Understanding p16INK4a regulation in vivo after exposure to noxious stimuli

 

Trainee:

Jessica Sorrentino

sorrentino

Research Mentor:

Dr. Norman Sharpless, MD

sharpless

Clinical Co-mentor:

Dr. Hyman Muss, MD

muss
Home Department Toxicology
Project Description

In mammals, expression of p16INK4a is delicately balanced. Overexpression increases cellular senescence, which can promote aging. Underexpression permits aberrant proliferation, sometimes leading to tumorigenesis. Many noxious agents, specifically DNA damaging agents such as UV light, chemotherapy, oxygen radicals, ionizing radiation, and telomere dysfunction can sometimes lead to an increase in p16INK4a expression. This increase will lead to hypoproliferation and sometimes, cellular senescence. The precise mechanism of p16INK4a regulation with cancer and aging in vivo is poorly understood. In vitro systems are of limited utility for this question as the act of culture potently induces p16INK4a expression in most mammalian cell types. In vivo analyses (e.g. IHC) have been limited by low expression of this potent tumor suppressor gene, as well as a lack of good reagents for detections (e.g. antibodies). For these reasons, we hereby propose to generate a p16INK4a reporter allele to facilitate our understanding of p16INK4a regulation with cancer and aging.

Through various techniques such as GFP imaging, quantitative PCR, immunohistochemistry, western blotting and flow cytometry, I will assess the correlation between p16Ink4a induction and GFP expression in our model system. These studies will determine the sensitivity and accuracy of measuring p16INK4a induction using an in vivo GFP reporter system. Although another p16INK4a reporter model has been generated in the Sharpless lab, the p16INK4a-GFP allele has the advantage that it will allow for the identification and purification of p16INK4a expressing cells through fluorescent-activating cell sorting (FACS) and fluorescent imaging. The generation of the p16INK4a -GFP murine model will help quantify p16INK4a levels in response to the sustained toxicity of various DNA damaging agents, as well as help me develop a better assay for measuring p16INK4a expression in blood. These studies can bring us closer to understanding how these noxious agents lead to advanced aging and better ways to decrease toxicity from prolonged drug treatment. Moreover, the murine data using the p16INK4a -GFP allele will inform our future human clinical trials and help develop a better assay. The new assay will make it easier to perform large, statistically powered human clinical trials. These studies could help the decisions of chemotherapy treatments or IR exposure by using the individual’s cellular physiology, to plan therapy.