1992;52:5780-5787. Cancer Res   Yong J. Lee, Zi-Zheng Hou, LindaLi Curetty, et al.   Development of Chronic Thermotolerance in Murine L929 CellsAbsence of HSP28 Synthesis and Phosphorylation during the

  Updated version

  http://cancerres.aacrjournals.org/content/52/20/5780

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  .permissions@aacr.orgDepartment at

To request permission to re-use all or part of this article, contact the AACR Publications

on March 10, 2014. © 1992 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from on March 10, 2014. © 1992 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

[CANCER RESEARCH 52, 5780-5787, October 15. 1992]

Absence of HSP28 Synthesis and Phosphorylation during the Development ofChronic Thermotolerance in Murine L929 Cells1

Yong J. Lee,2 Zi-Zheng Hou, LindaLi Curetty, Michael J. Borrelli, and Peter M. Corry

Department of Radiation Oncology, William Beaumont Hospital, Royal Oak, Michigan 48073

ABSTRACT

We investigated the correlation between chronic thermotolerance development and phosphorylation, synthesis, or expression of the HSP28family in murine L929 cells. Chronic thermotolerance developed duringheating at -11.5 ( as indicated by a biphasic survival curve. However,heat-induced phosphorylation of HSP28 was not detected. Furthermore,we failed to detect HSP28 synthesis during chronic heating by usingtwo-dimensional polyacrylamide gel electrophoresis. The lack ofHSP28 synthesis was also confirmed in acute thermotolerance. Similarresults were observed in NIH 3T3 cells. Although Southern blots clearlydemonstrated the presence of the HSP28 gene in genomic DNA, Northern blots failed to demonstrate its expression. Unlike HSP28, the expression of constitutive and inducible HSP70 genes, along with thesynthesis of their proteins, were stimulated during chronic heating at41.5°Cin L929 cells. These results suggest that HSP28 synthesis and

its phosphorylation are not required to develop both chronic and acutethermotolerance in L929 cells.

INTRODUCTIONHyperthermia (temperatures above 41°C)has been intermit

tently used as an agent for cancer therapy for several centuries.During this period experimental and clinical studies have demonstrated that hyperthermia combined with radiation/chemotherapeutic agents can be useful in cancer therapy (1, 2).However, recent clinical studies demonstrate that heating non-superficial tumors above 42°Cis technically difficult and com

monly leads to pain toxicity (3-6). These factors suggest thatheating at lower temperatures (<42°C) may be more effica

cious. However, chronic thermotolerance develops in cells during continuous heating at temperatures below 42.5°C(7-10).

Therefore, the mechanisms by which chronic thermotoleranceis induced and expressed should be clarified if lower-temperature heating protocols are to be used in cancer therapy.

Heat shock induces five major HSPs3 (Mr 110,000, 90,000,70,000, 28,000, and 8,500) in mammalian cells (11-13). Manystudies suggest that these polypeptides play a role in thermotolerance (12, 14-17). Recently, Crete and Landry (17), andLandry et al. (18) reported that intracellular concentration ofHSP28 and/or its phosphorylation correlated with the induction of acquired thermal resistance (thermotolerance) and heatprotection in mammalian cells. The development of thermotolerance is accompanied by an increased synthesis of HSP28, andHSP28 returns to control levels as thermotolerance decays

Received 5/14/92; accepted 8/7/92.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 in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed, at Department of Radiation Oncology, Research Laboratories, William Beaumont Hospital, 3601 W. Thirteen Mile Road, Royal Oak, Michigan 48073.

2 Supported by National Cancer Institute Grants CA48000, CA44S50. andCA49715 and William Beaumont Hospital Research Institute Grants 90-06 and91-10.

3 The abbreviations used are: HSP, heat shock protein; CHO, Chinese hamsterovary; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis;cDNA. complementary DNA; HSTF, heat shock transcription factor, SSC, standard saline citrate.

(19, 20). Landry et al. (21) also reported that Chinese hamster023 cells, which were transfected with a human HSP28 gene inplasmid pHS2711, contained an elevated level of HSP28 andbecame intrinsically resistant to thermal killing (21).

The small-molecular-weight heat shock protein HSP28 consists of several isoforms, some of which are phosphorylatedfollowing stimulation with heat shock, Ca2+ ionophore, sodiumarsenite, cycloheximide, or cytokines (17, 22-25). Previousstudies have demonstrated that these phosphorylated formsmay play an important role in the regulation of cell thermosen-sitivity (17). However, recent observations indicate that theratio of phosphorylatedrunphosphorylated isoforms of HSP28rather than phosphorylated HSP28 alone may be an importantfactor in determining the ability of cells to survive hyperthermia(19). Interestingly, Hepburn et al. (24) reported that tumornecrosis fator a-stimulated phosphorylation of HSP28 was notdetected in murine L cell lines. These results led us to use themurine L-cell system to investigate whether phosphorylatedHSP28 has an important role in the development of thermotolerance. We also postulated that the absence of phosphorylated HSP28 isoforms in murine L-cells may result from thelack of HSP28 protein synthesis and gene expression.

MATERIALS AND METHODS

Cell Culture and Survival Determination. Murine L929, NIH 3T3,Swiss 3T3, and CHO cells were cultured in McCoy's 5a medium

(Cellgro) containing 26 HIMsodium bicarbonate and 10% iron-supplemented calf serum (HyClone). T-25/75 flasks or 35-mm culture dishescontaining cells were kept in a 37°Chumidified incubator with a mix

ture of 95% air and 5% CO2. For survival determination after varioustreatments, cells were trypsinized, counted, and plated at appropriatedilutions. X-irradiated feeder cells (25 Gy) were used to maintain theplated cell density at 4000 cells/cm2 (26, 27). After 1-2 weeks of incubation at 37°C,colonies were stained and counted.

