correlationbetween the inductionof heat shock protein 70 and … · heat shock1 kandace j....

[CANCER RESEARCH 49, 2735-2742, May 15, 1989]

Correlationbetween the Inductionof Heat Shock Protein 70 and Enhanced ViralReactivation in Mammalian Cells Treated with Ultraviolet Light andHeat Shock1

Kandace J. Williams,2 Bryan E. Landgraf, Narda L. Whiting, and Joanne Zurlo3

Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03756

ABSTRACT

Enhanced viral reactivation (EVR) is considered to be one manifestation of an inducible response to DNA damage in mammalian cellsanalogous to the SOS response in Escherichia coli. EVR is characterizedby the increased survival of ultraviolet (UV)-irradiated virus in cellswhich have been pretreated with DNA-damaging agents or by another

type of cellular stress, heat shock (HS). In this study, we have analyzedthe induction of nuclear proteins from Vero cells treated with either UVor HS, with the goal of identifying the protein(s) which mediate the EVRresponse. Results of 2-dimensional protein gel electrophoresis and fluo-rographic analysis of [3SS]methionine-labeled nuclear proteins showed

that UV-irradiation caused the increased synthesis of five proteins at 4-9 h after treatment. At 19-24 h, one of these proteins was still beingsynthesized at a higher level in l \ -irradiated cells, and there were nine

additional proteins whose syntheses were enhanced over control levels.In contrast, HS induced only one M, 72,000 nuclear protein whosesynthesis was maximal during the 4-9-h labeling period and correspondedto one of the proteins induced by UV at 19-24 h. Subsequent Western

and Northern blot analyses have confirmed that this protein is a memberof the heat shock protein (hsp) 70 family. Elevated nuclear levels of thisprotein correlated temporally with the maximum EVR response inducedby each treatment (4 h after HS and 24 h after UV). Since the kineticsof EVR is different following UV and HS and parallels the difference inthe induction of nuclear levels of hsp70 following each treatment, theresults suggest that hsp70 may be involved in mediating the EVRresponse. In addition, this protein may also play a role in the recovery ofDNA synthesis in UV-irradiated cells.

INTRODUCTION

Treatment of Escherichia coli with agents which damageDNA and block replication causes the induction of the SOSresponse (3, 4). This response is regulated by the RecA andLexA proteins with the cleavage of the lexA repressor allowingfor the subsequent expression of at least 17 genes which mediatethe response (3, 4). Among the manifestations of the SOSresponse are increased DNA repair, induction of stable DNAreplication, mutagenesis, prophage induction and filamenta-tion. Another phenomenon detected in SOS-induced cells is theincreased survival of UV-irradiated bacteriophage which is

termed Weigle reactivation (5). In addition, there is an increasedmutation frequency in the surviving phage, referred to as Weiglemutagenesis (3). The SOS response thus results in increasedsurvival of the cells but also represents a mechanism to intro-

Received 5/25/88; revised 10/6/88, 1/6/89; accepted 2/7/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by USPHS Grant CA-42419 (J. Z.), NIEHS Training Grant ES-

07104 (K. J. W.), the Ryan Foundation (K. J. W., B. E. L.). and the HitchcockFoundation of the Dartmouth-Hitchcock Medical Center. Cancer Center CoreGrant CA-23108 to the Norris Cotton Cancer Center supported the availabilityof shared equipment, services, and facilities necessary to conduct this research.Presented in part at the 78th and 79th Annual Meetings of the AmericanAssociation for Cancer Research (1,2).

1 Present address: Joseph Gottstein Memorial Cancer Research Laboratory,

Department of Pathology, University of Washington School of Medicine, Seattle,WA98195.

' To whom requests for reprints should be addressed.

duce mutations into the genome.The presence of an inducible response to DNA damage in

mammalian cells, analogous to the SOS response, has beeninvestigated (4, 6-9). The strongest evidence for such a systemin mammalian cells has been obtained using viral probes toelucidate cellular functions (6). An inducible process analogousto Weigle reactivation occurs in mammalian cells treated withUV light, X-rays, or chemical carcinogens (4, 10-13). Subsequent infection of such pretreated cells with UV-irradiatedHerpes virus (14), SV40 (15), adenovirus (16), or parvovirus(17) results in an increased survival of the virus. This process,referred to as EVR,4 has been shown to be dependent upon de

novo protein synthesis (18, 19) and has been demonstrated tooccur in cells from various sources (14-17), including primarycells (20). One discrepancy which exists in the analogy betweenWeigle reactivation in bacteria and EVR in mammalian cells iswhether the latter is accompanied by increased viral mutagenesis (4, 11, 12, 17, 21-25). In studies where mutagenesis hasbeen observed, there are indications that, as in bacteria, the twoprocesses may be discrete (22, 23). The presence of such aninducible mutator function in mammalian cells would representone mechanism by which the carcinogenic process could beinitiated.

