hsp27 as a mediator of confluence-dependent...

ICANCERRESEARCH57. 2661-2667. July I. 19971

ABSTRACT

Resistance of colorectal cancer cells to chemotherapeutic drugs increases as cells reach confluence. Here we show that the small stressprotein HSP27, which has been described to block necrotic and apoptoticcell death, accumulates in confluent human colorectal cancer cell linesHT-29 and Caco2. Cell confluence also induces HSP27 phosphorylationand changes in its intracellular distribution. We also show that overexpression of human HSP27 by transfection of HT-29 cells increased theresistance of cells to doxorubicin or cisplatin and prevented drug-inducedapoptosis. Interestingly, nonconfluent HSP27-transfected cells and confluent control cells in which HSP27 is expressed at the same level displayeda similar drug resistance. HSP27-transfected cells did not exhibit anenhanced resistance when they reached confluence, nor was there anincreased accumulation of HSP27. We have previously shown that HSP27expression blocks tumor necrosis factor-induced cell death as a result ofdecreasing Intracellular reactive oxygen species (ROS). Here we show that

HSP27 overexpression in HT-29 cells, obtained either by transfection orby growing the cells at high density, correlated with a significant ROSdecrease. We conclude that cell confluent-dependent HSP27 accumulation, probably due to its ability to decrease ROS levels, is essential for theestablishment of the resistance of colorectal cancer cells when reachingconfluence.

INTRODUCTION

Cells respond to environmental stress by preferential synthesis andaccumulation of a conserved family of proteins referred to as heatshock proteins or stress proteins and by the acquisition of a dramatically increased capacity to survive subsequent hyperthermic stresses(1). To this group of proteins belongs the sHSP family.3 This familyis composed of different proteins varying in size from 15 to 30 kDa,depending on the species, that share sequence homology. Recent datahave shown that elevated expression of sHSP efficiently protectedmammalian cell death induced by heat (2), certain commonly usedanticancer drugs (3â€”5),oxidative stresses (6, 7), or the antitumoralcytokine TNF (8). The biochemical function of the sHSP family thatresults in an enhanced cellular protection against different forms ofstress is not yet clear, but it may be a consequence of the ability of

these proteins to act in vitro as molecular chaperones enhancing therenaturation of denatured polypeptides in an AlP-independent manner (9). In this capacity, they have been implicated as regulators ofactin cytoskeleton organization (10, 11). However, we have recentlyobserved that sHSPs protect against TNF-a-induced cell death bymodulating ROS via a glutathione-dependent pathway (12).

HSP27 shares biochemical properties with the other members of the

Received I2/10/96; accepted 4/30/97.â€˜I'hecosts 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 with18 U.S.C. Section 1734 solely to indicate this fact.

I Supported by the Conseil Regional de Ia Bourgogne, the Ligue Bourguignonne contre

Ic Cancer, Association pour Ia Recherche sur Ic Cancer Grant 601 1, Institut National pourla Sante et Ia Recherche Medicale Grant 930.501, and the Region RhÃ´ne-Alpes.

2 To whom requests for reprints should be addressed. Phone: 33-03-80-67-1788.

3 The abbreviations used are: sHSP, small heat shock protein; HSP, heat shock protein;

ROS, reactive oxygen species; TNF, tumor necrosis factor; ECL, enhanced chemiluminescence; CDDP, cisplatin; DXR, doxorubicin; HE, hydroethidine; EB, ethidium bromide.

sHSP family, such as oligomerization and phosphorylation. HSP27exists as large, dynamic cytoplasmic aggregates, with molecular native molecular masses ranging from 100â€”800kDa. Numerous stimuli(stress, treatment with TNF, and arsenite) as well as changes in cellphysiology (serum starvation) were found to alter the oligomerization

pattern of HSP27 (13â€”16).A key feature of HSP27 is its rapid and pronounced phosphoryla

tion, which occurs in different cellular conditions. HSP27 phosphorylation is observed after exposure to a variety of stresses, differen

tiating agents, mitogens, inflammatory cytokines such as TNF-cs andinterleukin 1, hydrogen peroxide, and other oxidants (17). Although

not studied in every case, HSP27 phosphorylation seems to occur atthe same serine residue of HSP27 (18), suggesting the involvement ofthe same kinase (MAPKAP kinase 2) that may be activated bydifferent signal transduction mechanisms. The role played by thismodification in the structure/function of HSP27 is not yet clear. Forexample, depending on the stimuli, phosphorylation correlates witheither a reduced (TNF-a and phorbol ester; Refs. 15 and 19) or anincreased (serum stimulation; Ref. 14) oligomerization of this protein.Other studies using directed mutagenesis of the HSP27 phosphorylated serine residues have also led to perplexing observations (20, 2 1).

