Copyright 0 1990 by the Genetics Society o f America
Genetic Differences in the Duration of the Lymphocyte Heat Shock Response in Mice Virginia K. Mohl,* Gregory D. Bennettt and Richard H. Finnell*’T *Program in Genetics and Cell Biology and tDepartment of Veterinary and Comparative Anatomy, Pharmacology and Physiology, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164-6520 Manuscript received September 19, 1989 Accepted for publication December 22, 1989
ABSTRACT Lymphocytes from adult mice bearing a known difference in genetic susceptibility to teratogeninduced exencephaly (SWV/SD, and DBA/2J)were evaluated for changes in protein synthesis following an in vivo heat treatment. Particular attention was paid to changes indicative of the heat shock response, a highly conserved response to environmental insult consisting of induction of a few, highly conserved proteins with simultaneous decreases in normal protein synthesis. The duration of heat shock protein induction in lymphocytes was found to be increased by 1 hr in the teratogensensitive SWV/SDstrain as compared to the resistant DBA/2J strain. Densitometric analysis revealed a significant decrease in the relative synthesis of at least two non-heat shock proteins (36 kD and 45 kD) in the SWV/SD lymphocytes as compared to DBA/2J cells. The increased sensitivity of protein synthesis to hyperthermia in the SWV/SD lymphocytes were lost in the FI progeny of reciprocal crosses between SWV/SD and DBA/2J mouse strains. Sensitivity to hyperthermia-induced exenceis recessive to resistance in these crosses. The relationship between altered protein synthesis phaly ~, and teratogen susceptibility is discussed. (MITCHELLa n d LIPPS 1978; MITCHELLet al. 1979), NVESTIGATION of the heat shock response beNaegleria gruberi (WALSH 1980) and yeast (KURTZ et gan with the discovery of a unique set of puffs in al. 1986).Despitethisscrutiny,theprecise role of the heat-pulsed salivary gland chromosomes of Drothese proteins in resistance t o cellular stress and their sophila busckii whichwerelaterassociated with the importanceduringdevelopmentremainsunclear production of specific proteins (RITOSSA1962; TIS(SCIANDRA and SUBJECK1984; PETKOand LINDQUIST SIERES, MITCHELL a n d TRACY 1974).Subsequent 1986; PRIMMETT et al. 1989). of a similar studies revealed the transient induction A common approach utilized in the elucidation of set of proteins known variously as heat shock or stress the function of a gene or gene product is the use of proteins, to be common to both eukaryotic and prokaryotic heat-treated cells (SCHLESINGER, ASHBURNER mutations, three types of which currently are being actively applied in heat shock research. Mutant Eschand TISSIERES 1982; LINDQUIST1986). T h e highly erichia coli strains that are defective in the positive conservednature of theheat shockproteingenes (MORANet al. 1982;LOWE,FULFORDand MORAN regulation of the heat shock response (COOPERand 1983; HUNT andMORIMOTO1985), and their unique RUETTINGER1985; GROSSMAN, ERICKSONand GROSS regulation during periods cellular of stress (TISSIERES, 1984) have been used to demonstrate the importance of these proteins in cell growth at elevated temperaMITCHELLand TRACY 1974), led to the exploitation tures (YAMAMORIand YURA 1982). A strain of Dicof the heat shock response as a model systemwith tyostelium which fails to produce several specific heat which to probe mechanisms of gene induction and shockproteinshas a decreasedabilitytogrowat regulation (NEIDHARDT, VANBOGELELand VAUGHN elevated temperatures (LOOMISand WHEELER1982) 1984; BIENZ1985).Heatshockproteinshavealso been implicated in the acquisition of thermotolerance while in some hydra strains, synthesis of a heat shock (YAMAMORI and YURA 1982; LI and WERB 1982; LI protein correlated with thermotolerance (BOSCHet al. and MAK 1985; BOSCHet al. 1988), andin the recovery 1988). In an attempt to specifically define the funcof normal cellular processes following the application tionsof a low molecular mass heatshockprotein, of a stress(YOST and LINDQUIST1986). These proteins PETKO and LINDQUIST(1986) created deletion and have been correlated with developmental changes in disruption mutations in the 26 kD heat shock protein Drosophila melanogaster organisms as diverse as gene (hsp 26) of yeast. They demonstrated that hsp 26 was notrequiredforseveraldifferentcellular T h e public;ltion costs o f this anicle were partly defrayed by the payment functions includingthermotolerance,germination, o f page cllargrs. Thisxl-ticlt. 111ust therefore bc hereby marked “ n d w r f u e m m t ” in accol-dlnce with 1 X U.S.C. 8 1794 solely to indicate this fact. andgrowthatelevatedtemperatures(PETKOand
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V. K. Mohl. G. D. Bennett and R. H. Finnell
LINDQUIST1986).Unfortunately,similarmutations have yet be described in higher eukaryotes. Lacking specific known mutations, theuse of organisms with known differences in their biological responses to heat stress may provide insight into any possible homeostaticrolesoftheseproteins.Ithas by FINNELLand colrecentlybeendemonstrated leagues (1986) that a strain dependent hierarchy of susceptibility t o a hyperthermia-induced neural tube defect (exencephaly) exists among inbred strains of mice. While the molecular basis of this strain variation is presently unknown, the response is clearly under genetic control. When susceptible (SWV/SD) animals areoutcrossedtomoreresistantstrains(LM/Bc, C576L/6J and DBA/ZJ; FINNELLet al. 1986; V. K. MOHLand R. H. FINNELL, unpublished data), the F1 hybridprogenywereresistanttohyperthermiainducedexencephaly. The degree ofresistancedepended upon the outcross strain, yet all three outcross strains demonstrated that sensitivity was recessive t o resistance toheat-inducedexencephaly.Reciprocal cross experiments indicate that the genotype of the embryo, rather than thephysiological response of the mother, is the major factor in determining susceptibility toheat-inducedexencephaly(FINNELL et al. 1986). T h e discovery of one highly susceptible strain (SWV/SD),severalmoderatelysusceptible(LM/Bc, SWR/J) and resistantstrains (DBA/ZJ, C57BL/6J) provides a unique model in which t o investigate the importance of the heat shock response to embryonic development in a mammalian system. Our working hypothesis has been that an environmental insult capable of disrupting normal mammalian development should have a demonstrable effect on maternal tissues. Previous results from our laboratorysupported thishypothesis by demonstrating induction of heat shock proteins in both embryonic tissues and maternal lymphocytes(BENNETT, MOHL and FINNELL 1990). While investigations of the early embryo provide direct evidence for the teratogenic effectsof theheattreatment, the severelylimited amount of available tissueand its rapid differentiation complicate biochemical analysis of cell function. The lymphocyte assay complements the embryonic tissue studies by providing a sample tissue that is readily isolated with little trauma to the cells and which has well defined culture characteristics and functionalactivity assays. In the studies reported here, the lymphocyte heat shock assay was expanded to examine the kinetics of protein synthesis followingin vivo hyperthermia. Cells from adultmice bearinga known genetic susceptibility for to heat-induced exencephaly were evaluated changes in protein synthesis following a single in vivo heat treatment. In thekinetic study described herein, a heat treatment previously shown to induce devel-
