Vol. 186, No. 1, 1992

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July 15, 1992

Pages 166-172

INDUCTION OF THERMOTOLERANCE IN T CELLS PROTECTS NUCLEAR DNA TOPOISOMERASE I FROM HEAT STRESS Richard P. Ciavarra 1" , Weijia Duvall 2, and FrankJ. Castora 2 D e p a r t m e n t s of 1Microbiology and Immunology and 2Biochemistry, Eastern Virginia Medical School, Norfolk, VA 23507-1696 Received May 18, 1992

In this study, w e have demonstrated that t o p o i s o m e r a s e I DNA relaxing activity i p r o t e c t e d against a severe heat shock in T cells m a d e thermotolerant by a prior modes heat treatment. However, following a severe heat-shock challenge and incubation a 37°C, t o p o i s o m e r a s e activity in the control p o p u l a t i o n eventuaiiy returned to level similar to those detected in thermotolerant cells. This recovery of topoisomeras, activity appears to result from the renaturation of heat-inactivated enzyme rather that from synthesis of n e w p r o t e i n because the rate of recovery of catalytic activity was no inhibited by the presence of the protein synthesis inhibitor, cycloheximide. ~ z992 Academic

Press,

Inc.

Elevated temperatures are k n o w n to induce a transient resistance to s u b s e q u e n heat e x p o s u r e in cells from a variety of organisms. This p h e n o m e n o n , t e r m e d acquire¢ thermotolerance, is associated with the rapid and preferential synthesis of a small set c highly conserved proteins, called hsps (reviewed in 1). Hsps are i n d u c e d by a n u m b e of apparently unrelated environmental stresses such as exposure to heavy metals alcohol, and oxidative stress, and have, therefore, also b e e n referred to as stres proteins. The diverse group of environmental insults that can induce hsp synthesie suggest that a c o m m o n induction signal, such as protein denaturation or alterec folding of newly m a d e proteins, is responsible for hsp gene activation. Numerou,, studies have implicated m e m b e r s of the hsp70 family in protecting cells from therma damage and/or e n h a n c e d recovery from thermal stress (reviewed in 2). In addition tc protecting cells from environmental insults, m e m b e r s of the hsp70 family have also b e e n s h o w n to provide important functions in nonstressed cells such va depolymerization of clathrin from coated vesicles (3), and facilitation of transpor across e n d o p l a s m i c reticulum and mitochondrial m e m b r a n e s (4,5).

*To w h o m correspondence should be addressed. Abbreviations: CHX, cycloheximide; c o n A, concanavalin A; hsps, heat shock proteins; hsc70, 70 k cognate2 or cons/itutively p r o d u c e d m e m b e r of the hsp70 family; PEG, polyethylen I glycol;~H-TdR, JH-thymidine. ooo6-291x/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Previous studies from one of our laboratories (6) d e m o n s t r a t e d that T cells .*xposed to a m o d e s t heat shock (induction temperature, 42.5°C for 60 min) were :apable o f r e s u m i n g DNA synthesis (T cell proliferation) at an accelerated rate relative o control cells (induction temperature, 37°C for 60 min) following a subsequent ;evere heat-shock challenge (44-45°C for 30 rain). The d e v e l o p m e n t of h e r m o t o l e r a n c e in T cells was associated with the induction of hsp synthesis ~articularly hsp90, hsc70 and the highly inducible hsp70. The close temporal tssociation b e t w e e n induction of hsp synthesis and protection of T cell proliferation tgainst heat stress suggests that hsps may be involved in protecting the cell's DNA ,Tnthetic machinery. H o w this might occur remains to be clarified. We have speculated hat hsps m a y protect a critical enzyme(s) required for DNA synthesis from stressn d u c e d damage. In this regard, DNA topoisomerases have b e e n implicated as m p o r t a n t enzymes in both DNA replication and transcription. Replication and ~ranscription are associated with conformational and topological changes in DNA and ~opoisomerases have evolved to a c c o m m o d a t e these changes. Two types of :opoisomerases have b e e n identified; one that produces transient protein-bridged tingle-strand breaks in DNA (type I topoisomerase) and another that produces double;trand breaks (type II topoisomerase, reviewed in 7 ). Hsps m a y interact with these m z y m e s during hyperthermic stress which may minimize thermal damage and/or tccelerate restoration of their native conformation and enzymatic activity. We have, therefore, b e g u n a study to assess whether protection of DNA synthesis against thermal injury in t h e r m o t o l e r a n t cells is associated with protection of topoisomerase I activity. MATERIALS AND METHODS G r o w t h a n d i n d u c t i o n o f t h e r m o t o l e r a n c e in T cells: Purified T cell .ymphoblasts were prepared by Con A stimulation of purified splenic T cells following 3ur previously r e p o r t e d p r o c e d u r e (6). In this study Con A activated T lymphoblasts will be referred to a T cells. After 48 -72 hrs of stimulation, T cells w e r e exposed for one hour to a control (37uC) or modest (42.50C) heat-shock t e m p e r a t u r e which we had previously d e m o n s t r a t e d induces a transient thermotolerant p h e n o t y p e (8). Following a recovery period of a/.p~roximately 12 hrs at 37°C, both populations were given a heatshock challenge at 44 C for 30 min. Cells were collected and either aliquoted for immediate extract preparation or evaluated for their ability to r e s u m e DNA synthesis. In some experiments CHX (50 uM, final concentration) was included in the m e d i a during the heat-shock challenge and recovery period. ,. P r e p a r a t i o n o f extracts: T cells (5-30x10 °) were washed once with o n e ml PBS and lysed with high salt (300 ul 5xPBS). DNA and cell debris were precipitated with the addition of 150 ul 18% PEG containing 1.0 M sodium chloride and r e m o v e d by a 10 min microfugation. The 300-400 ul of supernatant was recovered and assayed for topoisomerase activity. T y p e I T o p o i s o m e r a s e assay: Type I topoisomerase assays w e r e p e r f o r m e d with supercoiled pUC 19 plasmid DNA as substrate essentially as described in Lazarus et al. (9). Agarose gel electrophoresis was used to separate DNA of differing superhelical densities. O n e unit of topoisomerase relaxing activity removes 50% of the supercoils from the substrate in 30 min at 30°C. Data are presented as % of control following o u r previously r e p o r t e d calculation (6). T cell p r o l i f e r a t i o n assay: DNA ~ n t h e s i s was evaluated by incubating T cells in the presence of Con A (1.0 ug/ml) and aH-TdR (1.0 uCi/well) following o u r previously r e p o r t e d p r o c e d u r e (6). Following an overnight incubation (18-20 hours), cells were 167

