aberrant expression of photoreceptor-specific calcium-binding … · gatt-39and antisense primer...

[CANCER RESEARCH 60, 1914–1920, April 1, 2000]

Aberrant Expression of Photoreceptor-specific Calcium-binding Protein (Recoverin)in Cancer Cell Lines1

Akiko Maeda,2 Hiroshi Ohguro, Tadao Maeda, Ikuo Wada, Noriyuki Sato, Yoshio Kuroki, and Takashi NakagawaDepartments of Ophthalmology [A. M., H. O., T. M., T. N.], Biochemistry (Section 2) [I. W.], Pathology (Section 1) [N. S.], and Biochemistry (Section 1) [Y. K.], Sapporo MedicalUniversity School of Medicine, Sapporo 060-8543, Japan

ABSTRACT

Cancer-associated retinopathy (CAR) is an ocular manifestation of aparaneoplastic syndrome whereby immunological reactions to retinal an-tigens aberrantly expressed in tumor cells lead to the degeneration ofretinal photoreceptor cells. In our previous study (H. Ohguroet al., Invest.Ophthalmol. Vis. Sci.,40: 82–89, 1999), recoverin, a retina-specific calci-um-binding protein, and heat shock cognate protein 70 (hsc 70) wereidentified as autoantigens recognized by sera from patients with CAR.Therefore, we suggested that autoimmune reactions against both recov-erin and hsc 70 might be involved in the pathogenesis of CAR. Toelucidate the initial step of the molecular pathology of CAR, we examinedthe expression of recoverin and hsc 70 by reverse transcription-PCR andWestern blot using cell lines of several kinds of cancers, including lungsmall cell carcinoma, lung adenocarcinoma, gastric cancer, pancreaticcancer, breast cancer, uterine cervical cancer, endometrial cancer, andleukemia. Recoverin was expressed in 21 of the 31 cancer cell lines. Theexpression levels of hsc 70 were significantly higher in cancer cell linesthan in noncancerous cell lines. However, no difference in the expressionlevels of hsc 70 was observed between recoverin-positive and -negative celllines. Immunofluorescence labeling by the affinity-purified recoverin an-tibody revealed the immunoreactivity to recoverin as a granular patternwithin the cancer cells. Lung adenocarcinoma A549 cells, which did notexpress recoverin, exhibited a significant reduction in cell proliferationupon transfection with human recoverin cDNA. Taken together, ourpresent data suggest that the retina-specific calcium-binding protein re-coverin is expressed in more than 50% of a variety of cancer cells and mayplay a significant role in the cell proliferation of these tumor cells.

INTRODUCTION

A variety of neurological disorders called paraneoplastic syn-dromes are known to be associated with malignant tumors, althoughthe tumor or its metastases have not invaded the nervous system. Thisso called “remote effect” of cancer is considered to be mediated on anautoimmune basis. That is, the expression of a tumor antigen presum-ably triggers immunological responses which in turn recognize thesame antigen or shared epitope in the nervous system, resulting inneuronal cell damage. In the peripheral nervous system, Lambert-Eaton myasthenic syndrome is known to be associated with autoan-tibodies to the calcium channel of the neuromuscular junction, whichinterfere with the release of acetylcholine and cause proximal muscleweakness (1). In the central nervous system, paraneoplastic cerebellardegeneration has been identified to be caused by autoantibodiesagainst Purkinje cell antigen (called Yo antigen) detected in someindividuals with gynecological tumors (2). CAR3 has been identified