The elucidation of the nature of the inducible processes whichmanifest themselves as EVR in mammalian cells is dependenton the determination of the nature and function of inducedgene products. Several studies (7, 26-28) have focused on theinduction of proteins in human fibroblasts following treatmentwith UV light, mitomycin C, and the tumor promoter 12-0-tetradecanoylphorbol-13-acetate. Genes induced by theseagents include metallothionein Ha, collagenase and c-fos (1).Schorpp et al. (27) have also demonstrated the existence of aUV-inducible extracellular protein termed EPIF (extracellularprotein synthesis inducing factor), which mimicks the UV induction of gene products. Other studies have demonstrated theinduction of p53 tumor antigen (29), c-myc (30), plasminogenactivator (31), and DNA ligase II (32) by DNA-damagingagents. However, the role of these proteins in mediating aninducible DNA repair response is not known.

Previous studies by us (33) and others (34-35) have demonstrated that another type of cellular stress, i.e., HS, can alsoinduce cellular processes which are manifested by EVR inmammalian cells although in two reported studies (35, 57),EVR induced by HS was not accompanied by mutagenesis. Heatshock (along with other chemical and biological stresses) isknown to induce the expression of a small set of heat shockgenes (36). The induction of these genes is rapid and transientand appears to be regulated at both the transcriptional and

4The abbreviations used are: EVR, enhanced viral reactivation; HSV-1, herpes

simplex virus type I; HS, heat shock; hsp70, a family of heat shock proteins witha molecular weight of 70,000; pi, isoelectric pH; FBS, fetal bovine serum; PBS,phosphate buffered saline; 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; Ix SSC, standard saline citrate, (0.15 M NaCl, 0.015 M, sodiumcitrate); SDS, sodium dodecyl sulfate; HEPES, 4-(2-hydroxyethyl-l-piperazine-ethanesulfonic acid.

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EVR AND INDUCTION OF hsp70 BY UV AND HS

translational levels. Recent studies in E. coli (37), yeast (38),and Drosophila (39) have shown that heat shock and DNArepair genes can be coordinately regulated. The purpose of thepresent study was to determine whether there are nuclear proteins induced by both UV-irradiation and HS in Vero cells,with the goal of identifying proteins which mediate the EVRresponse. We have found that both UV-irradiation and HSinduce a protein with a 72,000 molecular weight subsequentlyidentified as a member of the hsp70 family. Our findingsindicate that this protein may play a role in the EVR response.

MATERIALS AND METHODS

Virus, Plasmids, Cell Culture and Treatments, and EVR Studies. HS V1, strain F, was obtained from American Type Culture Collection,Rockville, MD, and was propagated as described previously (20). Plas-mid pH 2.3, which was kindly provided by Dr. Richard Morimoto(Northwestern University, Evanston, IL) (40), was cleaved with BamHland Hindlll [Boehringer-Mannheim Biochemicals (BMB), Indianapolis, IN] to produce a 2.3-kilobase fragment containing the entire codingregion of the human hsp70 gene. Plasmid pFF435C which contains thecoding region of histone H3 was kindly provided by Dr. Janet Stein(University of Massachusetts Medical School, Worcester, MA). The2.5-kilobase EcoRl-Hindlll (BMB) fragment was used as a probe.

Vero cells (African green monkey kidney epithelial cell line) wereobtained from American Type Culture Collection and were cultured inMedium 199 (GIBCO Laboratories, Grand Island, NY) supplementedwith 5% FBS (Hyclone, Logan, UT) and gentamycin (Sigma ChemicalCo., St. Louis, MO) at 10 Mg/ml. The cells were maintained as anasynchronous population in monolayer culture and were treated withHS or UV when they were 80% confluent.

