[CANCER RESEARCH 55, 3016-3021, July 15, 1995]

Enhanced Expression of Calreticulin in the Nucleus of Radioresistant SquamousCarcinoma Cells in Response to Ionizing Radiation1

Priya Ramsamooj,2 Vicente Notario, and Anatoly Dritschilo

Department of RuiìiaiìonMedicine, Georgetown University Medical Center, Washington, D.C. 20007

ABSTRACT

Ionizing radiation has been shown to modulate gene and protein expression as well as cellular signal transduction pathways. However, thebiochemical and molecular mechanisms that underlie the cellular response to radiation are not fully understood. The effects of ionizingradiation on the expression of nuclear proteins have now been investigatedin radioresistant human head and neck squamous carcinoma cells (SQ-20B cells) using the techniques of two-dimensional PAGE, silver staining,and computer-assisted quantitative analysis. Radiation (600 cGy) induced

the expression of 10 proteins and suppressed the expression of 5 proteinsin the nuclei of SQ-20B cells as detected 4 h after treatment. Electroelutionand NH2-terminal amino acid sequence analysis revealed that one of theradiation-induced proteins was the ( :i ' ' -hiiulin«protein calreticulin. The

expression of calreticulin was increased approximately 6-fold in the nucleiof irradiated SQ-20B cells. Calreticulin and the other proteins whose

expression was affected by radiation may contribute to the radioresistancephenotype of SQ-20B cells.

INTRODUCTION

Radiation therapy plays a significant role in the treatment of cancerpatients. Determination of the effects of radiation on tumor cells at thebiochemical and molecular levels is vital to our understanding thecellular radiation response. Several studies have shown that radiationmodulates the expression of specific genes. Thus, radiation increasesthe expression of various cytokine and growth factor genes includingthose for tumor necrosis factor (1, 2), interleukin-1 (3), platelet-

derived growth factor, and fibroblast growth factor (4). Radiation alsoincreases the expression of nuclear factor K B (5), Gadd45 (6), andEF-1S (7). Sherman et al. (8) have shown that ionizing radiationincreases the expression oic-jun, c-fos andjun-B at the transcriptionallevel, and Hallahan el al. (9-11) have demonstrated that ionizing

radiation increases the expression of the transcription factor EGRIand that the induction of both EGRI and c-jun expression is mediated

by protein kinase C. Finally, Wilson et al. (12) have shown thationizing radiation increases the expression of c-myc in primary human

B cells, whereas Boothman and colleagues (13, 14) have characterizedseveral X-ray-induced transcripts as well as transcription factors and

DNA binding sites that may mediate the induction of these transcripts.It is thought that expression of these various genes is related to thecellular capacity for surviving radiation injury. The effects of ionizingradiation on cellular signal transduction pathways have also beeninvestigated. Thus, radiation activates phospholipase D (15), stimulates tyrosine kinases in human B lymphocytes (16), and specificallyinduces the tyrosine phosphorylation of p34cdc2 (17).

Other investigations have focused on changes in protein expressionlevels induced by exposure of cells to radiation. Lambert and Borek(18) compared the effects of X-rays on normal and oncogene-trans-

formed rat cells and detected 14 proteins that were differentiallyexpressed between the normal and oncogene-transformed cells.

Received 7/14/94; accepted 5/10/95.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 with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by NIH Grant CA45408 to A. D.2 To whom requests for reprints should be addressed, at Georgetown University

Medical Center, TRB E-207, 3970 Reservoir Road North West. Washington, D.C. 20007.

Boothman et al. (19) showed that exposure of human melanoma cellsto X-rays induced the expression of several specific polypeptides. Inaddition, a DNA-binding protein that is activated as a result ofexposure of lymphoblastoid cells to y-radiation has been character

ized (20) and purified (21). Although these studies have detectedseveral proteins whose expression is modulated by radiation, the rolesof these proteins in the cellular response have not been elucidated.

We have applied the powerful combination of two-dimensional

PAGE, silver staining, and quantitative computer analysis to characterizechanges in the expression of specific nuclear proteins in response toionizing radiation. We have studied human head and neck squamouscarcinoma cell line SQ-20B, which has been extensively characterized asrelatively radioresistant (22-24), to focus on those proteins that may

contribute to the phenotypic expression of radioresistance.