as a paraneoplastic syndrome of the visual system (3–5). CAR isfound in patients with small cell carcinoma of lung and other malig-nant tumors and is clinically characterized by photopsia, progressivevisual loss with a ring scotoma, attenuated retinal arterioles, andabnormalities of the a- and b-waves of electroretinogram. Histopa-thology revealed that loss of photoreceptor cells occurs primarily inthe retinas of CAR patients (5, 6). It was found that CAR is caused byan autoimmune reaction against a photoreceptor-specificMr 23,000calcium-binding protein called recoverin (7, 8). Functionally, recov-erin was found to play a major role in light and dark adaptation byregulating rhodopsin phosphorylation and dephosphorylation in acalcium-dependent manner (9, 10). In terms of the generation ofautoantibody to recoverin, it was identified that recoverin is aberrantlyexpressed in the cancer cells or cell lines obtained from CAR patients,and this may trigger the autoimmune reaction (11–13). However,preliminary studies have revealed that such aberrant expression ofretinal-specific recoverin is not seen in cancer cells without retinop-athy. These observations suggested that aberrant expression of recov-erin in cancer cells is an initial and critical step in the cause ofretinopathy. We still do not know the molecular mechanisms thatcause the aberrant expression of recoverin in cancer cells. This knowl-edge is a key to understanding the molecular pathology of CAR andto designing an effective treatment for retinopathy.

In addition to recoverin, other retinal antigens includingMr 65,000protein, enolase (Mr 46,000 protein), and neurofilament (Mr 58,000–62,000,Mr 145,000, andMr 205,000 proteins) are also recognized bythe sera of some CAR patients (14–27). Among these retinal autoan-tigens, recoverin alone or a combination of recoverin andMr 65,000protein has most frequently been reported as the immunoreactive bandby Western blot analysis. We have recently identified theMr 65,000protein as hsc 70 and have suggested that both anti-recoverin andanti-hsc 70 antibodies are involved in the pathogenesis of CAR (28).These observations allowed us to speculate that hsc 70 may beinvolved in the aberrant expression of recoverin.

In the present study, to test our hypothesis, we examined mRNAexpression of recoverin and hsc 70 in several kinds of cancer cell linesand found that more than 50% of them expressed recoverin.

MATERIALS AND METHODS

Cell Lines. The 33 cell lines used in this study are summarized in Table 1.The SSTW-2, HST-2, HMC-1, HMC-2, HC-MA, OSC40, OSC70, LHK-2,and transformed cells were provided by our laboratory, lung cancer cell lineswere provided by Dr. M. Hirasawa (Department of Internal Medicine, Section3, Sapporo Medical University School of Medicine, Sapporo, Japan), cervicaland endometrial cancer cell lines were provided by Dr. M. Koizumi (Depart-ment of Gynecology, Sapporo Medical University School of Medicine), andC1R cells provided by Dr. M. Takiguchi (Kumamoto University, Kumamoto,Japan) were basically established as described previously (29, 30). None of thedonors of the tumor cells described above had episodes of visual symptoms ofCAR. The other cell lines were either provided by the Japanese Collection ofResearch Bioresources (Tokyo, Japan) or purchased from American TypeCulture Collection. These cell lines were maintained in RPMI 1640 containing10% fetal bovine serum and antibiotics.

Normal Tissues and Neonatal Thymuses.Normal tissues (stomach, smallintestine, colon, spleen, liver, lung, kidney, prostate, pancreas, and heart) and

Received 9/7/99; accepted 2/3/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from the Japanese Ministry of Health, Naito Memorial Foun-dation, Ciba-Geigy Foundation for the Promotion of Science, The Mochida MemorialFoundation for Medical and Pharmaceutical Research, Uehara Memorial Foundation, andJapanese Retinitis Pigmentosa Society Research Foundation.

2 To whom requests for reprints should be addressed, at Department of Ophthalmol-ogy, Sapporo Medical School of Medicine, S-1 W-16, Chuo-ku, Sapporo 060-8543,Japan. Fax: 81-11-613-6575; E-mail [email protected].

3 The abbreviations used are: CAR, cancer-associated retinopathy; hsc 70, heat shockcognate protein 70; RT-PCR, reverse transcription-PCR; GFP, green fluorescent protein.

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six newborn thymuses were generously donated by Drs. M. Imamura and S.Yokoyama of the Hokkaido Children’s Hospital and Medical Center (Hok-kaido, Japan) who gave consent for participation after the study procedures,which are in accordance with the tenets of the Declaration of Helsinki, wereexplained to them.

