Kristie I. Aamodt and Dr. Kim L. O’Neill, Microbiology and Molecular Biology

The initial proposal for this project outlined the study of the link between DNA damage and aggressiveness of cancer cells. A major portion of the project involved developing a more time-efficient method of measuring DNA repair using a fluorescent staining technique that quantifies the amount of DNA that is damaged in each sample. The staining process involves using terminal deoxytransferase (tDt), an enzyme which adds nucleotides to DNA at the 3′ end of single-strand breaks. By incubating the samples with fluorescein-conjugated dUTP in addition to the enzyme, one fluorescent molecule is incorporated for each single-strand break. The amount of fluorescence in the each sample is determined by FACS analysis. By plotting the amount of fluorescence in each sample over time, the repair trends can be visualized and compared. In order to determine the effectiveness of this method we used an established protocol to damage the cells with hydrogen peroxide which induces single-strand breaks and collected samples at various time points.

While initial trends looked promising, I continued having problems with my undamaged control samples showing unusually high staining levels. So, I kept running samples through the assay changing all variables that may have caused the controls to have artificially high staining (number of washes, amount of enzymes, fluorescein, etc…). Unfortunately, the more samples I ran, the less consistent the results seemed to be. All the data showed was that staining was proportional to the number of washes and was not dependent on actual DNA damage. The assay was originally created to quantify apoptosis – programmed cell death – which occurs when DNA damage is too extensive to repair, so I decided to see if the assay would work if we induced apoptosis by damaging the cells beyond repair by increasing the hydrogen peroxide concentration and exposure time. By doing this I discovered that the staining did not work even for this purpose. Upon contacting the company that marketed the fluorescein stain I discovered that they were removing the stain from their product line. This discovery that my assay was not usable was a huge setback that actually completely shifted the focus of my project from DNA damage to studying the proteins involved in cell regulation, including apoptosis.

Specifically I began studying the interactions of thymidine kinase 1 (TK1) in carcinogenesis and tumorigenesis. TK1 is an essential nucleotide salvage pathway enzyme involved in DNA synthesis and repair. In normal cells TK1 levels increase only during cell proliferation. However, in most malignant conditions, TK1 is over-expressed significantly raising TK1 serum levels (sTK1) in cancer patients. As a result, sTK1 has been widely developed as a marker for use in cancer diagnosis. Although several studies have looked at sTK1, not much is known about how intracellular TK1 levels change during carcinogenesis or what interactions it has with well-established cancer pathways. Specifically I have been investigating how the presence of tumor protein 53 (p53)—an important tumor suppressor protein—influences TK1, allowing us to understand how TK1 is related to the p53 pathway. I was able to accomplish this by using two cell lines—TK-6 and W-TK1. These cell lines are p53 wild-type and p53 mutant, respectively. I measured TK1 levels in each of the cell lines while incubating them with pifithrin-α, a drug that knocks out p53 function, and resveratrol, a molecule that enhances p53 expression. Measuring TK1 levels has been my challenge over these past few months. Much of my time has been spent developing a direct ELISA test to measure TK1 concentration in the samples. After determining a reliable protocol for the ELISA I collected samples and am currently still in the process of analyzing TK1 levels using the ELISA as well as a well-established radioassay to measure TK1 enzyme activity.

In addition to the samples from the drug incubations, I have also analyzed apoptotic levels of the cells upon incubation and preliminary results indicate that p53 wild-type cells treated with resveratrol undergo more apoptosis than cells with mutant p53 in a time and concentration dependent manner. While I still need more data to confirm it, the results also seem to confirm that resveratrol acts through the p53 pathway, but in the absence of p53 some apoptosis was still detected indicating that other pathways may be activated when p53 is not available.

I presented some of the TK1 results at the 2007 Annual Meeting of the American Association for Cancer Research and have an abstract submitted to present at the 2008 Annual Meeting as well. Upon completion of the project I am planning on submitting it to a peer-reviewed journal for publication.