opmentalabnormalities(FINNELL et al. 1986)produced differences in the heat shock response between highly inbred strains of mice susceptible (SWV/SD) and (DBA/ZJ) resistant to heat-induced exencephaly. MATERIALS AND METHODS Two inbred mouse strains (SWV/SD, and DBA/2J) were maintained on a 12-hr light cycle in the College Hall Vivarium at Washington State University, Pullman. Reciprocal cross experiments were performed using the FI generation progeny of matings between the highly susceptible SWV/ SD and the resistant DBA/2J strains. Healthy, pathogenfive per polycarbonate cage and free micewerehoused allowed free access to Purina rodent chow and tap water. Animals 60 to 120 days of age were randomly assigned to treatment groups. All animal procedures were designed to minimizeanystress and discomfort to the experimental animals. Treatment of the adult animals consisted of a single, 10min incubation in either a 43" waterbath for heat stressed animals or a 38" waterbath for control animals. This treatet al. 1986). ment is described in detail elsewhere (FINNELL Core temperatures were recorded at one minute intervals using a YSI telethermometer and small animal probe (Yellow Springs Instruments, Yellow Springs, Ohio). By changing the depth of the restraining chamber in the waterbath, it is possible to control closely therate of temperature increase and thus insure uniformity of heat treatment among individual animals. Therefore, the core body temperature did not vary among the parental or hybrid strains. Heat stressed animals not sacrificed immediately following treatment were patted dry and placed into a 38" warm air incubator for twenty minutes to allow the animal's body temperature to gradually return to its normal basal level. N o age or strain related differences in the kinetics of the temperature curve were noted. For each strain, a minimum of five animals were evaluated at each time point. Following treatment, the mice were sacrificed by cervical dislocation, the spleen aseptically removed and placed into cold Hank's balanced salt solution (HBSS, GIBCO, Grand Island, New York). The spleen was then gently teased apart with mouse-toothed forceps and the cell containing supernatant collected. The number of nucleated cells was determined using a hemocytometer. Both the cells and the buffer were kept on ice throughout the procedure.Cell viability as determined by trypan blue exclusion (MISHELL and SHIICI 1980) ranged between 98% and 96% viable cells. To determine changes inspecific proteins synthesized following an in vivo heat treatment, spleen cells were harvested at 0, 1, 2, and 3 hr following a 10-min treatment at either control (38") or heat shock (43") temperatures. A volume of cell suspension containing between 2 and 5 million cells was collected for labeling with ["S]methionine. Each sample was spun down in a Beckrnan microfuge, the supernatant discarded and the resultant pellet resuspended into 200 pl of cold HBSS. Twenty microcuries [JsS]methionine (NEN Research Products, Boston, Massachusetts) was added and the suspension was allowed to incubate for one hour in a 37" water bath. Following incubation, the cells were washed twice with cold HBSS to remove any unincorporated methionine and to minimize the potential degradation of newly synthesized heat shock proteins (MITCHELL, PETERSON and BUZIN1985). The cells were then lysed with 100 pI LAEMMELI'S (1970) sample buffer and boiled for 10 min. To determine the amount of radioactivity that was incorporated into the cells, duplicate aliquots of sample were
Lymphocyte Response Heat Shock
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counted i n a Beckman scintillation counter with 10 ml Aquasol (Amersham, Arlington Heights, Illinois)as the scintillant. The remainder of the sample was either used immediately for gel electrophoresis or frozen at 70". Cells were considered to express a heat shock response if induction or enhancement of selected protein bands of appropriate molecular weights were detected on autoradiograms of samples separated by SDS-PAGE. The time of 45 w harvesting was selected to cover the period of greatest 451 change in protein synthesisbased on visual inspection of 36) 36 > autoradiograms. Preliminary experiments indicated no significant induction of heat shock proteins due to this lymphocyte isolation and protein labeling procedure (V. K. MOHL and R. H. FINNELL, unpublished results). " " e Electrophoresis was accomplished using the mini gel ap30O 43' 43O 43'43' trt. 43' trt. '38" 4 3 O 43'43' paratus (Idea Scientific, Corvallis, OR) after the manner of 0 0 hr. 1 2 3 0 3 0 1 2 hr. LAEMMELI (1 970). For each sample, equal amounts of radioactivity as determined by scintillation counting was loaded SWV/SDT x DBA/2Jd D DBA/2J9 x SWV/SDd onto gradient SDS polyacrylamide gels (12-20% acrylamkDa kDa ide). Gradient gels were poured seven at a time using a gradient gel pouring stand (Idea Scientific, Corvallis, Oregon) and gravity feed. After electrophoresis, gelswere 110. stained with Coomassie blue, dried, and exposed to X-ray 97 * 70 68 film (Kodak MNl X-ray film, Rochester, NY) forthree days. Molecular weightswere estimated based on the migra45 b 36 * tion of standard markers (nonradioactive markers: Sigma 36 Chemical Co.; St.Louis,Missouri; radioactive markers: N E N Research Products, Boston,Massachusetts). Autoradiograms were evaluated visually and then run on a densiD tometer (Bio-Rad, Richmond, California). trt. 38' 43O 43'43"43O T o facilitate between strain comparisons and to minimize hr. 0 0 1 2 3 hr. 0 0 1 2 3 the problems inherent in gel to gel comparisons, heattreated samples werecompared to control samples from the same experiment run on the samegel. This was accomFIGURE1 .-Autoradiograms. Fachgel represents one experiplished by dividing the integrated area of the treated peaks ment with a separate mouse treated for each time point. Lymphoof interest (36,45,68-70,97 and 1 10 kD) by that of control cytes were isolated at the indicated time following treatlnent and peaks of the same molecular mass. incubated for 1 hr with ["S]~nethionine a s described in the methods Factorial analysis of variance was used to determine the sections. Cells were lysed and equal anlounts of radio;Ictivity run effects of genotype and time after heat treatment on the 011 each lane of an SDS polyacryl;lmide gel. The protein bmds of various parameters measured. Significant differences beinterest to this study are indicated on the left side of the autoraditween the strains and among the time points were further ogram. The approxinxlte molecular weights were estimated by the analyzedusing a Student-Newman-Keds comparisonof inclusion of marker proteins with known molecular weights. The sample means (SOKAL andROLF 1969; STEELand TORRIE first lane of each autoradiogram contains the 38" control s m p l e 1980). Tests for significance were set at a = 0.05 unless to which the other lanes nlay be colnpred. A. SWV/SD. The smile otherwise noted. four bands seen with the DBA/2.J ;Inim;lls are apparent in cells from
c
RESULTS
Immediately following the heat shock treatment, new protein bands appeared in all mouse strains at 68, 70, 97, and 1 10 kD, with an occasional band also appearing at approximately 50 kD (Figure I , A-D). T h e greatest difference between the strains in heat shock protein synthesis visible by autoradiography was the duration of active synthesis of the 68 and 70 kD heat shock proteins. In the DBA/2J strain, synthesis of hsp 68 and 70 is limited in its duration to no more than 1 hr post treatment. In the susceptible SWV/SD strain, there continued to be evidence of active synthesis of hsp 68 and 70 until at least 2 hr post treatment in all experiments. Synthesis of the 97 kD protein appeared enhanced in all strains throughout the experimentalperiod. The 110 kD proteindemonstrated the greatestvariation in terms of its detectable
I
SWV/SD ;Inim;lls. However. induction of the 68- and 70-kD band continues to be expressed in the 4 3 " + 2 hr samples in all experiments. This was a consistent finding of five repeated experiments. B, DBA/2J. Induction of at least four prominent bands (68.70, 97 and I10 kD) is apparent at 43" + 0 hr. and a t 43" I hr. By the 43" 2 hr time point, synthesis of the 68- and 70-kD proteins has returned to control levels. Wlis w a s a consistent finding offive repeated experiments. C, The F, offspring ofmatingsbetween Sb'V/SD females and DBA/2J mdcs. Intiuction of the four protein bands followed the pattern described for the DBAILJ aninlals. D, the F Ioffspring of matings between the DBA/2J females and SWV/ S D nlales. Results resembled Figure 2. A and C, in four of five experiments. I n one experiment, the 68- and 70-kD bands were present at 43" 2 hr.
