EXPERIMENTAL
CELL
RESEARCH
202,
9-16
(19%)
uction in Hea hock Gene Expressio rmosensitivity in Senescent The
Wistar
Institute
oj Anatomy
and Biology,
36th
The expression of three major classes of heat shock genes was examined in human diploid cells at differing in vitro ages. Metabolic labeling of cellular proteins following a brief heat shock showed that the synthesis of heat shock proteins was significantly reduced in latepassage cells. Northern blot analyses revealed that the reduced expression of beat shock proteins in old cells correlated with a reduced accumulation of heat shockspecific transcripts. The attenuation of heat shock gene activity in senescent cells was not unique to thermal stress since exposure of cells to sodium arsenite (lo-50 PM) elicited a similar response. The reduced expression of heat shock gene products correlated with an increased thermal lability in late-passage cells following acute hyperthermic (49°C) exposure. The preinduction of heat shock genes protected cells against the lethal effects of acute hyperthermia and abolished the increased thermal lability observed in senescent cells. The reduced expression of the heat shock response demonstrates that old cells possess a diminished ability to withstand adverse environmental conditions and maintain homeostasis. 8 1992 Academic Press, Inc.
INTRODIJCTION
The progressive loss of replicative potential and generalized degeneration of human cells in culture appear to be universal phenomena of all nontransformed cell types and have been extensively studied as a model of cellular aging. Although the specific mechanisms responsible for the limited lifespan of cells in culture are unknown, a progressive reduction in these cells’ ability to respond to environmental signals (e.g., mitogenic stimulation) is characteristic of the senescent phenotype. We have examined the ability of human diploid fibroblast-like cells to respond to environmental stress
’ Present address: Department of Molecular Biology, Roche Biomedical Laboratories, Research Triangle Park, NC 27709. ’ To whom reprint requests should be addressed at Center for Gerontological Research, Medical College of Pennsylvania, 3300 Henry Ave., Philadelphia, PA 19129.
and Spruce
Street,
Philadelphia,
Pennsyluania
19104
during their in vitro lifespan by measuring heat shock gene expression. All cells respond to stress through various changes in gene expression‘ Some of these changes are presumably induced to help the cell cope with adverse environmental conditions and thereby maintain homeostasis. One of the best studied stress responses is the heat shock response (HSR). The HSR has been demonstrated in all organisms studied, from bacteria to mammals [I]. It involves the elevated expression of a small set of highly conserved proteins upon exposure to a variety of environmental stresses. In addition to induction by stress, many heat shock proteins (HSP) are ex tutively and during normal developmental processes, suggesting other roles for these proteins besides protection from environmental stress [4 j. Althougln many different HSPs have been described, only for the major HSP (i.e., HSP 70) has any intracellular function been truly demonstrated. Studies by ies et al. [a] and Chirico et ~1. [3] showed that an 70 protein is involved in the targeting and translocation of proteins across intracellular membranes Add~tio~la~ly, HSP 70 has been implicated in some aspects of normal cell cycle progression since it is induced in quiescent cells following serum stimulation [4] and during transition through the G,/S boundary of the cell cycle [5]. Although some functions of HSP 70 have been demonstrated, other roles for this protein, as well as other HS not understood. One of the most apparent aspects of t HSPs is their ability to protect celis against potentially life-threatening stress Chinese hamster cells, which constitutively express SPs at high levels, are unusually resistant to elevated temperatures [E;]. Similarly, the preinduction of HSPs by mild hyperthermic exposure bestows thermotolerance to a normaliy lethal temperature in a wide variety of cell ty es [a-91. Moreover, conditions other than hyperthermia which also induce HSPs also convey thermotolera 0 cells [a, 10, Il]. Although most investigations of induction andthe acquisition of thermotolerance are only correlative, the presence of HSPs is most probably involved in protecting the cell against adverse env~~o~rnent~~ conditions. 9 All
Copyright 0 1992 rights of reproduction
QOL4-4827/92 $5.00 by Academic Press, Inc. in any form reserved.
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As the HSR is seemingly induced to protect cells against adverse conditions, the extent of expression of heat shock genes allows for the molecular analysis of how these cells retain the ability to maintain homeostasis during cellular aging. Moreover, the heat shock response can serve as an ideal model system for investigations of changes in gene regulation which accompany cellular aging due to the relative ease of manipulating the expression of these genes. MATERIALS
AND
METHODS
Materials. C3H]Leucine (118 Ci/mmol) was obtained from ICN (Irvine, CA) and [ol-32P]dCTP (3000 Ci/mmol) was purchased from NEN (Boston, MA). Minimum essential medium minus leucine was obtained from Flow Laboratories (McLean, VA). The plasmid pH2.3 1121 contains the entire coding sequence of a human HSP 70 gene and was obtained from the American Type Culture Collection (Rockville, MD). The plasmids pHS811 and pHS208 [13] are human cDNA clones of HSP 90 and HSP 28, respectively, and were obtained as a generous gift from Drs. L. Weber and E. Hickey (University of South Florida). The plasmid pB,M [14] eontains coding sequences of the human ~~-~croglobulin gene and was obtained from Dr. R. Baserga (Temple University). Cell culture conditions. The buman fibroblast-like cell line WI-38 was maintained in culture and serially passaged as previously described [15]. Young cell cultures were those which had completed less than 50% of their in vitro lifespan and old cell cultures were those which had completed greater than 90% of their lifespan. Subcultures used in these investigations achieved a cumulative population doubling level of approximately 65 doublings. Cultures were routinely tested for mycoplasma contamination as described 1161. No mycoplasma contamination was detected in any of the sublines used in this study. Viability of cells following acute hyperthermic exposure was determined using an erythrosine 13 dye exclusion assay [17]. Induction of the HSR by thermal stress was performed by seeding young or old cells at 3 X lo4 cells/cm’ (unless otherwise indicated) into t-75 (for RNA extraction) or t-25 (for protein labeling) tissue culture flasks. Approximately 48 h later, the culture medium was removed and replaced with medium which had been prewarmed to a temperature of 43°C. The culture vessels were then immediately submerged in a circulating water bath maintained at a constant temperature of 43*C for the indicated lengths of time. Following the heat shock, the cells were washed three times in phosphate-buffered saline (PBS) and’processed for either RNA isolation or protein metabolic labeling. Induc$ion of the HSR by sodium arsenite was achieved by seeding cells as described above and replacing the medium 48 h later with fresh medium containing lo-50 @f sodium arsenite. Cultures were then analyzed as described for heat shock-treated cells. Protein ~beli~ and ~~~-po~yac~~rnide gel e~etrop~res~. Cellular proteins were radiolabeled following heat shock or sodium arsenite exposure by washing the cells three times in PBS and adding minimum essential medium minus leucine which contained 10% dialyzed fetal bovine serum and 10 pCi/ml [3H]leucine. Following a 2-h incubation at 37”C, the cells were washed three times in PBS and removed from the substratum by trypsinization [15]. The trypsin was in~bited by the addition of 5 mi of complete medium and the cells pelleted by centrifugation at 75g for 10 min. The cells were washed three times by resuspending the pellet in 5 ml of PBS and centrifuging as described above. The final cell pellet was resuspended in 1.50 ~1 of lysis buffer (20 mM Tris, pH 7.4; 1 mM EDTA; 1 mM 2-mercaptoethanol; I mA4 phenylmethylsulfonyl fluoride; 1% SDS) and the ceils were lysed by three cycles of freezing and thawing. The cell lysate was then heated for 10 min at 65°C and insoluble material re-
