Back from the DEAD?

Life and death aren’t necessarily absolute states? The cellular world has its own rules…

We often envision growth as a linear path from life to death. It’s true that the cells that make up your body are growing and dividing, but at the same time cells are also dying. In fact, this delicate balance of cellular growth and death is critical for health. For example, if cells stop dying and just proliferate, this can result in overgrowths and cancer. Cellular death is necessary to remove cells that are injured or abnormal. Alternatively, if cells stop growing and don’t replace the ones that are dying off then, eventually, there wouldn’t be enough cells for our organs to maintain their structure and function, and could lead to degenerative diseases. For these reasons, cellular death is as important to life as cellular growth. Just as cells have systems and mechanisms in place to enable their division, they also have specific mechanisms that cause their death. 

One mechanism of cellular death, called apoptosis, is named after a Greek word that means “falling to death”. During apoptosis, a cell literally kills itself. In this process, cells produce proteins called caspases that chew up the cell’s DNA and destroy the cell from the inside out. It was previously believed that production of caspases by a cell was basically a point-of-no-return beyond which the cell could not survive. After all, it would be logical to assume that if all of a cell's DNA is broken up, it's pretty much finished.  However, a fascinating research study demonstrated that cells can actually reverse the apoptosis process of cellular death, even at late stages including DNA damage and destruction of mitochondria, which are the parts of the cell that produce energy.  

High levels of a toxic substance like ethanol can cause cells to begin dying by apoptosis. When cells begin this apoptosis process, they sort of shrivel up and start breaking apart with bubble-like protrusions, called blebbing. The nucleus, where the cell’s DNA is kept, can also appear smaller and broken-up or fragmented. 

Here, the researchers were studying a few different types of cells: liver cells, heart cells, and fibroblasts - a type of skin cell. They exposed the cells to a toxic substance for several hours, and observed these classic signs of apoptotic cell death. But shockingly, after the toxic substance was washed away, the cells began regenerating and recovering. 

After 24 hours, over 90% of the cells had completely recovered. Videos of individual cells taken by the researchers show how the cells begin to die, and even begin to break apart. However, after the toxic substance is removed, they somehow manage to rebuild themselves and survive. The authors named this phenomenon "anastasis" which is a Greek word meaning “rising to life”.  But what exactly is going on in the cell during this recovery process? 

As mentioned earlier, when the apoptosis process of cellular death is triggered, proteins called caspases destroy the cell. Here, the authors observed that one such protein, caspase-3, was activated in the presence of toxic levels of ethanol, but after washing the ethanol away, caspase-3 was deactivated. These so-called “executioner caspases” are present in healthy cells, but held in an inactive state. Toxins or other cues can trigger a chain reaction within the cell that results in the activation of caspases. 

Inactive caspases in healthy cells are called procaspases. The procaspase has to be cleaved or cut to function. You can think of this sort of like a vicious dog on a leash. When it's muzzled on the leash, it’s held back and can't really do anything. But if you cut the leash, it's free to attack. It was previously believed that after caspase activation, a cell is doomed to die. But the discovery of anastasis contradicted this view. 

To further understand what is going on inside the cell during anastasis, the authors genetically engineered cells to produce a caspase biosensor. To do this, they inserted a gene into the cell to produce a special protein. This protein contained a portion that tethered it in the cytoplasm, the main inner portion of the cell surrounding the nucleus where DNA is kept. This tether was connected to a fluorescent molecule that could be taken up by the nucleus. If active caspases were present, they could cut the tether and the fluorescent molecule could then move into the nucleus. Living, healthy cells that have a fluorescent signal in the nucleus would mean that they had active caspases at one time, but then recovered. For example, when the authors treated cells with a toxin for a few hours, they saw the fluorescent color move from the cytoplasm into the nucleus, along with the blebbing and other signs of cell death. After removing the toxin and washing, many cells retained the fluorescent molecule in the nucleus. This indicates that cells can indeed recover from active caspases. Still, as mentioned earlier, DNA is broken up and destroyed during apoptosis. The researchers detected significant DNA damage after application of toxins. How then, could these cells survive? 

Upon a closer look, the scientists discovered that surviving cells had repaired much of their DNA, but some damage remained. Specifically, they noted that many cells contained micronuclei – multiple small nuclei instead of one large central nucleus for storing DNA. This could be thought of like a library full of books organized on shelves. The books are your DNA. Then, someone takes the books and throws them into lots of different boxes. The books are still there, but maybe some are damaged and the situation isn't as nice. The breaking apart of the cellular DNA during apoptosis and then putting it back together during recovery can cause massive genome rearrangements. The authors noted that a fraction of cells that had recovered from death displayed characteristics of cancer cells. So recovery from cellular death could indeed be a way that cancer could form. Still, even if cells could survive with damaged and mutated DNA, active caspases also chew up the cell’s proteins. How can all of the cell’s internal contents be rebuilt?

Unbelievably, the researchers were able to detect transcription inside the cell during recovery. Transcription is the process of making RNA from DNA. RNA is used to make proteins. The scientists then applied a drug that prevents the cell from making RNA. This greatly hindered the ability of the cell to recover from apoptosis. This suggests that the cell needs to be able to actively start producing new proteins in order to rebuild itself and recover. 

The authors speculated several intriguing implications from this study. For example, the phenomenon that anastasis induced massive genetic changes could help explain why repeated injury or alcohol abuse appears to increase cancer risk. More disturbingly, this could also be a means through which cancer cells survive chemotherapy and radiation treatments to become more aggressive or even acquire resistance. On the other hand, the anastasis recovery phenomenon might not be all bad. For example, this process could preserve injured cells that are hard to replace, like neurons or heart cells. Future research will likely uncover many exciting prospects as we learn more about the incredible abilities of the cellular world. 

Reference for the paper discussed in this article:

Tang, Ho Lam, et al. "Cell survival, DNA damage, and oncogenic transformation after a transient and reversible apoptotic response." Molecular biology of the cell 23.12 (2012): 2240-2252.