Hyperthermic Treatment. For hyperthermic treatment, T-25/75flasks or 35-mm Petri dishes were sealed with paraffin film and heatedby total immersion in a circulating water bath maintained within±0.05°Cof the desired temperature.

Labeling and Gel Electrophoresis. To examine the synthesis ofHSP28, cells were labeled with 100-200 ¿iCi/mlpHjleucine (specificactivity, 160 Ci/mmol; Amersham) during chronic heating (41.5°Cor42°C)or after acute heating (45.5°Cfor 10 min). After the cells werelabeled, they were washed twice with cold Hanks' balanced salt solution

and lysed in sample buffer. For two-dimensional PAGE, samples weresolubilized in sample buffer containing 8 M urea, 1.7% Nonidet P-40,and 4.3% /3-mercaptoethanol. Proteins were first separated in isoelec-tric focusing gels (pH 3.5-10). These gels are then laid across the top ofa SDS gradient (10-18%) slab gel (28). After electrophoresis, gels werefixed in 30% trichloracetic acid for 30 min. For fluorography, gels weredehydrated by washing for 15 min in each of 25% acetic acid, 50%acetic acid, and glacial acetic acid, consecutively. After fixation, gelswere placed in 125 ml 20% (w/v) 2,5-diphenyloxazole in glacial aceticacid for 2 h. The solution was removed, and the gel was shaken gentlyovernight in distilled water and dried in a slab gel dryer (Model 483;Bio-Rad, Richmond, CA) for 2.5 h at 60°C.The gel was placed into acassette with Kodak SB-5 X-ray film and placed in a -70°C freezer.

5780

on March 10, 2014. © 1992 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

HSP28 AND CHRONIC THERMOTOLERANCE

A L929

•¿�i

i

1O°9

10-O 3 6 9 12 15

HOURS AT 41.5°C2 4 6 8 1O

HOURS AT 42°C

Fig. 1. Survival curve at 41.5'C in L929 cells (A) or at 42'C in CHO cells (B).

The data are a compilation of three separate experiments.

After an optimum exposure, the fluorograph film was developed withKodak GBX developer and fixed with Kodak GBX fixer.

To observe the phosphorylation of HSP28 by heat shock, L929 orCHO cells were labeled with 120 ¿iCi/mlHy'2PO4 (carrier free, 285

Ci/mg; ICN) in full medium for 8 h during heating at 41.5°Cor 42°C,

respectively. After the cells were labeled, they were washed and lysed inthe sample buffer as described above. After electrophoresis, gels werefixed and dried for 1.2 h at 80°Cand placed into a stainless steel

cassette with intensifying screen. Gels were autoradiographed on KodakSB-5 X-ray film. The cassette was placed in a —¿�70°Cfreezer. After

exposure, autoradiographic film was developed as described above.Expression of the HSP70 or HSP28 Gene. Relative levels of HSP70

and HSP28 mRNA were determined using the Northern blot technique.Total cellular RNA was extracted by the LiCl-urea method of Tushinskiet al. (29). For RNA analysis, 30 Mg/ml of total RNA were electro-phoresed in a 1% agarose-formaldehyde gel (30). The RNA was blottedfrom the gels onto nitrocellulose membrane and baked at 80°Cfor 2 hin a vacuum oven. Membranes were prehybridized at 42°Cin 50%formamide, lx Denhardt's solution. 25 mm KPO4 (pH 7.4), 5x SSC

(lx SSC = 150 nui NaCl, 15 nut Na3C6H5O7), and 50Mg/ml denaturedand fragmented salmon sperm DNA. Hybridizations were carried out at42°Cin prehybridization solution containing 10% dextran sulfate and

radiolabeled HSP70 cDNA probes (both constitutive HSP70 and in-ducible HSP70 cDNAs were obtained from R. Morimoto, Northwestern University) or HSP28 cDNA probes (StressGen BiotechnologiesCorp.) at a concentration of 1.5 x IO6 cpm/ml. After hybridization,

blots were washed twice in 2x SSC for 15 min at room temperature andwashed in O.Sx SSC and 0.1% SDS for 25 min at 50°Cand twice in

L929 CHO

Fig. 2. Two-dimensional SDS-polyacrylam-ide gel electrophoretic analysis of proteins.L929 or CHO cells were labeled with H.V«PO4for 8 h during heating at 41.5'C or 42'C, re

spectively. Lysates from cells were analyzed,and phosphorylated proteins were detected byan autoradiograph. A, unheated control L929cells; B, heated L929 cells: C, unheated controlCHO cells; lì,heated CHO cells. Only a section of autoradiograph is shown. The locationsof HSP28a (a), HSP28b (b), and HSP28c (c)are identified.

A

37 °C

B

41.5°C

U

37°C

U

42°C

L929

A

37 °C

B

CHO

C

37°C

D

42°C

c b aY f •¿�»

9 P

Fig. 3. Two-dimensional SDS-polyacrylam-ide gel electrophoretic analysis of proteins. L929or CHO cells were labeled with 200 wCi/ml |'H]-leucine for 8 h during heating at 41.5°Cor 42°C,

respectively. Lysates from cells were analyzed,and 3H-labeled proteins were detected by a fluorograph. A. unheated control L929 cells; B,heated L929 cells; C, unheated control CHOcells; D, heated CHO cells. Only a section offluorograph is shown. The locations of HSP28a(a), HSP28b (ft), HSP28c (c), and actin (A) areidentified.

5781

on March 10, 2014. © 1992 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

HSP28 AND CHRONIC THERMOTOLERANCE

24 b45.5°C-10 mm >45.5°C

A L929 B CHO10'

io-

IO'

10-

NO TT

IO'

io-

1O"

NO TT

0.0 O.5 1.0 1.5 O.O O.5 1.O 1.5 2.O

HOURS AT 45.5°C

Fig. 4. Induction of thermotolerance by acute heating in L929 (A) or CHO (B)cells. Thermotolerance (TT) was observed when cells were heated for 10 min at45.5'C and then incubated at 37'C for 24 h before heating at 45.5'C. NO TT.survival cune of control cells heated at 45.5'C.