+
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induction period, with no consistent difference noted between the strains. When similar experiments were repeated on the FI generation of DBA/2J X SWV/SD reciprocal crosses, lymphocytes from theseanimals responded very much like the heat-resistant DBA/2J parental mice. In par-
V. K. Mohl, G . D. Bennett and R. H. Finnell
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FIGURE2.-A densitometric analysis comparing synthesis of two taon-hr;~tsllock proteins among the strains. FJch bar represents the relative incorpor;ation of methionine by the heat-treated samples dividrtl I)! the control s;~mple and averaged for five separate experiments. Rars extending below the "1." less than 100% of control, indicate treated cells synthesized less protein of that molecular weight than did control cells. Bars extending above the " I ,"greater th;ao 100% of control, represent a relative induction of proteins at that nlolecu1;ar III;ISS. St;atistic;llly significant depression of both the 3ci-kD ;and the 4.5-kD proteins was found in the SWV/SD samples at 2 hr post heat-treatment when compared to the similarly nornxalized rate of protein synthesis in the DBA/2J strain and the FI recipror;al crosses. A, The 36-kD non-heat shock protein; B, 45-kD non-heat shock protein. (*) Significant at a = 0.05, Student-NewIII>IWKCUIStest (S.N.K.).
ticular, the enhanced synthesis of the 68- and 70-kD proteins is observed immediately following heat treatment and by 2 hrs has returned to control levels. In fact, active synthesis of 68- and 70-kD heat shock proteins at 2 hr was detected in only one of the ten reciprocal cross experiments performed. Densitometry provided a method to quantify the changes in protein synthesis seen on autoradiograms. Figures 2 and 3 summarize the effects of hyperthermia on thesynthesis of heat shock and non-heat shock proteins relative to control levels of synthesis among the mouse strains. Factorial analysis of variance was
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FIGURE 3.-Densitometric analysis of four heat-shock proteins: 68-70 kD,97 kl). and 1 IO kD. The same comparison described in Figure 2 is presented in this figure for the heat-shock proteins. A, Methionine incorporation into the 68-70-kD protein was significantly increased in the SWV/SD and F, (DBA/2J female X SWV/ SD male) relative to the DBA/2J and FIreciprocal cross (SWV/SD female X DBA/2J male) at 1 hr post heat-treatment. B. Synthesis of the 97-kD protein was significantly increased in the SWV/SD strain relative to the othergenotypes at 3 hr post-treatment. C , N o significant differences were detected among the strains in synthesis ofthe 1 IO-kD protein. (*) Significant at a = 0.05, S.N.K.
Lymphocyte Heat Shock Response
performed for each of three heat shock protein peaks (1 10 kD, 97 kD and 68-70 kD) and two non-heat shock protein peaks (45 kD, 36 kD). The 68 and 70 kD protein peaks were pooled as our densitometry system did not allow for complete separation of such closely migrating peaks. The two non-heat shock peaks were chosen based on their consistent appearance in both strains and at all treatment time points. Examination of the effects of hyperthermia on nonheat shock proteins (36 kD, 45 kD; Figure 2, A and B) indicates in vivo hyperthermia treatment decreased synthesis of these lymphocyte proteins. Immediately following treatment, synthesis of the 36-kD protein was decreased by 20% (DBA X SWV) to 40% (SWV/ SD) relative to controls. The 45-kD protein was also affected at this time point, with a 10% (DBA/2J) to 50% (SWV/SD) reduction of protein synthesis. Synthesis of both the 36-kD and 45-kD proteins was significantly lower in the SWV/SD lymphocytes relative to lymphocytes from the DBA/2J or F1 offspring at 2 hr post heat treatment (Figure 2). In contrast to the 36-kD and 45-kD proteins, synthesis of the 68, 70, 97, and 110 kD proteins was enhanced by the43"exposure. Immediately after treatment, synthesis of the 68-70-kD proteins doubled the control levels as determined by densitometric analysis (Figure 3A) with average synthesis 180% (DBA/2J) to260% (DBA X SWV) of 38"treated controls from the respective genotypes. Lymphocytes from the DBA/2J strain returned to control levels by 1 hr while at this time the SWV/SD cells still demonstrated at least a 2-fold increase in synthesis of the 68-70-kD proteins.Inthe reciprocal cross experiments, the SWV/SD (M) X DBA/2J (P) resembled the DBA/2J cells at 1 hr post treatment with synthesis of the 68-70-kD protein significantly less than that of the SWV/SD cells or the other F1 cross (DBA X SWV). This densitometry result is anomalous as it does not reflect the results found by visible inspection of the autoradiograms and there were no statistically significant differences between the reciprocal cross experiments' densitometry results (factorial analysis of variance). Synthesis of the 97-kD protein was increased by about25% immediately following treatment inall experiments, with synthesis peaking in the 1-2-hr samples and remaining slightly enhanced in lymphocytes from all animals throughout the experimental period (Figure 3B). However, at the 3 hr time point, relative synthesis of the 97-kDprotein was significantly increased in the SWV/SD lymphocytes as cornpared to all other cells examined. Densitometry analysis found synthesis of the 110kD protein to be highly variable as was the case for its visible induction o n autoradiograms. No significant differences were noted among the experiments. Syn-
953
thesis of the 1 IO-kD protein peaked from 1 to 2 hr post heat treatment (Figure 3C). Changes in cellular protein synthesis as visualized on autoradiograms and verified by densitometry were accompanied by a change in the amount of radiolabel uptake by the cells. Immediately following the heat treatment in all three inbred strains, the amount of cellular radioactivity was significantly less than that found following control treatments (Figure 4). Two hours post treatment, no significant differences between control and heat shocked cells were detected in any of the strains. In one of the F1 crosses (Figure 4), methionine incorporation at 3 hr actually exceeded control levels. It was noted that the SWV/SD cells incorporated less radioactivity on averagethandid the SWV/SD cells. However, there was no significant difference in cellular methionine incorporation of the controls among the different lymphocytes studied. DISCUSSION
These experiments demonstrate that a heat treatment sufficient to induce developmental abnormalities in murineembryos is also capable of altering protein synthesis in at least one differentiated cell type. T h e changes seen in lymphocyte protein synthesis include the induction or enhancement of proteins of at least four different molecular masses (68, 70,97 and 110 kD) following in vivo treatment at 43" for 10 min. Synthesis of at two non-heat shock proteins (36 and 45 kD) was depressed by the heat treatment, as determined by densitometrictracings of autoradiograms. A generalized decrease in cellular [35S]methionine uptake immediately following hyperthermia exposure was also noted. These results are consistent with the heat shock response as previously described (SCHLESINCER, ALIPERTIand KELLEY1982; SUBJECK and SHYY1986). Genetic variation among the inbred strains following hyperthermic shock included differences in the kinetics of their lymphocyte heat shock response. Visual inspection of autoradiograms demonstrated a consistently longer period of induction of the 68 and 70 kD heat shock proteins in the SWV/SD as compared tothe DBA/2J mouse strains. Densitometry confirmed this difference between the inbred strainswith SWV/SD having thegreater induction relative to control levels in their lymphocytes, particularly at 1 hr post treatment. The offspring of reciprocal crosses between these two strains were found to behave most like the parental DBA/2J strain with visual inspection of theautoradiograms.However,thedensitometry results for the F1 reciprocal cross experiments were inconclusive. The inconsistency between the visual inspection of the autoradiograms and the densitometry results in the F1 experiments indicate that increased duration of heat shock protein synthesis alone
V. K. Mohl, G. D. Bennett and R. H. Finnell
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is insufficient for evaluating the genetically determined differences in cellular response to hyperthermia between these mouse strains. The affect of hyperthermia on normal protein synthesis had a more consistent correlation with sensitivity to teratogen-induced exencephaly in these experiments. Synthesis of both of the normal proteins investigated was most depressed in the SWV/SD lymphocytes, particularly at 2 hr post heat treatment. This continued depression of protein synthesis was not found in the F1 reciprocal experiments, indicating the more rapid recovery is a dominant trait within the SWV/SD and DBA/2J genotypes. Thus, the inbred mouse strain whose spleen cells seem least affected by hyperthermia (DBA/2J) is the strain with the greatest resistance to heat-inducedexencephaly (FINNELL et al. 1986) and this resistance trait is also dominant (V. K. MOHL and R. H. FINNELL, unpublished results). While the similarities in the segregation of the alleles controlling the lymphocyte heat shock response differences and that of susceptibility to hyperthermiainduced neural tubedefects are intriguing, it is uncertain at present whether the differences in lymphocyte protein synthesis reported here can be directly related to abnormal embryonic development.GERMAN (1984) hypothesized that preferential synthesis of heat shock protein could inhibit the synthesis of other proteins that are essential for normal development. However, the mere synthesis of heat shock proteins alone does not appear to be sufficient to lead to developmental defects in mouse embryos (BENNETT, MOHL and FINNELL 1990). Induction of these proteins may, in fact, protect the embryo from a later, more severe environmental stress (WALSHet al. 1987; MIRKES1987). The
"-"
FIGURE4.-Quantification of total methionineuptake by the cells. T h e total amount of radioactivity incorporated by the cells from DBA/ 25, SWV/SD and the F I offspring of reciprocal crosses between these two strains was determined in lysates from both control (38" + 0 hr) and heat-treated (43" = 0, 1 , 2 and 3 hr) animals. Following treatment, cells werelabeled with ["'SJmethionine for 1 hr. Labeled cells were washed with excess cold HBSS toremove unincorporated methionine. T h e total amount of radioactivity present in the cells was determined using liquid scintillation as described in the nlethods section. Uptake of the radiolabel was depressed immediately following heat-treatment in all experiments. Acid precipitableradioactivity was approximately 25% of the totalradioactivity in the lysates. (*) Significant at a = 0.05, S.N.K.