HSP27 cellular localization can also be modulated by severalstimuli such as heat, TNF-a, or serum deprivation (16, 17). HSP27 isconstitutively expressed at low levels in the cytosol of most humancells (22). Within minutes of heat treatment, HSP27 is phosphorylated, shifts from a nonionic detergent-soluble to -insoluble cellularcompartment, and relocalizes from the cytoplasm to within or aroundthe nucleus (10, 13, 23). It has been suggested that these modificationsin HSP27 properties may be part of a mechanism that activates the(protective)functionoftheprotein.

We have previously reported that for most anticancer agents, theirefficacy against colon cancer cells decreases progressively as cellsreach confluence (24â€”26).This occurrence, which we have calledconfluence-dependent resistance, was attributed to a decrease in intracellular drug accumulation and/or a decrease in the sensitivity ofthe cells to the different drugs in the confluent population. Interestedin knowing whether HSP27 could participate in the confluence

dependent resistance, we have studied the regulation of HSP27 cxpression by cell culture density in HT-29 and Caco2 human colorectal

cancer cells. We have found that HSP27 mRNA and protein accumulate as cell confluence increases. In contrast, no difference of expression between confluent cells and exponentially growing cells wasfound for the other major HSPs (HSP6O, HSP7O, and HSP89). Cellconfluence also induced HSP27 phosphorylation and changes in itsintracellular distribution. These modification provoked by cell confluence were not due to the nonproliferative status of the cells in thehigh-density cultures (i.e., most cells were in the@ phase of the

cellular cycle), because serum starvation had a very different effect inHSP27 properties. Finally, we show data concerning HSP27 protective activity and the ability to decrease ROS that suggest a determi

nant role for HSP27 in the establishment of the resistance of colorectalcancer cells when they reached confluence.

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HSP27 as a Mediator of Confluence-dependent Resistance to Cell Death Induced byAnticancer Drugs1

Carmen Garrido,2 Patrick Ottavi, Annie Fromentin, Arlette Hammann, AndrÃ©-PatrickArrigo, Bruno Chauffert, andPatrick Mehlen

Institut National de Ia Sante et de Ia Recherche MÃ©dicaleCJF 94-08. FacultÃ©de Medecine, 7 Boulevard Jeanne d'Arc, 21033 Dijon Cedex, France fC. G.. P. 0., A. F., A. H..B. CI, and Laboratoire du Stress Cellulaire, Centre National de Ia Recherche Scientifique UMR-5534, Unis'ersitÃ©Claude Bernard Lyon-I. 69622 Villeurbanne. France (A-P. A..P.M.J

Research. on September 13, 2018. © 1997 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

HSP27 AND CELL CONFLUENCE

MATERIALS AND METHODS

Cells. HT-29 and Caco2 human colon carcinoma cells were provided bythe American Type Culture Collection (Rockville, MD). We recently describedstable transformants of human HT-29 cells that carry the gene encoding humanHSP27 (4). One of the characterized clones (53) was used in this work.Another cell clone that carried the hygromycin resistance gene and plain vector(C1) was used as control.Cells were grown as monolayersin a controlledatmosphere (37Â°C,5% CO2) using Ham's F-b medium supplemented with10% FCS. The effect of serum starvation was studied in cells grown during48 h in the absence of serum. Nonconfluent HT-29 or Caco2 populations

(exponentially growing cells) were obtained by seeding 5 X l0@or 2.5 X l0@cells, respectively, in the wells of 96-well microculture plates and by seeding2 X 106 or I x 106 cells in a 50-mi Falcon flask (Falcon, Lincoln Park, NY).About 35% confluence was obtained after a 48-h culture. Confluent populations were obtained by seeding 10 X 10â€•or 5 X l0@ cells/well in 96-well

microculture plates and by seeding 10 X 106 or 5 x 106 cells in a 50-ml Falconflask.

