Mingyu Zhang

Date of Award

6-16-1998

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Michael J. Blake

Abstract

Virtually all cells respond to heat stress by increased expression or induction of the highly conserved heat shock proteins (HSPs). However rat Nb2-11 cells, a prolactin-dependent preT-cell line displayed an unusual heat shock response following exposure to heat stress (41$\sp\circ$C, 1 hr). There was no evidence of inducible HSP70 expression. Moreover, expression of the constitutive HSP70s and HSP90s decreased during the heat stress apparently reflecting a decrease in mRNA stability. Gel shift assays revealed that heat shock factor (HSF) was activated although its DNA binding rapidly deteriorated. Immunoblotting, using an antibody specific to HSF1, indicated that proteolysis of HSF1 may be responsible for this rapid termination of HSE-binding. CCAAT-binding, a component of constitutive HSP70 expression, was also reduced by heat stress in Nb2-11 cells and may account for the decline in constitutive HSP70 expression. Prolactin pretreatment prevented the fragmentation of HSF1, protected HSE- and CCAAT-binding, prevented the decline in constitutive HSP70 and HSP90 expression, and restored a modest expression of inducible HSP70 following heat treatment. Sodium butyrate (NaBT), a differentiating agent, rendered Nb2-SFJCD1 cells, a prolactin-independent cell line, more sensitive to heat-induced HSF proteolysis, resulting in a corresponding loss of inducible HSP70 expression. Further studies identified preexisting cysteine proteases that might be responsible for the proteolysis of HSF. Apparently, the heat shock response in Nb2 cells is attenuated by a mechanism that involves the premature deactivation of HSF by selective proteolysis.It has been well documented that activation of proteases particularly cysteine proteases are involved in the apoptotic process. Whether HSF is a specific target for activated proteases in cells undergoing apoptosis has been investigated. Apoptosis was invoked by heat treatment in both Nb2-11 and Nb2-SFJCD1 cell lines as determined by DNA fragmentation on agarose gels and flow cytometry. Other apoptotic stimuli such as vinblastine, cycloheximide, and thapsigargin, were all potent stimulators of HSF proteolysis and apoptosis in both cell lines. PRL correspondently inhibited both HSF proteolysis and apoptosis induced by these stimulators. Iodoacetamide, a cysteine protease inhibitor that blocks HSF fragmentation, was also an effective inhibitor of apoptosis. Therefore the proteolysis of HSF is associated with apoptosis in Nb2 lymphoma cells. HSF proteolysis and the concurrent loss of an appropriate heat shock response appear to contribute to apoptotic mechanisms in these cells. These studies may provide insight into the mechanisms of tumor progression from hormone dependency to malignancy and into possible treatment strategies for hormone-dependent tumors.

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