MATERIALS AND METHODS

Cell Culture, Radiation Treatment, and Nuclei Preparation. The headand neck squamous carcinoma cell line SQ-20B was established and characterized as described previously (22-24). Radiation dose-response analysis(radiation survival curves) showed a D„value of 2.4 Gy for SO-20B cells, and

clinical correlations were made on the basis of the response of the tumors fromwhich SQ-20B cells were derived to radiation therapy (recurrence or radio-

curability; Refs. 23 and 24). Both criteria indicated a relatively radioresistantphenotype of SQ-20B cells.

Cells were maintained in culture medium consisting of DMEM, 10% fetalbovine serum, 2 mM glutamine, 2 HIMpenicillin-streptomycin, and 0.4 fig/mlhydrocortisone and were routinely passaged at 70-80% confluency (approximately 3-4 days after subculturing). Cells were grown to confluency (asjudged by light microscopy, approximately 5-7 days after subculturing), andthe following morning irradiated (600 cGy) with a 6"Co irradiator (Theratron-

80). Control cells were not exposed to radiation. Cells were harvested 4 h afterirradiation by washing with PBS, trypsinization (0.05% trypsin, 0.53 mMEDTA; GIBCO-BRL), and centrifugation at 2500 X g. Nuclei were then

prepared from cell pellets as previously described (25) and frozen. Proteinconcentration was determined with the Pierce BCA protein assay system.

FACS.3 Control and irradiated SQ-20B cells were harvested in duplicate0-25 h after irradiation, and 1 X IO6 cells were resuspended in FACS buffer

[250 mM sucrose, 40 mM trisodium citrate (pH 7.6), 5% DMSO). Staining ofcells with propidium iodide (26) and DNA histogram analyses were performedin the FACS Core Facility of the Lombardi Cancer Research Center. FACSanalysis revealed the percentage of cells in various stages of the cell cycle.

2D-PAGE. Nuclei from control and irradiated SQ-20B cells were analyzedusing 2D-PAGE (27) with the ISO-DALT 2D-PAGE System (Large Scale

Biologicals, Inc.). The procedure represented a slightly modified version ofthat of Anderson and Anderson (28) as described previously (22). Frozennuclei were thawed and solubilized in a solution containing 9 M urea, 4%NP40, 2% ampholines (pH 8-10), and 1% DTT. Approximately 50 ju,gprotein

were applied to ID tube gels (25.5 cm) containing 3.5% polyacrylamide, 9 Murea, 2% ampholines (one part pH 3.5-10, three parts pH 4- 8), and 2% NP40.

The gels were prefocused at 200 V for 2 h with 0.085% phosphoric acid in thelower chamber and 0.02 N NaOH in the upper chamber. Molecular mass andpi standards (Sigma) were included in at least one of the tube gels in eachbatch. Isoelectric focusing was performed at 1200 V for approximately 20 h.The ISO-DALT 2D-PAGE System allows the simultaneous running of sam

ples of 20 tube gels (and subsequently 20 second-dimension slab gels), thus

•*The abbreviations used are; FACS, fluorescence-activated cell sorting; 2D-PAGE,

two-dimensional PAGE; pi, isoelectric point.

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RADIATION AND CALRETICULIN EXPRESSION

allowing the advantage of reproducibility. After isoelectric focusing, the tubegels were cut (1.85 cm from the acidic end, 5.2 cm from the basic end) andlayered on slab gels (17.8 x 17.8 cm, 10% polyacrylamide). Electrophoresiswas performed at 80 V for 20 to 22 h with constant cooling (RGB 300 coolingwater bath; Hoefer Scientific). The gels were then placed in fixative (40%methanol, 10% acetic acid) for at least 2 h.

Silver Staining. Fixed 2D-PAGE gels were subjected to silver staining

as previously described (22, 29). Briefly, gels were equilibrated for 30 minin 10% ethanol and 5% acetic acid (two changes of solution), washed withMilli-Q water (Millipore filtration unit. Model CDOFO 1205), incubatedwith DTT (15 jag/ml) for 30 min, washed three times in Milli-Q water, and

then exposed to 0.1% silver nitrate for 30 min. The gels were rinsed withMilli-Q water and developed with 3% sodium carbonate and 0.02% form

aldehyde for 5 to 15 min. Development was terminated by placing the gelsin 5% acetic acid.