CRISTOFALO moved by centrifugation at 14,OOOg for 5 min. Detection of radioactivity present in trichloroacetic acid-precipitable material was performed as described [lS].. and aliquots containing 50,000 cpm were loaded onto 10% SDS-polya~~lamida gels and electrophoresed at a constant 30 mA for 5 h. Gels were Auorographed using acetic acid and 2,5-diphenyloxazol as described by Skinner and Griswold [19]. Dried gels were placed against Kodak X-Omat X-ray film and stored at -70°C for 3-5 days. RNA isolation. Following heat shock or sodium arsenite exposure, cellular RNA was isolated using modifications of the procedure described by Bowman et aZ. (20). Cells were washed three times with PBS and lysed by the addition of 1.5 ml of RNA harvesting buffer (0.25 M NaCl; 30 mM Tris, pH 7.4; 5 mM EDTA; 1% SDS; and 1 mg/ml proteinase K). The lysate was incubated at 37’C for 30 min, after which time DNA was sheared by passing the lysate several times through a 26-gauge syringe needle. Cellular protein was removed by phenol/chloroform extraction, and the phases were separated by a lo-min ~entrifugation at 3OOOg. The resulting aqueous phase was extracted once with an equal volume of chloroform. Nucleic acids were precipitated by the addition of 3 vol of ethanol to the aqueous phase and allowing the mixture to incubate overnight at -20°C. The nucleic acids were collected by centrifugation at 10,OOOg for 20 min. To the precipitate was added 4 ml of a solution containing 2 M LiCl/IO mM EDTA and the mixture was extensively mixed over a 20-min period. High-mole~ular~weight RNA was isolated by ~entri~gation at 10,OOOg for 20 min, and the resulting pellet was resuspended in 10 mM Tris, pH 8.0; 1 mM EDTA. Northern blotting, hybridization, and washing conditions. Cellular RNA was denatured by glyoxylation and electrophoretically resolved on 1.0% agarose gels [21]. The RNA was transferred to Nytran nylon filters (Scheicher & Schuell, Keene, NH) by capillary blotting and fixed to the membrane by baking the filters in uucuo at 80°C for 2 h. Prior to prehybridization, glyoxal adducts were removed by placing the filters in boiling 30 mM Tris, pH 8.3, and allowing the solution to come to room temperature. Prehybridization was performed at 43°C for 2 h as described 1221. Hybridization was initiated by the addition of 50 ng of plasmid insert which had been radiolabeled with [cu-~‘P]dCTP by random primer extension. Hybridization was performed at 43°C for 16--18 h. After hybridization, filters were washed for 1 h at room temperature with 2X SSC, 0.2% SDS (1X SSC contains 0.15 MNaCl, 0.015 M sodium citrate) followed by a l-h wash at 55°C with 5X SSC, 0.2% SDS. The final wash was performed at 50°C for 15 min with 0.1X SSC, 0.2% SDS, after which time the filters were placed against Kodak X-Omat X-ray film. Autoradiography occurred at -70°C for 2-24 h. Densitometric scanning was performed using an ACD-18 automatic computing densitometer (Gelman Sciences, Ann Arbor, MI). Removal of hybridization signals prior to rehybridization with other probes was achieved by washing the filters in 0.1X SSC, 1.0% SDS at 80°C for 30 min.
RESULTS Induction
of HSPs
The exposure of WI-38 cells to mild hyperthermic conditions resulted in a pronounced change in the protein synthetic pattern of these cells. Figure 1 shows that the expos’ ee of cells to a temperature of 43°C caused the rapid induction of two proteins, one with an apparent molecular mass of 90 kDa and one of 70 kDa. The ‘IO-kDa protein was induced to a significantly greater extent and is consistent with the fact that HSP 70 is the
HEAT
SHOCK
RESPONSE
kDa
90 70 -
FIG. 1. Induction of beat shock proteins following hyperthermia. Young ceil cultures (lanes 1-3) or old cell cultures (lanes 4-6) were incubated at 43°C for either 20 min (lanes 2 and 5) or 40 min (lanes 3 and 6) and prepared for metabolic !abeling with [3H]leucine as described under Materials and Methods. Aliquots containing 50,000 cpm were applied to a 10% polyacrylamide gel and electrophoresed at a constant 30 mA for 5 h. The gel was fluorographed and placed against X-ray film for 3 days. Lanes 1 and 4 represent control cultures which were not beat shocked.
predominate heat shock protein synthesized following exposure to stress conditions [I]. The expression of these major HSPs was evident after as little as a 2O-min heat shock and was significantly larger after a 40-min heat shock. Although these HSPs were induced in both young and old cells, there was a disproportionate reduction in the expression of these proteins in old cells. In addition to thermal stress, exposing cultures to sodium arsenite also resulted in a change in the protein synthetic pattern of these cells (Fig. 2). A 2-h exposure to sodium arsenite caused two proteins of molecular weights 70,000 and 90,000 to be induced in a concentration-d ependent manner. Since both hyperthermia sodium arsenite have been previously shown to result in the induction of HSPs [ll]; we believe that these HSPs represent the same proteins that were induced following thermal stress. Moreover, old cell cultures showed a dem.onstrable reduction in the expression of these stress proteins at all sodium arsenite concentrations examined. The reduced expression of HSPs by old cells following sodium arsenite exposure was similar to that observed following beat shock and suggests that a similar mechanism is operating in response to both these inducers. The reduced expression of these HSPs was not the result of increased degradation of these proteins by old cells. Pulse-chase analysis of radiolabeled HSPs showed no differences between young and old cells in the decay of these proteins (not shown).
IN
AGING
Induction
HUMAN
CELLS
of Heat Shock Transcripts
Since the expression of HS s is known to be re lated at both the transcriptional and the kranslatio level in eucaryotie cells [I], nucleic acid hybridization studies were performed to determine if the r’educed expression of HSPs was the result of a reduction in transcriptional activity of these genes. Northern blot analysis of total cellular RNA from young cells showed that increased HSP 70 transcripts could be detected as soon as 10 min after a temperature shift to 43°C. Densitometric scanning showed that by 40 min transcripts had increased to over 50 times bas In contrast, old cells showed a reduced expression of HSP 70 mRNA with only a PO-fold increase after an identical 40-mainexposure to the tem~erat~~r~ sbift. Similarly, expression of SP 90 and HSP 2 also showed a generalized reduced i~d~ct~o~ by old cells. To ensure that the differences in heat shock r&WA levels were a true reflection of differences in the amount of specific transcripts, and not the result of diflerenees in the relative mRNA pools between young and old cells, filters were hybridized to cDNA fragment containing sequences of human &lobulin. As shown in Fig. 3, the amount of this m was essentially identical between young and old and remained constant throughout the heat shock. Although the isolation of RN was performed immediately following thermal stress, it is possible that an enhanced rate of heat shock-specific mRNA decay was
kDa
90 70 -
and
FIG. 2. Induction of heat shock proteins following exposure to sodium arsenite. Young cells (lznes 1-3) or old cells (lanes 4-6) were exposed to 25 PLM (lanes 2 and 5) or 50 ~IW sodium arsenite (lanes 3 and 6) for 2 h and cellular proteins were radiolabeled and analyzed as described in the legend to Fig. 1. Lanes I and 4 show controls cells which were not exposed to sodium arsenite.
