Kevin Mayer Senor Editor Genetic Engineering & Biotechnology News

Epigenetic Swings Can Maintain Quiescence or Induce Proliferation

Quiescent cells. We tend to ignore them because they are in the biological boondocks, far from the actively growing and dividing cells that enliven our cell cultures. Yet quiescent or conditionally nondividing cells not only account for the majority of cells in living tissues, they also retain the ability to reenter the cell cycle.

Ordinarily, quiescent cells maintain stability. They tap into their intrinsic regenerative capacities only as much as is needed to repair damage, battle infection, or carry life forward to successive generations. Occasionally, however, quiescent cells get out of control. They may grow and divide so heedlessly that they threaten the integrity of our tissues; weaken systems that respond to injury or prevent accelerated aging; or—perhaps worst of all—promote cancer.

When quiescent cells defy expectations, we may be tempted to denounce them as so many “deplorables.” But the wisest among us respond differently. They work harder to understand quiescent cells. For example, scientists have already established that while quiescent cells are reproductively inactive, they remain metabolically active. These scientists have shown that quiescent cells, so-called “sleeping cells,” are vigilant, epigenetically speaking.

Quiescent cells remain reproductively inactive only so long as the right epigenetic mechanisms remain engaged. As soon as these mechanisms break down or are superseded, the sleepers awaken and are roused to action. The results can be hard to predict.

As hard to read as quiescent cells may seem, they follow discernible rules. For example, quiescence is entered, sustained, or exited in accordance with epigenetic mechanisms that appear to be conserved across species, and are thus amenable to study. These mechanisms may be susceptible to manipulation. That is, they may prove to be so many buttons waiting to be pushed.

Some buttons may cause quiescent cells to go rogue. Others may shepherd quiescent cells back into the fold. Still others may activate or subdue quiescent stem cells, which typically account for less than one percent of the cells in our tissues. To learn which buttons to push for therapeutic purposes, scientists are conducting polls and surveys among cellular subpopulations.

Remembering the Forgotten Cells

Quiescent cells are often underrepresented in the petri dish, the conventional model of cellular behavior. Because it is limited to two dimensions, ordinary cell culture gives all cells unlimited access to nutrients and oxygen. As a result, it does a poor job of representing tumors, which are fed by blood vessels that have grown chaotically and distribute resources unevenly.

Real tumors contain regions in which cancer cells are starved and divide slowly or not at all, while managing to stay viable. These dormant cancer cells happen to be insensitive to standard chemotherapy, which mainly targets actively dividing cells. If dormant cancer cells survive chemotherapy, they can resume growth. They may even lead to cancer relapse.

If laboratory cell models are to mimic tumors more accurately—and help us prevent the resurgence of supposedly suppressed cancers—they need to incorporate not only dividing cells, but also quiescent cells. Laboratory cell models may do so, report scientists based at Uppsala University, by growing cancer cells in three dimensions. Three-dimensional cultures are essentially small tumors. They harbor interior cells that have a relatively limited supply of oxygen and nutrients.

In a paper that recently appeared in Cell Chemical Biology, the Uppsala team described a high-throughput gene expression profiling platform that featured “a tumor spheroid-based drug-screening assay to identify context-dependent treatment responses.” Essentially, the Uppsala scientists used their platform to monitor a gene-expression response to drugs of thousands of 3D cultures in parallel.

“As a proof of concept, we examined drug responses of quiescent cancer cells to oxidative phosphorylation (OXPHOS) inhibitors,” wrote the paper’s authors. “Use of multicellular tumor spheroids led to discovery that the mevalonate pathway is upregulated in quiescent cells during OXPHOS inhibition, and that OXPHOS inhibitors and mevalonate pathway inhibitors were synergistically toxic to quiescent spheroids.”

Although this combination of drugs—a common antiparasitic and a cholesterol-lowering agent—was effective in the model, it is still of uncertain safety. Nonetheless, it shows how three-dimensional modeling of tumors could be a useful tool in the hunt for new drugs that are toxic to dormant cancer cells.

Sustaining the Reserve Army of Stem Cells

Sidelined cells return to the cell cycle depending on the body’s ups and downs—whether, for example, the body is recovering from chemotherapy or radiation, which needs to be strong enough to eliminate cancer cells, yet restrained enough to spare healthy cells, including cells that take the lead in regenerating damaged tissue. This balancing act attracted the interest of scientists based at the University of Pennsylvania. They were particularly interested in learning how chemotherapy- and radiation-damaged intestinal tissue calls on its reserve of quiescent stem cells.

The scientists reported in the Journal of Cell Biology that stem cells in the intestinal epithelium resist injury because they stay in a dormant state during treatment. These stem cells, the scientists established, can be pushed out of dormancy and into the cell cycle by RNA-binding proteins in the Musashi family.

In earlier work, the Penn researchers had studied how Musashi proteins drive colon cancer. This work revealed that when Musashi proteins are inhibited in mice, the animals are less likely to get cancer.

“We knew that these Musashi proteins were important oncogenes and that if we inhibited them we could inhibit cancer, but now we see the downside,” said Chris J. Lengner, Ph.D., assistant professor in School of Veterinary Medicine’s Department of Biomedical Sciences at Penn and the senior author on the new study. “The downside is, when you delete them, the reserve stem cells can’t become activated and tissue cannot regenerate in the face of injury.”

This finding, Dr. Lengner added, confirms a common theme: Tumor suppression comes at the cost of regenerative capacity.