For HS treatment, HEPES buffer (pH 7.0) was added to the culturesto a final concentration of 15 HIMand the cultures were incubated at45Â°Cfor 45 min. After HS, the medium was replaced with fresh mediumat 37Â°C.For UV irradiation, the medium was removed from the

cultures, then the monolayers were rinsed once with PBS. The cellswere then UV irradiated using a germicida! lamp at 254 nm at anaverage fluence rate of 1.2 J/m2/s and fresh medium was then addedto the plates. Control cultures were sham-treated by either addingHEPES and incubating at 37Â°Cfor 45 min (HS controls) or by

removing the medium and rinsing the monolayers with PBS (UVcontrols). There was no observable difference in response between thesetwo control groups and for most experiments, only the UV controlswere included.

For EVR studies, Vero cells were seeded and treated with UV or HSas described above. At various times after treatment, the cells wereinfected with control or UV-irradiated (350 J/m2) HSV-1 at a low

multiplicity of infection (<0.1 plaque forming unit/cell), at dilutionscalculated to produce a sufficient number of plaques using a directplaque assay. These methods have been described in detail previously(20). Plaques were counted 3-4 days after infection. The level of EVRis expressed as the reactivation factor which is the ratio of the survivingfraction of UV-irradiated virus in UV-irradiated cells to that in controlcells. Reactivation factors greater than 1 reflect EVR.

Pulse Labeling and Nuclear Protein Isolation. For [3sS]methionine

labeling of cellular proteins, the medium was removed from the cellcultures, the monolayers were rinsed with PBS and the medium wasreplaced with methionine-free medium (MEM Select-AminÃ©kit;GIBCO) supplemented as above to which 1.5 mg unlabeled methionineper liter was added. The cells were pulse labeled for 5 h with 1.5 mCi[35S]methionine (>800 Ci/mmol; Amersham, Arlington Heights, IL)per 150-mm plate at 4-9 and 19-24 h after treatment of cells. Followingthe labeling period, the medium was removed and the cultures wererinsed twice with PBS. The cells were scraped in PBS and the suspensions were centrifuged at 100 x g for 5 min. Nuclei were isolated by amodification of the method of Hay and Aloni (41). Ice-cold hypotoniebuffer containing 50 mM Tris-HCl, pH 7.9; 1.5 HIM MgCl2; 5 mMEDTA; 1 mM phenylmethylsulfonyl fluoride (Sigma); 0.1 mM trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64; Sigma); 0.1

UM pepstatin (Sigma) was added to the cell pellets which were thenresuspended by gentle pipetting. Nucleic were recovered following threerounds of centrifugation (1000 x g for 2 min) and resuspension. Thefinal nuclear pellets were quick-frozen in an ethanol-dry ice bath andstored at â€”¿�80Â°C.Nuclear proteins were isolated by a modification of

two methods (42, 43). Briefly, the nuclear pellets were resuspended in150 n\ of a solution of micrococcal nuclease (9 units/ml; Sigma) in 20mM Tris-HCl, pH 8.8; 2 mM CaCli and passaged through a 28-gaugeneedle four to five times. The nuclei were then treated with 0.12 volumesof a solution containing 3% SDS and 10% 2-mercaptoethanol (Sigma)and again passaged twice through the 28-gauge needle. Solid urea(Ultrapure; Schwarz/Mann, Cleveland, OH) was added to 9 M followedby 1 volume of first-dimension lysis buffer (42, 44) [9.5 M urea; 2% w/v Nonidet P-40 (Sigma); 1.6% pH 5-8 ampholine and 0.4% pH 3.5-9.5 ampholine (LKB, Paramus, NJ); 5% 2-mercaptoethanol] and thesamples were quick frozen until used for 2D-PAGE.

2D-PAGE. Aliquots of each nuclear protein lysate containing anequivalent number of cpm, as determined by trichloroacetic acid precipitation (44), were subjected to 2D-PAGE essentially as described(42, 44) except that in one series of experiments, the ampholineconcentrations in the first-dimensional isoelectric focusing gels werealtered to contain 1.3% pH 3.5-5; 0.7% pH 5-8 in order to betterresolve proteins with an acidic pi. pH gradients in the first dimensionwere measured on 1-cm sections of duplicate blank gels using a standardpH meter. The second dimension was performed on exponential, 10-16% gradient polyacrylamide gels. Molecular weights in the seconddimension were determined by running aliquots of l4C-methylated

standard proteins (Amersham) in a single well near the edge of the slabgel. The gels were subjected to fluorography using Enhance (NEN,Boston, MA) and XAR-5 film (Eastman Kodak, Rochester, NY).