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HSP
70
HSP
90
HSP
28 -
P2M
-
FIG. 3. Induction of heat shock transcripts following hyperthermia. Cells were heat shocked and RNA was isolated as described under Materials and Methods. Total cellular RNA was isolated from cells after a lo-, 20-, or 40-min hype~hermic exposure (lanes 2-4 and 6-8, respectively). Total cellular RNA (3.5 pg) was electrophoret~cally resolved, transferred onto nylon filters, and sequentially hybridized to the probes pH2.3, pHS811, pHS208, and pB,M. Lanes 1-4 represent young cell cultures and lanes 5-8 represent old cell cultures. Lanes 1 and 5 show control cultures which were not heat shocked.
CRISTOFALO
for heat shock gene induction has been altered in senescent cells. Although it is possible that changes in the primary structure of these genes have occurred in latepassage cells, we feel that this is an unlikely explanation since these putative changes would have had to occur at three distinct loci corresponding to the three heat shock genes examined.
The most demonstrative characteristic of senescent cells in culture is their loss of proliferative capacity. It is possible that many of the phenotypic changes which occur during the limited lifespan of cells in culture are a direct consequence of this loss of mitotic potential. In order to determine if the reduction in the inducibility of heat shock genes in senescent cells is related to the proliferative nature of these cells, young cells were rendered quiescent by contact inhibition and tested for their ability to induce heat shock genes. As seen in Fig. 5, quiescent cells possess a capacity to express heat shock-specific transcripts equal to that of proliferating young cell cultures. The ability of growth-arrested cultures to induce heat shock genes to the same extent as that of proliferating cells shows that the reduced expression of the HSR in old cells is not a function of their proliferative state and is a consequence of some age-related change in their ability to regulate gene expression.
occurring in old cells, resulting in a reduced accumulation of these transcripts. However, recently we have shown that the turnover of heat shock transcripts is greatly reduced in old cells, ruling out the possibility 12345678 that the diminished expression of these gene products in old cells is the result of increased mRNA decay kinetics (Lute and Cristofalo, manuscript in preparation). Thus, the quantitative differences in heat shock transcripts between young and old cells appears to be the result of an inherent change in the inducibility of these genes during in ~ktro cellular aging. In order to determine whether the reduced ability of old cells to express heat shock-specific transcripts was unique to thermal stress, young and old cell cultures were exposed to varying concentrations of sodium arsenite and total cell RNA was isolated as described above. Figure 4 shows that treatment of cells with increasing concentrations of sodium arsenite resulted in a concomitant increase in HSP 70 transcripts. Similarly, HSP 90 and HSP 28 mRNAs were also induced in a concentration-dependent manner. However, as previP2M ously shown with thermal stress, old cells showed a diminished ability to induce heat shock-specific transcripts in response to sodium arsenite. Comparison of FIG. 4. Induction of heat shock transcripts following exposure to sodium arsenite. Northern blot analysis and hybridizations were perFigs. 3 and 4 shows that the relative differences in the formed as described in the legend to Fig. 3. RNA was isolated from inducibility of HSP genes between young and old cells young cells (lanes I-4) or old cells (lanes 5-8) after a 2-h exposure to are essentially equivalent whether cells are subjected to 10, 25, or 50 PM sodium arsenite (lanes 2-4, respectively). Lanes 1 heat shock or to sodium arsenite. These data suggest and 5 show control cultures which were not exposed to sodium arsenthat the intracellular signal mechanism(s) responsible ite.
HEAT !
HSP
70 -
HSP
90
HSP
28
2
SHOCK
RESPONSE
34
FIG. 5. Effect of the replicative state of cultures on the induction of heat shock transcripts. Young cells were inoculated at a density of 3 X lo4 cells per cm’ (lanes 1 and 2) or 1 X lo5 cells/cm’ (lanes 3 and 4). Forty-eight hours later the cells were heat shocked for 40 min (lane 2 and 4), and RNA was isolated as described under Materials and Methods. Northern blot analysis was performed as described in the legend to Fig. 3. Lanes 1 and 3 show control cultures which were not heat shocked.
Effect uf Cellular Hyperthermia
Age on Viability
following
Acute
One of the most apparent functions of heat shock proteins is their ability to protect cells against potentially lethal environmental stress. In order to examine how these cells respond to acute hyperthermia (both under normal growth conditions and after preinduction of HSPs) cultures were observed shortly after a rapid temperature shift. Figure 6 shows that the exposure of ei.ther young or old cells to a temperature of 49°C for 40 min caused pronounced changes in cell morphology and initiated the detachment of cells from the substratum (Figs. 6A and 6C). However, prior induction of heat shock proteins by a 2-h treatment with 25 PM sodium arsenite, followed by an acute hyperthermic exposure identical to that described above, resulted in an increased ability of these cells to maintain normal morphology and reduced the tendency of these cells to detach from the substratum (Figs. 6B and 6D). Similarly, the preinduction of HSPs by mild hyperthermia (40 min at 43°C) also conveyed a protective effect to these cells as indicated by an increased ability to resist changes in morphology following acute hyperthermic exposure (not shown). These data demonstrate that conditions which induce EISPs are able to allow these cells to resist morphologic changes which are associated with exposure to acute hyperthermia.
IN
AGING
HUMAN
CELLS
13
As the preinduction of HSPs appeared to convey a protective effect to these cells (as i tenance of morphologic integrity), performed to ascertain whether a feet is observed against cell death. Sine conditions are widely known to per&u thetic activity 111, we necessarily chose assay, ertbrosine B dye exclusion as a which would be independent of the i~bibitory effects of heat shock on de nova protein synthesis. Moreover, after detailed examination of the effects of beat shock on protein synthesis, clear differences in how young and old cells responded to thermal stress were revealed. At a wide range of temperatures (44-49”C), old cells demonstrated a greater sensitivity to the i~bibitory effects of heat shock on protein synthesis (not shown). Therefore, to avoid any exaggerated killing effects that might appear in old cells if cessation of protein synthesis was used as a viability index, a dye exclusion was chosen as the most definitive assay for measuring the immediate killing response following acute hyperthermia. Figure 7 shows the results of four i~de~e~~e~~ experiments in which cultures were expos of49”C. lengths of time to a temperature ature, young cells showed a near linear number of surviving cells as a function of time, with an average LD,, occurring at approximately 60 min. Senescent cells also showed a time-dependent decrease in the number of surviving cells; however, their abiiity to withstand this temperature was significantly reduced from examined, that of young cells (P < 0.05 at all t e points as determined by Student’s 1 test). oreover, the prein(by a prior treatment duction of beat shock proteins with 25 p*M sodium arsenit not only stabilized cell mosthe viabil.ity of cells followphology, but also increase ing acute hyperthermic e sure (compare 60 min with 60 min preinduced). It is interesting to note that both young and old cells were simil protected from this e feel that this is most stress by preinduction of IISPs. likely the result of the preinduction treatment yielding sufficient HSPs in both cultures to convey a similar protective effect. However, in the absence of preinduction, senescent cells were unable to synthesize heat shock gene products as fast, and to the same levels, as young cells and succumbed to the lethal efIects of acute hyperthermia to a greater extent.
