The Molecular Mechanisms that Regulate Translational Repression Under Hypoxia and Glucose Deprivation

The inefficient tumor vasculature and high glucose metabolism often found in tumor cells will likely result in tumor microenvironments that have limited availability of oxygen and glucose. Our studies showed that prolonged exposure to hypoxia (>16 hr, 1.0% 02) results in pronounced translational repression in PC3 cells. This delay in translational repression reflects the time needed for glucose to be depleted, since we found that both hypoxia and glucose deprivation (HGD) are required for translational repression. This HGF mediated translational repression is primarily modulated by elF2a phosphorylation, which in turn blocks ternary complex formation and suppresses translation by > 80%. The work presented in this thesis focuses on the molecular events that are responsible for the phosphorylation of elF2a during HGD. We found the PKRlike endoplasmic reticulum kinase (PERK) is responsible for the phosphorylation of elF2a during HGD. This PERK activation correlates with a 10-fold reduction of ATP compared to glucose deprived and glucose replete normoxic cells. Glucose addition or reoxygenation of cells exposed to HGD results in restoration of ATP levels, inactivation of PERK, reduction in elF2a phosphorylation, and translational recovery. To determine if the accumulation of mis-folded proteins in the lumen of the endoplasmic reticulum (ER) causes activation of PERK during HGD, PC3 cells were treated with the protein synthesis inhibitor cycloheximide prior to HGD. Our data demonstrates that inhibition of protein synthesis (which will prevent the accumulation of mis-folded proteins in the ER) did not prevent PERK activation during HGD. This data strongly suggests that PERK activation during HGD is independent of the accumulation of mis-folded proteins in the ER and is most likely a consequence of ATP depletion. Since tumors commonly have regions that are deprived of oxygen and glucose, understanding the pathways activated in response to these dual stresses may provide pertinent information on the growth and progression of tumors.