Western Blot Analysis. For this study, 2D-PAGE was performed onnuclear protein samples as described above and the proteins weretransferred to nitrocellulose (Schleicher and Schnell, Kcene. NH) usinga Transphor TE-50 electroblot unit (Hoefer, San Francisco, CA).Antibody 7.10 to Drosophila hsp70 was a gift of Dr. Susan Lindquist,University of Chicago. The screening protocol was a modification ofpublished protocols (45). The protein blots were blocked with 20%heat-inactivated FBS and then incubated with antibody for 1 h. Thefilters were then incubated with biotin conjugated sheep anti-rat IgGfollowed by an avidin-biotinylated-horseradish peroxidase complex(Vectastain ABC; Vector Laboratories, Inc., Burlingame, CA). Thecomplexes were detected by incubating the filters with horseradishperoxidase substrate (BioRad, Richmond, CA).

Northern Blot Analysis. For isolation of RNA, control, UV-irradiatedor heat shocked cells were removed from the culture dishes by scrapingand the cells were pelleted by slow speed centrifugation. The pelletswere resuspended in a 4 M guanidinium isothiocyanate solution andthe resulting lysate was layered over a cushion of CsCl as described byChirgwin et al. (46). The RNA was dissolved in H2O treated with 0.1 %diethylpyrocarbonate (Aldrich Chemical Co., Milwaukee, WI). ForNorthern blots, 20 Mgtotal RNA was denatured with formaldehyde andformamide and run on 1% agarose gels as described (47). RNA wastransferred to Gene Screen Plus (NEN) according to methods recommended by the manufacturer, and the membranes were baked for 2 hat 80Â°Cto remove the formaldehyde. The filters were prehybridized at42Â°Cin a solution recommended by the manufacturer which contained

50% formamide (Aldrich) and hybridized to probes prepared from pH2.3 to hsp70 and pFF435C to histone H3 labeled with [a-32P]dCTP(>3000 Ci/mmol, NEN) by the oligodeoxyribonucleotide-primed elongation method as described (48, 49). The filters were washed 3x5 minwith Ix SSC, 1% SDS at room temperature, twice for 30 min with thesame solution at 55Â°C,and twice for 20 min with 0.1 x SSC, 0.1%SDS at room temperature. The filters were exposed to Kodak XAR-5film with Cronex intensifying screens (E. M. Parker, Brookline, MA).The autoradiographs were quantitated using a scanning densitometer(E-C Apparatus Corp., St. Petersburg, FL) equipped with a Hewlett-Packard 3390-A Integrator (Hewlett-Packard, Palo Alto, CA).

RESULTS

EVR following UV Irradiation or HS Treatment of Vero Cells.Vero cells were infected with HSV-1 at 1,4, 12, or 24 h after

2736




UV or HS treatment to the cells. Fig. 1 demonstrates the levelof EVR, expressed as reactivation factor, at the times andtreatments indicated. The points represent the mean Â±SD ofduplicate experiments and were calculated from the ratios obtained from the average of duplicate plates for each experiment.

2-

1-

B -H _Â«Â«-"â€”'"

/"** ^Â«MMHVffMH

10 20 30

Figs. 2-7. Representative autoradiograms of replicate two-dimensional gels.Vero cells were seeded onto six 150-mm plates and when ~80% confluent,duplicate plates were either treated as control, UV irradiated (10 J/mJ), or HStreated (45'C, 45 min). During either the 4-9 h or 19-24 h period after treatmentone plate from each group was labeled with 1.5 mCi of ("S]methionine (>800Ci/mmol) and then immediately harvested for 2D-PAGE, which was done asdescribed in "Materials and Methods." Equal acid-precipitable cpms of each

nuclear lysate were applied to all gels. The gels were dried and subjected tofluorography.

Fig. 2. Selected area of autoradiograms from 2D-PAGE analyses of [3!S]-methionine labeled nuclear proteins at 4-9 h posttreatment from control I I), UVirradiated (B), and HS treated (C) Vero cells showing protein X with a pi of 5.8and a molecular weight of 72,000.

TIME OF INFECTION(HOURS AFTER TREATMENT)

Fig. 1. Time course of EVR in Vero cells following UV (12 J/m2) or HS(45'C, 45 min). Cells were infected with control or UV-irradiated (350 J/m2)HSV-1 at the indicated times after treatment. The reactivation factor is the ratioof the surviving fraction of UV-irradiated HSV-1 in UV or HS-treated cells tothat of control cells. This graph represents the mean Â±SD of duplicate experiments; where no error bars appear, the SD was within the symbol.