The results of this investigation have shown that the induction of three major classes of heat shock genes following exposure to environmental stress is attenuated in senescent human fibroblasts. The reduced expression of heat shock gene products by old cells correlates with a diminished ability to withstan oreover, the prior inlethal hyperthermic conditions.
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CRISTOFALO
FIG. 6. Protection from acute hyperthermic exposure by preinduction of heat shock proteins. Young cells (A and B) or old cells (C and D) inverted microscope and Kodak were exposed to a temperature of 49°C for 40 min and cultures immediately photographed usin, u a Nikon Tri-X film. B and D show cells which were t~reated with 26 J& sodium arsenite for 2 h immediately before an acute hyperthermic exposure identical to that described above.
duction of HSPs conveys a protective effect against lethal hyperthermic conditions, establishing a correlation between HSPs and the ability to withstand stress in these cells. These data suggest that one mechanism by which senescent cells lose their ability to maintain homeostasis is through a reduced ability to express heat shock gene products. A relationship between acquired thermotoleran~e and heat shock protein synthesis has been well documented in several species of eucaryotic organisms (for review see [ 11). Although many previous investigations have shown an association between thermotoleran~e and HSP synthesis, direct involvement of HSPs and protection against thermal damage had not established. However, Riabowol et al. [23] showed direct involvement of HSP 70 and the ability of cells to withstand heat shock by use of a neutralizing antibody against HSP 70. Similarly, Johnston and Kucey [24] showed that cells containing a high copy number of HSP 70 5’ flanking sequences displayed a greatly reduced ability to express HSP 70 and a corresponding increase in thermosensiti-
vity. A species of Hydra which lives in a thermally stable environment, and which has lost the ability to express an HSP ‘IO-like product, is significantly more thermolabile than a similar species which is able to express this major heat shock protein 1251. Moreover, only the species of Hydra which is able to induce an HSP 70-like protein is able to acquire thermotolerance. Although the molecular mechanisms by which HSP 70, and possibly other HSPs, is able to confer protection against stress are unknown, their presence is most probably participating in a basic cellular defense system which allows cells to cope with potentially damaging environmental conditions. Just as the mechanisms of action of HSPs are largely unknown, the intracellular signals which initiate expression of heat shock genes are also presently not understood. Although the inducers of the heat shock response are numerous and seeming diverse, one common characteristic most share is the ability to create altered intracellular proteins. A ~~0~0~~~~ strain which synthesizes mutant forms of actin constitutively expresses
HEAT
SHOCK
RESPONSE
IN
AGlNG
~~~A~
CELLS
15
cells in our ~~vest~gatio~* However9 it is ~oss~~~e that the level of abnormal proteins beheved to be present in senescent cells is insufficient to evoke the heat shock response. Studies of the stress response 0fTer opportunities to describe in molecular terms how organisms are able to resist environmental change and maintain homeostasis as they age. A diminished abiiity to ‘ntain homeostasis has been described as a primary raeteristic of aging 1321.However, i~vest~gat~o~s of changes in gene expression which result in a decrease ability to rn~~~t~~~ homeostasis have, for t,he most art, been lacking. Tsuiji. et al. 1331resorted that at a e passage, e-hebuman fibroblast cell line TfG-I retained the ability to synthesize beat-~~~~~~~~e polypeptides. Lui et al. [34,35] showed that senescen.t fibroblasts expressed lower amounts of products following exposure to cers, and that this 0 20 40 60 60 P.I. reduced expression was attribu to tra~s~~~~~i~~~~ Minutes at 49°C mechanisms Similarly, Fargnol . [36] ~-e-portedthat FIG. 7. Cell viability following acute hyperthermic conditions. rat fibrob~asts &owed a reduced a~~~~tyto elicit, the heat Cells were exposed to a temperature of 49°C for the indicated length shock response as a ~~~~~~~~of the age of the ani of time and ~mmed~at~Iy removed from the substratum by trypsinizafrom which the cells were obtained. These data ar tion. Cells were collected by a IO-mm ce~trifugat~oll at 75g and resussig~i~can~e since they suggest that a reduced abihty to pended in the medium which was used during the hyperthermic expoexpress heat shock genes may be an inherent. charactersure to reintroduce cells which may have detached under this condition. Cells were centrifuged as described above and resuspended in istic of organismal aging and not merely an artifact asPBS, and cell viability was measured using an erythrosine B dye exsociated with fibroblasts cultured in u&e. In another clusion as described under Materials and Methods. Shown are the series of in vim experiments, Fleming et al. [37] showed mean results of four independent experiments (&I SEM). The results by two-dimensional gel electrophosesis that old Droshown for 60 PI are cultures in which HSPs were preinduced with sodium arsenite as described in the legend to Fig. 6 followed by a sophila synthesize a Barges number of Bow-molecuiar60-min exposure to a temperature of 49°C. Slashed bars represent weight heat shock proteins than young flies fo~~~~~~g young cell cultures and the closed bars show old cell cultures. exposure to elevated tempera es Such an observation is interesting in that it HSPs [ZSJ. Sirn~~~rly, prod~~t~o~ of large amounts of changes (i.e., never gene ~rodn~ts~ during aging. HOWever, it may not address t,he ~~e~~~~~of a reduced ability abnormal proteins by ~~~~~~~~~i~ co& activates transcription of heat shock genes [27]. Ananthan et aE. [28] for adaptive change in response to stress, since most studies have implicated the major ~~gb,~rn~~e~~~~~showed that rni~~~~~je~tio~ of conformationally altered SB (i.e., HSP 70) as being responsible for proproteins into Xenopus oocytes could induce the heat tecting cells against stress [B-25]. shock response, providing direct evidence that abnorTo our knowledge, the results presented in this study mal proteins can serve as an intracellular signal to inare the first to suggest that the expression of genes reduce this response in eucaryotic cells. sponsible for maintaining homeostasis is reduced to a The fact that altered proteins can elicit the HSR is significant in light of our results showing reduced ex- physiologically significant extent (as shown by the differential sensitivity between young and se~es~e~~cells pression of heat shock genes in senescent cells. Many to acute hype~the~mia~ during cehular aging. Since our changes in protein synthesis and post-translational processes during cellular aging have been described. Re- investigation showed that a red~~~~~ in three major duction in the fideelity of protein synthesis [%I, postclasses of heat shock genes occurred ~~~~~~rn~~~~t~yin translational ~odi~~atio~s such as deamination 1301, late-passage cells, it is likely that the rn@~~a~~srnresponand changes in protein conformation [31] have all been sible for signal t~a~~~~ct~o~ f~‘olfowing stress is attenuated during aging. It is interestingto note that an addishown to occur during aging of cultured cells. The intracellular accumulation of altered proteins could theretional cellular defense system rn~~~~~~t~~o~e~~inducfore potentially lead to the induction of the heat shock tion, is also significantly reduced in senescent human response as cells senesce. In light of these observations, cells following exposure to toxic met.& ions [38, 391. It it is interesting that an increase in the basal level ex- may therefore be a ~~~~ra~~~e~~~e~o~~e~~~ of diminpression of heat shock genes was not evident. in senes- ished signaling pathways which leads to a reduced abilcent
16
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ity of old cells to readily ment. This work was supported Institutes of Health.
adapt to a changing
by Grant
AG 00131-03
from
AND
environ-
3.
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CRISTOFALO
Mech.