0.2X SSC and 0.1% SDS for l h at 50°C.Blots were placed into a

stainless steel cassette with intensifying screen and autoradiographed asdescribed above. To remove the hybridized probe from the nitrocellulose membrane prior to hybridization with another probe, the membrane was washed with sterile boiling water for 2 min, and then prehy-

bridization was started immediately.Southern Blot. Monolayer cells in a T-150 flask were trypsinized

and pelleted by centrifugation at 4°Cfor 10 min at 90 x g. Cell pellets

were resuspended with ice-cold Tris-buffered saline (137 HIMNaCl, 2.7ITIMKCI, 25 HIMTris, pH 7.4) and repelleted by centrifugation for 5 minat 300 x g. The Tris-buffered saline washes were repeated twice. Cellswere resuspended in 10 miviTris, 1 mm EDTA (pH 8.0) at a concentration of 5 x IO7 cells/ml. Samples were transferred into a 50-ml

conical tube. The method used to prepare genomic DNA is described bySambrook et al. (31). In brief, cells were treated with extraction buffer(10 mMTris, 100 mut EDTA, 20 Mg/ml pancreatic RNAase, 0.5% SDS,pH 8.0) and proteinase K (final concentration, 100 Mg/ml).The sampleswere then extracted with phenol. The extracted samples were dialyzedat 4°Cfour times against 4 liters of a solution of 50 mM Tris (pH 8.0)

and 10 mM EDTA (pH 8.0) until the absorbence (A27o)of the dialysate

was less than 0.05. The DNA solution (30 Mgof DNA) was tranferredinto a microcentrifuge tube and diluted to 900 n\ with 10 mM Tris (pH8.0) and then added with 100 M' of lOx restriction enzyme buffer(Hindlll, EcoRI, or Pstl). After incubation for 3 h on ice, 30 units ofrestriction enzyme were added to the DNA solution. The mixture wasincubated for l h at 37°C.The digestion reaction was stopped by adding

0.5 M EDTA (pH 8.0) to a final concentration of 10 mM. The digestedDNA was concentrated by precipitation with 0.6 volume of 5 Mammonium acetate and 2 volumes of ethanol overnight at -20°C. The sam

ples were centrifuged for 5 min at 12,000 x g. The pellets were dissolvedwith 30 M!of 10 m.\i Tris, 1 ITIMEDTA (pH 8.0) and 5 M'of bromphenolblue and then heated for 2 min at 56°Cbefore loading. The DNA

fragments were separated by electrophoresis through an agarose gel(0.7% gel in 0.5X Tris-borate/EDTA electrophoresis buffer). After electrophoresis was completed, the gel was stained with ethidium bromide(2.5 Mg/ml) for 15 min and destained with autoclaved deionized waterfor 2 h. To denature the DNA, the gel was soaked for 45 min in NaOHsolution (1.5 MNaCl, 0.5 NNaOH). The gel was rinsed with autoclaveddeionized water and then neutralized by soaking for 30 min in Trisbuffer solution [1 MTris (pH 7.4), 1.5 MNaCl]. The DNA was blottedfrom the gels onto nitrocellulose membrane, which was then baked at80°Cfor 2 h in a vacuum oven. Membranes were prehybridized at 42°Cin 50% formamide, 5x Denhardt's solution, 25 m\i KPO4 (pH 7.4), 5x

SSC (lx SSC = 150 mM NaCl, 15 mM NajC6H5O7), and 50 Mg/mldenatured and fragmented salmon sperm DNA. Hybridizations werecarried out at 42°C,in prehybridization solution containing 10% dex-

tran sulfate, 1% SDS, and radiolabeled HSP28cDNA probe (StressGenBiotechnologies Corp.). After hybridization, blots were washed in 2xSSC for 15 min at room temperature, followed by 2x SSC for 15 minat 42°C,and washed in 0.5x SSC and 0.1% SDS for 25 min at 50°C,and twice in 0.2x SSC and 0.1% SDS for 40 min at 50°C.Blots were

placed into a stainless steel cassette with an intensifying screen andautoradiographed as described above.

RESULTS

Development of Thermotolerance during Chronic Heating.Fig. 1 shows L929 or CHO cell survival following a 41.5°Cor42°Cexposure, respectively. Both cell lines developed chronic

thermotolerance during heating as indicated by the biphasicsurvival curves. The chronic thermotolerance development inCHO cells was consistent with observations from previous experiments (32, 33).

L929 CHO

Fig. 5. Two-dimensional SDS-polyacrylam-ide gel electrophoretic analysis of proteins fromL929 (left) or CHO (right) cells. Cells wereheated at 45.5'C for 10 min and labeled with100 ^Ci/ml [-'HJIeucine during incubation at37'C for 24 h. Lysates from cells were analyzed,and 3H-labeled proteins were detected by a flu-orograph. A. unhcated control cells; B. heatedcells. Only a section of each gel is shown. Thelocations of HSP28a (a). HSP28b (b). HSP28c(c), and actin (I! are identified.

37°CT5

c b at• r A

B45.5°C

A* 4

- «

c b a

5782

on March 10, 2014. © 1992 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

HSP28 AND CHRONIC THERMOTOLERANCE

NIH3T3

45.5°C-10 mm24h>45.5°C

10

o 20MINUTES AT 45.5°C

Fig. 6. Induction of thermotolerance by acute heating in NIH 3T3 cells. Ther-motolerance (7T) was observed when cells were heated for 10 min at 45.5°Candthen incubated at 37'C for 24 h before heating at 45.5'C. NO TT, survival curveof control cells heated at 45.5'C. The data are a compilation of two separate

experiments.