Antibodies. Anti-bovine recoverin rabbit IgG was prepared using a proteinG-Sepharose column chromatograph (Pharmacia Biotech, Uppsala, Sweden)according to the method described previously (31). The purity and proteincontents were determined by SDS-PAGE and spectrophotometry, respectively.

Western Blot. Western blot analysis was carried out as described previ-ously (32). Briefly, the protein fraction isolated by ISOGEN reagent accordingto the manufacturer’s procedure (Nippon Gene, Tokyo, Japan) was analyzedby SDS-PAGE using a 12.5% polyacrylamide gel. Separated proteins in a gelwere electrotransferred to polyvinylidene difluoride membranes in 10 mM

bis-Tris phosphate buffer (pH 8.4) containing 10% methanol. After blockingwith 5% skim milk in PBS, the membrane was probed successively withanti-recoverin antibody and horseradish peroxidase-labeled anti-rabbit IgG(Funakoshi Co., Tokyo, Japan). Immunoreactive bands were visualized by anenhanced chemiluminescence system (Amersham Pharmacia Biotech, Buck-inghamshire, United Kingdom) according to the method described by themanufacturer.

RT-PCR Analysis. Total RNA from cell lines was isolated using ISOGENreagent according to the procedure described by the manufacturer (NipponGene) and reverse-transcribed by using Superscript II with oligo(dT) primer(Life Technologies, Inc., Rockville, MD). The incubation was carried out at42°C for 50 min and then at 70°C for 15 min. The PCR amplifications wereperformed using 4.4ml for recoverin or 2.2ml for hsc)(70 andb-actin from theRT reaction mixture in 50ml of PCR mixture containing 50 pmol of sense andantisense primers. After the initial incubation at 94°C for 4 min, 30 cycles ofamplification were conducted with denaturation at 94°C for 1 min, annealing at55°C for 1 min, and extension at 72°C for 2 min. The following primer pairs wereused for RT-PCR analysis: (a) sense primer 59-TGTGTTCCGCAGCTTC-GATT-39 and antisense primer 59-TGAGGCTCAACTAACTGGATCAG-39 for

Fig. 1. Expression of mRNA for recoverin in various tumor cell lines. Twomg of RNAfrom 31 tumor cell lines and from 2 EBV-transformed B lymphocytes (controls) werereverse-transcribed to generate cDNA pools, and then 4.4ml from a 22-ml cDNA poolwere used for PCR, using specific primers as described in “Materials and Methods.” PCRproducts were evaluated by agarose gel electrophoresis and ethidium bromide staining.

Fig. 2. Western blot analysis for recoverin. Whole cell lysates containing approxi-mately 20mg of proteins were loaded on SDS-PAGE gel, followed by electrotransfer toa polyvinylidene difluoride membrane. Western blot analysis was performed using theaffinity-purified anti-recoverin polyclonal antibody (1:400 dilution). The details of theWestern blot are described in “Materials and Methods.” Western blots of selectedrecoverin-positive and -negative cell lines by PCR are shown in thetopandbottom panels,respectively. The results of Western blot analysis of the other tumor cells are summarizedin Table 1.

Table 1 Summary of recoverin expression in various cancer cell lines

Origin Cell line Histology

Recoverin expression

RT-PCR Western blot

Lung SBC-1 Small cell carcinoma 1 1SBC-2 Small cell carcinoma 2 2SBC-5 Small cell carcinoma 2 2LU-134-A-H Small cell carcinoma 2 2A549 Adenocarcinoma 2 2LC-81 Adenocarcinoma 2 2LC-133 Adenocarcinoma 6 2LC-142 Adenocarcinoma 6 2LHK-2 Adenocarcinoma 1 1

Cervix HCA-1 Undifferentiated 1 1TMCC-1 Adenocarcinoma 6 6SiHa Squamous cell carcinoma 1 1Caski Squamous cell carcinoma 2 2CAC-1 Adenocarcinoma 1 1

