Over the course of the next seven weeks, I will frequently post "in lab" as an away message or respond as such if I am called during working hours on Monday, Wednesday, and Friday. So what am I doing in lab? Allow me to explain (both the why and what such a question might address):
As a first-year cancer biology grad student at the University of Chicago, I am required to complete two 10-week (whole quarter) rotations or time-periods in a research laboratory run by a professor on the cancer biology committee (though not necessarily exclusively so). Shortly before I arrived on campus last September, I was surfing through faculty research pages in search of a potential PI (principal investigator) to rotate with. I found Eileen Dolan's page and just needed to see "identify genetic determinants contributing to cellular susceptibility to chemotherapeutic agents" before I was hooked. I immediately emailed Dr. Dolan and set up an appointment with her just two days after I would arrive in Chicago. Our meeting went well, and she agreed to let me rotate with her in the spring.
The months passed amazingly quickly and before I knew it, spring break was over and spring quarter was starting! The week before break I attended lab meeting (for those who don't know, these are weekly sessions in which a lab member or members present their work and receive feedback on it from the rest of the lab, especially the PI) and learned that I would continue a project started by another grad student who was just finishing her rotation. I owe much already to Nora for doing a lot of the grunt work (optimizing a protocol...more on that later) and letting me have all the fun.
My, rather our, project is to decrease the expression of a particular drug metabolizing enzyme, CYP1B1, in lymphoblastoid cell lines that highly express that enzyme and see if these cells become more sensitive to the chemotherapy agent daunorubicin. The hypothesis is that high expression of this drug clearing enzyme allows these cells to render the chemo useless (breaking it down very quickly, etc) and thus escape cell death. If we can decrease the amount of the enzyme in these cells (by decreasing the mRNA which in turn would decrease protein levels), then these cells should die more readily (at lower drug concentrations) than they do otherwise. I'll go later into the science specifics of how we decrease expression and dose with the drug.
Why is this project relevant, you might ask? The vast majority of chemotherapy agents must be processed by the liver in order to become biologically active. Many over-the-counter and prescription drugs work the same way. When the drug enters the body, it reaches the bloodstream through intestinal lining (unless administered intravenously) and then travels through the liver before continuing on to the rest of the body. The liver is such an important organ (and not surprisingly the largest internal organ) because it processes all the circulating blood in the entire body. It detoxifies and cleanses blood continuously (which is why the only solution for a hangover is time; the liver can only process all that alcohol, i.e. toxin, so fast). There is a superfamily of enzymes found primarily in the liver known as the Cytochrome P450 (CYP450) family. These enzymes handle the vast majority of toxins and foreign agents the liver processes, including drugs.
Proper drug formulation is crucial for its activity since these enzymes modify the compounds in certain ways and in short try their best to inactivate or destroy them (hard for the body to distinguish friend from foe: while you can train your eyes to tolerate contact lenses you can't train your liver to be nice to certain things and attack others). Knowing this, drug companies formulate their drugs such that they usually must be modified in some way to actually work. Just popping the pill in your mouth does nothing; it must travel through the liver and then reach its target through the bloodstream. You can determine how long this takes by measuring the time between you take a painkiller and when you start feeling relief (it takes me about an hour with Advil, but I have no idea if that's typical...) Anyway, chemo agents must be processed by the liver to work.
The catch, however, is that not all livers can process drugs the same way because each person has a slightly different set or variation of the CYP450 enzymes. This variation is caused by the genetics that make us 99.97% alike and yet still 0.03% different. As a result, while some people can process a particular drug well such that it gains proper activity, others cannot process it and that drug does not work for them and can potentially build up to unsafe concentrations in their body (chemo agents in particular are very nasty; you don't really want them at the therapeutic concentration much less anything higher, especially when they're not really working anyway!) The enzyme we're working on, CYP1B1, is a member of this family and studies have implicated its role in conferring resistance to daunorubicin. Thus, we postulate, if we can lower the activity of this enzyme we can let the dauno be processed the way it should to properly kill the cell. Make sense?
