This page was produced as an assignment for Genetics 677, an undergraduate course at UW-Madison.
Methotrexate
Figure 1.
PubChem was first used to look for any small molecules that could target and bind MSH2. None were found, so PubMed was used to look drugs that could potentially be useful in MSH2 deficient HNPCC patients and I found the drug methotrexate [1-2]. PubChem was then used to learn about methotrexate specifically [3].
Methotrexate is an antineoplastic antimetabolite with immunosuppressant properties. It is an inhibitor of tetrahydrofolate dehydrogenase and prevents the formation of tetrahydrofolate, necessary for synthesis of thymidylate, an essential component of DNA. For this reason, methotrexate inhibits DNA replication and cell growth and has been used to treat many types of cancers in the past [4]. Methotrexate can also be quite toxic because it also affects other tissues that proliferate besides tumors. The 2- and 3-dimensional structure of methotrexate is shown in Figure 1.
Methotrexate treatment has also recently been found to cause the accumulation of potentially lethal 8-hydroxy-2’-deoxyguanosine (8-OHdG) oxidative DNA lesions (Figure 2). These 8-OHdG lesions, in the presence of methotrexate, are incorporated into newly replicated DNA. Wild type MSH2 is able to identify and remove these 8-OHdG lesions, resulting in a healthy cell. Cells lacking MSH2 or with nonfunctional MSH2 cannot remove these 8-OHdG toxic lesions, resulting in the accumulation of these lesions and everntually leading to cell death [1-4].
Figure 2. Shows how methotrexate can alter DNA to form 8-OHdG lesions.
Question: Can low doses of methotrexate prevent the development of colon cancer in high-risk HNPCC patients?
Hypothesis: I hypothesize that methotrexate is capable of causing accumulation of 8-OHdG lesions in MSH2 deficient cells, resulting in the cell death of these specific cells while MSH2 proficient cells are unharmed. This specific targeting of MSH2 deficient cells may prevent colon cancer in high-risk HNPCC patients.
Experiment:
A mouse model will be utilized to investigate the prophylactic capability of methotrexate. MSH2-/- mice either will be treated with a low dose of methotrexate 3 times per week from birth or will be untreated. After a certain period of time, perhaps a month, the mice will be sacrificed and the number of colonic polyps will be counted. A diagram of this experimental design is shown in Figure 3.
Figure 3. Diagrams the proposed methotrexate mouse experiment.
Expected Results:
I would expect that MSH2-/- mice that are treated with methotrexate from birth would have a fewer number of colonic polyps compared to untreated MSH2-/- mice. This would indicate that methotrexate is capable of selectively targeting MSH2 deficient cells for cell death before they can proliferate and form a tumor or cancer.
Analysis
The proposed experiment will begin to investigate the prophylactic capabilities of methotrexate. One drawback of the proposed experiment is that the number of colonic polyps that usually results in HNPCC patients with colon cancer is low, normally only a few. This may make is difficult to see a difference in the number of colonic polyps between the two treatment groups. More colonic polyps may be able to be induced by having a null MLH1 mutation in the background. This would make it easier to see a treatment effect.