Northern Blot Analysis. RNA was extracted using the guanidium isothiocyanate-cesium chloride method. Twenty @gof total RNA were separated byelectrophoresis through 1% agarose-formamide gels followed by capillary

transfer to nitrocellulose filters. The HSP27 probe was generated by PCR andcontained the cDNA fragment coding for the 98 COOH-terminal amino acids(27). The actin probe was a 0.5-kb PvuII-XbaI fragment from pHMa A-l (28).The purified fragments were labeled to high specific activity (2â€”4X l0@dpm4@g) with a random primer labeling system (Amersham, ArlingtonHeights, IL). Hybridization was done by standard procedure. Labeled probeswere removed from filters by incubating them in water at 85Â°Cfor 1 h. Beforehybridization with a second probe, removal was assessed by autoradiography.Two criteria were used to control for the amount of RNA loaded on the gel: (a)the intensity of the stained EB 185 and 28S RNAs; and (b) the signal given byhybridization of the filter with an actin probe. Both signals did correlate well,indicating that the cellular level of cytoskeletal actin mRNA remained rela

tively constant during the course of the experiment. Each RNA analysis wasrepeated at least twice. Blots were quantitated and normalized for actin usingan Image Store analyzer (UVP, London, United Kingdom).

Cell Fractionation: Detergent Lysis and Hypotonic Lysis. Cells growingin 50-ml Falcon flasks were washed with PBS, scraped from the flasks, andpelleted at 1,000 x g for 5 mm. Lysis was performed at 4Â°Cin a buffercontaining 10 mistTris (pH 7.4), 1 mistMgCl2, 10 mistNaCI, and 0.1% TritonX-l00. The lysates were centrifuged at 2,000 X g for 10 mm (pellet P2). The

resulting supernatants were centrifuged at 20,000 X g for 10 mm (pellet P20and supematant 5). Before loading into the gel, subcellular fractions wereboiled in Laemmli sample buffer. For hypotonic lysis, we used the protocoldescribed above, except that the lysis buffer was devoid of Triton X-l00.

Gel Electrophoresis and Immunoblotting. For immunoblouing, wholecell lysates were prepared by lysing the cells with 2% SDS in 137 mMNaCl,2.7 mist KC1, 8 mM Na2HPO4, I .5 mist @â€˜2@4'and NaC1IP (pH 7.4) at 68Â°Cfor 5 mm. Protein concentration was measured in the supernatant by using themicro BCA protein assay (Pierce, Asnieres, France). Equal amounts of protein(100 @g)were separated in an 11% SDS-polyacrylamide gel. Proteins weretransferred to a nitrocellulose filter by electroblotting. Blots were blocked with5% bovine albumin in NaCI/P1 (pH 7.4) and 0. 1% Tween 20 and probed withmouse monoclonal antibodies (I :1000) to human recombinant HSP27, HSP6O,HSP7O,and HSP9O(StressGen, Victoria, Canada). After three 5-mm washes inNaCI/Pi, pH 7.4, 0. 1% Tween 20 (PBST), the blots were incubated withperoxidase-conjugated antimouse IgG. The ECL Western blotting analysis

system (Amersham, Les Ullis, France) was subsequently used for proteindetection. Blots were repeated twice and quantified using the Image Storeanalyzer. Two-dimensional electrophoresis was performed as described previously (29). As a control in the same two-dimensional Western blot in whichHSP27 isoforms from human colorectal cancer cells were determined, HSP27isoforms from HeLa cells extracts were also analyzed. A Bioprofil system(Vilber Lourmat) was used to quantify the different HSP27 isoforms. Theanalysis was performed within the range of proportionality of the film. Tocompare different samples, the percentage of each isoform was determined bydividing the value of each isoform by the sum of the values of all isoforms.

Drug Cytotoxicity Assay. Cells were treated with increasing concentrations of CDDP or DXR (Roger Bellon, Neuilly, France) for 4 h. The culture

medium was then replaced by drug-free medium for 6 additional days. Thislong posttreatment period was chosen to give the cells enough time to die anddetach from the well. The number of surviving cells was measured with a

methylene blue colorimetric test (24).Cell Morphological Studies. The percentageof apoptoticcells was deter

mined by fluorescence microscopy after staining for 10 mm at 37Â°CwithHoechst 33342 at a final concentration of 5 @.&g/ml.Detached cells wererecovered by centrifugation of cell culture supernatant and mixed with the

adherent cells recovered by trypsinization. Cells were judged apoptotic on thebasis of characteristic chromatin hypercondensation. Cell viability was determined by trypan blue exclusion. Three hundred cells were counted in triplicate,and the percentages of apoptotic and dead cells were evaluated.