Computerized Quantitative Analysis. Analysis of the protein profilesobtained using 2D-PAGE was performed by densitometric scanning of thesilver-stained gels, digitization of the image, computer-assisted comparison of

protein profiles, quantitation of protein differences, and determination of themolecular masses and pi values of the proteins of interest. These procedureswere implemented with a Sun Microsystems 3/110 computer interfaced to aPhotometries scanner and digitizer (PS200 power supply, 1035 x 1320 assay).The gels were scanned with a Nikon f2.8 macro lens, and the output wasdigitized in such a manner that the background noise (attributable to variationsin the light box used to illuminate the image) was subtracted from the proteinspot signal, which was then assigned a value of 0 (white) to 250 (black). Thedata were stored on the main computer, and comparative analysis of the proteinprofiles was performed with the ELSIE-5 software program, an updated

version of a program previously described by Olson and Miller (30). Thenuclear protein patterns of control and irradiated SQ-20B cells were compared,

and the quantitative differences were estimated by normalization of the spotvalues to that of the internal standard actin (43 kDa, pi 5.3). The molecularmass and pi values were determined with the ELSIE-5 program on the basis of

internal protein standards in combination with the additional molecular massand pi standards included in each 2D-PAGE experiment.

Microsequencing. Two proteins shown by 2D-PAGE analysis to be induced by radiation treatment were purifed for NH2-terminal sequencing analysis. Nuclear proteins prepared from irradiated SQ-20B cells were separatedusing 2D-PAGE, and the resultant gels stained by colloidal Coomassie blueG-250 which, unlike silver staining, does not interfere with the subsequent

microsequencing (31, 32). The two proteins of interest were excised from thegels and electroeluted with an Amicon Centrilutor. The electroeluted proteinswere subjected to SDS-PAGE (33) and then transferred overnight to a poly-vinylidene difluoride membrane (Millipore) in Tris-glycine-methanol bufferwith a Bio-Rad Transblot Cell at 30 V and 4°C(34). The membrane was

stained with 0.1% Commassie blue R-250 in 50% methanol and destained with

50% methanol and 0.1% acetic acid, and the bands of interest were excised andsubjected to microsequencing by Dr. Jane Walent at the University ofWisconsin Biotechnology Center.

RNA Isolation and Northern Blot Analysis. Total RNA from control andirradiated (600 cGy) SQ-20B cells was isolated with the RNAzol B method(Tel-Test, Inc.). Briefly, cells were lysed with RNAzol B reagent, and the RNAwas extracted by addition of chloroform and centrifugation (12,000 X g, 4°C).The RNA was isolated by precipitation (isopropanol for 15 min at 4°C),centrifugation (12,000 X g, 4°C),and ethanol washing. Twenty ;n.gRNA from

each sample were electrophoresed on 1.4% agarose gels under alkaline conditions (18% formaldehyde) and transferred to nitrocellulose. After baking at80°Cunder vacuum for 2 h, the blot was hybridized at 42°Cin 50% formam-ide, 5X SSC, IX Denhardt's solution, 50 mM Na,HPO4 (pH 6.5), 1% glycine,250 fig/ml tRNA, and 32P-labeled cDNA probe. The calreticulin cDNA probe

(1.8 kb) was isolated from a pBluescript plasmid containing the calreticulininsert, which was kindly provided by Dr. Marek Michalak. The glyceralde-hyde-3-phosphate dehydrogenase cDNA probe (1.4 kb; Ambion, Inc.) wasused as a loading control. Both cDNA probes were labeled with [a-32P]dCTP

(New England Nuclear) using a megaprime end-labeling kit (Amersham).

Hybridized blots were washed sequentially with 2X SSC and 0.1% SDS, 0.1XSSC, 0.1% SDS, and 0.1X SSC and then autoradiographed (10 days for thecalreticulin cDNA probe, 2 days for the glyceraldehyde-3-phosphate dehydro

genase cDNA probe).

RESULTS

FACS Analysis of Confluent Cells. To investigate possible differences in cell cycle distribution in confluent cultures of controlversus irradiated SQ-20B cells, we performed FACS analysis 0-25 hafter irradiation. The percentage of cells in G0-G1; S-phase, and G2-M

did not differ significantly between control (Fig. 1A) and irradiated(Fig. Iß)SQ-20B cells. Thus, the use of confluent cells in this study

avoided complications due to cell cycle redistribution (for example,G2 arrest) in the interpretation of results.

