ANALYSIS OF NELFINAVIR-INDUCED ENDOPLASMIC RETICULUM STRESS

Nelfinavir (Viracept (R)) is an HIV protease inhibitor that has been shown to induce the endoplasmic reticulum (ER) stress reaction in human cancer cells. Although the presumed drug doses needed for an efficient ER stress reaction and ensuing apoptosis in cancer cells is somewhat higher than those prescribed for HIV-infected persons, nelfinavir represents one of the few clinically applicable ER stress-inducing agents, and is currently being tested in clinical studies on cancer patients. Therefore, this chapter describes how to obtain and use nelfinavir for in vitro and in vivo studies. In addition, methods are described that might facilitate the analysis and monitoring of the nelfinavir-induced ER stress response either in cancer cells in cell culture or in cancer tissue biopsies. These methods include various fluorescence-based ER staining techniques and the expression analysis of primary and secondary ER stress markers by immunoblotting and RT-PCR analysis. Among the several methods presented, the analysis of an unconventional XBP1 splicing, caused by the ER stress sensor IRE1, is shown to present the most sensitive and most specific marker for nelfmavir-induced ER stress. Primers and PCR conditions suitable for XBP1 PCR and splicing analysis are presented. Such a PCR-based XBP1 splicing analysis might not only be suitable to monitor nelfinavir-induced ER stress, but could also be applied in drug screening programs to test for other ER stress-inducing agents with similar activities or synergistic activities with nelfinavir.