What I Do in Lab, continued

It's interesting, but sometimes I feel as though the New York Times and I are strangely in sync with one another, especially in regards to their articles about science topics. Just yesterday I found these two articles, Genes Explain Race Disparity in Response to a Heart Drug and A Genetics Pioneer Sees a Bright Future, Cautiously
that made me pause for a moment. It was pharmacogenomics in mainstream news. Hooray! See? What I'm working on isn't so obscure after all. For current "real-world" applications of the kind of stuff I'm working on, please read these articles.

Anyway, back to my work. So I talked about how we aim to decrease the levels of a particular drug-clearing enzyme to make the cells we treat more sensitive to our chemo agent. The next thing I should explain is how we actually go about manipulating levels of said enzyme.

First, a brief lesson in molecular biology. DNA, or deoxyribonucleic acid, is considered to be the "code of life" as it prescribes nearly all the information required for organisms in all five kingdoms of life to fulfill the requirements of life, i.e. grow, reproduce, survive, etc. Living things, however, are not made of DNA alone (obviously). The information coded in DNA proceeds along the path to proteins (what we actually see and feel in living things, i.e. skin, hair, etc) through an intermediary molecule known as RNA, or ribonucleic acid. DNA and RNA are very closely related but have significantly different properties which suits them for their particular roles. RNA is much less stable (it's single-stranded instead of double-stranded) and is usually rapidly degraded after it's produced (compared to DNA which lasts until the cell dies). The type of RNA I'm interested in in particular is messenger RNA, or mRNA. The "messenger" bit comes from a famous molecular biologist who likened that molecule to a "messenger from God" for it's role in transducing DNA's message (so much for all scientists being determinedly irreligious!). What does the RNA send its divine message to? Structures called ribosomes which are synthesis stations in which mRNA is used as a template off of which proteins are built.

If you consider the percent composition of living things, we are possibly 99.9980% protein, 0.0015% RNA, and 0.0005% DNA. Do not take these percentages for fact; I literally made them up just for a sense of scale. But I digress.

If we want to knockdown protein levels, there are three conceivable ways we can go about doing that. One is to alter the gene (DNA) to make a substandard protein that doesn't do its job so well. Another would be to break down the final protein or inhibit its activity somehow by making it stick to another protein or small molecular (this is actually how many new fancy drugs work). A final way, and the way we're using, is to go after the mRNA and reduce its levels such that the protein isn't made in normal quantities in the first place.

Back in the early 1990s, two labs headed by molecular biologists Craig Mello and Andrew Fire discovered an incredibly potent viral defense mechanism in the roundworm C. elegans in which foreign RNA (many viruses have RNA instead of DNA as their primary "code of life"; bit of a misnomer here since viruses are technically not considered living things) was rapidly degraded by a unique type of RNA produced by the worm's cells. This defense mechanism had already been identified in plants, but what made Mello and Fire special (and eventually Nobel laureates) was their insight that could harness that natural defense mechanism to attack any RNA of their choice. This technique, called RNAi (short for RNA interference), won them the Nobel Prize in 2006 (just over a decade after their discovery) and is an extremely common and invaluable laboratory technique. (If you care to find an earlier post I made back in winter 2007, you can read about my experience of actually meeting Dr. Mello at the Field Museum here in Chicago).

Back to the lab. The siRNA experiment is actually quite simple:
1) I siphon off the volume of cell suspension I'll need from the flasks in which they grow (the cells we use, lymphoblastoid cells, do not grow flat on plates but suspended in media...think Guild Navigators for you Dune fans, except these cells fortunately can't bend space).
2) I then spin down the cells to form a tiny pellet that I then wash with a simple pH neutral solution to remove any remaining media.
3) Spin again to make a new, cleaner pellet.
4) Now I add in a "nucleofection solution" which basically makes lots of tiny holes in the cells such that stuff can get in, but they still stay alive.
5) I carefully plate these hole-ridden cells onto a dish full of tiny wells.
6) Quickly, else the cells will die, I add in my siRNA solution that is full of those attacking RNA molecules. Since I wish to knockdown the CYP1B1 protein, the siRNA I use is specific for the mRNA for that protein.
7) Once the cells have been dosed with the siRNA, I place them in a machine where they are "zapped" with an electric pulse to make the cells take up the siRNA. This takes seconds.
8) Once the cells are zapped, I give them some warm media and stick them in the incubator for some minutes to let them recover from the shock (a pun, haha).
9) Once they've relaxed a bit, I plate them out onto a new dish and add some more media so that they will be happy.
10) At a predetermined time point, in my case 24 and 48 hours after plating, I'll scoop up those cells and go on to the next experimental step: RNA isolation.