V o l . 186, N o . 1, 1992

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extent of DNA synthesis d e t e r m i n e d by liquid scintillatic

RESULTS Fig. 1 shows the results of a typical assay for type I topoisomerase. Groups A, l and C are triplicate samples of serial dilutions (A, neat; B, 1/3; C, 1/9) of a cellul," extract p r e p a r e d from T cells grown at 37°C. The u n d i l u t e d sample (group A) sho~ the greatest a m o u n t of topoisomerase activity as evidenced by the nearly complel conversion o f supercoiled form I DNA into m o r e relaxed forms (Ir). Groups B and exhibit decreased a m o u n t s of topoisomerase-generated I r DNA and increased a m o u n t of supercoiled DNA as the extract is diluted. Groups D, E, and F are triplicates c equivalent serial dilutions of T cells given a heat shock (44 °C) for 30 minutes. There i a r e d u c t i o n in topoisomerase activity from the T cells exposed to 44 °C as manifested b the r e d u c e d conversion of highly supercoiled DNA to m o r e relaxed forms in groups E E, and F relative to those comparable samples in groups A-C. Although there is som, cell death associated with the hyperthermic exposure, the results have b e e n n o r m a l i z o to equivalent n u m b e r s of viable cells. As n o t e d above, t h e r m o t o l e r a n t T cells are able to r e s u m e DNA replication at aJ accelerated rate relative to control cells following a severe heat-shock challenge. It wa therefore of interest to determine whether this ability to r e s u m e DNA synthesi following t h e r m a l injury is associated with protection of type I topoisomerase. In ordel to test this possibility, thermotolerant and control T cells were subjected to thq indicated heat-shock challenge temperatures and t h e n immediately assayed for residua t o p o i s o m e r a s e activity. Aliquots of cells were also removed and tested for their abilir to reinitiate Con A-induced proliferation (i.e., r e s u m p t i o n of DNA replication). It car be seen from the results presented in Figure 2 (left panel) that prior m o d e s t heat shoc~ /

protected cellular DNA synthesis from thermal injury. We also observed marked p r o t e c t i o n of topoisomerase I activity in thermotolerant cells. For example, following ; 45°C heat shock, approximately 6X more enzyme activity was detected in the t h e r m o t o l e r a n t cells versus control T cells (Figure 2, right panel). These data suggesl

A

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Figure 1. Gel electrophoretic separation of the products of topoisomerasecatalyzed relaxation of plasmid DNA. The first lane is control, untreated pUC19 DNA.. The subsequent grouos of lanes labeled A, B, and C are triolicate samples treated with neat, 1/3, and 1/9 diluted extract, respectively, from ceils grown at 37°C. Groups labeled D, E, and F are triplicate samoles treated with neat, 1/3, and 1/9 diluted extract from cells given a 44°C heat shock. DNA species labeled I, II, and I r are highly supercoiled,nicked, and enzymaticaUy relaxed circular DNA, respectively. 168

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Figure 2. Relationship between resumption of DNA synthesis and type I topoisomerase activity following heat stress. T lymphoblasts were subjected to an induction temperature of either 37°C (open circles) or 42.5 °C (closed circles), rested and then incubated for 30 minutes at the indicated heat shock temperatures. Immediately following heat shock, an aliquot of cells was removed to determine their ability to resume DNA synthesis (left panel). The remaining ceils were used to prepare extracts for type I topoisomerase activity determinations (right panel). :hat t h e ability o f t h e r m o t o l e r a n t

cells t o r e p l i c a t e DNA f o l l o w i n g h e a t stress m a y

J e p e n d , at l e a s t in p a r t , o n p r o t e c t i o n o f this critical e n z y m e . B e c a u s e c o n t r o l cells d o n o t r e c o v e r t h e i r ability to s y n t h e s i z e DNA f o l l o w i n g aeat stress e v e n a f t e r 24 h o u r s o f i n c u b a t i o n at 3 7 ° C , w e a s k e d w h e t h e r this loss w a s :he r e s u l t o f c o n t i n u e d i n a c t i v a t i o n o f t o p o i s o m e r a s e I. This h y p o t h e s i s w a s t e s t e d b y nonitoring :hallenge.

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Figure 3. Effect of heat stress on type I topoisomerase activity and kinetics of recovery. T cells were incubated either at 37°C or 42.5°C and then rested for 10 hours at 37°C. Following this rest period, cells were given a 30 minute severe heat-shock challenge (44°C, 30 minutes) and either tested immediately (t=0) or at the times indicated for topoisomerase activity. 169

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Induction of thermotolerance in T cells protects nuclear DNA topoisomerase I from heat stress.

In this study, we have demonstrated that topoisomerase I DNA relaxing activity is protected against a severe heat shock in T cells made thermotolerant...
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