DBAXSWV
beneficial effect of the heat shock proteins in protecting normal development is reminiscent of the proposed role of these proteins in cellular homeostasis (SCIANDRA and SUBJECK1984; LI and WERB 1982), and may better explain the relationship between the cellular effects described here and susceptibility to hyperthermia-induced exencephaly. For example,experimental work conducted in cellculture suggest that cell survival is positively correlated with the extent to which normal protein synthesis is inhibited, the duration of heat shock protein induction and the time required to return to normal protein synthesis (reviewed in SUBJECK and SHYY1986). The ability of the cells to retain normal cellular functions even during periods of stress appears to play a key role in preventing cell death (WELCHand SUHAN 1985). Thus, evaluating normal cellular functions such as protein synthesis as well as the induction of heat shock proteins would seem likely to provide the best estimate of the response of cells to environmental stress. In summary, the lack of appropriate animal models has hindered our understanding of the role of heat shock proteins in mammalian homeostatic responses to hyperthermia. As part of a series of experiments utilizing a strain hierarchy of susceptibility to hyperthermia-induced neural tube defects, we have discovered a difference in the heat shock response of lymphocytes from two inbred mouse strains. Our results indicate that the relative synthesis of normal proteins is most depressed in the SWV/SD strain, the strain most susceptible toheat-inducedexencephaly. Together with the increased durationofheat shock protein synthesis, this indicates that lymphocytes from the SWV/SD strain are responding in a more dramatic
Lymphocyte Heat Shock Response
mannertothehyperthermic insult than are those from the other strains used in this study. These effects are lost in the F1 hybrid progeny of susceptible SWV/ SD and the more resistant DBA/2J strain. This work was supported i n part by a National Science Foundation predoctoral fellowship to V.K.M., and by research grant ES 04326 from theNational InstitutesofHealth to R.H.F. Theauthors wish to express their appreciation to MICHAELVAN WAESfor his critical review of the manuscript, PATRICIA AGER for her technical R. for editorial assistance, and C . SMITH,P. PERRON and THOMPSON and clerical assistance.
LITERATURECITED BmNwT, (;. D., V. K. M o m a n d R. H. FINNELL, 1990Embryonic and maternal heat shock responses to known teratogenic insults. Reprod. Toxicol. ( i n press). BIENz, M., 1985 Transient and developnlental activation of heatshock genes. Trends Biol. Sci. 00: 157-161. H. R. BODEand R. E. STEELE, BOSCH,T . C . G., S . M. KRYLOW, 1988 Therniotoletance and synthesis of heat shock proteins: These responses are present in Hydra attenuate but absent in Hydra oligactis. Proc. Natl. Acad. Sci. USA 85: 7927-7931. COOPER,S., and T . RUETTINGER,1975 A temperature sensitive nonsense mutation affecting the synthesis of a major protein ofEscherichia coli K 1 2 . Mol. Gen. Genet. 139: 167-176. FINNELL, R. H., S. P. MOON, L. C. ABBOTT,J. A. GOLDEN andG. F. CHERNOFF, 1986 Straindifferences in heat-inducedneuraltube defects i n mice. Teratology 33: 247-252. J., 1984 Embryonicstresshypothesisofteratogenesis. GERMAN, Am. J. Med. 76: 293-301. GROSSMAN, A. D., J. U'. ERICKSONandC. A. GROSS, 1984 The htpR gene product of E. coli is a sigma factor for heat shock promoters. Cell 38: 383-390. HUNT,C., and R. I . MORIMOTO, 1985 Conserved featuresof eukaryotic hsp 70genes revealed by comparison with the nucleotide sequence of human hsp 70. Proc. Natl. Acad. Sci. USA 82: 6455-6459. S., J . ROW, L. PETKOand LINDQUIST,1986 An ancient KURT%, developnlental induction:Heat-shock proteins induced i n sporulation and oogenesis. Science 231: 1 1 54-1 157. LAEMMIJ, U . K.,1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T 4 . Nature 227: 680685. LI, G. C., and J. V . MAK,1985 Induction of heat shock protein synthesis in murine tumors during the development of thermotolerance. Cancer Res. 45: 3816-3824. 1.1, G . C., and Z. WERR,1982Correlation betweensynthesis of heat Shock proteins and development of thermotolerance i n Chinese hamster fibroblasts. Proc. Natl.Acad. Sci. USA 79: 321 8-3222. LINQUIST,S . , 1986 The heat shock response. Annu. Rev. Biochenl. 55: 1 1 5 1- 1 19 1 . LOOMIS,W . F., at1d S . A. WHEELER,1982 Chromatin-associated heat shock proteins of Dictyosteliu. Dev. Biol. 90: 4 12-4 18. LOWE, D. G., W. D. FULFORD at1d L. A . MORAN, 1983 Mouse a11d Drosophila genes encoding the ~ni?jor heat shock protein (Ilsp 70) are highly conserved. Mol. Cell. Biol. 3: 1540-1 543. MIRKES, 1'. E., 1987Hyperthernlia-inducedheat shock response m d thcr~notolel.;l~~ce i n postilnplantatiol~ratembryos. Dev. Biol. 119: 115-122. MISHELL, B. B., m d S . M. SHIIGI, 1980 Selected Methods in Cellular Immunology. W. H. Freenlan, San Francisco.