Ok, so that's a good bit of background. On my next post I'll explain how I will go about testing our hypothesis. Cheers!
As a first-year cancer biology grad student at the University of Chicago, I am required to complete two 10-week (whole quarter) rotations or time-periods in a research laboratory run by a professor on the cancer biology committee (though not necessarily exclusively so). Shortly before I arrived on campus last September, I was surfing through faculty research pages in search of a potential PI (principal investigator) to rotate with. I found Eileen Dolan's page and just needed to see "identify genetic determinants contributing to cellular susceptibility to chemotherapeutic agents" before I was hooked. I immediately emailed Dr. Dolan and set up an appointment with her just two days after I would arrive in Chicago. Our meeting went well, and she agreed to let me rotate with her in the spring.
The months passed amazingly quickly and before I knew it, spring break was over and spring quarter was starting! The week before break I attended lab meeting (for those who don't know, these are weekly sessions in which a lab member or members present their work and receive feedback on it from the rest of the lab, especially the PI) and learned that I would continue a project started by another grad student who was just finishing her rotation. I owe much already to Nora for doing a lot of the grunt work (optimizing a protocol...more on that later) and letting me have all the fun.
My, rather our, project is to decrease the expression of a particular drug metabolizing enzyme, CYP1B1, in lymphoblastoid cell lines that highly express that enzyme and see if these cells become more sensitive to the chemotherapy agent daunorubicin. The hypothesis is that high expression of this drug clearing enzyme allows these cells to render the chemo useless (breaking it down very quickly, etc) and thus escape cell death. If we can decrease the amount of the enzyme in these cells (by decreasing the mRNA which in turn would decrease protein levels), then these cells should die more readily (at lower drug concentrations) than they do otherwise. I'll go later into the science specifics of how we decrease expression and dose with the drug.
Why is this project relevant, you might ask? The vast majority of chemotherapy agents must be processed by the liver in order to become biologically active. Many over-the-counter and prescription drugs work the same way. When the drug enters the body, it reaches the bloodstream through intestinal lining (unless administered intravenously) and then travels through the liver before continuing on to the rest of the body. The liver is such an important organ (and not surprisingly the largest internal organ) because it processes all the circulating blood in the entire body. It detoxifies and cleanses blood continuously (which is why the only solution for a hangover is time; the liver can only process all that alcohol, i.e. toxin, so fast). There is a superfamily of enzymes found primarily in the liver known as the Cytochrome P450 (CYP450) family. These enzymes handle the vast majority of toxins and foreign agents the liver processes, including drugs.
Proper drug formulation is crucial for its activity since these enzymes modify the compounds in certain ways and in short try their best to inactivate or destroy them (hard for the body to distinguish friend from foe: while you can train your eyes to tolerate contact lenses you can't train your liver to be nice to certain things and attack others). Knowing this, drug companies formulate their drugs such that they usually must be modified in some way to actually work. Just popping the pill in your mouth does nothing; it must travel through the liver and then reach its target through the bloodstream. You can determine how long this takes by measuring the time between you take a painkiller and when you start feeling relief (it takes me about an hour with Advil, but I have no idea if that's typical...) Anyway, chemo agents must be processed by the liver to work.
The catch, however, is that not all livers can process drugs the same way because each person has a slightly different set or variation of the CYP450 enzymes. This variation is caused by the genetics that make us 99.97% alike and yet still 0.03% different. As a result, while some people can process a particular drug well such that it gains proper activity, others cannot process it and that drug does not work for them and can potentially build up to unsafe concentrations in their body (chemo agents in particular are very nasty; you don't really want them at the therapeutic concentration much less anything higher, especially when they're not really working anyway!) The enzyme we're working on, CYP1B1, is a member of this family and studies have implicated its role in conferring resistance to daunorubicin. Thus, we postulate, if we can lower the activity of this enzyme we can let the dauno be processed the way it should to properly kill the cell. Make sense?
Ok, so that's a good bit of background. On my next post I'll explain how I will go about testing our hypothesis. Cheers!
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