body are quiescent: temporarily out of the proliferative cell cycle, waiting for a signal to wake up and become active again. Cells grown in culture will also enter such a state, either because they’re too crowded or have run out of nutrients. Princeton University’s Hilary Coller recently found that some of these cells are surprisingly metabolically active, even while not proliferating.
Previous work on lymphocytes, Coller says, suggested that quiescence is associated with a “sleepy metabolic state” during which cells take up less glucose and excrete fewer waste products, such as lactate. Whereas a proliferating cell has to replicate all of its contents in order to divide, quiescent cells don’t make new proteins, lipids or organelles, and aren’t replicating DNA.
Coller says that quiescence must be “fundamentally important,” because many cells normally spend long periods in this state, awaiting activation. Immunological memory depends on resting lymphocytes being ready to proliferate when exposed to a familiar antigen, fibroblasts slumbering in the skin need to activate to heal wounds, liver regeneration depends on activation of quiescent hepatocytes, and germ cells can spend many years waiting for their moment. Even some tumor cells can remain quiescent, and thus resistant to most chemotherapy, for a long time. Despite this, Coller says we have a “poor understanding” of what quiescence means, or how it works. She’s also interested in the medical implications of understanding quiescence, although she admits she could be biased: “If you have a hammer everything looks like a nail. I study quiescence, so lots of diseases look like quiescence pathologies to me!” Nonetheless, her hunch that there might be more to quiescence than meets the eye paid off when her group discovered unexpected metabolic activity in quiescent fibroblasts. Keck School of Medicine researcher Lucio Comai, who evaluated Coller’s paper (PLoS Biol, 8:e1000514, 2010) for Faculty of 1000, says a lot of people would have thought, “Why bother doing these experiments?” but he added that Coller’s finding “changes a paradigm.”
Coller and her colleagues coaxed cultured fibroblasts into quiescence by growing them until they were tightly packed, and then, instead of transferring them to new culture flasks as a cell biologist would normally do, kept them in these crowded conditions. This induced contact inhibition, and the cells stopped growing. In other experiments she deprived cells of nutrients to encourage quiescence.
From microarray analysis, she found that the quiescent cells increased expression of some genes involved in metabolism. This was a surprise, as she expected that metabolic pathways would shut down, as they do in lymphocytes. Coller contacted metabolism expert Joshua Rabinowitz, who came to Princeton at around the same time as she did. He helped her identify and quantify different metabolites, and determined metabolic flux through these cells by measuring glucose consumption and amounts of glycolytic intermediates using chromatography and mass spectroscopy.
Although nucleotide biosynthesis is reduced in quiescent fibroblasts (because they do not have to make DNA), Coller was surprised to find that the Krebs cycle seemed to be operating at an increased rate. The pathway that provides ribose for nucleotide synthesis, the pentose phosphate pathway (PPP), was also running quickly.
Contact-inhibited fibroblasts also produce large amounts of extracellular matrix proteins, such as lamin and collagen. Coller suggests that quiescent cells might turn into protein factories that generate material for the rest of the body, as they do not need to create more copies of themselves. She also thinks quiescent cells might be continuously recycling proteins and membrane components to ensure that damaged macromolecules do not accumulate. “These cells are geared up to maintain a very healthy cellular environment,” Comai agrees.
Coller is now interested in assaying enzymes to probe differences in the PPP between proliferating and quiescent fibroblasts. Her group found that inhibiting the function of a PPP enzyme, glucose-6-phosphate dehydrogenase, induced apoptosis in quiescent cells, sparking Coller’s curiosity as to why this pathway is so important. She thinks it could be because the enzymes that are turned on in quiescent cells generate NADPH, which helps regenerate the antioxidant glutathione. Without the protection of the NADPH-producing pathway, the cells apoptose and die. This has led Coller to think about anticancer treatments. Because chemotherapies tend to target actively growing cells, quiescent tumor cells will survive standard treatments. But if Coller can find an “Achilles heel” in quiescent cells, she hopes it can be used to slay hard-to-kill tumor cells.