in Vivo Fluorescent Measurement of Intracellular ROS. A fluorescentprobe, HE (Molecular Probe-Interchim, MontluÃ§on,France), the NaBH4-reduced form of EB, was used to measure the intracellular content of ROS inliving cells (30). Cells were washed twice with PBS and incubated for 10 mmwith 40 @ig/mlHE. The flow cytometry analysis was performed using aFACScan flow cytometer (Becton Dickinson, Le Pont de Claix, France) using488 nm excitation. Emission filter was 610 nm bandpass for oxidized HE (EB)fluorescence.

Cell Cycle Analysis. Confluentcells were treatedfor 4 h with CDDP andleft to recover for different periods of time in drug-free medium. Cells wereharvested after trypsinization (2.5 mg/mI trypsmn and 0.2 mg/mI EDTA inHank's medium without Ca2@or Mg21. Monoparametric cell cycle analysisusing propidium iodide was performed using a Becton Dickinson fluorescence

activated cell analyzer after nuclear staining with the cell cycle DNA reagentkit (Becton Dickinson). Data were interpreted using CELLFIT software andthe SOBR mathematical model provided by the manufacturer. Results represent a minimum of 10â€•cells assayed for each determination.

Intracellular Platinum Accumulation. Cells (2 X 10@S)were seeded into5-cm Petri dishes, incubated at 37Â°Cfor 48 h, and exposed to 5 @g/mlCDDPfor 4 h. After three washes in ice-cold PBS, the cells were scraped off thedishes, suspended in 0.5 ml ofPBS, and stored at â€”20Â°C.After sonication, thetotal cellular content of CDDP was determined by atomic absorption spectroscopy (Perkin-Elmer Corp.). Platinum levels were expressed as nanograms ofplatinum/milligrams of protein.

RESULTS

CellConfluenceInducesChangesin HSP27Expressionin TwoHuman Colorectal Cancer Cell Lines. HSP27 steady-state proteinexpression was examined by immunoblot analysis in HT-29 andCaco2 cells cultured at different cell densities. HSP27 accumulated ascultures reached confluence. For both cell lines, HSP27 content incells of the stationary phase was 10â€”15times higher (depending onthe cells used) than that in exponentially growing cells (Fig. 1A).Asa control, we examined in parallel the topoisomerase II content in thecultures. As described previously (26), its expression was higher inexponentially growing cells than it was in confluent cultures. In

contrast, no significant differences in protein content were seen for themajor heat shock proteins HSP6O, HSP7O, and HSPS9 (Fig. 1, A andB). Increased HSP27 steady-state protein in confluent cells seemed tobe transcriptionally mediated, because HSP27 mRNA levels were alsohigher in the confluent populations (Fig. 10.

HSP27 Phosphoinoforms Accumulate in Confluent Cultures.HSP27 phosphoisoforms, identified by their isoelectric point (14, 16,

21 , 30) using whole extracts from HeLa cells as a control, werequantified in cells cultured at high density and in exponentiallygrowing cells. The two-dimensional immunoblots presented in Fig. 2show that in exponentially growing Caco2 cells, HSP27 is recoveredat the level of the basic unphosphorylated isoform (Fig. 2, a) and themonophosphorylated isoform (Fig. 2, b) at 69 and 31%, respectively.Interestingly, in confluent cultures, the unphosphorylated isoformstrongly decreases (about 14%) on behalf of isoform b and c, detectable at 50 and 36%, respectively. Hence, high cell density inducedHSP27 phosphorylation.

2662




Caco2

NC CC

47 83.141 10,6

HT-29 Caco2NCCC NCCC

HSP27 in the particulate P2 fraction (up to 40%, Fig. 3A). For bothcultures, no HSP27 was detected in the particulate fractions when the

cellular fractionation was performed in the presence of 0. 1% of thenonionic detergent Triton X-lOO (Fig. 3B). To analyze whether thechange of cellular distribution of HSP27 was specific, the intracellulardistribution of HSP7O, a known cytoplasmic and nuclear gene product, was analyzed under the same conditions. HSP7O was equallyrecovered in the three fractions in both exponentially growing cellsand confluent cells. No changes were observed when fractionationwas performed in the presence of Triton X-lOO (Fig. 3, C and D). This

suggests that HSP27 and HSP7O have specific cellular locales inhuman colorectal cancer cells and that only the localization of HSP27is modified by cell culture density.