Mech.
of Aging,
of Aging,
pp. 639-
Ageing
Deu. 36,
Li,
B. (1989)
F., and Li, B. (1989)
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T., Fornance, A. J., Schneider, E. L., and Proc. Natl. Acad. Sci. USA 87, 846-850. J. K., Dubitsky, R., and Bensch, Sci. USA 85, 4099-4103. M. C. (1988) C. L. (1992)
K. G.
In Vitro Exp.
Cell.
Geron-
CELL
RESEARCH
202,
9-16
(19%)
uction in Hea hock Gene Expressio rmosensitivity in Senescent The
Wistar
Institute
oj Anatomy
and Biology,
36th
The expression of three major classes of heat shock genes was examined in human diploid cells at differing in vitro ages. Metabolic labeling of cellular proteins following a brief heat shock showed that the synthesis of heat shock proteins was significantly reduced in latepassage cells. Northern blot analyses revealed that the reduced expression of beat shock proteins in old cells correlated with a reduced accumulation of heat shockspecific transcripts. The attenuation of heat shock gene activity in senescent cells was not unique to thermal stress since exposure of cells to sodium arsenite (lo-50 PM) elicited a similar response. The reduced expression of heat shock gene products correlated with an increased thermal lability in late-passage cells following acute hyperthermic (49°C) exposure. The preinduction of heat shock genes protected cells against the lethal effects of acute hyperthermia and abolished the increased thermal lability observed in senescent cells. The reduced expression of the heat shock response demonstrates that old cells possess a diminished ability to withstand adverse environmental conditions and maintain homeostasis. 8 1992 Academic Press, Inc.
INTRODIJCTION
The progressive loss of replicative potential and generalized degeneration of human cells in culture appear to be universal phenomena of all nontransformed cell types and have been extensively studied as a model of cellular aging. Although the specific mechanisms responsible for the limited lifespan of cells in culture are unknown, a progressive reduction in these cells’ ability to respond to environmental signals (e.g., mitogenic stimulation) is characteristic of the senescent phenotype. We have examined the ability of human diploid fibroblast-like cells to respond to environmental stress
’ Present address: Department of Molecular Biology, Roche Biomedical Laboratories, Research Triangle Park, NC 27709. ’ To whom reprint requests should be addressed at Center for Gerontological Research, Medical College of Pennsylvania, 3300 Henry Ave., Philadelphia, PA 19129.
and Spruce
Street,
Philadelphia,
Pennsyluania
19104
during their in vitro lifespan by measuring heat shock gene expression. All cells respond to stress through various changes in gene expression‘ Some of these changes are presumably induced to help the cell cope with adverse environmental conditions and thereby maintain homeostasis. One of the best studied stress responses is the heat shock response (HSR). The HSR has been demonstrated in all organisms studied, from bacteria to mammals [I]. It involves the elevated expression of a small set of highly conserved proteins upon exposure to a variety of environmental stresses. In addition to induction by stress, many heat shock proteins (HSP) are ex tutively and during normal developmental processes, suggesting other roles for these proteins besides protection from environmental stress [4 j. Althougln many different HSPs have been described, only for the major HSP (i.e., HSP 70) has any intracellular function been truly demonstrated. Studies by ies et al. [a] and Chirico et ~1. [3] showed that an 70 protein is involved in the targeting and translocation of proteins across intracellular membranes Add~tio~la~ly, HSP 70 has been implicated in some aspects of normal cell cycle progression since it is induced in quiescent cells following serum stimulation [4] and during transition through the G,/S boundary of the cell cycle [5]. Although some functions of HSP 70 have been demonstrated, other roles for this protein, as well as other HS not understood. One of the most apparent aspects of t HSPs is their ability to protect celis against potentially life-threatening stress Chinese hamster cells, which constitutively express SPs at high levels, are unusually resistant to elevated temperatures [E;]. Similarly, the preinduction of HSPs by mild hyperthermic exposure bestows thermotolerance to a normaliy lethal temperature in a wide variety of cell ty es [a-91. Moreover, conditions other than hyperthermia which also induce HSPs also convey thermotolera 0 cells [a, 10, Il]. Although most investigations of induction andthe acquisition of thermotolerance are only correlative, the presence of HSPs is most probably involved in protecting the cell against adverse env~~o~rnent~~ conditions. 9 All
Copyright 0 1992 rights of reproduction
QOL4-4827/92 $5.00 by Academic Press, Inc. in any form reserved.
10
LUCE
AND
As the HSR is seemingly induced to protect cells against adverse conditions, the extent of expression of heat shock genes allows for the molecular analysis of how these cells retain the ability to maintain homeostasis during cellular aging. Moreover, the heat shock response can serve as an ideal model system for investigations of changes in gene regulation which accompany cellular aging due to the relative ease of manipulating the expression of these genes. MATERIALS
AND
METHODS
Materials. C3H]Leucine (118 Ci/mmol) was obtained from ICN (Irvine, CA) and [ol-32P]dCTP (3000 Ci/mmol) was purchased from NEN (Boston, MA). Minimum essential medium minus leucine was obtained from Flow Laboratories (McLean, VA). The plasmid pH2.3 1121 contains the entire coding sequence of a human HSP 70 gene and was obtained from the American Type Culture Collection (Rockville, MD). The plasmids pHS811 and pHS208 [13] are human cDNA clones of HSP 90 and HSP 28, respectively, and were obtained as a generous gift from Drs. L. Weber and E. Hickey (University of South Florida). The plasmid pB,M [14] eontains coding sequences of the human ~~-~croglobulin gene and was obtained from Dr. R. Baserga (Temple University). Cell culture conditions. The buman fibroblast-like cell line WI-38 was maintained in culture and serially passaged as previously described [15]. Young cell cultures were those which had completed less than 50% of their in vitro lifespan and old cell cultures were those which had completed greater than 90% of their lifespan. Subcultures used in these investigations achieved a cumulative population doubling level of approximately 65 doublings. Cultures were routinely tested for mycoplasma contamination as described 1161. No mycoplasma contamination was detected in any of the sublines used in this study. Viability of cells following acute hyperthermic exposure was determined using an erythrosine 13 dye exclusion assay [17]. Induction of the HSR by thermal stress was performed by seeding young or old cells at 3 X lo4 cells/cm’ (unless otherwise indicated) into t-75 (for RNA extraction) or t-25 (for protein labeling) tissue culture flasks. Approximately 48 h later, the culture medium was removed and replaced with medium which had been prewarmed to a temperature of 43°C. The culture vessels were then immediately submerged in a circulating water bath maintained at a constant temperature of 43*C for the indicated lengths of time. Following the heat shock, the cells were washed three times in phosphate-buffered saline (PBS) and’processed for either RNA isolation or protein metabolic labeling. Induc$ion of the HSR by sodium arsenite was achieved by seeding cells as described above and replacing the medium 48 h later with fresh medium containing lo-50 @f sodium arsenite. Cultures were then analyzed as described for heat shock-treated cells. Protein ~beli~ and ~~~-po~yac~~rnide gel e~etrop~res~. Cellular proteins were radiolabeled following heat shock or sodium arsenite exposure by washing the cells three times in PBS and adding minimum essential medium minus leucine which contained 10% dialyzed fetal bovine serum and 10 pCi/ml [3H]leucine. Following a 2-h incubation at 37”C, the cells were washed three times in PBS and removed from the substratum by trypsinization [15]. The trypsin was in~bited by the addition of 5 mi of complete medium and the cells pelleted by centrifugation at 75g for 10 min. The cells were washed three times by resuspending the pellet in 5 ml of PBS and centrifuging as described above. The final cell pellet was resuspended in 1.50 ~1 of lysis buffer (20 mM Tris, pH 7.4; 1 mM EDTA; 1 mM 2-mercaptoethanol; I mA4 phenylmethylsulfonyl fluoride; 1% SDS) and the ceils were lysed by three cycles of freezing and thawing. The cell lysate was then heated for 10 min at 65°C and insoluble material re-