Phosphorylation of the HSP28 Family. To investigate thephosphorylation of Afr 28,000 heat shock protein (HSP28) during chronic heating, L929 or CHO cells were labeled withH332PO4 for 8 h at 41.5°Cor 42°C,respectively. Fig. 2D shows

that the phosphorylation of HSP28 occurred during heat shockin CHO cells. Two of the phosphorylated isoforms (HSP28band HSP28c) were confirmed as HSP28 family members by asilver staining method (data not shown). Unlike CHO cells, thisphosphorylation was not observed in L929 cells (Fig. 2B).

Synthesis of HSP28 Family. To determine whether the absence of phosphorylated HSP28 in L929 cells was due to thelack of synthesized HSP28 during heating at 41.5°C,L929 cellswere labeled with [3H]leucine, and proteins were analyzed ontwo-dimensional PAGE (Fig. 3B). Similar experiments werealso performed during heating at 42°Cin CHO cells (Fig. 3D).

The fluorographs in one- and two-dimensional PAGE showthat five major HSPs (Mr 110,000, 87,000, 70,000, 28,000, and8,500) were preferentially synthesized during heating at 42°Cin

CHO cells (Fig. 3D; data not shown). The HSP70 family contains constitutive HSP70 (Mr 70,000) and inducible HSP70(MT 68,000). In L929 cells, three major HSPs (A/r 110,000,87,000, and 70,000) and two minor HSPs (A/r 73,000 and57,000) were preferentially synthesized during heating at 41.5°Cin L929 cells (Fig. 3Ä;data not shown). Unlike CHO cells,

unphosphorylated HSP28a with two of the phosphorylated isoforms (HSP28b and HSP28c) was not observed in L929 cells(Fig. IB).

Acute Thermotolerance and HSP28 Synthesis. To determine whether HSP28 synthesis was required to develop acutethermotolerance in L929 cells, cells were heated for 10 min at45.5°C,incubated for 24 h at 37°C,and then either challengedto 45.5°C(Fig. 4A) for survival determination or lysed in sam

ple buffer for protein profiles (Fig. 5B, left). Similar experiments were also performed in CHO cells (Fig. 4B; Fig. 50, left).

Acute thermotolerance was observed in both cell lines(Fig. 4). Thermotolerance ratios at 10~2 isosurvival levels were

3.3 and 4.7 for L929 or CHO cells, respectively. Fig. 5 (A andB, right) shows a two-dimensional PAGE of proteins from un-heated and heated CHO cells, respectively. The levels of allthree proteins of the HSP28 family, HSP28a along with its twophosphorylated isoforms (HSP28b,c), increased during incubation at 37°Cfor 24 h after heating at 45.5°Cfor 10 min. Unlike

CHO cells, the lack of HSP28 synthesis was observed in L929cells (Fig. 5B, left). Similar results were observed in NIH 3T3cells (Fig. 6). When NIH 3T3 cells were heated at 45.5°Cfor 10min, they became thermotolerant to a heat treatment at 45.5°C

administered 24 h later (Fig. 6). The thermotolerance ratio at10~' isosurvival level was 2.6. As in L929 cells, a lack of HSP28

synthesis was observed in NIH 3T3 cells (Fig. 7).Expression of the HSP28 Family. To investigate whether the

lack of HSP28 synthesis resulted from the deficiency of HSP28gene expression in L929 cells, experiments were performed toquantify the levels OÕHSP28encoding mRNA during heating at41.5°C.Northern blot data illustrated that there was no detectable HSP28 mRNA during heating at 41.5°Cin L929 cells

(Fig. 8A). In contrast, the level of HSP70 mRNA (inducibleHSP70 and constitutive HSP70) increased rapidly within 4 h at41.5°C(Fig. 8B) in L929 cells. The level of inducible HSP70

mRNA decreased quickly; no inducible HSP70 mRNA wasdetected 8 h after initiating heating at 41.5"C (Fig. 8B). The

level of constitutive HSP70 mRNA also decreased within 8 hduring heating; however, the level of this mRNA remainedequivalent to the control level. Similar results were observed in

NIH3T3

Fig. 7. Two-dimensional SDS-polyacrylam-ide gel electrophoretic analysis of proteins fromNIH 3T3 cells. Cells were heated at 45.5'C for10 min and labeled with 100 nCi/m\ [3H]leucineduring incubation at 37°Cfor 24 h. Lysatesfrom cells were analyzed, and 3H-labeled proteins were detected by a fluorograph. A, un-heated control cells; B, heated cells. Only a section of each gel is shown. The locations ofHSP28a (a), HSP28b (b), HSP28c (c), constitutive HSP70 (a), inducible HSP70 (è),HSP90,HSP110, and actin (•¿�()are identifiai.

A37°C*( 90-*g-j

^'n-•"•'

•¿� 28 'C ^bB45.5°CV90-»^^^b

70"»

•¿� A*""•

A^c

ba28 ^ »• »•

5783

on March 10, 2014. © 1992 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

HSP28 AND CHRONIC THERMOTOLERANCE

A

\HSP28

GO oo co

oO 4|.5UC

OIO

trated that the failure of detection was unlikely to be due totechnical problems. A human HSP28 cDNA probe recognizedhamster as well as mouse mRNA for HSP28 (Figs. 8A and 10).

