Proteolysis of heat shock transcription factor is associated with apoptosis in rat Nb2 lymphoma cells.

Abstract

Previously, we reported that prolactin (PRL)-dependent Nb2 lymphoma cells exhibit an aberrant heat shock response because of cysteine protease-mediated fragmentation of the heat shock transcription factor (HSF). Moreover, exposure of the cells to PRL abrogated heat-induced HSF proteolysis. The present study was conducted to investigate whether HSF proteolysis is a component of the apoptotic process in this model. Initially, the effect of heat stress (41 degrees C for 1 h) on apoptosis, determined by agarose gel electrophoresis and flow cytometric analysis, was evaluated in PRL-dependent Nb2-11 cells and in an autonomous subline (Nb2-SFJCD1). Heat was found to induce HSF proteolysis concomitant with activation of apoptosis in each cell line; treatment with PRL blocked these effects. To determine whether HSF proteolysis occurred as a generalized phenomenon associated with apoptosis, the effects of other activators of this process were evaluated. Vinblastine, cycloheximide, and thapsigargin stimulated fragmentation of HSF and hydrolysis of DNA in each cell line. The addition of PRL blocked the effects of vinblastine but was ineffective in cells treated with either cycloheximide or thapsigargin. Iodoacetamide, a cysteine protease inhibitor that blocks HSF fragmentation, also inhibited apoptosis. In addition, Z-VAD, a general caspase antagonist, blocked vinblastine-induced fragmentation of HSF and DNA, suggesting that the enzyme responsible for proteolysis of the transcription factor was likely a caspase family member. The results suggest that proteolysis of HSF reflects the action of one or more caspases activated as a consequence of stimulation of cell death. It is concluded that HSF may represent a previously unrecognized substrate for caspases or other cysteine proteases activated during apoptosis.