Radiation-induced Changes in Protein Expression. We havecompared the 2D-PAGE nuclear protein profiles of control (Fig. 2A)

100

8tH

UJ(0

Q.

Ill

U

u

UJU

111<0<XQ_

UJ

uu

u

60-

40 -

20-

10 15 20 25

TIME (HOURS)

B100

~ 80-

60 -

40 -

20 -

10 15 20 25

TIME (HOURS)

Fig. 1. Effect of irradiation on Ihe cell cycle distribution of confluent SO-20B cells.The percentage of cells in various stages of the cell cycle for control (A) and irradiated (B)SQ-20B cells was delermined by FACS analysis at various times after irradiation (600cGy). Differences in the percentages of cells in Gn-G, (A), S-phase (•),and G2-M (•)between control and irradiated samples were not statistically significant (P ^ 0.05,Student's / test; Ref. 35). Values are means of two independent determinations.

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RADIATION AND CALRETICULIN EXPRESSION

lt ^.10

0

r •6 a—. 8 6 8

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•b/ v°Fig. 2. 2D-PAGE protein profiles of nuclei from control (A) and irradiated (B) SQ-20B cells. Nuclei were prepared from confluent control and irradiated (600 cGy) cells 4 h after

irradiation, and 50 fig nuclear protein were applied to 2D-PAGE gels. The gels were silver stained, and the protein profiles compared by quantitative computer analysis. Ten proteinswere found to be induced with radiation (proteins 1-10), and five were found to be suppressed with radiation treatment (proteins a-e). Left, acidic proteins; right, basic proteins. Large

arrows, actin (43 kDa; pi 5.3).

and irradiated (Fig. 2ß)SQ-20B cells in order to focus on those

proteins that may play a role in the regulation of gene expression orin DNA damage and repair. The 2D-PAGE system used has the

advantage of allowing the simultaneous analysis of up to 20 samples,with minimal variation in the protein profiles as detected by silverstaining. To ensure that any observed differences in protein expressionwere due to radiation treatment, we analyzed control and irradiatedsamples on triplicate 2D-PAGE gels. The degree of induction or

suppression (fold change) of protein expression by irradiation wascalculated by normalization of the computer-derived spot value (obtained by computer digitization of the silver-stained gel) for each

protein to that of the internal standard actin (43 kDa, pi 5.3) from thesame gel. The mean value for the actin spot was 131 ±28 arbitraryunits for each 2D-PAGE gel in the analysis. The resulting normalized

spot/actin value was then used to determine the fold change between

control and irradiated samples. This 2D-PAGE analysis system reli

ably assesses proteins within the molecular mass range from 15 to 150kDa and the pi range between 4 and 1.

We detected 10 proteins (molecular mass, 24-108 kDa; pi,4.3-6.5) whose expression was increased 1.4-10-fold by ionizingradiation (proteins 1-10) and five proteins (molecular mass, 23-29kDa; pi, 4.8-5.7) whose expression was decreased 2.3-55-fold(proteins a-e, Table 1).

Purification and Microsequencing of Radiation-induced Proteins. To identify the radiation-induced proteins detected by 20-PAGE, we applied the techniques of electroelution and NH2-terminal

amino acid sequence analysis. We initially chose proteins 1 and 2(Fig. 2 and Table 1) for further analysis for two reasons: (a) both werestainable with Coomassie blue and present in sufficient quantities forthe subsequent manipulations, and (b) both migrated to positions in

Table 1 Proteins differentially expressed in control versus irradiated SQ-20B cells

Spot/actin value"

Spot Control 600 cGy Fold change Size (kDa) PiRadiation-inducedproteins12345678y10Radiation-suppressedabCde0.1

00±0.0300.200±0.0300.020±0.0020.060±0.0050.040±0.0070.010

±0.0060.050±0.0020.007±0.0010.050±0.0030.010+0.001proteins0.300

+0.0100.600+0.0300.200

±0.0060.700+0.0220.400±0.0100.400

±0.0601.100±0.0200.200

+0.0500.150±0.0200.200

±0.0600.050±0.0020.200±0.0030.010±0.0080.150+0.0040.040+0.0100.100

+0.0500.011+0.0030.011

±0.0030.300±0.0060.1

10 ±0.00746102.55541.434360202.54100634445603055247210826232925244.94.36.06.55.34.56.25.25.55.34.94.85.45.65.7" Values are means of triplicate samples, each normalized against actin. The differences between control and irradiated samples were statistically significant (P •&0.05, Student's

/ test; Ref. 35).