Table 1 Summary of 2D-PAGE/fluorographic analysis of the induction ofnuclear proteins in Vero cells by UV light

Vero cells were UV-irradiated at 10 J/m2 and pulse-labeled at the timesindicated with ["S]methionine. Nuclear proteins were subjected to 2D-PAGEfollowed by fluorography as described in "Materials and Methods." The proteins

listed showed enhanced synthesis following I V irradiation when compared tocontrol (untreated) samples. These results were obtained following analysis oftwo to three replicate gels for each treatment and time point.

Labeling Period (hours after treatment)

4-9hprotein

piA

6.7B6.8C6.6D5.27*

5.0M,68,00049,00045,00048,00046,000proteinXÂ°1234561"8919-24

hPi5.85.45.95.86.05.45.25.04.94.4M,72,00035,00060,00044,00020,00068,00045,00046,00038,00043,000

" This protein was the only protein whose synthesis was enhanced during the4-9-h labeling period following HS treatment.

'' This synthesis of this protein was enhanced over control levels during both

labeling periods.

V-- - â€¢¿�

19 iÂ«hrpÂ«t

BH-lMrdnuclc.rp.

IÂ» 14 hr po*( lint.

Fig. 3. Selected area of autoradiograms from 2D-PAGE analyses of [35S]methionine-labeled nuclear proteins at 19-24 h posttreatment from control (A),UV-irradiated (B), HS-treated (C) Vero cells showing protein X with a pi of 5.8and a molecular weight of 72,000.

2737




Fig. 4. Selected area of autoradiogramfrom 2D-PAGE analysis of [3!S]methionine-labeled nuclear proteins at 4-9 h posttreatmentfrom control (A), UV-irradiated Vero cells (B)showing proteins A, B, and C (see Table 1).

BUV-lrradl*lÂ«d(10J/Â«z)

r>5 l**led IWClÂ«V prolf ms

4-9 tir pc-it -treÂ«tm*fit

Fig. 5. Selected area of autoradiogramfrom 2D-PAGE analysis of ["SJmethionine-labeled nuclear proteins at 4-9 h posttreatmentfrom control (A) and UV-irradiated (B) Verocells showing protein D and protein 7.

Control<><Â»ijb*im nuclev protet

4-9 hr post-trtatment

Maximum EVR was achieved by infecting HS-treated Vero

cells 4 h after treatment, although EVR is still observed whenthe cells were infected at 12 and 24 h after HS treatment. UV-irradiated Vero cells exhibited highest EVR 24 h after treatment. There was no further enhancement of EVR beyond the24 h time point, up to 48 h, in UV-irradiated Vero cells (results

not shown).2D-PAGE Analysis of 35S-labeled Nuclear Proteins from UV-

and HS-treated Vero Cells. 2D-PAGE was performed on nuclear proteins isolated from control, UV- and HS-treated Verocells which had been pulse labeled at 4-9 h or 19-24 h followingtreatment. Figs. 2-7 are representative autoradiograms of replicate gels. Our results showed that while there were manynuclear proteins induced by UV treatment, there was only oneprotein which appeared to be induced by both UV and HStreatments (designated X; Figs. 2 and 3). This protein, with anapproximate molecular weight of 72,000 and a pi of 5.8, exhibited elevated synthesis during the early labeling period in HS-treated cells (Fig. 2) and during the later period in UV-irradiated cells (Fig. 3). Protein "X" was the only nuclear protein to

exhibit elevated synthesis in HS-treated cells.

The approximate pi and molecular weight of the additionalnuclear proteins whose synthesis was enhanced over controllevels by UV are summarized in Table 1. Only those proteinswhich showed enhancement in multiple gels from separateexperiments are included in this table. The results show thatduring the 4-9-h labeling period after UV irradiation, 5 nuclearproteins exhibited enhanced synthesis over that of control levels(designated A-D, 7; Figs. 4 and 5). During the 19-24-h labelingperiod, the synthesis of 10 nuclear proteins was enhanced inthe UV-irradiated cells as compared to control cells (designatedX, 1-9; Figs. 3, 6, and 7). The synthesis of only one of the 5nuclear proteins which was induced in the early labeling periodremained elevated over control levels in the later time (protein7; Figs. 5-7).