Presence of the HSP28 Gene. To determine whether the absence OÕHSP28mRNA from L929 cells was due to the absenceof the HSP28 gene, genomic DNA was isolated from L929,CHO, or Swiss 3T3 cells and partially digested with restrictionenzyme (///Will, EcoRl, or PstI). These DNA fragments wereseparated and then hybridized with HSP28 cDNA probe.Southern blot data demonstrated that only />sfl-digested DNA

fragment was detected. Nonetheless, the data from Fig. 11clearly show that the HSP28 gene was present in all three celllines. The size of the DNA fragment containing the HSP28gene was approximately 3.5 kilobases. The results from Fig. 11also illustrate that DNA fragments that were cleaved by Hindlllor £c0RI might not migrate on the 0.7% agarose gel. Thismight be due to the large size of DNA fragments. Anotherpossible reason for the failure to detect the Hindlll or EcoRlfragments was that the enzymes did not cut the DNA.

BHSP70

a- .<

co•¿�vj

CO

oo co

\

<5•¿�O

°

t*

OIo

JIro

Fig. 8. Northern blots of mRNA extracted from L929 or CHO cells. L929 cellswere heated at 41.5"C for 4, 8, or 13 h. HSP28 mRNA from CHO cells whichwere heated at 45.5'C for 10 min and incubated at 37*C for 12 h (CHO-12H) were

used as a reference, because we previously observed that HSP28 mRNA fromCHO cells was well recognized by HSP28 cDNA probe (20). RNA was isolated,and an equal amount of RNA (30 ng) was loaded onto each lane of the agarose-formaldehyde gel for separation. After separation, RNA was blotted from the gelonto nitrocellulose membrane and hybridized with HSP28 cDNA probe (A) orHSP70 cDNA probe (fi). 37°CRNA from unheated L929 cells. Arrows (a and ¿>),

inducible HSP70 mRNA and constitutive HSP70 mRNA, respectively. Note thathybridization of HSP28 cDNA probe and that of HSP70 cDNA probe wereperformed consecutively in the same nitrocellulose membrane (see "Materials andMethods" for details).

NIH 3T3 cells (Fig. 9). There was no detectable HSP28 mRNAduring incubation at 37°Cfor 2-24 h following heat shock at45.5°Cfor 10 min (Fig. 9A). Unlike in HSP28, the expression

of constitutive and inducible HSP70 genes was clearly observedafter heat shock (Fig. 9B). In Swiss 3T3 cells, the expression ofthe HSP28 gene, along with the synthesis of its protein, wasobserved during heating at 41.5°C(Fig. 10; data not shown).

Northern blots of mRNA extracted from CHO cells (see LaneCHO-I2H in Fig. 8A) or murine Swiss 3T3 cells (Fig. 10) illus

DISCUSSION

Several conclusions can be drawn upon consideration of thedata presented. While chronic thermotolerance was developedduring heating at 41.5°Cin L929 cells, heat-induced phosphor-

ylation, synthesis, and expression ofHSP28 were not observed.The lack of HSP28 synthesis was also confirmed in the development of acute thermotolerance after heating at 45.5°Cfor 10

min. Unlike L929 cells, preferentially synthesized HSP28 andits phosphorylated isoforms (HSP28b and HSP28c) were detected during chronic heating at 42°Cin CHO cells. Neverthe

less, the expression of both the constitutive and inducibleHSP70 genes, along with the synthesis of their proteins, wasstimulated during chronic heating at 41.5°C in L929 cells.

These results suggest that HSP28 synthesis and its phosphor-ylation are not required to develop chronic thermotolerance inL929 cells. These results were consistent with the observationfrom acute thermotolerance in L929 (Figs. 4 and 5; Ref. 34),NIH 3T3 cells (Figs. 6 and 7), HT1080 (35), or HSP26 gene-deleted yeast cells which develop thermotolerance normally(36). In yeast cells, HSP26 is considered to play no vital role inthermotolerance (37). Of course, we should consider other possibilitéssuch as compensatory interactions between HSP70 andHSP28 or between a-crystallin and HSP28. Klemenz et al. (38)reported that aB-crystallin was induced by heat shock in NIH3T3 cells. This protein is known to share similar biochemicaland physical properties with small heat shock proteins, e.g.,HSP28.

Chronic thermotolerance is expressed in two different ways(39, 40). First, cells are continuously heated at lower temperatures (<42.5°C), and chronic thermotolerance develops as in

dicated by a biphasic survival curve (Fig. 1; Refs. 7-9, 32, 33).Second, cells are preheated at temperatures below 42.5°C,andthey become resistant to challenge heating at 43°Cor higher(41-43). Metabolic labeling studies with [35S]methionine have

suggested that preferentially synthesized HSPs (A/r 110,000,87,000, and 70,000) may play an important role in the development of chronic thermotolerance (44-46). Nevertheless, ourdata clearly demonstrate that chronic thermotolerance can develop in the absence of HSP28 synthesis (Figs. 1 and 3). Moreover, several researchers have proposed that multiple mechanisms exist for the development of thermotolerance (16, 47,

5784

on March 10, 2014. © 1992 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

HSP28 AND CHRONIC THERMOTOLERANCE

NIH3T3

Fig. 9. Northern blots of tnRNA extractedfrom NIH 3T3 cells. Cells were heated at45.5'C for 10 min and incubated at 37°Cforthe intervals (2-24 h) indicated at the bottomof each lane. RNA was isolated, separated, andthen hybridized with HSP28 cDNA probe(A) or HSP70 cDNA probe (B). 37'C, RNAfrom unheated cells. Arrows (a and b), induc-ible HSP70 mRNA and constitutive HSP70mRNA, respectively.

A HSP28 B HSP70

a-»-

b—w ro ^ Oí -* pò-J 3- 3- 3- ^ ^

O ^ 3-O L_ _1

45.5°C-10min —¿�

CO NJ3- 3- 3" ro

j

45.5°C-10min —¿�

SWISS3T3

41.5°C

Fig. 10. Northern blots of mRNA extracted from Swiss 3T3 cells. Swiss 3T3cells were heated at 41.5°Cfor 4, 8, or 13 h. RNA was isolated, separated, andthen hybridized with HSP28 cDNA probe. 37'C, RNA from unheated cells.

