Human health and risk for disease ultimately depend on the integrity of our DNA, the genetic material that provides the body’s blueprint for manufacturing proteins that carry out the function of cells and organs. Changes in DNA during life are believed to trigger cancer and many other chronic diseases. Such changes can result from exposure to certain chemicals found in the workplace and others in the diet and medications, and to sunlight in outdoor workers. CROET research on DNA is focused on the mechanisms that can lead to prevention and therapeutic strategies for reversing DNA damage relevant to the workplace.
Scientists from the CROET laboratory of Stephen Lloyd, in collaboration with scientists from OHSU, the National Institutes of Health, the University of Arkansas and Vanderbilt University, have developed a high-throughput drug-screening assay to find a new class of novel anti-cancer drugs. The results of this work were detailed in a pilot study published in the October, 2012 issue of the open access journal, PLoS one. What is novel about the assay is that it is designed to look for drugs that inhibit a specific cellular process known to render cancer cells resistant to a commonly used class of chemotherapeutic agents.
Many chemotherapeutic agents, including drugs such as mitomycin C, cisplatin and nitrogen mustard, target tumor cells by virtue of their ability to chemically cross-link the double-stranded DNA that comprises chromosomes. DNA cross-linking interferes with DNA strand separation, which is vital to a variety of cellular functions, such as DNA replication and cell division. Because cancer cells are rapidly dividing cells, and cross-linked DNA inhibits DNA replication, the chemotherapeutic drugs induce affected cancer cells to die via a pre-programmed cell suicide mechanism known as apoptosis.
So, how do cancer cells become resistant to these chemotherapeutic drugs? It is a process known as translesion DNA synthesis (TLS), mediated by a DNA replication enzyme called pol κ, which is able to traverse cross-linked DNA and complete the replication cycle, allowing cells, including cancer cells, to survive and reproduce. Although TLS is an essential process for normal cells to survive limited genotoxic stress, the ability of pol κ to bypass DNA cross-links can limit the efficacy of chemotherapeutic agents that act by the mechanism described here. Therefore, drugs that inhibit pol κ may reverse the resistance of cancer cells to these chemotherapeutic agents.
One type of cancer, gliomas, are the most common form of primary brain cancer and represent what is currently a generally incurable tumor in humans. These tumors are highly resistant to current treatment strategies, including chemotherapy with drugs that induce DNA cross-linking, leading to median survival of patients with high-grade gliomas of only one year post-diagnosis. Significantly, the amount of pol κ is increased in tumors from glioma patients, and its level is highly correlated with the grade of disease and the prognosis for cure. Thus, the identification of inhibitors that target pol κ may be crucial for improving the therapeutic efficacy of chemotherapeutic agents.
The assay developed by the Lloyd lab is actually quite simple. Moreover, it is conducted in a system comprised of 1536 tiny petri dishes, or wells, pictured here, which allows over a thousand prospective pol κ inhibitors to be tested per assay. In each well is a fluid medium holding a specialized length of double-stranded DNA containing a fluorescent marker. When pol κ is introduced, it begins to replicate the DNA molecule and consequently displaces another DNA strand that can be measured. Specifically, the DNA is designed so that, when pol κ reaches a certain spot in its replication run, another short segment of DNA containing the fluorescent marker is released from the original double strand. When released, the fluorescent marker is activated and the well glows. If a prospective pol κ inhibitor drug, also introduced into the test well, is active against pol κ, release of the fluorescent marker is prevented and the test well remains dark. Therefore, a positive test is one in which no fluorescence is detected.
Using this new assay, the scientists were able to test a library of almost 16,000 bioactive molecules at 7 different concentrations. From the original 16,000 molecules, they identified 60 prospective “hits” for further validation as pol κ inhibitors. Three of these 60 were selected as proof-of-principle compounds and further characterized for their specificity toward pol κ. Although the three compounds selected would, for a variety of reasons, not ultimately be candidates for new anti-cancer drugs, the strategy for finding such drugs was validated in this pilot study. This study has moved the research effort one step closer to the development of pol κ-targeted novel combination cancer therapeutics. The next step in the process is to expand testing to a larger molecular library of over 400,000 compounds.