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MITCHELL,H. K., and L. S. LIPPS, 1978 Heat shock and phenocopy induction i n Drosophila. Cell 15: 907-918. MITCHELL, H. K., N. S. PETERSON and C. H. BUZIN,1985 Self degradation of heat shock proteins. Proc. Natl. Acad. Sci. USA 82: 4969-4973. MITCHELL, H. K.,G. MOLLER, N. S. PETERSON and L. S. LIPPSSARMEINTO, 1979Specific protection from phenocopy induction by heat shock. Dev. Genet. 1: 181-192. MORAN, I>. A., M. CHAUVIN, M. E. KENNEDY, M. KORRI, D. G. LOWE,R. C. NICHOLSON and M.D. PERRY, 1982 The major heat-shock protein (hsp 70) gene family: Related sequences i n mouse, Drosophila and yeast. Can. J. Biochem. Cell Biol. 61: 488-499. NEIDHARDI, F. C., R. A. VANBOCELELand V. VAUGHN, 1984 The genetics and regulation of the heat shock proteins. Annu. Rev. Genet. 18: 295-329. PETKO,L.,and S. LINDQUIST,1986 Hsp26 is notrequiredfor growth at high temperatures, nor for thernlotolerance, spore development, o r germination. Cell 45: 885-894. D. R. N., W.E. NORRIS, G. J. CARLSON. R. J. KEYNES PRIMMETT, and C. D. STERN, 1989 Periodic segmental anomalies induced by heat shock in the chick embryo are associated with the cell cycle. Developrnent 105: 119-130. RITOSSA,F., 1962 A new puffing pattern induced by temperature shock and DNP in Drosophila. Experientia 18: 571-573. M . J., G. ALIPERTI and P.M. KELLEY, 1982 T h e SCHLESINGER, response of cells to heat shock. Trends Biochem. Sci. 7: 222225. SCHLESINGER, M. J., M. ASHRURNERandA . TISSIERES (Editors), 1982 HeatShock, from Bacteria to Man. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. SCIANDRA, J. J., and J. R. SUBJECK, 1984 Heat shock proteins and protection of proliferation and translation in nlanllnalian cells. Cancer Res. 44: 5 188-5 194. SOKAL,R. R., and F.J. ROHLF,1969 Biometry, The Principles and Practise ofStatistics in Biological Research. W.H. Freenlan, San Francisco. STEEL,R. C . D., and J. H . TORRIE, 1980 Principles and Procedures and Statistics. McGraw-Hill, New York. SURJECK, J. K., and T. T . SHYY, 1986 Stress protein systems of mammalian cells. Am. J. Physiol. 2 5 0 CI-CI 7. TISSIERES, A , , H. K. MITCHELL and U . M. TRACY, 1974 Protein synthesis in salivary glands of Drosophila melanogaster: Relation to chromosome puffs. J. Mol. Biol. 84: 389-398. WALSH,C., 1980 Appearance of heat shock proteins during the induction of multipleflagella i n Naegleriagruberi. J. Biol. Chenl. 255: 2629-2632. WALSH,D. A . , N. W.KLEIN, L. E. HIGHTOWERand M. J. EDWARDS, 1987 Heat shock and thermotolerance during early embryo development. Teratology 36: 18 1- 19 I . WELCH,W. J., and J. P. SUHAN, 1985 Morphological study o f t h e nlamnlalianstressresponse: Characterimtionof ch;mges in cytoplasmic organelles, cytoskeleton, and nucleoli and appearance of intranuclearactin filaments i n rat fibroblasts after he;ltshock treatment. J. Cell Biol. 101: 1 198-1 2 11. YAMAMORI,T.,and T. YURA,1982 Genetic control of heat shock protein synthesis and its bearing on growth and ther111al resistSci. USA 79: ance in Escherichia coli K12. Proc.Natl.Acad. 860-864. YOST, H. J.. and S . IJNDQUIST, 1986 R N A splicing is interrupted by heat shock and is rescued by heat shock protein synthesis. Cell 45: 185-193. Communicating editor: R. E:. GANSCHOW
Genetic Differences in the Duration of the Lymphocyte Heat Shock Response in Mice Virginia K. Mohl,* Gregory D. Bennettt and Richard H. Finnell*’T *Program in Genetics and Cell Biology and tDepartment of Veterinary and Comparative Anatomy, Pharmacology and Physiology, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164-6520 Manuscript received September 19, 1989 Accepted for publication December 22, 1989
ABSTRACT Lymphocytes from adult mice bearing a known difference in genetic susceptibility to teratogeninduced exencephaly (SWV/SD, and DBA/2J)were evaluated for changes in protein synthesis following an in vivo heat treatment. Particular attention was paid to changes indicative of the heat shock response, a highly conserved response to environmental insult consisting of induction of a few, highly conserved proteins with simultaneous decreases in normal protein synthesis. The duration of heat shock protein induction in lymphocytes was found to be increased by 1 hr in the teratogensensitive SWV/SDstrain as compared to the resistant DBA/2J strain. Densitometric analysis revealed a significant decrease in the relative synthesis of at least two non-heat shock proteins (36 kD and 45 kD) in the SWV/SD lymphocytes as compared to DBA/2J cells. The increased sensitivity of protein synthesis to hyperthermia in the SWV/SD lymphocytes were lost in the FI progeny of reciprocal crosses between SWV/SD and DBA/2J mouse strains. Sensitivity to hyperthermia-induced exenceis recessive to resistance in these crosses. The relationship between altered protein synthesis phaly ~, and teratogen susceptibility is discussed. (MITCHELLa n d LIPPS 1978; MITCHELLet al. 1979), NVESTIGATION of the heat shock response beNaegleria gruberi (WALSH 1980) and yeast (KURTZ et gan with the discovery of a unique set of puffs in al. 1986).Despitethisscrutiny,theprecise role of the heat-pulsed salivary gland chromosomes of Drothese proteins in resistance t o cellular stress and their sophila busckii whichwerelaterassociated with the importanceduringdevelopmentremainsunclear production of specific proteins (RITOSSA1962; TIS(SCIANDRA and SUBJECK1984; PETKOand LINDQUIST SIERES, MITCHELL a n d TRACY 1974).Subsequent 1986; PRIMMETT et al. 1989). of a similar studies revealed the transient induction A common approach utilized in the elucidation of set of proteins known variously as heat shock or stress the function of a gene or gene product is the use of proteins, to be common to both eukaryotic and prokaryotic heat-treated cells (SCHLESINGER, ASHBURNER mutations, three types of which currently are being actively applied in heat shock research. Mutant Eschand TISSIERES 1982; LINDQUIST1986). T h e highly erichia coli strains that are defective in the positive conservednature of theheat shockproteingenes (MORANet al. 1982;LOWE,FULFORDand MORAN regulation of the heat shock response (COOPERand 1983; HUNT andMORIMOTO1985), and their unique RUETTINGER1985; GROSSMAN, ERICKSONand GROSS regulation during periods cellular of stress (TISSIERES, 1984) have been used to demonstrate the importance of these proteins in cell growth at elevated temperaMITCHELLand TRACY 1974), led to the exploitation tures (YAMAMORIand YURA 1982). A strain of Dicof the heat shock response as a model systemwith tyostelium which fails to produce several specific heat which to probe mechanisms of gene induction and shockproteinshas a decreasedabilitytogrowat regulation (NEIDHARDT, VANBOGELELand VAUGHN elevated temperatures (LOOMISand WHEELER1982) 1984; BIENZ1985).Heatshockproteinshavealso been implicated in the acquisition of thermotolerance while in some hydra strains, synthesis of a heat shock (YAMAMORI and YURA 1982; LI and WERB 1982; LI protein correlated with thermotolerance (BOSCHet al. and MAK 1985; BOSCHet al. 1988), andin the recovery 1988). In an attempt to specifically define the funcof normal cellular processes following the application tionsof a low molecular mass heatshockprotein, of a stress(YOST and LINDQUIST1986). These proteins PETKO and LINDQUIST(1986) created deletion and have been correlated with developmental changes in disruption mutations in the 26 kD heat shock protein Drosophila melanogaster organisms as diverse as gene (hsp 26) of yeast. They demonstrated that hsp 26 was notrequiredforseveraldifferentcellular T h e public;ltion costs o f this anicle were partly defrayed by the payment functions includingthermotolerance,germination, o f page cllargrs. Thisxl-ticlt. 111ust therefore bc hereby marked “ n d w r f u e m m t ” in accol-dlnce with 1 X U.S.C. 8 1794 solely to indicate this fact. andgrowthatelevatedtemperatures(PETKOand