Effect of Growth Arrest by Serum Starvation on HSP27 Properties. To analyze whether the effect of cell confluence in HSP27 wasdue to the lack of actively dividing cells, we analyzed cell growtharrested by serum starvation. Confluent cells and serum-starved cells

A CC NC

P2P2OS P2 P20 S

F'1SP27@

I P2 P20 S P2 P20 SCC NC

B CC NC

P2P2OS P2 P20 S

HSP27

C NC CC

P2P2OS P2P20 S

HSP7O@

DH S P 7 0@

Fig. 3. Cell confluence induces changes in HSP27 subcellular distribution. Confluent(CC) and nonconfluent (NC) HT-29 cells, 0.2 X l0@'cells and I X 106 cells, respectively,were hypotonically lysed in the absence (A) or presence (B) of 0.1% Triton X-l00 andfractionated as described in â€œMaterialsand Methods.â€•The distribution of HSP27 in thedifferent fractions (P2, 2,000 X g pellet; P20 and S. 20,000 X g pellet and supernatant.respectively) was analyzed in immunoblots probed with HSP27 antibody. The accumulation of HSP27 in the different fractions was measured by densitometry. The sameexperiment was performed to study HSP7O distribution. In this case, I X l0@cells werelysed in the absence (C) or presence (D) of Triton X-lOO and probed with HSP7Oantibody. Autoradiographs of ECL-revealed immunoblots are presented. One representative cell fractionation analysis, of the three performed. is shown.

AHT-29

NC CC

%GOIGI 51.9 71

%S 38 18

HSP27@@ â€” -

HSP70@@@@ â€”

T0 P0 I1@@ 4

B C

NC CC

HSP9O hsp27 â€¢â€¢ â€¢I

HSP6O

HSP27@ actin

Fig. I. Cell confluence induces HSP27 expression in two human colon cancer celllines.A,HSP27,HSP7O,andtopoisomeraseII steady-stateproteinslevelsfromnonconfluent (NC) and confluent (CC) HT-29 and Caco2 cells were examined by immunoblotanalysis. The percentages of G0-G, and S-phase cells in each sample are indicated. B,immunoblotanalysisof HSP9O,HSP6O,and HSP27proteinsin nonconfluent(NC)andconfluent (CC) HT-29 cells. C, HSP27 steady-state RNA levels in HT-29 and Caco2 cellswere determined by Northern blot. To verify that equal amounts of RNA were loaded, theblot was rehybridized with an actin probe. RNA and protein analysis was repeated threetimes. One representative experiment is shown.

cb aV V V

NC

T,7@@@

@;@y vCC@

Fig. 2. Quantitative analysis of HSP27 phosphoisoforms in nonconfluent and confluentcells. Two-dimensional immunoblots probed with anti-H5P27 serum of the total proteinsof 5 X l0@nonconfluent (NC) and I X l0@confluent (CC) Caco2 cells. Autoradiographsof ECL-revealed immunoblots are presented. The acidic end is to the left. Arrowheads a,b, and c, the three major HSP27 isoforms. The a isoform represents the unphosphorylatedform of the protein. Two-dimensional immunoblots were repeated twice. One representative experiment is shown.

HSP27 Subcellular Distribution Is MOdified @flCells Culturedat Confluence.We next analyzedthe intracellulardistributionofHSP27 in high-density stationary phase and exponentially growing

Caco2 and HT-29 cells. After lysis of exponentially growing cells,HSP27 is mainly recovered in the 20,000 X g supernatant fraction,whereas the particulate fractions P2 (2,000 X g) and P20 (20,000 X g)contained less than 5% of its total cellular content (Fig. 3A). Adifferent distribution of HSP27 was observed in the cells lysed fromthe confluent culture. In this case, we found an accumulation of

2663




suggests that increased HSP27 content, either by transfection or bycell density, is related to the increased cell survival in the drugs.Supporting the notion that the increase in HSP27 content participatesin the resistance phenotype of confluent cells, a similar resistance tothe drugs is found between confluent and nonconfluent HSP27-transfected cells in which HSP27 is expressed at the same level (Fig. 5).