48). One develops independently of protein synthesis (type I),and the other (type II) requires protein synthesis. Therefore,other possibilities, e.g., that chronic thermotolerance can evendevelop in the absence of all HSP synthesis, should be considered (49).

Of the five major stress proteins (A/r 110,000, 87,000,70,000, 28,000, and 8,500) in mammalian cells, most attentionhas been paid to the high-molecular-weight HSPs (HSP70,HSP90, and HSP 110). Considerably less attention has beenpaid to the low-molecular-weight HSPs (HSP28 and HSP8.5).Particularly, very little information is available on the biochemical properties of HSP28. Because HSP28 in mammalian cellscontains little or no methionine residues, it is often missed inmetabolic labeling studies which use [-15S]methionine (11, 12).However, recent studies which use [3H]leucine (17), anti-

HSP28 (19), or silver staining technique (20) have demonstrated a correlation between the intracellular concentration ofHSP28 and the development of thermotolerance in Chinesehamster cells. Nevertheless, our results clearly indicated that

this was not the case in L929 cells. The absence of phosphory-lated HSP28 was observed during chronic heating at 41.5'C(Fig. 2B) or after acute heating at 45.5°Cfor 10 min (Fig. 5Ä,

left) in L929 cells. These observations are consistent with results from treatment with tumor necrosis factors. Tumor necrosis factor has been shown to stimulate the phosphorylationof HSP28 in human dermal fibroblasts, HeLa D98/AH2, ME180, and bovine aortic endothelial cells but not in L929 cells(24, 25).

The data in Fig. 8/1 illustrate that the absence of the HSP28protein family from L929 cells is not caused by a defect in thetranslational machinery or in posttranscriptional modification.It is due to the lack of HSP28 mRNA. In addition, the absenceof HSP28 mRNA does not result from the absence of theHSP28 gene (Fig. 11). It is due to the failure of HSP28 genetranscription. What remains unknown is whether this transcription failure is caused by a defect in the HSP28 promoter orin the HSP28 transcription factor. It has been well known that

•¿�*-»

CDroCD

COHCO

O

Fig. 11. Southern blots of genomic DNA extracted from L929, Swiss 3T3. orCHO cells. Genomic DNA was isolated, partially digested with restriction enzyme (//»uflll, EcoRl. or Pstl). separated, and then hybridized with HSP28cDNA probe. Only the Pjfl-digested DNA fragment was detected. Only a sectionof the autoradiograph is shown. 0X117 RF DNA///aeIII fragments were used asmolecular weight standards (data not shown).

5785

on March 10, 2014. © 1992 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

HSP28 AND CHRONIC THERMOTOLERANCE

the transcriptional induction of heat shock genes in eukaryotesis mediated by the HSTF (50-55). This protein contains atranscriptional activation domain whose activity is repressedunder nonshock conditions and activated upon heat shock(56-59). The transient activity of HSTF is associated withphosphorylation (60). In Drosophila and HeLa cells, the activated HSTF binds to the promoters which contain heat shockelement (61) and then stimulates transcription (56-58). In yeastcells, however, HSTF is already bound to the heat shock elements prior to heat shock. These observations suggest that thesimple binding of HSTF to the heat shock element is not sufficient for transcriptional activation. A modification of HSTF isrequired to stimulate transcription (54, 58, 62). Obviously, further studies at the molecular level are necessary to demonstratewhether the lack of HSP28 gene expression is due to the failureof the activation of HSTF for HSP28 gene.

REFERENCES

1. Meyer, J. L. The clinical efficacy of localized hyperthermia. Cancer Res., 44(Suppl.):4745s-4751s, 1984.

2. Stewart, J. R., and Gibbs. F. A. Hyperthermia in the treatment of cancerperspectives on its problems. Cancer (Phila.), 54: 2823-2830, 1984.

3. Kapp, D. S., Fessenden. P., Samulski, T. V.. Bagshaw, M. A., Cox, R. S., Lee,E. R., Lohrbach, A. W., Meyer, J. L., and Frionas, S. D. Stanford UniversityInstitutional report. Phase I evaluation of hyperthermia equipment for Inperthermic treatment of cancer. Int. J. Hyperthermia, 4: 75-116, 1988.

4. Sapozink, M. D., Gibbs, F. A., Gibbs, P., and Stewart, J. R. Phase I evaluation of hyperthermia equipment-university of Utah institutional report. Int.J. Hyperthermia, 4: 117-132, 1988.

5. Shimm. D. S., Celas, T. C., Olesen, J. R., Casady, J. R., and Sim, D. A.Clinical evaluation of hyperthermia equipment: the University of Arizonainstitutional report for NCI hyperthermia equipment evaluation contract.Int. J. Hyperthermia, 4: 39-52.

6. Corn. P. M., Jabboury. K., Kong, J. S., Armour. E. P., McCraw, J. F., andLeDuc, T. Evaluation of equipment for hyperthermia treatment of cancer.Int. J. Hyperthermia. 4: 53-74, 1988.

7. Palzer, R. J., and Heidelberger, C. Studies on the quantitative biology ofhyperthermic killing of HeLa cells. Cancer Res., 33: 415-421. 1973.

8. Dewey, W. C., Hopwood, L. E.. Sapareto, S. A., and Gerweck, L. E. Cellularresponses to combinations of hyperthermia and radiation. Radiology. 123:463-474, 1977.

9. Gerweck, L. E. Modification of cell lethality at elevated temperatures. ThepH effect. Radiât.Res., 70: 224-235, 1977.

10. Urano, M. Kinetics of thermotolerancc in normal and tumor tissues: a review. Cancer Res., 46: 474-482, 1986.

11. Landry, J.. Bernier, D.. Chretien, P., Nicole, L. M., Tanguay, R. M., andMarceau, N. Synthesis and degradation of heat shock proteins during development and decay of thermotolerance. Cancer Res., 42: 2457-2461, 1982.