“We demonstrated that Msi proteins are dispensable for normal homeostasis and self-renewal of the active intestinal stem cells, despite their being highly expressed in these cells,” wrote the authors of the Journal of Cell Biology paper.” In contrast, Msi proteins are required specifically for activation of reserve intestinal stem cells, where Msi activity is both necessary and sufficient to drive exit from quiescence and entry into the cell cycle.”

Essentially, the authors determined that intestinal stem cells protect themselves by maintaining quiescence. The so-called reserve cells manage to resist radiation, the authors speculated, because they are not cycling. “There is some evidence,” Dr. Lenger noted, “that in this state the DNA is very compact and thus physically resistant to damage.”

“If you were to protect the cells and keep them in a more dormant state at the time of radiation,” Dr. Lenger continued, “you would expect the patient to avoid some of those nasty gastrointestinal complications of the treatment.”

Yet this dormancy might also work against cancer therapy. Some researchers believe that some cancers may originate from a cancer stem cell that quietly lurks in tissues and causes malignancies to reemerge long after an otherwise apparently successful treatment. It’s possible this activation could be governed in part by Musashi proteins.

Joining the Metabolic and Energetic Precariat

Those who endure material deprivation may experience so much stress that they age before their time. The problem is, stress may activate responses that bring relief in the short term, but spell disaster in the long term.

That’s what a team of scientists at Drexel University found when they studied how quiescent cells contribute to aging. The scientists modeled quiescence in cultured fibroblasts by imposing nutrient restrictions. Then they squeezed the fibroblasts yet further by instituting ATP deprivation.

These measures, the scientists reported in Frontiers in Genetics, caused the fibroblasts to adopt a signaling profile previously associated with inflammation. “Analysis of the transcription factor (TF) landscape induced by this treatment revealed alterations in several signal transduction nodes beyond the expected biosynthetic adaptations,” wrote the article’s authors. “These included increased abundance of NF-κB regulated TFs and altered TF subsets regulated by Akt and p53.”

Activation of the NF-κB pathway, achieved in the current study by stressing quiescent cells, has been linked to viral and bacterial infections, immune development, and inflammatory diseases. In addition, reduced ATP abundance and attendant upregulation of NF-κB signaling has been reported in aging and in a wide spectrum of diseases. So, it seems that aging cells respond to stress in the same way they may respond to an infection.

Stressed quiescent cells also downregulated protein p53 in the energy-starved cells. While low abundance of this protein prevents programmed cell death, it also impairs genome repair, which presents further evidence that aging cells essentially trade in their virility to survive.

“Cellular ‘instincts’ to survive acute challenges are not well suited for long-term chronic stresses,” said Andres Kriete, Ph.D., the study’s principal investigator, associate director and teaching professor in Drexel’s School of Biomedical Engineering, Science and Health Systems. “If cells are energetically stressed over longer periods of time, the genome maintenance could be impaired, which may explain why age is a risk factor for cancer.”

The authors asserted that their energy restriction model provides “a benchmark for the investigation of cell survival pathways and related molecular targets that are associated with restricted energy supply associated with biological aging and metabolic diseases.” In other words, the energy restriction model can expose a link between energy and aging that is usually beyond the reach of conventional models, which employ cells that are constantly proliferating.

Hunkering Down or Living Large

A team of biologists at Cold Spring Harbor Laboratory (CSHL) presented evidence that most cells cannot survive in a quiescent state unless an epigenetic mechanism called RNA interference (RNAi) is up and running. RNAi and other epigenetic processes induce changes in where and when specific genes are expressed without altering their genetic code.

Using simple fission yeast (Schizosaccharomyces pombe), a model organism for both epigenetics and quiescence, the team discovered that mutants lacking RNAi were unable to enter, maintain, or exit quiescence. Yeast without RNAi could survive only if they were in the process of dividing.

The scientists, led by CSHL’s Robert A. Martienssen, Ph.D., reported their results in Science. RNAi, the article’s authors indicated, promotes proper chromosome segregation during entry into quiescence. It also prevents inappropriate silencing of the ribosomal DNA (rDNA) during this reproductively dormant stage.

“We propose,” the scientists wrote, “a model in which RNAi promotes RNA polymerase release in both cycling and quiescent cells: (i) RNA pol II release mediates heterochromatin formation at centromeres, allowing proper chromosome segregation during mitotic growth and G0 entry, and (ii) RNA pol I release prevents heterochromatin formation at rDNA during quiescence maintenance.”

“The dual role of RNAi in the active cell cycle and in quiescence may therefore stem from a similar mechanism of RNA polymerase release, with a different consequence for heterochromatin formation depending on the genomic context and cellular state,” the authors elaborated. “Throughout eukaryotic evolution, RNAi and H3K9 methylation are always found together, indicating a codependency.”

A CSHL-issued release emphasized that the release of RNA polymerase I specifically from regions of the genetic material occupied by rDNA is of central importance: “rDNA is the genetic material that encodes the RNA that helps make up ribosomes, protein factories found in all cells. rDNA, much like the DNA that encodes heterochromatin, is highly repetitive and occurs in many copies across the genome. The action of RNAi releases RNA polymerase I from rDNA regions, and in so doing prevents the over-accumulation of heterochromatin during the maintenance of quiescence.”

“This research may explain the key role that RNAi plays in stem cells, which are quiescent for much of their life; and also in cancer, which, after all, is the stimulation of cells that are normally quiescent to begin dividing and proliferating. That transition, interestingly, is often accompanied by mutations in RNAi,” Dr. Martienssen concluded.

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