CRISTOFALO moved by centrifugation at 14,OOOg for 5 min. Detection of radioactivity present in trichloroacetic acid-precipitable material was performed as described [lS].. and aliquots containing 50,000 cpm were loaded onto 10% SDS-polya~~lamida gels and electrophoresed at a constant 30 mA for 5 h. Gels were Auorographed using acetic acid and 2,5-diphenyloxazol as described by Skinner and Griswold [19]. Dried gels were placed against Kodak X-Omat X-ray film and stored at -70°C for 3-5 days. RNA isolation. Following heat shock or sodium arsenite exposure, cellular RNA was isolated using modifications of the procedure described by Bowman et aZ. (20). Cells were washed three times with PBS and lysed by the addition of 1.5 ml of RNA harvesting buffer (0.25 M NaCl; 30 mM Tris, pH 7.4; 5 mM EDTA; 1% SDS; and 1 mg/ml proteinase K). The lysate was incubated at 37’C for 30 min, after which time DNA was sheared by passing the lysate several times through a 26-gauge syringe needle. Cellular protein was removed by phenol/chloroform extraction, and the phases were separated by a lo-min ~entrifugation at 3OOOg. The resulting aqueous phase was extracted once with an equal volume of chloroform. Nucleic acids were precipitated by the addition of 3 vol of ethanol to the aqueous phase and allowing the mixture to incubate overnight at -20°C. The nucleic acids were collected by centrifugation at 10,OOOg for 20 min. To the precipitate was added 4 ml of a solution containing 2 M LiCl/IO mM EDTA and the mixture was extensively mixed over a 20-min period. High-mole~ular~weight RNA was isolated by ~entri~gation at 10,OOOg for 20 min, and the resulting pellet was resuspended in 10 mM Tris, pH 8.0; 1 mM EDTA. Northern blotting, hybridization, and washing conditions. Cellular RNA was denatured by glyoxylation and electrophoretically resolved on 1.0% agarose gels [21]. The RNA was transferred to Nytran nylon filters (Scheicher & Schuell, Keene, NH) by capillary blotting and fixed to the membrane by baking the filters in uucuo at 80°C for 2 h. Prior to prehybridization, glyoxal adducts were removed by placing the filters in boiling 30 mM Tris, pH 8.3, and allowing the solution to come to room temperature. Prehybridization was performed at 43°C for 2 h as described 1221. Hybridization was initiated by the addition of 50 ng of plasmid insert which had been radiolabeled with [cu-~‘P]dCTP by random primer extension. Hybridization was performed at 43°C for 16--18 h. After hybridization, filters were washed for 1 h at room temperature with 2X SSC, 0.2% SDS (1X SSC contains 0.15 MNaCl, 0.015 M sodium citrate) followed by a l-h wash at 55°C with 5X SSC, 0.2% SDS. The final wash was performed at 50°C for 15 min with 0.1X SSC, 0.2% SDS, after which time the filters were placed against Kodak X-Omat X-ray film. Autoradiography occurred at -70°C for 2-24 h. Densitometric scanning was performed using an ACD-18 automatic computing densitometer (Gelman Sciences, Ann Arbor, MI). Removal of hybridization signals prior to rehybridization with other probes was achieved by washing the filters in 0.1X SSC, 1.0% SDS at 80°C for 30 min.
RESULTS Induction
of HSPs
The exposure of WI-38 cells to mild hyperthermic conditions resulted in a pronounced change in the protein synthetic pattern of these cells. Figure 1 shows that the expos’ ee of cells to a temperature of 43°C caused the rapid induction of two proteins, one with an apparent molecular mass of 90 kDa and one of 70 kDa. The ‘IO-kDa protein was induced to a significantly greater extent and is consistent with the fact that HSP 70 is the
HEAT
SHOCK
RESPONSE
kDa
90 70 -
FIG. 1. Induction of beat shock proteins following hyperthermia. Young ceil cultures (lanes 1-3) or old cell cultures (lanes 4-6) were incubated at 43°C for either 20 min (lanes 2 and 5) or 40 min (lanes 3 and 6) and prepared for metabolic !abeling with [3H]leucine as described under Materials and Methods. Aliquots containing 50,000 cpm were applied to a 10% polyacrylamide gel and electrophoresed at a constant 30 mA for 5 h. The gel was fluorographed and placed against X-ray film for 3 days. Lanes 1 and 4 represent control cultures which were not beat shocked.
predominate heat shock protein synthesized following exposure to stress conditions [I]. The expression of these major HSPs was evident after as little as a 2O-min heat shock and was significantly larger after a 40-min heat shock. Although these HSPs were induced in both young and old cells, there was a disproportionate reduction in the expression of these proteins in old cells. In addition to thermal stress, exposing cultures to sodium arsenite also resulted in a change in the protein synthetic pattern of these cells (Fig. 2). A 2-h exposure to sodium arsenite caused two proteins of molecular weights 70,000 and 90,000 to be induced in a concentration-d ependent manner. Since both hyperthermia sodium arsenite have been previously shown to result in the induction of HSPs [ll]; we believe that these HSPs represent the same proteins that were induced following thermal stress. Moreover, old cell cultures showed a dem.onstrable reduction in the expression of these stress proteins at all sodium arsenite concentrations examined. The reduced expression of HSPs by old cells following sodium arsenite exposure was similar to that observed following beat shock and suggests that a similar mechanism is operating in response to both these inducers. The reduced expression of these HSPs was not the result of increased degradation of these proteins by old cells. Pulse-chase analysis of radiolabeled HSPs showed no differences between young and old cells in the decay of these proteins (not shown).
IN
AGING
Induction
HUMAN
CELLS
of Heat Shock Transcripts
Since the expression of HS s is known to be re lated at both the transcriptional and the kranslatio level in eucaryotie cells [I], nucleic acid hybridization studies were performed to determine if the r’educed expression of HSPs was the result of a reduction in transcriptional activity of these genes. Northern blot analysis of total cellular RNA from young cells showed that increased HSP 70 transcripts could be detected as soon as 10 min after a temperature shift to 43°C. Densitometric scanning showed that by 40 min transcripts had increased to over 50 times bas In contrast, old cells showed a reduced expression of HSP 70 mRNA with only a PO-fold increase after an identical 40-mainexposure to the tem~erat~~r~ sbift. Similarly, expression of SP 90 and HSP 2 also showed a generalized reduced i~d~ct~o~ by old cells. To ensure that the differences in heat shock r&WA levels were a true reflection of differences in the amount of specific transcripts, and not the result of diflerenees in the relative mRNA pools between young and old cells, filters were hybridized to cDNA fragment containing sequences of human &lobulin. As shown in Fig. 3, the amount of this m was essentially identical between young and old and remained constant throughout the heat shock. Although the isolation of RN was performed immediately following thermal stress, it is possible that an enhanced rate of heat shock-specific mRNA decay was
kDa
90 70 -
and
FIG. 2. Induction of heat shock proteins following exposure to sodium arsenite. Young cells (lznes 1-3) or old cells (lanes 4-6) were exposed to 25 PLM (lanes 2 and 5) or 50 ~IW sodium arsenite (lanes 3 and 6) for 2 h and cellular proteins were radiolabeled and analyzed as described in the legend to Fig. 1. Lanes I and 4 show controls cells which were not exposed to sodium arsenite.