All in all the nucleofection takes approximately 2 hours from start to finish. Not too bad, considering I performed an RNA isolation today which took approximately 4.5 hours, so I'm not exactly in the mood to describe that right now...but I will do so tomorrow! Stay tuned!

Spring Quarter Rotation, or What I Do in Lab

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!

To catch up: Finals Week(s)

Losing Eve six weeks ago hurt, a lot. That loss along with being truly and insanely busy with finals (enough that I probably lost about five pounds and made myself sick), rather took blogging further off my mind than unfortunately it normally is (unfortunate for those few yet faithful readers). In return, I promise to be more diligent and keep this updated every few days.

So, how did winter quarter end? I survived, to say the least, and managed to pull out a few surprises for myself. I scored two A's and one B, which is an improvement over my one A and two B's from last quarter. Guess what I plan to get this spring J? The B came from Fundamentals of Molecular Biology, which is actually quite sad considering how relatively elementary that class was compared to Cell Biology of autumn quarter (what I consider quite possibly the hardest class I've ever taken, period, except perhaps orgo one and two). The A's were in Cancer Biology II, in which a B would have been not only downright embarrassing but pathetic! My A was a pretty good one nonetheless, certainly one of the top three in the class (a rare statement from me about a science class since high school; to be fair I've done but seldom superlatively well unlike in some of my English classes).

The second A was in Signal Transduction which went from being possibly the easiest to the scariest to the easiest class on Earth all in the space of a few short weeks. To begin with, the three cancer biology students in that class (myself, Kelly, and Tanmayi) were there because we were told there would only be one exam and a take-home at that. Score! This is what we thought for about the first half of the quarter, a thought that allowed me to guilt-free zone out a bit during some of the more tedious lectures. Then, after checking with the TA that "this class really is just going to have a take-home final, right?" We're told, "no, we're changing it this year since so many people skipped class last year." Frak! The next few weeks went by in a bit of a panic that was accompanied by furious and diligent note-taking. Now comes the week before finals. Tuesday is the review session and Thursday the in-class final. I bring with me all 8 weeks of notes which were derived from the wood pulp of an entire acre. Instead of our usual classroom, we find ourselves in a conference room with a video from a Nobel Prize-winning lecturer ready to begin. What was going on? To allay our fears, our professor informs us that due to "excellent attendance", they've decided to make the final take-home after all. A collective sigh of relief exhaled from the students and suddenly the day become a whole lot brighter. I was saved! The final ended up being five questions and required just five lectures (out of 16) to answer. All in all it probably took me just over two hours, and most of that time was spent flipping through pages to find the pathway I needed. Total length? Just fewer than three pages (compared to the eight of my cancer biology take-homes). I don't know the final distribution for that class (there were only eight of us in it), but I must say I am grateful for my A despite not being as attentive (or smart) as most of the other people in there.

As for my other classes, Can Bio II wrapped up well yet rather hectically. My poster presentation went fine (I could have been more prepared, but still did well) though getting my final in nearly gave me a heart attack. The Friday before we were informed that our final would be due no later than 9 a.m. on Tuesday, March 18 (the poster session was the 17^th). Since I mostly worked on the poster/studied for Mol Bio over the weekend, I didn't devote much time to the final except doing some background research on the questions. So I get home on the 17^th around 1:15 to find an email from my TA asking that our finals be sent in no later than 3 p.m. so she can distribute them to our professors. Needless to say, I almost had a cardiac infarction and immediately informed my TA of what our professor said. I went on to spend the next nearly 12 hours typing away almost without break. The final product wasn't my best, but at least it was done. Considering I headed into the final with almost the highest grade in the class, I wasn't too worried…

Anyway, enough about classes. Next post: spring break!

Blogging with Microsoft Word…

Who would have thought that two huge rivals, Google and Microsoft, could team up (at least on some level) to let users of both companies' products do something fun and useful? Blogger, a very popular blogging resource owned by Google is compatible with a new "Blog Post" feature in Microsoft Word 2007. This means I can type any document I want in Word (like what I'm doing right now), click "publish," and voila! My doc gets posted to my Blogger page smoothly and beautifully.

Why does this matter? For one, it lets me write and save blog posts before I publish them in a secure and familiar program. Instead of a normal Word doc, something I've used before, now when I'm ready I can post from Word directly instead of copying my text into Blogger's "add post" feature. Probably the best part of this feature, though, is my ability to publish anything I can normally put into a Word doc to my blog. By this I mean an Excel spreadsheet, Powerpoint graphics, Word tables and graphics, equations, other images, you name it, all without formatting troubles you often get by moving between different programs.

Why I am excited about this? It's because I'm a total geek and increasingly big fan of the new Microsoft Office 2007 suite. Vista, from what I've heard, was pretty much a total bust but Office '07 carries the day in my book…