Endometrium Ishikawa Adenocarcinoma 1 1SNG II Adenocarcinoma 6 2HEC-1-B Adenocarcinoma 1 1

Stomach SSTW-2 Signet cell carcinoma 1 1KATO III Signet cell carcinoma 6 2HST-2 Signet cell carcinoma 6 6

Pancreas MIA-PaCa-2 Adenocarcinoma 2 2BxPC-3 Adenocarcinoma 2 2Capan-1 Adenocarcinoma 2 2

Breast MCF-7 Adenocarcinoma 1 6HMC-1 Adenocarcinoma 6 6HMC-2 Adenocarcinoma 1 1

Esophagus HC-MA Squamous cell carcinoma 1 1Oral cavity OSC40 Squamous cell carcinoma 1 1

OSC70 Squamous cell carcinoma 1 1Erythrocyte K562 Leukemia 2 2Lymphocyte C1RA2402 B-cell lymphoma 1 6

YH EBV-BCLa Transformed cell 2 2KK EBV-BCLa Transformed cell 2 2

a EBV-BCL; EBV-transformed B lymphocyte from healthy individuals.

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ABERRANT EXPRESSION OF RECOVERIN IN CANCER CELLS



recoverin (expected PCR product, 369 bp); (b) sense primer 59-TGTGGCTTC-CTTCGTTATTGG-39 and antisense primer 59-GCCAGCATCATTCACCAC-CAT-39 for hsc70 (expected PCR product, 342 bp); (c) sense primer 59-CTGTCT-GGCGGCACCACCAT-39 and antisense primer 59-GCAACTAAGTCATA-GTCCGC-39for b-actin (expected PCR product, 254 bp). The amplified PCRproducts were electrophoresed on a 1.5% agarose gel containing ethidium bro-mide. The densitometric analysis of the bands was performed using Epi-LightUVF500 (Aisin Cosmos R&D Co., Ltd., Tokyo, Japan).

To confirm the identity of the bands, the PCR product for recoverin wascloned into pCRII vector with a TA cloning kit (Invitrogen, Carsbad, CA). Thenucleotide sequences of the clones were determined using an ABI Geneticanalyzer PRIM 310 and an AmpliCycle sequencing kit (Perkin-Elmer, FosterCity, CA).

Immunocytochemistry. Cells were cultured overnight on coverslips coatedwith 0.1% polylysin, fixed in ice-cold 3.7% formaldehyde for 10 min, andpermeabilized in methanol for 20 min at220°C. The coverslips were incubatedwith primary antibody for 30 min at 20°C, washed three times with 0.5% BSA inPBS for 5 min, and incubated with FITC-labeled secondary antibody for 30 minat 20°C. The coverslips were then washed as described above and mounted on aslide glass using Vectashield fluorescence mounting medium (Vector Laborato-ries, Inc., Burlingame, CA). The specific antibody binding was visualized on alaser scanning confocal microscope (Bio-Rad, Richmond, CA).

Transfection of Human Recoverin cDNA into A549 Cells.Human re-coverin cDNA was obtained from Dr. S. Kawamura (Department of Biology,Osaka University, Osaka, Japan). Transfection of human cDNA into A549cells was performed by the method described by Kawamotoet al. (33), withsome modifications. Briefly, 1mg of human recoverin cDNA inserted in thepIRES puro expression vector or GFP cDNA in the same vector (control) wasmixed with 4ml of LipofectAMINE in a total of 400ml of RPMI 1640 for 15min at room temperature. Each mixture was then added to 6-well plates ofA549 cells and incubated at 37°C. Twenty-four h after the incubation, 100mlof FCS and puromycin (final concentration, 20mM) were added to the mixture,

which was incubated for an additional 48 h at 37°C for selection of cellsexpressing the plasmids. For further incubation, RPMI 1640 containing 10%FCS and 2mM puromycin was used.

