Novel Functions And Physiological Significance Of Ral Interacting Protein - RLIP76 In In Vitro And In Vivo Models

This dissertation is divided into four parts. The first part is an introduction of energy dependent xenobiotic transporters and their relevance in multidrug resistance. However, the main focuse is the description of RLIP76 protein, non-ABC transporter, which it the most efficient transporter of glutathione conjugates and anti-cancer drugs. The second part describes materials and methods that were used in two investigated projects.

The third part explains the correlation of RLIP76 with signaling protein POB1 and an involvement of these two proteins in apoptosis of lung cancer cells. The effect of recombinant POB1 and its deletion mutant POB11-512, on transport activity of RLIP76 towards doxorubicin and dinitrophenyl-glutathione were studied. The results demonstrated that, along with an increased POB1/RLIP76 ratio, the transport of doxorubicin and dinitrophenyl-glutathione by RLIP76 decreased. Incubation of cancer cells with recombinant POB1 led to an arrest of their cell cycle. Augmentation of cellular POB1 caused increasing intracellular doxorubicin accumulation as well as a decreasing rate of doxorubicin efflux from cells. These results showed for the first time that augmented POB1 inhibits RLIP76 transport function, leading to apoptosis of cancer cells through the accumulation of endogenously formed GSH-conjugates. In the fourth part, it was demonstrated, that RLIP76, through participation in ligand-receptor complex endocytosis, can mediate insulin-resistance. Insulin sensitivity tests demonstrated that administration of increasing doses of recombinant RLIP76 resulted in development of insulin resistance in RLIP76 wild type (WT) mice. Double administration of insulin to RLIP76 knockout (KO) animals caused a dramatic increase of insulin sensitivity. Distribution of hydrocortisone, into RLIP76 KO animals did not have any effect on glucose level. Lack of RLIP76 in KO animals did not change an expression of phosphoenolpyruvate carboxykinase and fructose 1,6-bisphosphatase but specific activities of these enzymes were diminished. Cell culture studies showed that inhibition of RLIP76 resulted in increased glucose uptake, Akt1 phosphorylation, FOXO1 inactivation, and a reduction of endocytosis of insulin and epidermal growth factor. Augmenting RLIP76 by cell transfection caused an opposite effect. These studies demonstrated that RLIP76 antagonizes the effect of insulin, and link oxidative stress with the mechanisms which function to terminate insulin-signaling through ligand-receptor complexes endocytosis.