12. Li. G. C. Elevated levels of 70,000 dalton heat shock protein in transientlythermotolerant Chinese hamster fibroblasts and their stable heat resistantvarients. Int. J. Radial. Oncol. Biol. Phys.. //: 165-177, 1985.

13. Lee, Y. J., Kim, D., andCorry, P. M. Effect of histidine on histidinol-inducedheat protection in Chinese hamster ovary cells. J. Cell. Physiol., 144: 401-407, 1990.

14. Subjeck, J. R., Sciandra, J. J.. and Johnson, R. J. Heat shock proteins andthermotolerance: A comparison of induction kinetics. Br. J. Radiol.. 55:579-584. 1982.

15. Laszlo, A., and Li, G. C. Heat-resistant varients of Chinese hamster fibroblasts altered in expression of heat shock protein. Proc. Nati. Acad. Sci. USA,«2:8029-8033. 1985.

16. Lee, Y. J., and Dewey, W. C. Effect of cycloheximide or puromycin oninduction of thermotolerance by sodium arsenite in Chinese hamster ovarycells: involvement of heat shock proteins. J. Cell. Physiol., /32:41 -48, 1987.

17. Crete, P., and Landry, J. Induction of HSP27 phosphorylation and thermore-sistance in Chinese hamster cells by arsenite, cycloheximide, A23187, andEGTA. Radiât.Res., 121: 320-327, 1990.

18. Landry, J., Crete, P.. Lamarche, S., and Chretien P. Action of Ca2+-depen-dent processes during heat shock: role in cell thermoresistance. Radiât.Res.,113: 426-436. 1988.

19. Landry, J., Chretien, P.. Laszlo, A., and Lambert. H. Phosphorylation ofHSP27 during development and decay of thermotolerance in Chinese hamster cells. J. Cell. Physiol.. 147: 93-101, 1991.

20. Lee, Y. J.. Hou, Z., Curetty, L., and Corry, P. Expression, synthesis, andphosphorylation of HSP28 family during development and decay of thermotolerance in CHO plateau-phase cells. J. Cell. Physiol., ISO: 441-446. 1992.

21. Landry, J., Chretien, P., Lambert, H.. Hickey, E., and Weber, L. A. Heatshock resistance conferred by expression of the human HSP27 gene in rodentcells. J. Cell Biol., 109: 7-15, 1989.

22. Kim, Y-J., Shuman, J., Sette, M., and Przybyla, A. Nuclear localization andphosphorylation of three 25-kilodalton rat stress proteins. Mol. Cell. Biol., 4'468-474, 1984.

23. Welch, W. J. Phorbol ester, calcium ionophore, or serum added to quiescentrat embryo fibroblast cells all result in the elevated phosphorylation of two28,000-dalton mammalian stress proteins. J. Biol. Chem., 260: 3058-3062,1985.

24. Hepburn, A., Demolle, D., Boeynaems, J-M., Fiers, W., and Dumont, J. E.Rapid phosphorylation of a 27 kDa protein induced by tumor necrosis factor.FEBS L«tt.,227: 175-178, 1988.

25. Kaur, P., and Saklatvala, J. Interleukin 1 and tumour necrosis factor increasephosphorylation of fibroblast proteins. FEBS Lett., 241:6-10, 1988.

26. Highfield, D. P., Holahan, E. V., Holahan, P. K., and Dewey, W. C. Hyper-thermic survial of Chinese hamster ovary cells as a function of cellular population density at the time of plating. Radiât.Res., 97: 139-153, 1984.

27. Borrelli, M. J.. Thompson, L. L., and Dewey, W. C. Evidence that the feedereffect in mammalian cells is mediated by a diffusible substance. Int. J. Hyperthermia, 5: 99-103, 1989.

28. Walker, J. M. Gradient SDS polyacrylamide gel electrophoresis. in: J. M.Walker (ed.), Methods in Molecular Biology, Vol. 1, pp. 57-61. Clifton, NJ:Humana Press, 1984.

29. Tushinski, R., Sussman, P., Yu, L., and Bancroft, F. Pregrowth hormonemessenger RNA: glucocorticoid induction and identification in rat pituitarycells. Proc. Nati. Acad. Sci. USA, 74: 2357-2361, 1977.

30. Lehrach, H., Diamond, L., Wozney, J., and Boedtker, H. RNA molecularweight determinations by gel electrophoresis under denaturing conditions, acritical reexamination. Biochemistry, 16: 4743-4751, 1977.

31. Sambrook, J., Fritsch, E. F., and Maniatis, T. Molecular Cloning. A Laboratory Manual, Ed. 2. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989.

32. Lee, Y. J., Dewey, W. C., and Li, G. C. Protection of Chinese hamster ovarycells from heat killing by treatment with cycloheximide or puromycin: involvement of HSPs? Radiât.Res., ///: 237-253, 1987.

33. Lee, Y. J., Hou, Z., Curetty, L., Borrelli, M. J., and Corry, P. M. Correlationbetween redistribution of 26 kDa protein and development of chronic thermotolerance in various mammalian cell lines. J. Cell. Physiol., 145:324-332,1990.

34. Lee, Y. J., Hou, Z., Curetty. L., and Borrelli, M. J. Development of acutethermotolerance in L929 cells: lack of HSP28 synthesis and phosphorylation.J. Cell. Physiol., 152: 118-125, 1992.

35. Mivechi, N. F., Monson, J. M., and Hahn, G. M. Expression of HSP-28 andthree HSP-70 genes during the development and decay of thermotolerance inlekemic and nonleukemic human tumors. Cancer Res., 51:6608-6614, 1991.