Cell Proliferation Assay. The cell proliferation of A549 cells transfectedwith human recoverin cDNA was estimated by using a WST-1 assay accordingto the manufacturer’s guidelines (Boehringer Mannheim). A549 cells trans-fected with GFP cDNA were used as a control. This assay is based on thecleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenase inviable cells (34). Briefly, A549 transfectants (23 104 cells/well) were incu-bated with 100ml of culture medium in 96-multiwell plates. After 24 or 48 hof incubation, 10ml of solution containing 3.3 mg/ml WST-1 were added toeach well, and cells were incubated for an additional hour at 37°C. Thereafter,the absorbance at 450 nm of each well was measured by MPR-A4i (TOSOH,Tokyo, Japan).

RESULTS

In the present study, we examined aberrant recoverin expressionand determined the expression levels of hsc 70 mRNA in cancer cellsto understand the molecular pathology of the onset of CAR, using celllines derived from several types of cancers. Fig. 1 shows recoverinmRNA expression analyzed by RT-PCR using the 31 cancer cell linesand 2 EBV-transformed B lymphocytes as controls. Twenty-one of 33cell lines (1 of 4 lung small cell carcinoma cell lines, 3 of 5 lungadenocarcinoma cell lines, 3 of 3 gastric signet cell carcinoma celllines, 3 of 3 breast cancer cell lines, 5 of 6 cervical/endometrial cancercell lines, 3 of 3 oral squamous cell cancer cell lines, and 1 of 2leukemia cell lines) expressed recoverin mRNA (summarized in Table1). As shown in Fig. 2 and Table 1, this aberrant expression ofrecoverin in cancer cell lines was also confirmed by Western blot

Fig. 4. Expression of recoverin in neonatal thy-muses. cDNA pools derived from six neonatal thy-muses were investigated. We used 4.4ml from a22-ml cDNA pool for PCR, using specific primers asdescribed in “Materials and Methods.” PCR productswere evaluated by agarose gel electrophoresis andethidium bromide staining.

Fig. 3. Expression of recoverin in normal adulttissues. cDNA pools derived from normal adulttissues were examined. We used 4.4ml from a22-ml cDNA pool for PCR, using specific primersas described in “Materials and Methods.” PCRproducts were evaluated by agarose gel electro-phoresis and ethidium bromide staining.

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analysis using the affinity-purified anti-recoverin antibody. In concur-rence with the findings of Murakamiet al. (35), who reported thatrecoverin was exclusively expressed within photoreceptor cells andretinal bipolar cells, we also found no expression of recoverin innormal adult tissues (stomach, small intestine, colon, spleen, liver,lung, kidney, prostate, pancreas, and heart; Fig. 3). However, recov-erin was found to be expressed in three of six of newborn thymuses(Fig. 4). The identity of the bands from cancer cell lines and thymuseswas confirmed by sequencing the PCR products (Fig. 5).

To determine the expression level of hsc 70 mRNA in these cancercell lines and initially for confirmation of the semiquantitative RT-PCR analysis, the levels of amplification were measured after variousnumbers of PCR cycles. The intensity of the signal increased linearlyup to 18 cycles in both hsc 70 andb-actin, which was used as aninternal control (Fig. 6). The ratio of intensity of hsc 70:b-actin at 18cycles was calculated to estimate the expression of hsc 70. As shownin Table 2, hsc 70 was expressed at significantly higher levels incancer cells than in control cells (peripheral blood mononuclear cells)from five healthy individuals. However, when we compared thecorrelation of hsc 70 and recoverin expression levels, we found thatthere was no significant difference in hsc 70 expression betweenrecoverin-positive cancer cells and recoverin-negative cancer cells.

To elucidate the physiological and pathological aspects of theaberrant expression of recoverin, localization of recoverin within thecancer cells was determined by immunocytochemistry using the af-finity-purified anti-recoverin antibody. As shown in Fig. 7, the punc-tate structure at the perinuclear region of the cells was stained with theantibody, suggesting that recoverin is associated with endomembranesystems such as endosomes/lysosomes.