Analysis of Western Blots with Antibody to hsp70. Based onthe pi and molecular weight of protein "X" as well as on the

fact that it was the only protein whose synthesis was elevatedfollowing HS treatment of Vero cells, we performed Westernblot analysis of nuclear proteins from control, UV-irradiatedand HS-treated cells using antibody 7.10, a rat monoclonal Abraised against Drosophila hsp70.5 This analysis was done in

5S. Lindquist, personal communication.

2738




6.9 6.4 5.9

BFig. 6. Autoradiograms from 2D-PAGE analysis of ("S]methionine-labeled nuclear proteins, with a pi range of 6.9-4.4, from control (A) and UV-irradiated (A)

cells at 19-24 h posttreatment showing proteins X. I, 2, 3, 4 (see Table I).

P'Â» S.9 5.4 4.9 4.0

- 100 K

- 69

- 46

9

- 30

Fig. 7. Autoradiograms from 2D-PAGE analysis of ("Sjmethionine-labeled nuclear proteins with a pi range of 5.9-4.0, from control (A) and UV-irradiated (A)cells at 19-24 h posttreatment showing proteins 1, 5, 6, 7, 8, 9.

order to positively identify protein "X" as a member of the

hsp70 and to confirm the pattern of its induction following HSand UV. The results indicate that control cells express onemember of the hsp70 family constitutively during both the 4-9- and 19-24-h periods (Fig. 8), and confirmed that the spotcorresponding to hsp70 had the same A/r and pi as protein "X".

This is consistent with previous studies in human cells whichhave demonstrated that hsp70 is a constitutive, cell cycle-dependent protein (50). The Western blot analysis indicatesthat HS induced this hsp70 over control levels during the 4-9-h period and also the synthesis of another member of the hsp70family, which cross-reacts with antibody 7.10. This type ofanalysis clarifies and confirms our 2D-PAGE results by dem

onstrating that there is a newly synthesized hsp70-related protein appearing in Vero cells after HS treatment, in addition toenhanced synthesis of the constitutive hsp70. The Western blotanalysis of nuclear proteins from UV-irradiated cells also corroborates the 2D-PAGE studies showing that the constitutivehsp70 exhibits enhanced synthesis during the 19-24-h post-treatment period only, without synthesis of any other memberof the mammalian hsp70 family during either labeling period.

Northern Blot Analysis. Northern blots were performed inorder to confirm that hsp70 is specifically inducible by UVtreatment. RNA samples from Vero cells at various times afterUV or HS treatment as well as from untreated cells at the sametime points were hybridized simultaneously with 32P-labeled

2739




C 4-9C 19-24

HS 4-9 HS 19-24

UV 4-9

IUV 19-24

Fig. 8. Western blot analysis of nuclear proteins from control, UV, or HS-treated Vero cells at 4-9 and 19-24 h after treatment with antibody 7.10 to hsp70and visualization using a horseradish peroxiduse conjugated second antibodysystem as described in "Materials and Methods."

probes for hsp70 and histone H3. Both probes were used inorder to distinguish between constitutive, cell cycle (S-phase)dependent expression of hsp70 (50) and stress-induced expression of this gene. Histone 3A is expressed only during S-phase(51, 52) and thus the presence of mRNA for this gene indicatesthat a subpopulation of the cells is synthesizing DNA. A representative autoradiogram showing the results from these studies may be seen in Fig. 9. The lanes labeled C contain RNAfrom untreated cultures of asynchronous Vero cells. While theexpression of hsp70 and histone H3 appears to change depending on the time of RNA isolation (4, 10, and 24 h), the relativeratio of the signals for these two genes remained constant (Table2). The lanes labeled "HS" contain total cellular RNA fromcells at 1, 4, 10, and 20 hours after HS at 45Â°Cfor 45 min. HS

clearly induces the levels of steady state mRNA for hsp70 at 1and 4 h following treatment and these levels decrease by 10 h.In contrast, the expression of histone H3 in HS treated cells issuppressed until at least 4 h, but appears to resume by 10 hafter treatment. The ratios of hsp70/histone expression increaseat least 7-10-fold over control values at the early times, butdecrease in half or less than controls at the later times (Table2). UV-irradiation of Vero cells causes the induction of hsp70mRNA levels by 4 h after treatment and these levels remainedconstant during the 10- and 24-h time points (Fig. 9, UV lanes).