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V. K. Mohl. G. D. Bennett and R. H. Finnell
LINDQUIST1986).Unfortunately,similarmutations have yet be described in higher eukaryotes. Lacking specific known mutations, theuse of organisms with known differences in their biological responses to heat stress may provide insight into any possible homeostaticrolesoftheseproteins.Ithas by FINNELLand colrecentlybeendemonstrated leagues (1986) that a strain dependent hierarchy of susceptibility t o a hyperthermia-induced neural tube defect (exencephaly) exists among inbred strains of mice. While the molecular basis of this strain variation is presently unknown, the response is clearly under genetic control. When susceptible (SWV/SD) animals areoutcrossedtomoreresistantstrains(LM/Bc, C576L/6J and DBA/ZJ; FINNELLet al. 1986; V. K. MOHLand R. H. FINNELL, unpublished data), the F1 hybridprogenywereresistanttohyperthermiainducedexencephaly. The degree ofresistancedepended upon the outcross strain, yet all three outcross strains demonstrated that sensitivity was recessive t o resistance toheat-inducedexencephaly.Reciprocal cross experiments indicate that the genotype of the embryo, rather than thephysiological response of the mother, is the major factor in determining susceptibility toheat-inducedexencephaly(FINNELL et al. 1986). T h e discovery of one highly susceptible strain (SWV/SD),severalmoderatelysusceptible(LM/Bc, SWR/J) and resistantstrains (DBA/ZJ, C57BL/6J) provides a unique model in which t o investigate the importance of the heat shock response to embryonic development in a mammalian system. Our working hypothesis has been that an environmental insult capable of disrupting normal mammalian development should have a demonstrable effect on maternal tissues. Previous results from our laboratorysupported thishypothesis by demonstrating induction of heat shock proteins in both embryonic tissues and maternal lymphocytes(BENNETT, MOHL and FINNELL 1990). While investigations of the early embryo provide direct evidence for the teratogenic effectsof theheattreatment, the severelylimited amount of available tissueand its rapid differentiation complicate biochemical analysis of cell function. The lymphocyte assay complements the embryonic tissue studies by providing a sample tissue that is readily isolated with little trauma to the cells and which has well defined culture characteristics and functionalactivity assays. In the studies reported here, the lymphocyte heat shock assay was expanded to examine the kinetics of protein synthesis followingin vivo hyperthermia. Cells from adultmice bearinga known genetic susceptibility for to heat-induced exencephaly were evaluated changes in protein synthesis following a single in vivo heat treatment. In thekinetic study described herein, a heat treatment previously shown to induce devel-
opmentalabnormalities(FINNELL et al. 1986)produced differences in the heat shock response between highly inbred strains of mice susceptible (SWV/SD) and (DBA/ZJ) resistant to heat-induced exencephaly. MATERIALS AND METHODS Two inbred mouse strains (SWV/SD, and DBA/2J) were maintained on a 12-hr light cycle in the College Hall Vivarium at Washington State University, Pullman. Reciprocal cross experiments were performed using the FI generation progeny of matings between the highly susceptible SWV/ SD and the resistant DBA/2J strains. Healthy, pathogenfive per polycarbonate cage and free micewerehoused allowed free access to Purina rodent chow and tap water. Animals 60 to 120 days of age were randomly assigned to treatment groups. All animal procedures were designed to minimizeanystress and discomfort to the experimental animals. Treatment of the adult animals consisted of a single, 10min incubation in either a 43" waterbath for heat stressed animals or a 38" waterbath for control animals. This treatet al. 1986). ment is described in detail elsewhere (FINNELL Core temperatures were recorded at one minute intervals using a YSI telethermometer and small animal probe (Yellow Springs Instruments, Yellow Springs, Ohio). By changing the depth of the restraining chamber in the waterbath, it is possible to control closely therate of temperature increase and thus insure uniformity of heat treatment among individual animals. Therefore, the core body temperature did not vary among the parental or hybrid strains. Heat stressed animals not sacrificed immediately following treatment were patted dry and placed into a 38" warm air incubator for twenty minutes to allow the animal's body temperature to gradually return to its normal basal level. N o age or strain related differences in the kinetics of the temperature curve were noted. For each strain, a minimum of five animals were evaluated at each time point. Following treatment, the mice were sacrificed by cervical dislocation, the spleen aseptically removed and placed into cold Hank's balanced salt solution (HBSS, GIBCO, Grand Island, New York). The spleen was then gently teased apart with mouse-toothed forceps and the cell containing supernatant collected. The number of nucleated cells was determined using a hemocytometer. Both the cells and the buffer were kept on ice throughout the procedure.Cell viability as determined by trypan blue exclusion (MISHELL and SHIICI 1980) ranged between 98% and 96% viable cells. To determine changes inspecific proteins synthesized following an in vivo heat treatment, spleen cells were harvested at 0, 1, 2, and 3 hr following a 10-min treatment at either control (38") or heat shock (43") temperatures. A volume of cell suspension containing between 2 and 5 million cells was collected for labeling with ["S]methionine. Each sample was spun down in a Beckrnan microfuge, the supernatant discarded and the resultant pellet resuspended into 200 pl of cold HBSS. Twenty microcuries [JsS]methionine (NEN Research Products, Boston, Massachusetts) was added and the suspension was allowed to incubate for one hour in a 37" water bath. Following incubation, the cells were washed twice with cold HBSS to remove any unincorporated methionine and to minimize the potential degradation of newly synthesized heat shock proteins (MITCHELL, PETERSON and BUZIN1985). The cells were then lysed with 100 pI LAEMMELI'S (1970) sample buffer and boiled for 10 min. To determine the amount of radioactivity that was incorporated into the cells, duplicate aliquots of sample were
Lymphocyte Response Heat Shock
95 1