36. Petko, L., and Lindquist, S. Hsp26 is not required for growth at high temperature, nor for thermotolerance, spore development, or germination. Cell,«:885-894. 1986.

37. Susek, R. E., and Lindquist, S. L. Hsp26 of Saccharomyces cerevisiae isrelated to the superfamily of small heat shock proteins but is without ademonstrable function. Mol. Cell. Biol., 9: 5265-5271, 1989.

38. Klemenz, R.. Frohli, E., Steiger, R. H., Schafer, R., and Aoyama, A. aB-Crystallin is a small heat shock protein. Proc. Nati. Acad. Sci. USA, 88:3652-3656, 1991.

39. Jung, H. A generalized concept for cell killing by heat. Effect of chronicallyinduced thermotolerance. Radiât.Res., 127: 235-242, 1991.

40. Jung, H. Effect of chronically induced thermotolerance on thermosensitiza-tion in CHO cells. Int. J. Hyperthermia. 7: 621-628. 1991.

41. Henle, K. J., Karamuz, J. E., and Leeper, D. B. Induction of thermotolerancein Chinese hamster ovary cells by high (45") or low (40°)hyperthermia.Cancer Res., 38: 570-574, 1978.

42. Jung, H., and Kolling, H. Induction of thermotolerance and sensitization inCHO cells by combined hyperthermic treatments at 40 and 43"C. Eur. J.Cancer, 16: 1523-1528, 1980.

43. Spiro, I., Sapareto, S. A., Raaphorst, G. P.. and Dewey, W. C. The effect ofchronic and acute heat conditioning on the development of thermal tolerance.Int. J. Radiât.Oncol. Biol. Phys.. 8: 53-58, 1982.

44. Mivechi, N., and Li. G. C. Thermotolerance and profile of protein synthesisin murine bone marrow cells after heat shock. Cancer Res., 45: 3843-3849,1985.

45. Przybytkowski. E., Bates, J. H. T., Bates, D. A., and Mackillop, W. J.Thermal adaptation in CHO cells at 40'C: the influence of growth conditionsand the role of heat shock proteins. Radiât.Res., 107: 317-331, 1986.

46. Hatayama. T., Kano, E., Taniguchi, Y., Nitta, K., Wakatsuki, T., Kitamura,T., and Imanara H. Role of heat-shock proteins in the induction of thermotolerance in Chinese hamster V79 cells by heat and chemical agents. Int. J.Hyperthermia, 7:61-74, 1991.

47. Lee, Y. J., and Dewey, W. C. Thermotolerance induced by heat, sodiumarsenite, or puromycin: its inhibition and differences between 43'C and 45'C.J. Cell. Physiol., 135: 397-406, 1988.

48. Laszlo, A. Evidence for two states of thermotolerance in mammalian cells.Int. J. Hyperthermia, 4: 513-526, 1988.

49. Under. S. B., Price, B. D., Mannheim-Rodman, L. A., and Calderwood, S. K.Inhibition of heat shock gene expression does not block the development ofthermotolerance. J. Cell. Physiol., 151: 56-62, 1992.

50. Parker, C. S., and Topol, J. A Drosophila RNA polymerase II transcriptionfactor binds to the regulatory site of an hsp 70 gene. Cell, 37: 273-283, 1984.

5786

on March 10, 2014. © 1992 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

HSP28 AND CHRONIC THERMOTOLERANCE

51. Wu, C. An exonuclease protection assay reveals heat-shock element and 327:727-730, 1987.TATA-box binding proteins in crude nuclear extracts. Nature (Lond.), 317: 57. Kingston, R. E., Schuetz, T. J., and Larin, Z. Heat-inducible human factor84-87, 1985. that binds to a human hsp70 promoter. Mol. Cell. Biol., 7:1530-1534, 1987.

52. Wu, C, Wilson. S., Walker, B., Dawid, I., Paisley, T., Zimarino, V., and 53. Sorger, P. K., Lewis, M. J., and Pelham, H. R. B. Heat shock factor isUeda, H. Purification and properties of Drosophila heat shock activator regulated differently in yeast and HeLa cells. Nature (Lond.), 329: 81-84,protein. Science (Washington DC), 238: 1247-1253, 1987. ,9g7

53. Sorger, P. K., and Pelham, H. R. B. Purification and characterization of a J9 Nieto.So(e|0> j., wiederrecht, G., Okuda, A., and Parker, C. S. The yeastheat shock element bmdmg protem from yeast. EMBO J., 6: 3035-3041, hea( shock transcrjp,ion factor comains „¿�transcriptiOnal activation domain

54. Sorger, P. K., and Pelham, H. R. B. Yeast heat shock factor is an essential «*°*aetivit>' is rePressed under nonshock Conditions. Cell, 62: 807-817,

DNA-binding protein that exhibits temperature-dependent phosphorylation. I ,Cell 54- 855-864 1988 Sorger. P. K. Yeast heat shock factor contains separable transient and sus-

55. Wiederrecht, G., Shuey, D. J., Kibbe. W. A., and Parker, C. S. The Saccha- tained response transcriptional activators. Cell, 62: 793-805, 1990.romyces and Drosophila heat shock transcription factors are identical in size 61. Pelham, H. R. B. A regulatory upstream promoter element in the Drosophilaand DNA binding properties. Cell, 48: 507-515, 1987. Hsp 70 heat-shock gene. Cell, 30: 517-528, 1982.

56. Zimarino, V., and Wu, C. Induction of sequence-specific binding of Droso- 62. Jakobsen, B. K., and Pelham, H. R. B. Constitutive binding of yeast heat-phila heat shock activator protein without protein synthesis. Nature (Lond.), shock factor to DNA in vivo. Mol. Cell. Biol., 8: 5040-5042, 1988.

5787

on March 10, 2014. © 1992 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from