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RADIATION AND CALRETICULIN EXPRESSION

Fig. 3. SDS-PAGE gels of purified proteins 1 and 2. Proteins 1 and2 were excised and electroeluted from 2D-PAGE gels, subjected toSDS-PAGE, transblotted, and stained with Coomassie blue R-250.

Molecular weight markers, kDa. Arrows, bands corresponding toprotein 1 (apparent molecular mass, 100 kDa, A) and protein 2(apparent molecular mass, 63 kDa, B).

B

C'5•*-•o4-

a

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-200

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-97.4

-69

-46

paVYFKEQFLDgd

MLLSVPLLLGLLGLAVAEPAVYFKEQFLDGDGWTSRWIES

10 20 30

Fig. 4. Comparison of the amino acid sequence of protein 2 with that of calreticuiin.The upper 13 amino acids were detected by HPLC analysis of the residues released fromprotein 2 by automated Edman degradation (uppercase tellers, amino acids that werereadily assigned; lowercase letters, amino acids that were more difficult to assign). Asearch of the Swiss Protein Sequence Data Bank with the nine most reliable amino acids(uppercase tetters) resulted in a 100% match with amino acids 21-29 of the Ca2+-binding

protein calreticulin (lower sequence; Ref. 36).

the 2D-PAGE gels that were relatively unpopulated, thus increasingthe chances that we would excise a single protein for microsequenc-ing. It was necessary to prepare 20-50 2D-PAGE gels to accumulate

sufficient protein for subsequent manipulations. Bands containingelectroeluted proteins 1 and 2 were excised from Coomassieblue-stained polyvinylidene difluoride membranes (Fig. 3) andsubjected to NH2-terminal amino acid sequencing by automated

Edman degradation.The NH2 terminus of protein 1 was blocked, but we were able to

obtain amino acid sequence data for protein 2. We used the nine mostreadily identified amino acids (Fig. 4) to probe the Swiss ProteinSequence Data Bank. This search resulted in a 100% match of all nineamino acids with the protein calreticuiin. The 13 amino acids detectedby automated Edman degradation of protein 2 correspond to aminoacids 19—31of calreticuiin predicted from the cDNA sequence data

(36). Because calreticuiin is an endoplasmic reticulum membraneprotein, the NH2-terminal 18-amino acids, which constitute the signal

peptide, would have been cleaved to yield the mature protein and,therefore, undetectable by Edman degradation. Calreticuiin has anapparent molecular mass of 60-63 kDa in SDS-PAGE gels (Laemmlisystem) and is highly acidic (pi 4.14-4.67; Ref. 36). These values are

in good agreement with the corresponding values estimated from our2D-PAGE gels (Table 1).

Northern Blot Analysis. To determine whether the radiation-in

duced increase in calreticuiin protein expression was due to a corresponding increase in calreticuiin mRNA expression, we performed

Northern blot analysis on control and irradiated samples of SQ-20B

RNA (Fig. 5). The calreticuiin probe detected a band of 1.8 kb inSQ-20B cells (Fig. 5, top panel). The level of calreticulin mRNAexpression in SQ-20B cells was not increased up to 8 h after exposureof 600 cGy ionizing radiation. Rehybridization of the blot with glyc-eraldehyde-3-phosphate dehydrogenase was used as a loading control

(Fig. 5, bottom panel). These results confirmed that the observedincrease in calreticulin in the nucleus of SQ-20B cells was not the

result of increased levels of RNA transcripts.

DISCUSSION

The purpose of this study was to examine the changes in expressionof nuclear proteins induced by exposure of radioresistant cells toionizing radiation. Our goal was to characterize and identify suchproteins through 2D-PAGE analysis, computer-assisted quantitationof silver-stained gels, protein purification, and NH2-terminal sequenc

ing. We chose silver staining (29) rather than isotopie radiolabelingand autoradiography to detect proteins of interest on the basis of ourprevious studies showing that 2D-PAGE analysis of isotopically la

beled cellular extracts results in many artifacts that do not reflectchanges in protein expression (22). Moreoever, the proteins of interesthad to be stainable with Coomassie blue to ensure their expression insufficient (p,g) quantities for purification and automated Edmandegradation (32).

control 600 cGy

048 048

-GAPDH

Fig. 5. Time course of calreticuiin (CRT) mRNA expression in control and irradiated SO-20B cells. A Northern blot containing 20 M-g/lane total RNA was hybridizedto a 32P-labeled calreticulin cDNA probe (top panel). The level of expression of

calreticulin mRNA (1.8 kb) did not increase with radiation of 600 cGy as judged bycomparison of the control (¡eftlanes) and irradiated (right lanes) samples at 0, 4 and8 h after radiation exposure. As a loading control, the same blot was hybridized to a32P-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1.4 kb) cDNA

probe (bottom panel).