We have recently proposed HSP27 as a novel regulator of apoptosis, because its expression in murine L929 cells blocked apoptosisinduced by several stimuli (31). Interested in knowing more about theprotective effect of HSP27 against chemotherapeutic drugs, we havestudied whether HSP27 also inhibits apoptosis induced by CDDP.HSP27- and mock-transfected cells were treated with CDDP (5 p@g/mlfor 4 h), and 96 h after drug removal, cells were fixed and stained withHoechst 33242 for chromatin labeling. Overexpression of HSP27caused a 24 Â± 2.3% decrease in the number of apoptotic cellscompared to that of control transfectants (percentages were determined from 300 cells counted in triplicate). This result was confirmedby cell staining with deoxynucleotidyl transferase (Apoptag) to detectDNA fragmentation (32). Hence, HSP27 overexpression blocksCDDP-induced apoptotic cell death.

HSP27 Accumulation Induces Changes in Intracellular ROSLevels. We next tried to determine the molecular mechanisms thatallow HSP27 to decrease the cytotoxic effect of these drugs. BecauseHSP27 is known to interact with the cytoskeleton (33), it couldinfluence drug uptake and therefore explain the different sensitivity ofthe cells. However, we found that similar amounts of the drug weretaken up by the HSP27-transfected and the control-transfected cells(19.35Â±2.4ngCDDP/mgproteinaftera4-htreatmentwith5 p.g/mlCDDP, n = 4). ROS are known mediators of anticancer drug-inducedcell death (34). In previous studies, we demonstrated in L929 murinecells that stable expression of human HSP27 correlates with decreasedcellular content of ROS (12). This capacity of HSP27 to alter basalROS levels was associated with its protective activity against agents

2664

A

SS NC CC

HSP27@ ____

HSP7O@

B SS CC

P2P20 S P2P20 S

H S P 2 7@@ . â€”-=::

@uIJP2 P20 S P2 P20 S

ss ccFig. 4. Effect of growth arrest by serum starvation in H5P27 level and subcellular

localization. A, immunoblot analysis of total protein extracts from HT-29 nonconfluentcells serum-starved for 48 h (SS), nonconfluent proliferative cells (NC), and confluentcells (CC). Immunoblots were probed with either HSP27 antibody or HSP7O antibody. B,1 X 10Â°nonconfluent, serum-starved cells (55) and confluent cells (CC) were lysed in theabsence of nomonic detergent. The distribution of HSP27 in the different fractions wasanalyzed by immunoblot and (C) quantified by densitometry. Immunoblots were repeatedthree times.

had a similar cell cycle distribution with around 80% of the cells in theG0-G1phase. Nevertheless, as shown in Fig. 4A, serum starvation didnot provoke an increase in HSP27 content. Similarly, no accumulationof HSP27 in the P2 fraction was detected in the serum-starved cells(Fig. 4, B and C).

Overexpression of HSP27 Increases the Resistance of Cells toDXR and CDDP. We have recently shown that HSP27 participatesin the resistance of colorectal cancer cell line HT-29 to DXR (4).Using the stable HT-29 cell clones expressing human HSP27 and themock-transfected clones as a control described in previous work, weshow here that HSP27 expression also protects against CDDP treatment. The immunoblot presented in Fig. 5 shows the steady-state levelof HSP27 from nonconfluent and confluent cells from two representative clones of HSP27-transfected and mock-transfected HT-29 cells.Exponentially growing HT-29 cells transfected with human HSP27are more resistant to CDDP or DXR than are proliferating mocktransfected cells (Fig. 6). This resistance phenotype cannot be cxplaned by differences in the cell cycle distribution. The percentagesof HSP-27-transfected cells in G0-G1 are 48% in the nonconfluentcells and 70.5% in the confluent cells, respectively. The percentagesof mock-transfected cells in G0-G1 phase are 50.5% in the nonconfluent cells and 71% in the confluent cells, respectively. Interestingly,confluent mock-transfected cells and exponentially growing HSP27-transfected cells, which contain a similar HSP27 content, are equallyresistant to CDDP- or DXR-induced cell death. This result strongly

1 2 3 4

HSP27 CONTROLNCCC NC CC1 2 3 4

HS P2 7@ -:L::@@ @::â€˜

HS P7 0@ = :::::

:@ 180C

@ 150

I@Fig. 5. HSP27 steady-state level does not change with cell confluence in HSP27-

overexpressing cells. Immunoblot analysis of total protein extracts from nonconfluent(NC) and confluent (CC) HSP27-transfected (Lanes 1 and 2) and mock-transfected (Lanes3 and 4) HT-29 cells. hnmunoblots were probed with HSP27 antibody or HSP7O antibody.H5P27 content was quantified by densitometry. Immunoblots were repeated three times.