Comparison of the levels of steady state hsp70 mRNA withthose of histone H3 reveals that at 4 h following UV, histoneexpression is very low (comparable to levels 1 h after HS), butincreases with time as DNA synthesis in the cells resumes. Thisis best illustrated by comparing the ratios of hsp70/histoneexpression in UV-irradiated cells to the ratios obtained fromcontrol cells (Table 2). In the samples from UV treated cells,the ratios increase to 3.2 and 2.2 times the control values at 4and 10 h, respectively. In addition, while these ratios are atleast 3-4-fold less than those observed in the samples from theHS-treated cells, the relative pattern of expression of both genesfollowing each treatment is similar suggesting that hsp70 induction by UV is specific and not cell cycle related. At 24 hafter UV, the ratio of hsp70/histone decreases to below thecontrol value. This decrease in ratio reflects an increase inexpression of histone 3A, rather than a decrease in hsp70expression, which is still elevated at 24 h (Fig. 9).

DISCUSSION

EVR is shown to occur in mammalian cells in response toDNA damaging agents as well as to heat shock (6, 10-25, 33-

HS uy

kbT

4.4Â»-

2.4>

1.4Â»

hÂ»|l 4 10 20|| 4 10 24|| 4 10 24Â¡

fl fiÂ»

*

0.3"

Fig. 9. Northern blot analysis of RNA from control (C), HS, and UV-treatedVero cells, at the indicated number of hours after treatment, hybridized to probesp2.3 to hsp70 and to pFF435C to histone H3. The mRNA for hsp70 is 2.4kilobases and that for histone H3 is 0.6 kilobase.

Table 2 Relative expression of hsp70/histone H3 in control, HS-, or UV-treatedVero cells

Ratios of hsp70/histone H3 were calculated from the peak area values generated by the densitometric scans of the autoradiogram shown in Fig. 9.

Treatment"ControlHSUVTime(h)4102414102441024Relativeexpression,

hsp70/histoneH31.781.801.97>18*>15*0.920.685.814.101.22

" Vero cells were untreated, HS-treated at 45Â°Cfor 45 min, or UV-irradiatedat 12 J/m2 and RNA was isolated from the cells after treatment at the times

indicated.* These are minimum values since the exposure time exceeded the linearity of

the film for the hsp70 signals at 1 and 4 h after HS.

2740




35, 57). This process is analogous to the SOS-mediated Weiglereactivation in E. coli and has been used as evidence to suggestthe presence of a similar system in mammalian cells (6). Oneproperty of the EVR response in mammalian systems is that itis delayed, usually occurring maximally at 24-36 h after treatment and, in addition, the response is inhibited by cyclohexi-mide (6, 18-19). In contrast, the induction of EVR by HS does

not require such an extensive lag period (33), but the responseis also inhibited by cycloheximide (34). The goal of this studyhas been to assess the induction of nuclear proteins by twoagents which cause EVR in Vero cells, UV, and HS, and tocorrelate the appearance of commonly induced proteins withthe EVR response. We have determined that the only nuclearprotein induced by both treatments is protein "X", subsequently

identified as a member of the hsp70 family. Furthermore,enhanced synthesis of this protein, as measured by [35S]methi-

onine incorporation, occurred at different times following eachtreatment and correlated with the maximum EVR response byboth treatments.

The next series of experiments was designed to further characterize hsp70 induction by UV. Since the expression of hsp70is known to be cell cycle regulated with maximal expressionoccurring at the Gi/S boundary (50), the appearance of enhanced levels of hsp70 in nuclear lysates of Vero cells at 19-24

h after UV treatment could have simply been a cell cycle effectassociated with the recovery of DNA synthesis. In order todistinguish between UV-specific induction of hsp70 and cellcycle-mediated expression of this protein, we compared mRNA

levels of hsp70 and histone H3 at various times after eachtreatment using Northern blots. Histone H3 is specificallyexpressed during S phase (51, 52) and thus levels of its mRNAwould be a basis on which to assess the changes occurring inhsp70 expression relative to the cell cycle. In control cells weobserved that the ratio of expression of these two genes wasconstant. As expected, HS treatment caused a large inductionof hsp70 mRNA with, at early times after HS, a very low levelof histone H3 expression, yielding high hsp70/H3 mRNAratios. At 4 and 10 h after UV treatment, expression of hsp70mRNA was elevated without a concomitant increase in histoneH3 expression, suggesting that hsp70 was specifically inducedby UV. These results are consistent with observations that inE. coli (37), yeast (38), Drosophila (39), and rats (53), geneswhich are involved in the responses to heat shock and DNAdamaging agents can be coordinately regulated. At 24 h afterUV, the levels of hsp70 expression were still elevated, but bythis time, the level of histone 3A expression had also increased,suggesting that at least some of the cells had entered S piase.Thus, the elevated levels of hsp70 mRNA at this time pointmay reflect the normal cell cycle-dependent expression of this

