by Monya Baker

Healthy and cancerous stem cells protect their DNA

Recent studies in cancer treatments point to an ugly truth: the best cells to destroy are the most resistant to attack. Work in several human cancer types indicates that only a subset of cancer cells is capable of regenerating tumours when transplanted into mice. Michael Clarke of Stanford University in Palo Alto, California, one of the first researchers to identify such a subset in a solid tumour1, hypothesized that these cancer stem cells would be less susceptible to chemotherapies than the other cells in the tumour.

When radiation therapist Maximilian Diehn joined Clarke's lab as a postdoc in 2004, they decided to test this idea. Radiation works by causing DNA to splinter — this keeps cells from dividing and can trigger cell death. Tests of DNA damage in treated cells revealed that cancer stem cells survived relatively unscathed compared to nontumourigenic cells. Radiation breaks strands of DNA by generating unstable molecules called reactive oxygen species (ROS), so Diehn and Clarke reasoned that there had to be something about the cancer stem cells that made them more capable of dealing with ROS.

But there's a lot of work between having an idea and proving it. First, Clarke's lab had to establish whether and why ROS levels are lower in breast cancer stem cells. For that, they had to wait for suitable patient samples. There's no way to know when those will arrive, says Clarke, and each one has to be tested within 24 hours, requiring scientists to change plans with little warning. Even processing a sample in time still doesn't mean an experiment will work. "You can go in and find out that the pathologist didn't give you any cancer cells or not enough to get an analysis," says Clarke. "That happens all the time."

After finally collecting enough data they found that, compared with nontumourigenic cells, breast cancer stem cells expressed higher levels of genes that help scavenge ROS and had lower intracellular levels of ROS2. Measurements of DNA breaks and cell survival were all consistent with the hypothesis that cancer stem cells are better able to resist death by radiation damage.

To know what protective programs cells are using, researchers need to be absolutely certain what cells they are working with. As useful as they are, cell-sorting techniques produce mixed populations of cells in which a particular type is more common. But even then, cells within the sorted population can vary dramatically. The best way to tie a particular trait to a particular cell type is to work with a clonal population, in which all cells derive from a single cell. "To do this experiment, we needed a clonogenic in vitro assay which didn't exist," recalls Clarke. This was painstakingly developed by faculty member Robert Cho, who is, along with Diehn, first author on the paper.

Also essential to gathering data for the paper was the ability to examine the gene expression in individual cells, which researchers were able to do by using a technique developed by Stanford's Steve Quake. "With that we could unequivocally tell what we were looking at," says Clarke. "That's going to be a very, very powerful tool." This revealed more specifically how breast cancer stem cells resist radiation: they upregulate genes required to synthesize glutathione, an antioxidant thought to allow cancer cells to resist both radiation and drug therapies.

This pernicious trait is probably part of the body's ability for self-repair, says Clarke. Many cells trigger a self-destruct mechanism after they are mutated, but stem cells seem to run counteracting, protective mechanisms. "The body is trying to get rid of the damaged cells, but you need a pool of cells to regenerate the tissue," explains Clarke. Other labs have observed that healthy neural and haematopoietic stem cells have lower ROS levels than non-stem cells; Clarke and Diehn found the same was true for normal breast epithelial stem cells.

Clarke suspects that stem cells likely have several additional enhanced mechanisms to resist or repair damage and notes that a separate mechanism has already been identified in the brain3. The mechanisms likely vary from tissue to tissue, and it seems probable that cancer stem cells may not be able to make use of all the protective programs that their normal counterparts use. Clonogenic assays and single-cell techniques will help pinpoint the specific programs used by tissue-specific cancer stem cells, and that could lead to therapies. "If there's a subtle difference," says Clarke, "someone will find a way to exploit it."

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