טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentElbaz Yael
SubjectPost-Translational Regulation of iNOS in Normoxia and
Hypoxia in Macrophages and Tumor Cells
DepartmentDepartment of Medicine
Supervisors Professor Nitza Lahat
Professor Emeritus Haim Bitterman
Dr. Michal Rahat
Full Thesis textFull thesis text - English Version


Abstract

The tumor microenvironment, which is rich in chemokines, cytokines and growth factors, plays a critical role in tumor initiation and progression. Low oxygen tensions (hypoxia) characterize fast growing solid tumors, and it correlates with worse prognosis, higher invasiveness and resistance to chemo- and radiotherapy. Tumor associated macrophages (TAMS) that infiltrate the tumor and accumulate in these hypoxic areas, can potentially eradicate tumor cells by direct cytotoxicity or by initiating apoptosis via generation of high amounts of nitric oxide (NO). In contrast, low amounts of NO are pro-angiogenic and are associated with tumor progression. Although high expression of inducible nitric oxide synthase (iNOS), which produces NO, was found in TAMs, their cytotoxicity in vivo is often ineffective and they are associated with increased tumor aggressiveness. We have previously shown that hypoxia inactivates iNOS, without affecting its expression, by reversibly disrupting its interaction with the cytoskeletal protein α-actinin 4 (ACTN4), leading to its displacement from its intracellular localization at the sub-membranal region. Little is known about the actual amounts of NO produced within the tumor microenvironment. Our aim was to delinieate the hypoxic regulation of iNOS expression and activity in both macrophages and tumor cells. We show that IFNg and LPS induce iNOS expression not only in the mouse RAW 264.7 macrophage cell line, but also, to various degrees, in the mouse TRAMP-C1 and TRAMP-C2 prostate cancer cell lines, as TRAMP-C1 expressed low levels and TRAMP-C2 expressed high amonts of iNOS. In both tumoral cell lines hypoxia did not change iNOS expression, but it completely abolished production of NO and reoxygenation restored this production to its normoxic levels. As we have shown before, in both macrophages and tumor cell lines this hypoxia-induced inactivation of iNOS was associated with the displacement of iNOS from the sub-membranal areas, and disruption of its co-localization with the ACTN4, whose expression and intracellular localization at the periphery of the cells remained constant even in hypoxia. In addition, we show that production of NO depends on the integrity of the actin cytoskeleton, as cytochalazin B, but not nocodazole, which inhibit the polymerization of the actin and tubulin cytoskeleton, dose-dependently reduced NO production on both RAW 264.7 and TRAMP-C2 cells. We conclude that hypoxia post-translationally inhibit NO production in a mechanism which is common to macrophages and tumor cells, and we suggest that in the tumoral context these hypoxia-reduced amounts of NO may be pro-angiogenic rather than cytotoxic, and promotes tumor growth.