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RADIATION AND CALRETICULIN EXPRESSION

We examined confluent cells to avoid the effect of radiation-

induced cell cycle redistribution which may have impaired ourability to determine which proteins are differentially expressed incontrol versus irradiated cells as a direct consequence of radiation.FACS analysis confirmed that the percentage of cells in G()-G,,S-phase, and G2-M was not significantly altered by radiation

treatment. Furthermore, immunoblot experiments with antibodiesspecific for cell cycle-related proteins (cyclin A, cyclin Bl, and

proliferating cell nuclear antigen) revealed no differences in protein expression between control and irradiated SQ-20B cells (datanot shown). Therefore, it is unlikely that the radiation-inducedchanges in protein expression observed by 2D-PAGE analysis are

simply a consequence of cell cycle redistribution.We have shown that irradiation of SQ-20B cells increased the

expression of 10 proteins and decreased the expression of 5 proteins.The expression of these 15 proteins did not appear to be affected byirradiation in a radiosensitive cell line SQ-38 (D0 170 cGy; data not

shown). Since the analysis of a large population of radioresistant andradiosensitive cell lines necessary to statistically support this conclusion is impractical, we can only speculate that these radiation-induced

proteins play a role in expression of the radioresistance phenotype.We definitively identified one of the 10 radiation-induced proteins

in SQ-20B cells as calreticulin. The molecular mass (63 kDa) and pi(4.3) estimates obtained from our 2D-PAGE gels are consistent with

those published for calreticulin (reviewed in réf.36). Calreticulin is aCa2+-binding protein that was first described in the sarcoplasmic

reticulum of skeletal muscle cells. More recently, it has been identified in several nonmuscle cells and referred to by various names,including calregulin, reticuloplasmin, CRP55, CaBP3, and ERp60.Calreticulin is thought to be localized to the rough endoplasmicreticulum (36). However, Krause et al. (37) and Van Delden et al. (38)have provided evidence for the presence of calreticulin in specializedCa2+ storage and release vesicles, termed "calciosomes" (39), in

HL-60 cells. In addition, Opas et al. (40) have shown that calreticulin

may also be present in the nucleus and nuclear envelope, and, in fact,contains a putative nuclear targeting signal in the primary amino acidsequence (PPKIKDPD, residues 188-195; Ref. 36). Its primary

amino acid sequence also reveals that calreticulin is a member of theKDEL (endoplasmic reticulum retention sequence) family of proteins,which includes glucose-regulated protein 94, BiP (glucose-regulated

protein 78), and protein disulfide isomerase (41). We suggest that theapproximately 6-fold increase in calreticulin expression observed inthe nucleus of SQ-20B cells after exposure to ionizing radiation may

be attributable to translocation of this protein from the endoplasmicreticulum (or calciosomes) to the nucleus. Proteins of the KDELfamily have a high degree of sequence homology to the stress-induced

proteins heat shock protein 70 and heat shock protein 90/83. Furthermore, under stress conditions, both BiP and glucose-regulated protein

94 have been detected in the nucleus in addition to the endoplasmicreticulum (41). Relocalization of calreticulin to the nucleus couldtherefore represent a stress response of SQ-20B cells to ionizing

radiation.Calreticulin expression is increased in tumor cells and proliferating

cells although its function is unknown (36). Perhaps the observedincrease in calreticulin expression after radiation treatment was aconsequence of increased proliferation. Although we cannot rule outthis possibility, it seems unlikely that the increase in calreticulin in thenucleus was solely a consequence of increased cellular proliferation,because the cells were confluent before radiation treatment and thecell cycle distribution was essentially unchanged after radiation treatment (Fig. 1). In addition, cycloheximide treatment did not alter theobserved radiation-induced increase in calreticulin in the nucleus ofSQ-20B cells (data not shown). This suggests that calreticulin increase