100

80

60

40

20

0

coop (p.g/mI)100

80â€¢

60

40'

20'

-j

>>It

(0-J-Iw0

a2-J

>>It

Co-J-Jw0

0 5 10

DXR(pg/ml)

Fig. 6. Increased HSP27 content inhibits cell death induced by CDDP or DXR.Nonconfluent (0) and confluent (0) HSP27-transfected as well as nonconfluent () andconfluent (â€¢)mock-transfected HT-29 cells were treated with increasing concentrationsof DXR or CDDP for 4 h. Six days after drug removal, cell survival was measured. Dataare illustrated as the mean Â±SE (n = 4).

such as TNF-a. To study whether HSP27 also has this capacity inhuman colorectal cancer cells, we have compared human HSP27-expressing and mock-transfected HT-29 cell clones. As shown in Fig.7 there was a 1.6-fold decrease in ROS levels in HSP27-transfectedcells compared to that in mock-transfected cells. Remarkably, a decrease in ROS levels was also obtained when there was HSP27accumulation due to growing the cells at high density. ConfluentHT-29 cells had a 1.5-fold decrease in ROS content compared to thatof exponentially growing cells (Fig. 7, left panel). Hence, in the samecell line, a similar HSP27 level observed in two different contexts(i.e. , proliferative or confluent populations) was associated with asimilar intracellular ROS level.

HT-29 CELLS HT-29-TRANSFECTED CELLSFig. 7. HSP27 decreases ROS cellular level. Left panel, in vivoestimation of intracellular ROS levels in nonconfluent (NC) andconfluent (CC) HT-29 cells. As a control for ROS production, nonconfluent wild-type HT-29 cells were treated for 10 mm with 100 @u@imenadione. Rightpanel, in vivoestimation of intracellular ROS levelsin nonconfluent HSP27-transfected and mock-transfected (control)HT-29cells.As a controlfor ROSproduction,nonconfluentmocktransfected cells were treated with 100 psi menadione. Results arepresented as mean oxidized HE (EB) fluorescence indexes that werecalculated by dividing the mean EB fluorescence of each sample bythat measured in nonconfluent wild-type HT-29 cells. SDs are mdicated (n = 3).

1.g@

I .6â€¢

1.3

0.7

0.4

menadione NC CC menadione control HSP27

2665

DISCUSSION

In this paper, we show that cell culture density modulates HSP27expression and provokes posttranslational modifications that mayaffect its activity. HSP27 accumulates as cells reach confluence.Notably, when cells are growth-arrested in G0-G, phase by serumstarvation rather than by growing at high density, HSP27 levels do notincrease. This suggests that+1SP27 accumulation by cell confluence isnot cell cycle-dependent. The effect of cell confluence seems to bespecific for HSP27, because we found no changes in the intracellularcontent of the other major HSPs (HSP6O, HSP7O, and HSP89).Therefore, cell culture density does not induce a general stress response. Interestingly, it is HSP27 that has been specifically implicatedin the resistance to anticancer drugs (3â€”5,35) and therefore couldparticipate in the higher resistance of colorectal cancer cells whengrown at high density.

We also show that high-density cultures are associated with increased content of HSP27 phosphoisoforms and with dramaticchanges in the intracellular localization of this protein. In exponentially growing human colorectal cancer cells, HSP27 is mainly dispersed in the soluble phase of the cytoplasm. As cultures reachconfluence, HSP27 accumulates around the nucleus. This phenomenon was abolished when lysis was performed in the presence of thenonionic detergent Triton X-l00. It can be concluded that in highdensity stationary phase cells, an important fraction of HSP27 isprobably interacting with the nuclear membrane. This particular behavior of HSP27 is not due to the fact that around 80% of cells in the

confluent population are in the G0-G1 phase of the cell cycle, becauseserum starvation provokes a similar recruitment of@ -phase cellsand does not alter HSP27 localization in these cells. Because HSP27is known to interact with the cytoskeleton (10, 33), one hypothesiscould be that the particular distribution of HSP27 is a consequence ofcytomembrane rearrangements that take place in high-density cultures.