counted i n a Beckman scintillation counter with 10 ml Aquasol (Amersham, Arlington Heights, Illinois)as the scintillant. The remainder of the sample was either used immediately for gel electrophoresis or frozen at 70". Cells were considered to express a heat shock response if induction or enhancement of selected protein bands of appropriate molecular weights were detected on autoradiograms of samples separated by SDS-PAGE. The time of 45 w harvesting was selected to cover the period of greatest 451 change in protein synthesisbased on visual inspection of 36) 36 > autoradiograms. Preliminary experiments indicated no significant induction of heat shock proteins due to this lymphocyte isolation and protein labeling procedure (V. K. MOHL and R. H. FINNELL, unpublished results). " " e Electrophoresis was accomplished using the mini gel ap30O 43' 43O 43'43' trt. 43' trt. '38" 4 3 O 43'43' paratus (Idea Scientific, Corvallis, OR) after the manner of 0 0 hr. 1 2 3 0 3 0 1 2 hr. LAEMMELI (1 970). For each sample, equal amounts of radioactivity as determined by scintillation counting was loaded SWV/SDT x DBA/2Jd D DBA/2J9 x SWV/SDd onto gradient SDS polyacrylamide gels (12-20% acrylamkDa kDa ide). Gradient gels were poured seven at a time using a gradient gel pouring stand (Idea Scientific, Corvallis, Oregon) and gravity feed. After electrophoresis, gelswere 110. stained with Coomassie blue, dried, and exposed to X-ray 97 * 70 68 film (Kodak MNl X-ray film, Rochester, NY) forthree days. Molecular weightswere estimated based on the migra45 b 36 * tion of standard markers (nonradioactive markers: Sigma 36 Chemical Co.; St.Louis,Missouri; radioactive markers: N E N Research Products, Boston,Massachusetts). Autoradiograms were evaluated visually and then run on a densiD tometer (Bio-Rad, Richmond, California). trt. 38' 43O 43'43"43O T o facilitate between strain comparisons and to minimize hr. 0 0 1 2 3 hr. 0 0 1 2 3 the problems inherent in gel to gel comparisons, heattreated samples werecompared to control samples from the same experiment run on the samegel. This was accomFIGURE1 .-Autoradiograms. Fachgel represents one experiplished by dividing the integrated area of the treated peaks ment with a separate mouse treated for each time point. Lymphoof interest (36,45,68-70,97 and 1 10 kD) by that of control cytes were isolated at the indicated time following treatlnent and peaks of the same molecular mass. incubated for 1 hr with ["S]~nethionine a s described in the methods Factorial analysis of variance was used to determine the sections. Cells were lysed and equal anlounts of radio;Ictivity run effects of genotype and time after heat treatment on the 011 each lane of an SDS polyacryl;lmide gel. The protein bmds of various parameters measured. Significant differences beinterest to this study are indicated on the left side of the autoraditween the strains and among the time points were further ogram. The approxinxlte molecular weights were estimated by the analyzedusing a Student-Newman-Keds comparisonof inclusion of marker proteins with known molecular weights. The sample means (SOKAL andROLF 1969; STEELand TORRIE first lane of each autoradiogram contains the 38" control s m p l e 1980). Tests for significance were set at a = 0.05 unless to which the other lanes nlay be colnpred. A. SWV/SD. The smile otherwise noted. four bands seen with the DBA/2.J ;Inim;lls are apparent in cells from
c
RESULTS
Immediately following the heat shock treatment, new protein bands appeared in all mouse strains at 68, 70, 97, and 1 10 kD, with an occasional band also appearing at approximately 50 kD (Figure I , A-D). T h e greatest difference between the strains in heat shock protein synthesis visible by autoradiography was the duration of active synthesis of the 68 and 70 kD heat shock proteins. In the DBA/2J strain, synthesis of hsp 68 and 70 is limited in its duration to no more than 1 hr post treatment. In the susceptible SWV/SD strain, there continued to be evidence of active synthesis of hsp 68 and 70 until at least 2 hr post treatment in all experiments. Synthesis of the 97 kD protein appeared enhanced in all strains throughout the experimentalperiod. The 110 kD proteindemonstrated the greatestvariation in terms of its detectable
I
SWV/SD ;Inim;lls. However. induction of the 68- and 70-kD band continues to be expressed in the 4 3 " + 2 hr samples in all experiments. This was a consistent finding of five repeated experiments. B, DBA/2J. Induction of at least four prominent bands (68.70, 97 and I10 kD) is apparent at 43" + 0 hr. and a t 43" I hr. By the 43" 2 hr time point, synthesis of the 68- and 70-kD proteins has returned to control levels. Wlis w a s a consistent finding offive repeated experiments. C, The F, offspring ofmatingsbetween Sb'V/SD females and DBA/2J mdcs. Intiuction of the four protein bands followed the pattern described for the DBAILJ aninlals. D, the F Ioffspring of matings between the DBA/2J females and SWV/ S D nlales. Results resembled Figure 2. A and C, in four of five experiments. I n one experiment, the 68- and 70-kD bands were present at 43" 2 hr.
+
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induction period, with no consistent difference noted between the strains. When similar experiments were repeated on the FI generation of DBA/2J X SWV/SD reciprocal crosses, lymphocytes from theseanimals responded very much like the heat-resistant DBA/2J parental mice. In par-
V. K. Mohl, G . D. Bennett and R. H. Finnell
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FIGURE2.-A densitometric analysis comparing synthesis of two taon-hr;~tsllock proteins among the strains. FJch bar represents the relative incorpor;ation of methionine by the heat-treated samples dividrtl I)! the control s;~mple and averaged for five separate experiments. Rars extending below the "1." less than 100% of control, indicate treated cells synthesized less protein of that molecular weight than did control cells. Bars extending above the " I ,"greater th;ao 100% of control, represent a relative induction of proteins at that nlolecu1;ar III;ISS. St;atistic;llly significant depression of both the 3ci-kD ;and the 4.5-kD proteins was found in the SWV/SD samples at 2 hr post heat-treatment when compared to the similarly nornxalized rate of protein synthesis in the DBA/2J strain and the FI recipror;al crosses. A, The 36-kD non-heat shock protein; B, 45-kD non-heat shock protein. (*) Significant at a = 0.05, Student-NewIII>IWKCUIStest (S.N.K.).
ticular, the enhanced synthesis of the 68- and 70-kD proteins is observed immediately following heat treatment and by 2 hrs has returned to control levels. In fact, active synthesis of 68- and 70-kD heat shock proteins at 2 hr was detected in only one of the ten reciprocal cross experiments performed. Densitometry provided a method to quantify the changes in protein synthesis seen on autoradiograms. Figures 2 and 3 summarize the effects of hyperthermia on thesynthesis of heat shock and non-heat shock proteins relative to control levels of synthesis among the mouse strains. Factorial analysis of variance was
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FIGURE 3.-Densitometric analysis of four heat-shock proteins: 68-70 kD,97 kl). and 1 IO kD. The same comparison described in Figure 2 is presented in this figure for the heat-shock proteins. A, Methionine incorporation into the 68-70-kD protein was significantly increased in the SWV/SD and F, (DBA/2J female X SWV/ SD male) relative to the DBA/2J and FIreciprocal cross (SWV/SD female X DBA/2J male) at 1 hr post heat-treatment. B. Synthesis of the 97-kD protein was significantly increased in the SWV/SD strain relative to the othergenotypes at 3 hr post-treatment. C , N o significant differences were detected among the strains in synthesis ofthe 1 IO-kD protein. (*) Significant at a = 0.05, S.N.K.