in the nucleus of SQ-20B cells after radiation treatment is not the

result of increased protein expression.Radiation-induced changes in the expression of a Ca2+-binding

protein have not been previously described. Given that (a) calreticulincopurifies with an inositol 1,4,5-triphosphate-sensitive intracellularCa2+ storage compartment in HL-60 cells (37, 38), (b) diacylglycerol

resulting from inositol 1,4,5-triphosphate mobilization activates pro

tein kinase C (15), (c) protein kinase C is activated in response toradiation (11), and (d) radiation or Ca2"1"mobilization stimulates

phospholipase D (15), we speculate that calreticulin plays a role in thesignal transduction pathway that mediates the cellular response toradiation. Thus, the radiation-induced generation of free radicals and

consequent oxidation of membrane phospholipids may promotephosphoinositol turnover, resulting in calreticulin-mediated Ca2+ mobilization, translocation of this Ca2+-binding protein to the nucleus, and

protein kinase C activation. Since the cellular function of calreticulin isunknown, further studies are necessary to investigate this hypothesis.

ACKNOWLEDGMENTS

All 2D-PAGE experiments and computer-assisted quantitative analysiswere performed in the 2D-PAGE Core Facility of the Lombardi Cancer

Research Center at Georgetown University. Microsequencing was performedat the University of Wisconsin Biotechnology Center by Dr. Jane Walent. Wealso thank Dr. Marek Michalak for providing the pBluescript-CRT plasmid and

for many helpful discussions on calreticulin and radiation response.

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2. Hallahan, D. E., Spriggs, D. R., Beckett, M. A., Kufe, D. W., and Wcichselbaum,R. R. Increased tumor necrosis factor a mRNA after cellular exposure to ionizingradiation. Proc. Nati. Acad. Sci. USA, 86: 10104- 10107. 1989.

3. Woloschak, G. E., Chang-Liu, C-M., Jones, P. S., and Jones, C. A. Modulation of

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4. Witte, L., Fuks. Z., Haimonovitz-Freidman, A., Vlodavsky. !.. Goodman, D. S.. andEldor, A. Effects of irradiation on the release of growth factors from cultured bovine,porcine, and human endothelial cells. Cancer Res., 49: 5066-5072, 1989.

5. Brach. M. A., Hass, R., Sherman. M. L.. Gunji. H.. Weichselbaum, R., and Kufe, D.Ionizing radiation induces expression and binding activity of the nuclear factor KB.J. Clin. Invest., 88: 691-695, 1991.

6. Papathanasiou, M. A., Kerr, N. C., Robbins, J. H., McBride, O. W., Alamo, I. J.,Barrett, S. F., Hickson, I. D., and Fornace. A. J., Jr. Induction by ionizing radiationof the gadd45 gene in cultured human cells: lack of mediation by protein kinase C.Mol. Cell. Biol., //.- 1009-1016, 1991.

7. Jung. M., Kondratyev, A. D., and Dritschilo. A. Elongation factor 16 is enhancedfollowing exposure to ionizing radiation. Cancer Res., 54: 2541-2543, 1994.

8. Sherman. M. L., Datta, R., Hallahan, D. E., Weichselbaum. R. R., and Kufe, D. E.Ionizing radiation regulates expression of the c-jun protooncogene. Proc. Nati. Acad.Sci. USA, 87: 5663-5666, 1990.

9. Hallahan, D. E., Virudachalam, S.. Beckett, M. S., Sherman, M. L., Kufe. D., andWeichselbaum. R. R. Mechanisms of X-ray-mediated protooncogene c-jun expression in radiation-induced human sarcoma cell lines. Int. J. Radiât.Oncol. Biol. Phys.,21: 1677-1681. 1991.

10. Hallahan. D. E., Sukhatme, V. P., Sherman, M. L., Virudachalam, S., Kufe, D., andWeichselbaum, R. R. Protein kinase C mediates x-ray inducibility of nuclear signaltransducers EGRI and JUN. Proc. Nati. Acad. Sci. USA. 88: 2156-2160, 1991.

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1995;55:3016-3021. Cancer Res   Priya Ramsamooj, Vicente Notario and Anatoly Dritschilo  Ionizing RadiationRadioresistant Squamous Carcinoma Cells in Response to Enhanced Expression of Calreticulin in the Nucleus of

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