In an earlier post, I complained about trying to schedule a long experiment when sick. Now that I have a bit more time, I can explain what that experiment was all about.
This is a continuation of the leukemia studies I'm currently doing. I've been analyzing tumors to determine how certain genes promote leukemia, and I've gathered a fair amount of data about what the cancer cells look like and how they develop.
To understand the reason for my experiment, one must understand how cancers grow. Unfortunately, cancer is a widely variable disease, so there are a lot of characteristics that determine what sort of tumor you have and how it behaves. Fortunately, cancer has a set of identified "hallmark" characteristics that are absolutely critical for tumors to grow. Some of these include the ability to promote blood vessel growth within the tumor (angiogenesis) and the ability to prevent immune responses.
Two other characteristics are the ability of cells to divide forever (limitless replication) and ability to avoid death signals (evading apoptosis). The difference between these two is subtle but important. Normally, a cell has checkpoints that control when it divides and how long it can divided for. Similarily, if something goes wrong in a cell, there are checkpoints to prevent that cell from dividing and basically tell it to commit suicide. That means there are two ways for a cell to proliferate into a cancer: either divide at an alarming rate, or fail to die when they should.
To illustrate this idea, think of the number of people living in a town. You could get a population boom because people are zealously procreating with no second thought to contraception or family planning. In conrast, another town could reproduce at a normal rate, but due to longer lifespans, the old buggers don't die off. After a bit of time, both towns would be overpopulated. Essentially, the Baby Boomers represent both these scenarios...
That's right, I just called the Baby Boom generation a cancer...
So the question I have about my tumors is how they're growing: are they dividing too much, or just outliving their potential?
To answer that, you just have to look at how quickly cells are dividing. There are a number of ways to do this, but one common technique involves treating cells (in vitro or in vivo) with a green dye called CFSE. The dye gets taken up by cells, and each time a cell divides, the amount of green color inside that cell is reduced by half. Essentially, the green is diluted out. You can then tell how many times cells divide in a given amount of time based on how green (or not green) they are.
Another common technique is one I'm using called BrdU staining. Understanding this technique requires an understanding of what happens during cell division. For a dividing cell to ensure the right amount of genetic material ends up in the daughter cells, it makes a copy of all its DNA. When this DNA is replicated, it gets opened up and synthesized from molecules you may have heard of: A, T, C, and G. Bromodeoxyuridine (BrdU) is a an analog of thymadine (the "T" of A, T, C, and G), and can take its place during DNA synthesis. Thus, if you treat an organism with BrdU, dividing cells will incorporate it into its DNA in place of T, while non-dividing cells will not.
To detect the cells that have BrdU, you follow up with flow cytometry (which I described in my previous lesson). This lets you detect the BrdU with a fluorescent anti-BrdU antibody, so you can just look to see which cells are fluorescent to know which are in the process of dividing.
"But wait!" you say. "How can you detect DNA with an antibody? DNA is inside the cell! Antibodies can't get there!"
Yeah, well, that's an easy solution. Treating the cells with a permeabilizing reagent (often the detergent saponin) pokes holes in the cell membrane and lets antibodies in. Done.
So anyway, it looks like my cells are indeed rapidly dividing, because they have copious amounts of BrdU in them. Importantly, this doesn't rule out the possibility that the cells are also avoiding apoptosis. It's likely that both unchecked division and death avoidance is going on in these leukemias.
That's like having the baby boomers discover immortality. Scary.
Currently listening to: NPR - Science Friday
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