Lymphocyte Heat Shock Response
performed for each of three heat shock protein peaks (1 10 kD, 97 kD and 68-70 kD) and two non-heat shock protein peaks (45 kD, 36 kD). The 68 and 70 kD protein peaks were pooled as our densitometry system did not allow for complete separation of such closely migrating peaks. The two non-heat shock peaks were chosen based on their consistent appearance in both strains and at all treatment time points. Examination of the effects of hyperthermia on nonheat shock proteins (36 kD, 45 kD; Figure 2, A and B) indicates in vivo hyperthermia treatment decreased synthesis of these lymphocyte proteins. Immediately following treatment, synthesis of the 36-kD protein was decreased by 20% (DBA X SWV) to 40% (SWV/ SD) relative to controls. The 45-kD protein was also affected at this time point, with a 10% (DBA/2J) to 50% (SWV/SD) reduction of protein synthesis. Synthesis of both the 36-kD and 45-kD proteins was significantly lower in the SWV/SD lymphocytes relative to lymphocytes from the DBA/2J or F1 offspring at 2 hr post heat treatment (Figure 2). In contrast to the 36-kD and 45-kD proteins, synthesis of the 68, 70, 97, and 110 kD proteins was enhanced by the43"exposure. Immediately after treatment, synthesis of the 68-70-kD proteins doubled the control levels as determined by densitometric analysis (Figure 3A) with average synthesis 180% (DBA/2J) to260% (DBA X SWV) of 38"treated controls from the respective genotypes. Lymphocytes from the DBA/2J strain returned to control levels by 1 hr while at this time the SWV/SD cells still demonstrated at least a 2-fold increase in synthesis of the 68-70-kD proteins.Inthe reciprocal cross experiments, the SWV/SD (M) X DBA/2J (P) resembled the DBA/2J cells at 1 hr post treatment with synthesis of the 68-70-kD protein significantly less than that of the SWV/SD cells or the other F1 cross (DBA X SWV). This densitometry result is anomalous as it does not reflect the results found by visible inspection of the autoradiograms and there were no statistically significant differences between the reciprocal cross experiments' densitometry results (factorial analysis of variance). Synthesis of the 97-kD protein was increased by about25% immediately following treatment inall experiments, with synthesis peaking in the 1-2-hr samples and remaining slightly enhanced in lymphocytes from all animals throughout the experimental period (Figure 3B). However, at the 3 hr time point, relative synthesis of the 97-kDprotein was significantly increased in the SWV/SD lymphocytes as cornpared to all other cells examined. Densitometry analysis found synthesis of the 110kD protein to be highly variable as was the case for its visible induction o n autoradiograms. No significant differences were noted among the experiments. Syn-
953
thesis of the 1 IO-kD protein peaked from 1 to 2 hr post heat treatment (Figure 3C). Changes in cellular protein synthesis as visualized on autoradiograms and verified by densitometry were accompanied by a change in the amount of radiolabel uptake by the cells. Immediately following the heat treatment in all three inbred strains, the amount of cellular radioactivity was significantly less than that found following control treatments (Figure 4). Two hours post treatment, no significant differences between control and heat shocked cells were detected in any of the strains. In one of the F1 crosses (Figure 4), methionine incorporation at 3 hr actually exceeded control levels. It was noted that the SWV/SD cells incorporated less radioactivity on averagethandid the SWV/SD cells. However, there was no significant difference in cellular methionine incorporation of the controls among the different lymphocytes studied. DISCUSSION
These experiments demonstrate that a heat treatment sufficient to induce developmental abnormalities in murineembryos is also capable of altering protein synthesis in at least one differentiated cell type. T h e changes seen in lymphocyte protein synthesis include the induction or enhancement of proteins of at least four different molecular masses (68, 70,97 and 110 kD) following in vivo treatment at 43" for 10 min. Synthesis of at two non-heat shock proteins (36 and 45 kD) was depressed by the heat treatment, as determined by densitometrictracings of autoradiograms. A generalized decrease in cellular [35S]methionine uptake immediately following hyperthermia exposure was also noted. These results are consistent with the heat shock response as previously described (SCHLESINCER, ALIPERTIand KELLEY1982; SUBJECK and SHYY1986). Genetic variation among the inbred strains following hyperthermic shock included differences in the kinetics of their lymphocyte heat shock response. Visual inspection of autoradiograms demonstrated a consistently longer period of induction of the 68 and 70 kD heat shock proteins in the SWV/SD as compared tothe DBA/2J mouse strains. Densitometry confirmed this difference between the inbred strainswith SWV/SD having thegreater induction relative to control levels in their lymphocytes, particularly at 1 hr post treatment. The offspring of reciprocal crosses between these two strains were found to behave most like the parental DBA/2J strain with visual inspection of theautoradiograms.However,thedensitometry results for the F1 reciprocal cross experiments were inconclusive. The inconsistency between the visual inspection of the autoradiograms and the densitometry results in the F1 experiments indicate that increased duration of heat shock protein synthesis alone
V. K. Mohl, G. D. Bennett and R. H. Finnell
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SWVXDBA
is insufficient for evaluating the genetically determined differences in cellular response to hyperthermia between these mouse strains. The affect of hyperthermia on normal protein synthesis had a more consistent correlation with sensitivity to teratogen-induced exencephaly in these experiments. Synthesis of both of the normal proteins investigated was most depressed in the SWV/SD lymphocytes, particularly at 2 hr post heat treatment. This continued depression of protein synthesis was not found in the F1 reciprocal experiments, indicating the more rapid recovery is a dominant trait within the SWV/SD and DBA/2J genotypes. Thus, the inbred mouse strain whose spleen cells seem least affected by hyperthermia (DBA/2J) is the strain with the greatest resistance to heat-inducedexencephaly (FINNELL et al. 1986) and this resistance trait is also dominant (V. K. MOHL and R. H. FINNELL, unpublished results). While the similarities in the segregation of the alleles controlling the lymphocyte heat shock response differences and that of susceptibility to hyperthermiainduced neural tubedefects are intriguing, it is uncertain at present whether the differences in lymphocyte protein synthesis reported here can be directly related to abnormal embryonic development.GERMAN (1984) hypothesized that preferential synthesis of heat shock protein could inhibit the synthesis of other proteins that are essential for normal development. However, the mere synthesis of heat shock proteins alone does not appear to be sufficient to lead to developmental defects in mouse embryos (BENNETT, MOHL and FINNELL 1990). Induction of these proteins may, in fact, protect the embryo from a later, more severe environmental stress (WALSHet al. 1987; MIRKES1987). The
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FIGURE4.-Quantification of total methionineuptake by the cells. T h e total amount of radioactivity incorporated by the cells from DBA/ 25, SWV/SD and the F I offspring of reciprocal crosses between these two strains was determined in lysates from both control (38" + 0 hr) and heat-treated (43" = 0, 1 , 2 and 3 hr) animals. Following treatment, cells werelabeled with ["'SJmethionine for 1 hr. Labeled cells were washed with excess cold HBSS toremove unincorporated methionine. T h e total amount of radioactivity present in the cells was determined using liquid scintillation as described in the nlethods section. Uptake of the radiolabel was depressed immediately following heat-treatment in all experiments. Acid precipitableradioactivity was approximately 25% of the totalradioactivity in the lysates. (*) Significant at a = 0.05, S.N.K.
DBAXSWV
beneficial effect of the heat shock proteins in protecting normal development is reminiscent of the proposed role of these proteins in cellular homeostasis (SCIANDRA and SUBJECK1984; LI and WERB 1982), and may better explain the relationship between the cellular effects described here and susceptibility to hyperthermia-induced exencephaly. For example,experimental work conducted in cellculture suggest that cell survival is positively correlated with the extent to which normal protein synthesis is inhibited, the duration of heat shock protein induction and the time required to return to normal protein synthesis (reviewed in SUBJECK and SHYY1986). The ability of the cells to retain normal cellular functions even during periods of stress appears to play a key role in preventing cell death (WELCHand SUHAN 1985). Thus, evaluating normal cellular functions such as protein synthesis as well as the induction of heat shock proteins would seem likely to provide the best estimate of the response of cells to environmental stress. In summary, the lack of appropriate animal models has hindered our understanding of the role of heat shock proteins in mammalian homeostatic responses to hyperthermia. As part of a series of experiments utilizing a strain hierarchy of susceptibility to hyperthermia-induced neural tube defects, we have discovered a difference in the heat shock response of lymphocytes from two inbred mouse strains. Our results indicate that the relative synthesis of normal proteins is most depressed in the SWV/SD strain, the strain most susceptible toheat-inducedexencephaly. Together with the increased durationofheat shock protein synthesis, this indicates that lymphocytes from the SWV/SD strain are responding in a more dramatic
Lymphocyte Heat Shock Response
mannertothehyperthermic insult than are those from the other strains used in this study. These effects are lost in the F1 hybrid progeny of susceptible SWV/ SD and the more resistant DBA/2J strain. This work was supported i n part by a National Science Foundation predoctoral fellowship to V.K.M., and by research grant ES 04326 from theNational InstitutesofHealth to R.H.F. Theauthors wish to express their appreciation to MICHAELVAN WAESfor his critical review of the manuscript, PATRICIA AGER for her technical R. for editorial assistance, and C . SMITH,P. PERRON and THOMPSON and clerical assistance.
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