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Jessica Kaplunov
Microbiology and Molecular Genetics Program
B.S. 2006, Seton Hall University

Thesis Advisor: Utz Herbig, Ph.D.
Assistant Professor
Department of Microbiology and Molecular Genetics

Thursday, August 9, 2012
12:00 P.M., ICPH Auditorium


Cellular senescence is a stable growth arrest that can function as a potent tumor suppressing mechanism and also contribute to organismal aging. Although a variety of stresses can cause cells to become senescent, a primary cause for this irreversible growth arrest is the dysfunction of telomeres, the ends of linear chromosomes. Repeated rounds of DNA replication has been demonstrated to shorten telomeres, leading to telomere dysfunction, activation of DNA damage checkpoints, and subsequent cellular growth arrest.
Aging is characterized by a progressive loss of tissue structure and function; however, the cause of tissue degeneration remains poorly understood. Accumulation of senescent cells in ageing tissues might partially explain the decline in tissue function and regeneration observed in old age. Previous work in the Herbig laboratory found a significant association between senescent fibroblasts and age in baboon skin. Current work indicates that senescent cells displaying dysfunctional telomeres accumulate with age in the liver, adrenal gland, and pancreas, but not in heart, frontal cortex, lung, kidney or colon of aging baboons. Though the liver, adrenal gland and pancreas are proliferative tissues, dysfunctional telomeres are not preferentially shorter than other telomeres in the same cell suggesting that telomere erosion is not the primary cause of dysfunction. Our work reveals tissue specific age-associated telomere dysfunction in aging primates.
Short and dysfunctional telomeres have been proposed to play a critical role in the progression of human breast cancer. In the context of deregulated p53 and Rb signaling telomere dysfunction leads to chromosomal instability and cell death, a situation frequently observed in advanced breast cancer precursor lesions such as high grade ductal carcinoma in situ (DCIS. We demonstrate that ductal hyperplasias of the breast, benign breast cancer precursor lesions, are comprised almost entirely of cells that displayed markers of cellular senescence. Cells in ductal hyperplasias, but not in DCIS or invasive carcinomas, contained multiple dysfunctional telomeres and elevated levels of macroH2A, a protein involved in senescence associated heterochromatin formation indicating that these cells have undergone telomere dysfunction induced senescence (TDIS). To characterize the causes for TDIS in breast cancer precursor lesions, we asked whether oncogenic signaling, an early event in cancer development, affects telomere dysfunction. We demonstrate that oncogenic H-Ras-V12 (Ras) induced-DNA replication stress led to both non-telomeric and telomeric DNA damage foci. While non-telomeric foci were repaired and diminished over time, telomeric foci were not and persisted causing TDIS. We used drugs which specifically induce DNA replication stress in normal human fibroblasts and observed TDIS demonstrating that telomeres are vulnerable to DNA replication stress. Taken together, our data demonstrate that oncogene induced DNA replication stress causes TDIS and that TDIS in cells comprising ductal hyperplasia is a critical tumor suppressing mechanism
Oncogenic HRasV12 both induces an oncogene-induced DNA replication stress induced TDIS response and increased reactive oxygen species (ROS). Here, it is determined that ROS cause telomere dysfunction. Since Ras induces DNA replication stress preferentially at telomeres and Ras induced senescence has been demonstrated to be dependent on ROS production, we hypothesized that Ras induced ROS cause DNA replication stress at telomeres. We demonstrate that ROS generated by drug treatment or high oxygen conditions cause growth defects telomere dysfunction. Knockdown of oxidative damage repair proteins OGG1 and MTH1, which repair and eliminate 8oxodG, in normal human fibroblast (BJ) cells and hTERT-immortalized BJ cells, rapidly induces TDIS. Conversely, overexpression of these proteins protects against telomere dysfunction in BJ cells exposed to oxidative stress. 8oxodG has been proposed to play a role in mutagenesis and carcinogenesis, and consistent with this, we found that, co-expression of MTH1 and Ras reduces Ras induced DNA damage, telomere dysfunction, and DNA replication stress. Our data suggest that oxidative stress triggers TDIS and DNA replication stress and that suppression of 8oxodG formation diminishes oncogene-induced senescence.

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