The effects of chronic and acute toxicological exposures on the expression of the tumor suppressor p16INK4a

Sorrentino, Jessica A.

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March 22, 2019

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Sorrentino, Jessica A.
- Affiliation: School of Medicine, Curriculum in Toxicology

Abstract

In mammals, expression of p16INK4a is highly regulated. Excess expression can lead to cellular senescence and aging, while impaired activation is associated with cancer. The precise mechanism of p16INK4a regulation in vivo is poorly understood. In vitro systems have limited utility since proliferation in culture induces p16INK4a . Both extrinsic (chemotherapy and ionizing radiation) and intrinsic (telomere shortening and improper DNA damage repair) stimuli can induce p16INK4a , but the kinetics of and cellular responses to these genomic insults have not been examined in vivo. To address this question, we developed a murine strain with firefly luciferase `knocked-in' to the endogenous p16INK4a locus and under control of the p16INK4a promoter (p16LUC). To determine the expression of p16INK4a after chronic exposure, we exposed p16LUC mice to 50 ppm arsenic (As), 42% fat diet (HFD), 350 J/m2 Ultraviolet B light (UVB), or cigarette smoke (CS) for a minimum of 6 months. Every other month, p16LUC mice were imaged to measure luciferase induction. At 30 weeks of exposure, mice exposed to CS displayed 2 times higher levels of whole body luciferase activity than ambient air (AA) controls. Additionally, mice exposed to UVB exhibited 1.5 times higher levels of luciferase activity by 6 weeks of exposure that reached 8 times over control mice by 24 weeks. In As exposed mice, there was a slight, but statistically significant induction of p16INK4a by 24 weeks. We observed no differences in p16INK4a expression in mice on HFD compared to normal diet (ND, 4%) after 78 weeks. We can deduce the direct DNA damaging agents, CS and UVB, are leading to an induction of p16INK4a while in direct DNA-damaging agents (e.g. As) cause a slight induction of p16INK4a . However, non-DNA damaging agents, such as HFD, show no changes in expression when compared to ND controls. To determine the regulation of transient p16INK4a expression, mice were exposed to a high dose of UVB light (2000 J/m2). The mice were then imaged for luciferase induction at various time points over one month, allowing the burn to heal. To manipulate the induction of p16INK4a the mice were treated with pharmacological inhibitors, including dexamethasone (dex), clodrosome (clod), and compound A (cA). Dex, a known immunosuppression, showed a decrease in p16LUC expression, during the healing process along with clod, known to be toxic to macrophages, and cA, a p65 inhibitor. This data suggests that inflammation at the site of the burn can lead to the induction of p16INK4a through the recruitment of macrophages along the Nfκb pathway. The data generated from these experiments will demonstrate how environmental exposures are associated with expression of p16INK4a , a mediator of tumor suppression and aging.

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