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Tobacco, Nitrosamines, Damage, Repair, Mutations, and Cancer

Institution: Beckman Research Institute of the City of Hope
Investigator(s): Timothy O'Connor, Ph.D.
Award Cycle: 2008 (Cycle 17) Grant #: 17RT-0109 Award: $654,674
Subject Area: Cancer
Award Type: Research Project Awards

Initial Award Abstract
Despite major advertising campaigns, smoking remains a major public health concern and can lead to lung cancer. Lung cancer progressively develops from damage by chemicals to DNA in cells. Failure to repair DNA damage leads to mutations, until finally, the accumulation of mutations in critical genes results in cancer. Tobacco and tobacco smoke are intricate chemical mixtures that contain carcinogens, and the composition of these mixtures complicates their cancerous effects. To evaluate mixtures that form mutations, and consequently cancer, it is important first to understand how the components function independently to alter cellular DNA. Nitrosamines (NAs) are a class of potential carcinogens that are found in tobacco, mainstream tobacco smoke and side stream tobacco smoke. NAs are divided into two categories: those found in many products (e.g., cured meats, synthetic rubber, etc.) and those that are tobacco-specific. However, data on how these agents form mutations are lacking. Determining the ability of various NAs to induce mutations in cells is vital information toward understanding the development of lung cancer. Therefore, we will evaluate the steps leading to mutations caused by individual NAs in mammalian cells.

Human cells contain many genes that produce proteins to control cell division. The mutation process begins with modification of DNA by NAs. Repair of this DNA damage is generally efficient, but depending on the DNA sequence and location, some modifications can remain during the process of creating a new DNA copy prior to cellular division. The persistence of an unrepaired DNA modification can lead to a DNA mutation in one of the two new cells. Accumulation of mutations in the DNA of genes that control cell division results in unbridled growth and the formation of a cancer. Lung cancer occurs after many decades of exposure to the DNA damaging compounds in tobacco, and can be considered as a failure to repair DNA damage in cells. The progression from DNA modification to mutation is difficult to measure in normal cells, and it is even more difficult to study DNA damage. Although mutations can bestow a growth advantage on cells, cells with a single mutation are often indistinguishable from the original cells. Therefore, we will use two types of genes in our experiments.

One important growth control gene native to human cells will be used to monitor DNA damage and compare that to mutations found in human cancer. Since this native gene will generally not manifest differences with a single mutation, we will use another type of gene that we can take out of human cells to evaluate the mutations. The other gene found in a bacterial virus is not normally found in human cells, but we will insert it into human lung cell DNA so that we can study both damage and mutations.

First, we will map NA-induced damage in the native gene that is often mutated in lung cancer and in the viral gene embedded in the human lung cells. Then we will map the mutations caused by NAs at each base in a DNA sequence. The information obtained will include the percentage of cells that have mutations following NA exposure (mutation frequencies), the types of mutations formed (mutation signatures), and a map of the actual mutation positions in the DNA sequence. Thus, for single species of NAs, we can compare processes that are similar to the first steps leading to lung cancer. Human lung cells in culture are a better model than animal cells in culture, but still lack the structure found in lung tissue. To mirror more closely modifications to human lung, we will use a model that approximates human lungs and uses the same types of cells. We will then introduce cigarette smoke and its condensate to these systems to examine the effects of complex mixtures on DNA damage and mutations. These investigations will permit us to evaluate the damage and mutagenicity of each NA alone and compare those to complex mixtures.

We anticipate that this data will provide insights into the mechanism by which cells develop cancer, which can prove useful in the early diagnosis of mutations leading to cancer.