12
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12345678
HSP
70
HSP
90
HSP
28 -
P2M
-
FIG. 3. Induction of heat shock transcripts following hyperthermia. Cells were heat shocked and RNA was isolated as described under Materials and Methods. Total cellular RNA was isolated from cells after a lo-, 20-, or 40-min hype~hermic exposure (lanes 2-4 and 6-8, respectively). Total cellular RNA (3.5 pg) was electrophoret~cally resolved, transferred onto nylon filters, and sequentially hybridized to the probes pH2.3, pHS811, pHS208, and pB,M. Lanes 1-4 represent young cell cultures and lanes 5-8 represent old cell cultures. Lanes 1 and 5 show control cultures which were not heat shocked.
CRISTOFALO
for heat shock gene induction has been altered in senescent cells. Although it is possible that changes in the primary structure of these genes have occurred in latepassage cells, we feel that this is an unlikely explanation since these putative changes would have had to occur at three distinct loci corresponding to the three heat shock genes examined.
The most demonstrative characteristic of senescent cells in culture is their loss of proliferative capacity. It is possible that many of the phenotypic changes which occur during the limited lifespan of cells in culture are a direct consequence of this loss of mitotic potential. In order to determine if the reduction in the inducibility of heat shock genes in senescent cells is related to the proliferative nature of these cells, young cells were rendered quiescent by contact inhibition and tested for their ability to induce heat shock genes. As seen in Fig. 5, quiescent cells possess a capacity to express heat shock-specific transcripts equal to that of proliferating young cell cultures. The ability of growth-arrested cultures to induce heat shock genes to the same extent as that of proliferating cells shows that the reduced expression of the HSR in old cells is not a function of their proliferative state and is a consequence of some age-related change in their ability to regulate gene expression.
occurring in old cells, resulting in a reduced accumulation of these transcripts. However, recently we have shown that the turnover of heat shock transcripts is greatly reduced in old cells, ruling out the possibility 12345678 that the diminished expression of these gene products in old cells is the result of increased mRNA decay kinetics (Lute and Cristofalo, manuscript in preparation). Thus, the quantitative differences in heat shock transcripts between young and old cells appears to be the result of an inherent change in the inducibility of these genes during in ~ktro cellular aging. In order to determine whether the reduced ability of old cells to express heat shock-specific transcripts was unique to thermal stress, young and old cell cultures were exposed to varying concentrations of sodium arsenite and total cell RNA was isolated as described above. Figure 4 shows that treatment of cells with increasing concentrations of sodium arsenite resulted in a concomitant increase in HSP 70 transcripts. Similarly, HSP 90 and HSP 28 mRNAs were also induced in a concentration-dependent manner. However, as previP2M ously shown with thermal stress, old cells showed a diminished ability to induce heat shock-specific transcripts in response to sodium arsenite. Comparison of FIG. 4. Induction of heat shock transcripts following exposure to sodium arsenite. Northern blot analysis and hybridizations were perFigs. 3 and 4 shows that the relative differences in the formed as described in the legend to Fig. 3. RNA was isolated from inducibility of HSP genes between young and old cells young cells (lanes I-4) or old cells (lanes 5-8) after a 2-h exposure to are essentially equivalent whether cells are subjected to 10, 25, or 50 PM sodium arsenite (lanes 2-4, respectively). Lanes 1 heat shock or to sodium arsenite. These data suggest and 5 show control cultures which were not exposed to sodium arsenthat the intracellular signal mechanism(s) responsible ite.
HEAT !
HSP
70 -
HSP
90
HSP
28
2
SHOCK
RESPONSE
34
FIG. 5. Effect of the replicative state of cultures on the induction of heat shock transcripts. Young cells were inoculated at a density of 3 X lo4 cells per cm’ (lanes 1 and 2) or 1 X lo5 cells/cm’ (lanes 3 and 4). Forty-eight hours later the cells were heat shocked for 40 min (lane 2 and 4), and RNA was isolated as described under Materials and Methods. Northern blot analysis was performed as described in the legend to Fig. 3. Lanes 1 and 3 show control cultures which were not heat shocked.
Effect uf Cellular Hyperthermia
Age on Viability
following
Acute
One of the most apparent functions of heat shock proteins is their ability to protect cells against potentially lethal environmental stress. In order to examine how these cells respond to acute hyperthermia (both under normal growth conditions and after preinduction of HSPs) cultures were observed shortly after a rapid temperature shift. Figure 6 shows that the exposure of ei.ther young or old cells to a temperature of 49°C for 40 min caused pronounced changes in cell morphology and initiated the detachment of cells from the substratum (Figs. 6A and 6C). However, prior induction of heat shock proteins by a 2-h treatment with 25 PM sodium arsenite, followed by an acute hyperthermic exposure identical to that described above, resulted in an increased ability of these cells to maintain normal morphology and reduced the tendency of these cells to detach from the substratum (Figs. 6B and 6D). Similarly, the preinduction of HSPs by mild hyperthermia (40 min at 43°C) also conveyed a protective effect to these cells as indicated by an increased ability to resist changes in morphology following acute hyperthermic exposure (not shown). These data demonstrate that conditions which induce EISPs are able to allow these cells to resist morphologic changes which are associated with exposure to acute hyperthermia.
IN
AGING
HUMAN
CELLS
13
As the preinduction of HSPs appeared to convey a protective effect to these cells (as i tenance of morphologic integrity), performed to ascertain whether a feet is observed against cell death. Sine conditions are widely known to per&u thetic activity 111, we necessarily chose assay, ertbrosine B dye exclusion as a which would be independent of the i~bibitory effects of heat shock on de nova protein synthesis. Moreover, after detailed examination of the effects of beat shock on protein synthesis, clear differences in how young and old cells responded to thermal stress were revealed. At a wide range of temperatures (44-49”C), old cells demonstrated a greater sensitivity to the i~bibitory effects of heat shock on protein synthesis (not shown). Therefore, to avoid any exaggerated killing effects that might appear in old cells if cessation of protein synthesis was used as a viability index, a dye exclusion was chosen as the most definitive assay for measuring the immediate killing response following acute hyperthermia. Figure 7 shows the results of four i~de~e~~e~~ experiments in which cultures were expos of49”C. lengths of time to a temperature ature, young cells showed a near linear number of surviving cells as a function of time, with an average LD,, occurring at approximately 60 min. Senescent cells also showed a time-dependent decrease in the number of surviving cells; however, their abiiity to withstand this temperature was significantly reduced from examined, that of young cells (P < 0.05 at all t e points as determined by Student’s 1 test). oreover, the prein(by a prior treatment duction of beat shock proteins with 25 p*M sodium arsenit not only stabilized cell mosthe viabil.ity of cells followphology, but also increase ing acute hyperthermic e sure (compare 60 min with 60 min preinduced). It is interesting to note that both young and old cells were simil protected from this e feel that this is most stress by preinduction of IISPs. likely the result of the preinduction treatment yielding sufficient HSPs in both cultures to convey a similar protective effect. However, in the absence of preinduction, senescent cells were unable to synthesize heat shock gene products as fast, and to the same levels, as young cells and succumbed to the lethal efIects of acute hyperthermia to a greater extent.
