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"TUMOR-INDUCED BYSTANDER RESPONSES IN NORMAL NEURAL STEM/PROGENITOR CELLS: THE ROLE OF SECRETED FACTORS"

by
Neha Sharma
Molecular Biology, Genetics and Cancer Track
B.S. 2003, Gujarat University, India
M.S. 2005, Gujarat University, India


Thesis Advisor: Edouard I. Azzam, Ph.D.
Professor
Department of Radiology

Friday, June 24, 2016



Abstract

Neural stem cells are uncommitted, multi-potent cells generated throughout the life of an individual. They exist in embryonic, fetal and adult tissues, and give rise to rapidly amplifying progenitors responsible for proper formation and functioning of the central nervous system (CNS). Therefore, signaling events that affect their survival or self-renewal and differentiation, will likely impact development of the CNS, and may result in the emergence or exacerbation of progressive degenerative outcomes. In this project, we investigated the effect of soluble factors secreted by control or 137Cs-É◊-irradiated glioblastoma or medulloblastoma cells on redox-modulated endpoints in recipient human NSPs. We also investigated the modulating effects of the tumors°¶ microenvironment on the induced effects.
Glioblastoma multiforme is the most common and most aggressive primary brain tumor in adults. In contrast, medulloblastoma is the most common pediatric malignant brain tumour. Ionizing radiation is a mainstay of therapy for both tumors. Therefore, targeted or bystander effects induced by ionizing radiation that adversely affect the viability, self-renewal or differentiation of stem cells can contribute to the late debilitating effects observed after therapy.
In the first section of the Results, we report on the effects of diffusible factors secreted by glioblastoma and medulloblastoma cells on the induction of oxidative stress in bystander neural stem progenitors (NSPs). Growth medium harvested from the non-irradiated brain tumor cells, following 24 h of growth, induced prominent oxidative stress in recipient NSPs as judged by overall increases in mitochondrial superoxide radical levels (p<0.001), activation of c-jun N-terminal kinase, and decrease in the active form of FoxO3a. The induced oxidative stress was associated with phosphorylation of p53 on serine 15, a marker of DNA damage, induction of the cyclin dependent kinase inhibitors p21Waf1 and p27Kip1, and perturbations in cell cycle progression (p<0.001). These changes were also associated with increased apoptosis as determined by enhanced annexin V staining (p<0.001) and caspase 8 activation (p<0.05), and altered expression of critical regulators of self-renewal, proliferation, and differentiation. Surprisingly, exposure of the tumour cells to radiation only slightly altered the induced oxidative changes in the bystander NSPs, except for medium from irradiated medulloblastoma cells that was more potent at inducing apoptosis in the NSPs than medium from non-irradiated cells (p<0.001). The elucidation of such stressful bystander effects provides avenues to understand the biochemical events underlying the degenerative outcomes associated with brain cancers. It is also relevant to tissue culture protocols whereby growth medium conditioned by tumour cells is often used to support the growth of stem cells.
In the second section of the Results, we examined the role of c-Jun N-terminal kinase signaling in regulation of the redox homeostasis in human neural stem precursors.The goal was to gain greater understanding of the mechanisms that maintain healthy survival of NSPs, because at present little is known about the mechanism underlying the regulation of the redox environment in NSPs, which is essential for homeostatic cellular functions. The redox-modulated c-Jun N-terminal kinases (JNK) are a molecular switch in stress signal transduction and are involved in numerous brain functions. Using a selective but broad spectrum inhibitor of JNK1/2/3, we investigated the role of JNK in regulating the levels of reactive oxygen species in mitochondria, mitochondrial membrane potential, and survival in H9 neural stem/progenitor cells. Relative to control, incubation of H9 cells for 24 h with SP600125, an anthrapyrazolone inhibitor of JNK, resulted in increased abundance of mitochondrial superoxide radicals (p<0.001), concomitant with decreases in mitochondrial membrane potential (p<0.001), in the levels of active nuclear FoxO3a, and in survival (p<0.001). Together, the results support a role for JNK in the redox-homeostasis of neural stem/progenitor cells. Identifying regulators of the cellular redox environment will enhance our understanding of the mechanisms that modulate neural stem cell functions and optimize therapeutic applications targeting JNK.
In sum, this thesis project shows that regulation of the cellular redox environment in NSPs is exquisitely sensitive to regulation by soluble factors secreted by normal or tumor cells in their microenvironment. It also highlights a prominent role for the c-Jun N-terminal kinases in regulation of their redox homeostasis.


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