Recent studies indicate that phosphorylation plays a key role in theregulation of HSP27 function and its contribution to survival afterstress (10, 33). It has been proposed that phosphorylation induces

conformational changes in HSP27, resulting in a lower binding effi

ciency of HSP27 for the barbed end of microfilaments and consequentstabilization of the cytoskeleton. However, other results point out therelative importance of HSP27 phosphorylation in the modulation of itsprotective function(s) (20). Therefore, although we show here that cellconfluence modifies HSP27 phosphorylation and its intracellular localization, the biological significance of these changes in HSP27function(s) remains to be determined.

To understand the role of HSP27 accumulation during the devel

MEAN FLUORESCENCE INDEX




opment of the resistance of confluent cells against drug-induced celldeath, we have used HSP27-transfected HT-29 cells. Overexpressionof HSP27 inhibits human colorectal cancer cell death induced byDXR (4) and CDDP, as we have shown. This effect was not due todifferences in the intracellular accumulation of CDDP, because, asalso found in human testis tumor cells (5), similar amounts of the drugwere taken up by HSP27-transfected and control-transfected cells.The increase in the resistance to CDDP and DXR of proliferativeHSP27-overexpressing cells is similar to that observed when control

cells are grown at a confluent state. In other words, we can mimic the

level of resistance of confluent cells just by increasing the HSP27level in proliferative cells. HSP27-overexpressing cells do not furtherincrease HSP27 content when reaching confluence. This result is notsurprising, because in many different HSP27-overexpressing cells, no

increased accumulation of HSP27 is detected even after heat shocktreatment.4 Remarkably, the absence of induction of HSP27 accumulation when these cells reach confluence is accompanied by the lackof an enhanced cell resistance. Together these data suggest that theprotection conferred by HSP27 is strongly involved in the resistancephenotype of confluent colon carcinoma cells.

We have previously shown that expression of HSP27 blocked theapoptotic process generated by several stimuli in murine L929 fibroblasts (31). We show here that HSP27 accumulation also reduces the

apoptosic process induced by CDDP in human colorectal cancer cells.

Hence, HSP27 may represent a pleotropic inhibitor of apoptosis indifferent cell lines.

Concerning the protective activity of HSP27, we have demonstrated

in murine L929 cells that HSP27 decreased the cellular content ofROS (12). The intensity of the phenomenon depended on the concentration of the expressed human HSP27. This is probably a conservedproperty of HSP27, because we show here that HSP27 also decreasesROS levels in a very different cellular model, human colorectal cancercells. The ability of HSP27 to modulate intracellular redox can lead to

protective mechanisms that may not be restricted to oxidative stressresistance and may explain a more general protective role of this

protein. Elevated levels of HSP27, by buffering cellular ROS, may

protect cellular structures that are damaged by stress and particularlyactin microfilaments (36, 37). HSP27 expression, by inducing a prore

duced state, may also modify the metabolism and induce changes incellular structures. Because there is a decrease in ROS levels as cellsreach confluence and because we have been able to mimic this ROSdecrease in proliferative cells by overexpressing HSP27, we suggestthat HSP27 could be a modulator of ROS levels in the development ofthe resistance of confluent cells. Hence, it is tempting to speculate thatin the course of cell confluence, HSP27 accumulation may represent

a major event establishing a proredox state that, in turn, may at leastpartially explain the confluence-induced resistance against CDDP and

DXR, two well-known ROS producers (34).Confluent cultures are better models than actively growing cells for

growth conditions in a solid tumor. Our data support the participationof HSP27 in the inherent resistance of confluent cells to chemotherapeutic drugs. It would now be interesting to test whether drugs that

modulate HSP27 expression and/or posttranslational modificationscould also be used to reduce the resistance of confluent cells toanticancer drugs.

ACKNOWLEDGMENTS

We thank M. F. Michel, D. Pinard, and D. Guillet for their technicalassistance, and E. Solary, F. Martin, and K. Nason-Burchenal for their valuable

advice.

4 P. Mehlen and A-P. Arrigo. unpublished observations.

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1997;57:2661-2667. Cancer Res Carmen Garrido, Patrick Ottavi, Annie Fromentin, et al. Cell Death Induced by Anticancer DrugsHSP27 as a Mediator of Confluence-dependent Resistance to

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