The results of this investigation have shown that the induction of three major classes of heat shock genes following exposure to environmental stress is attenuated in senescent human fibroblasts. The reduced expression of heat shock gene products by old cells correlates with a diminished ability to withstan oreover, the prior inlethal hyperthermic conditions.
LUCE
AND
CRISTOFALO
FIG. 6. Protection from acute hyperthermic exposure by preinduction of heat shock proteins. Young cells (A and B) or old cells (C and D) inverted microscope and Kodak were exposed to a temperature of 49°C for 40 min and cultures immediately photographed usin, u a Nikon Tri-X film. B and D show cells which were t~reated with 26 J& sodium arsenite for 2 h immediately before an acute hyperthermic exposure identical to that described above.
duction of HSPs conveys a protective effect against lethal hyperthermic conditions, establishing a correlation between HSPs and the ability to withstand stress in these cells. These data suggest that one mechanism by which senescent cells lose their ability to maintain homeostasis is through a reduced ability to express heat shock gene products. A relationship between acquired thermotoleran~e and heat shock protein synthesis has been well documented in several species of eucaryotic organisms (for review see [ 11). Although many previous investigations have shown an association between thermotoleran~e and HSP synthesis, direct involvement of HSPs and protection against thermal damage had not established. However, Riabowol et al. [23] showed direct involvement of HSP 70 and the ability of cells to withstand heat shock by use of a neutralizing antibody against HSP 70. Similarly, Johnston and Kucey [24] showed that cells containing a high copy number of HSP 70 5’ flanking sequences displayed a greatly reduced ability to express HSP 70 and a corresponding increase in thermosensiti-
vity. A species of Hydra which lives in a thermally stable environment, and which has lost the ability to express an HSP ‘IO-like product, is significantly more thermolabile than a similar species which is able to express this major heat shock protein 1251. Moreover, only the species of Hydra which is able to induce an HSP 70-like protein is able to acquire thermotolerance. Although the molecular mechanisms by which HSP 70, and possibly other HSPs, is able to confer protection against stress are unknown, their presence is most probably participating in a basic cellular defense system which allows cells to cope with potentially damaging environmental conditions. Just as the mechanisms of action of HSPs are largely unknown, the intracellular signals which initiate expression of heat shock genes are also presently not understood. Although the inducers of the heat shock response are numerous and seeming diverse, one common characteristic most share is the ability to create altered intracellular proteins. A ~~0~0~~~~ strain which synthesizes mutant forms of actin constitutively expresses
HEAT
SHOCK
RESPONSE
IN
AGlNG
~~~A~
CELLS
15
cells in our ~~vest~gatio~* However9 it is ~oss~~~e that the level of abnormal proteins beheved to be present in senescent cells is insufficient to evoke the heat shock response. Studies of the stress response 0fTer opportunities to describe in molecular terms how organisms are able to resist environmental change and maintain homeostasis as they age. A diminished abiiity to ‘ntain homeostasis has been described as a primary raeteristic of aging 1321.However, i~vest~gat~o~s of changes in gene expression which result in a decrease ability to rn~~~t~~~ homeostasis have, for t,he most art, been lacking. Tsuiji. et al. 1331resorted that at a e passage, e-hebuman fibroblast cell line TfG-I retained the ability to synthesize beat-~~~~~~~~e polypeptides. Lui et al. [34,35] showed that senescen.t fibroblasts expressed lower amounts of products following exposure to cers, and that this 0 20 40 60 60 P.I. reduced expression was attribu to tra~s~~~~~i~~~~ Minutes at 49°C mechanisms Similarly, Fargnol . [36] ~-e-portedthat FIG. 7. Cell viability following acute hyperthermic conditions. rat fibrob~asts &owed a reduced a~~~~tyto elicit, the heat Cells were exposed to a temperature of 49°C for the indicated length shock response as a ~~~~~~~~of the age of the ani of time and ~mmed~at~Iy removed from the substratum by trypsinizafrom which the cells were obtained. These data ar tion. Cells were collected by a IO-mm ce~trifugat~oll at 75g and resussig~i~can~e since they suggest that a reduced abihty to pended in the medium which was used during the hyperthermic expoexpress heat shock genes may be an inherent. charactersure to reintroduce cells which may have detached under this condition. Cells were centrifuged as described above and resuspended in istic of organismal aging and not merely an artifact asPBS, and cell viability was measured using an erythrosine B dye exsociated with fibroblasts cultured in u&e. In another clusion as described under Materials and Methods. Shown are the series of in vim experiments, Fleming et al. [37] showed mean results of four independent experiments (&I SEM). The results by two-dimensional gel electrophosesis that old Droshown for 60 PI are cultures in which HSPs were preinduced with sodium arsenite as described in the legend to Fig. 6 followed by a sophila synthesize a Barges number of Bow-molecuiar60-min exposure to a temperature of 49°C. Slashed bars represent weight heat shock proteins than young flies fo~~~~~~g young cell cultures and the closed bars show old cell cultures. exposure to elevated tempera es Such an observation is interesting in that it HSPs [ZSJ. Sirn~~~rly, prod~~t~o~ of large amounts of changes (i.e., never gene ~rodn~ts~ during aging. HOWever, it may not address t,he ~~e~~~~~of a reduced ability abnormal proteins by ~~~~~~~~~i~ co& activates transcription of heat shock genes [27]. Ananthan et aE. [28] for adaptive change in response to stress, since most studies have implicated the major ~~gb,~rn~~e~~~~~showed that rni~~~~~je~tio~ of conformationally altered SB (i.e., HSP 70) as being responsible for proproteins into Xenopus oocytes could induce the heat tecting cells against stress [B-25]. shock response, providing direct evidence that abnorTo our knowledge, the results presented in this study mal proteins can serve as an intracellular signal to inare the first to suggest that the expression of genes reduce this response in eucaryotic cells. sponsible for maintaining homeostasis is reduced to a The fact that altered proteins can elicit the HSR is significant in light of our results showing reduced ex- physiologically significant extent (as shown by the differential sensitivity between young and se~es~e~~cells pression of heat shock genes in senescent cells. Many to acute hype~the~mia~ during cehular aging. Since our changes in protein synthesis and post-translational processes during cellular aging have been described. Re- investigation showed that a red~~~~~ in three major duction in the fideelity of protein synthesis [%I, postclasses of heat shock genes occurred ~~~~~~rn~~~~t~yin translational ~odi~~atio~s such as deamination 1301, late-passage cells, it is likely that the rn@~~a~~srnresponand changes in protein conformation [31] have all been sible for signal t~a~~~~ct~o~ f~‘olfowing stress is attenuated during aging. It is interestingto note that an addishown to occur during aging of cultured cells. The intracellular accumulation of altered proteins could theretional cellular defense system rn~~~~~~t~~o~e~~inducfore potentially lead to the induction of the heat shock tion, is also significantly reduced in senescent human response as cells senesce. In light of these observations, cells following exposure to toxic met.& ions [38, 391. It it is interesting that an increase in the basal level ex- may therefore be a ~~~~ra~~~e~~~e~o~~e~~~ of diminpression of heat shock genes was not evident. in senes- ished signaling pathways which leads to a reduced abilcent
16
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