Abstract
Virtually all cells respond to heat stress by increased expression or induction of one or more of the highly conserved cellular stress response proteins, heat shock proteins (HSPs). Here, we report the unusual property of rat Nb2-11 cells, a prolactin-dependent pre-T-cell line, to display reduced HSP expression following exposure to elevated temperature. After heat stress (41 degrees C, 1 h), there was no evidence of inducible members of the 70 kDa HSP family, a response common to other cell culture and tissue systems. Moreover, expression of constitutive members of the HSP70 and HSP90 families decreased during the heat stress, apparently reflecting a decrease in mRNA stability. Gel shift assays revealed that heat shock factor (HSF) was activated in spite of the lack of expression of inducible HSP70 transcripts, 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 heat shock element 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 heat shock element and CCAAT binding, prevented the decline in constitutive HSP70 and HSP90 expression, and restored a modest expression of inducible HSP70 following heat treatment. Results of this study describe a unique regulatory defect in HSP expression in Nb2-11 cells, possibly a common characteristic of other hormone-dependent tumors.
Highlights
At both the cellular and organismic level, heat and other metabolic and environmental stressors are known to cause a rapid and massive synthesis of a set of highly conserved proteins termed heat shock proteins (HSPs1; for review, see Refs. 1 and 2)
Total RNA was extracted from cells at each time point, and HSP70, HSP89␣, and HSP89 mRNA levels were determined by Northern blotting (Fig. 1)
The magnitude of expression of this transcript was consistently greater in Nb2-SFJCD1 cells. This transcript corresponds to a constitutively expressed HSP70 gene observed in other cells and tissues that hybridized with our cDNA probe, as it is similar in size and present in unstressed cells
Summary
At both the cellular and organismic level, heat and other metabolic and environmental stressors are known to cause a rapid and massive synthesis of a set of highly conserved proteins termed heat shock proteins (HSPs1; for review, see Refs. 1 and 2). Expression of the human constitutive HSP70 gene(s) is cell cycle regulated [5] and is induced by both serum [6] and the adenovirus E1a protein [7]. The multimerization of HSF confers DNA binding activity, which allows the complex to bind to a conserved and repetitive DNA sequence (heat shock element (HSE)) that has been identified in the promotor region of the HSP70 gene [13, 14]. The addition of picogram quantities of PRL to such stationary cells reinitiates cell cycle progression [19] This line has proven invaluable for the study of PRL receptor coupling to a growth response.
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