gene.It is not clear why we detected an increase in the steady state

level of hsp70 mRNA as early as 4 h after UV-irradiation whilethe results of the 2D-PAGE nuclear protein analysis did notshow an increased synthesis of the protein during the 4-9-hlabeling period. One possible explanation for this observationis that the newly synthesized hsp70 protein remained in thecytoplasm. Milarski and Morimoto (50) have shown that thesubcellular distribution of hsp70 in HeLa cells varies with thestage of the cell cycle, and that while the protein is distributeddiffusely throughout the cytoplasm and nucleus during GÃ¬andG2, it is almost exclusively localized in the nucleus during Sphase. Since DNA synthesis was not detected in UV irradiated

cells 4-9 h after treatment (33),6 it is possible that translation

of the hsp70 mRNA began early but that the protein remainedin the cytoplasm until the time of the second labeling period,19-24 h, when expression of histone H3 indicated that DNAsynthesis resumed and the protein migrated to the nucleus. Analternative explanation for this observation is that even thoughthere was an elevated steady-state level of hsp70 mRNA, increased translation was delayed. We plan to test both of thesepossibilities by conducting an analysis of cytoplasmic proteinsand employing in vitro translation of mRNA from cells 4 hafter UV-irradiation.

Incubation of HeLa cells at elevated temperatures causesinhibition of replicÃ³n initiation and elongation (54, 55). It hasbeen suggested that this inhibition is due to denaturation ofreplicative enzymes, and inhibition of protein synthesis (55).However, as we have noted previously (33) and in this report,the induction of EVR in HS-treated Vero cells does not requireas long a lag period as it does in cells treated with DNA-damaging agents. After HS, maximal EVR occurred beforethere was a significant increase in histone H3 mRNA. Incontrast, maximal EVR in UV-irradiated cells occurred at 24h when there was a recovery of DNA synthesis as evidenced bythe level of histone H3 expression. The results of the presentstudy show that one correlation between UV and HS inducedEVR in Vero cells is the increased level of hsp70 in nuclearextracts of treated cells. This observation supports a role forhsp70 in the EVR response and suggests that the EVR responsemay be discrete from other events which mediate the recoveryof DNA synthesis in HS treated cells.

A question which may be posed based on our observationsrelates to the possible role of hsp70 in DNA synthesis in thehost cells following UV treatment. We have demonstratedincreased nuclear levels of hsp70 at 19-24 h after treatment, atime when there are elevated levels of histone H3 mRNA,indicating that at least some of the cells have entered S phase.Thus, while it is not known whether hsp70 participates inmammalian DNA replication, our results, along with the observations that hsp70 transcription is induced by serum stimulation (56) and that the protein accumulates in the nucleusduring S phase (50) are consistent with a role for this proteinduring the recovery of DNA synthesis. Since the evidencepresented above supports a role for hsp70 in the survival ofUV-irradiated Herpes virus, it is possible that hsp70 maysomehow participate in the replication of UV-damaged cellularDNA. However, since our results have also shown there are atleast 13 additional nuclear proteins whose synthesis is enhancedby UV treatment in Vero cells, it may be that hsp70 alone isnot sufficient and that one or more of these additional proteinsmay also be necessary to allow DNA replication to resume.Experiments are currently under way to identify and determinethe functions of these inducible proteins.

ACKNOWLEDGMENTS

The authors would like to thank Drs. Richard Morimoto and JanetStein for their gifts of plasmids pH2.3 and pFF435C, respectively, Dr.Susan Lindquist for her gift of antibody 7.10, Drs. James D. Yager andJeffrey A. Silverman for their critical reading of the manuscript andDrs. Donald A. Young, Richard I. Morimoto, and Susan Lindquist forhelpful discussions during the course of this work.

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1989;49:2735-2742. Cancer Res Kandace J. Williams, Bryan E. Landgraf, Narda L. Whiting, et al. Ultraviolet Light and Heat ShockEnhanced Viral Reactivation in Mammalian Cells Treated with Correlation between the Induction of Heat Shock Protein 70 and

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