We next examined whether recoverin may have some particularphysiological role in the cancer cells besides that of an autoantigen.Because it has been reported that CAR patients had a preferable

Fig. 5. The identity of the bands was confirmed bysequencing the PCR product. RT-PCR amplification withRNA isolated from the SSTW-2 cell line demonstrated100% identity with the known human recoverin sequence189–574 (underlined).Arrows indicate the PCR primersused for amplification. The raw nucleotide sequences(number 100–160) obtained by ABI Genetic analyzerPRIM 310 were in agreement with recoverin sequence325–385 (shown in thebottom panel). All other PCRproducts from other tumor cell lines and thymus showedan identical nucleotide sequence.

Fig. 6. Semiquantitative RT-PCR for hsc 70. The levels of amplification were meas-ured after various numbers of PCR cycles (B; Refs. 43 and 44).A, the intensity of thesignal increased linearly up to 18 cycles in both hsc 70 andb-actin, which was used as aninternal control. The ratio of hsc 70:b-actin intensity at 18 cycles was calculated toestimate the expression of hsc 70.

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prognosis compared with cancer patients without retinopathy (36), wespeculated that aberrant expression of recoverin may affect the cellproliferation process. To test our hypothesis, human recoverin (Fig. 8)or GFP cDNA was transfected into A549 cells in which recoverin wasnot expressed, and cell proliferation rates were compared with eachother by WST-1 assay. The rates of cell proliferation of recoverintransfectants were reduced to 83.4% and 73.7% after 24- and 48-hincubations, respectively, as compared with GFP-transfectants (Fig.9). This result demonstrated that expression of recoverin modulatedthe cell proliferation of A549 cells.

DISCUSSION

Recoverin expressed exclusively within photoreceptor cells andretinal bipolar cells is known to be a highly pathogenic molecule,based on the fact that immunization with purified recoverin inducedhigh serum antibody titers to recoverin, the activation of immuno-competent T cells, and photoreceptor degeneration in rats (37). InCAR, aberrant expression of retina-specific recoverin in tumor cells is

suggested to be a possible mechanism of autoantibody production(11–13). Similar to this, a Purkinje cell antigen (Yo antigen) wasrecognized within the tumors in paraneoplastic cerebellar degenera-tion of patients with gynecological tumors (2). Nevertheless, such anantigen was not detected in similar tumors obtained from individualswithout neurological symptoms. Therefore, aberrant expressions ofneuron-specific molecules in tumor cells seem to be a key causalmechanism in the degeneration of the target neuronal regions. How-

Fig. 7. Immunocytochemistry for recoverin. Cellswere cultured overnight on coverslips coated with0.1% polylysin, fixed in ice-cold 3.7% formalde-hyde, and permeabilized in methanol for 20 min at220°C. The coverslips were incubated with primaryantibody for 30 min at 20°C and incubated withFITC-labeled secondary antibody for 30 min at20°C. The specific antibody binding was visualizedon a laser scanning confocal microscope (Bio-Rad).The details of the immunocytochemistry are de-scribed in “Materials and Methods.” This figureshows SSTW-2 as a representative result.

Fig. 8. Transfection of human recoverin cDNA to A549 cells. Human recoverin cDNAwas transfected into A549 lung adenocarcinoma cells. Expression of recoverin wasconfirmed by RT-PCR analysis.

Table 2 Expression level of hsc 70 in cancer cells and noncancerous cells

Twenty-one recoverin-positive cancer cell lines, 10 recoverin-negative cell lines (Table1), and peripheral blood mononuclear cells from five healthy individuals were examined.Because the intensity of the signal increased linearly up to 18 cycles of RT-PCR in bothhsc 70 andb-actin as an internal control, the ratio of hsc 70:b-actin at 18 cycles wascalculated to estimate the expression of hsc 70. The details are described in “Materials andMethods.” Data represent mean6 SE of each experiment.

Expression level of hsc 70(hsc 70:b-actin)

Cancer cell Recoverin (1) 0.976 0.49]]Recoverin (2) 0.836 0.16P , 0.01

Noncancerous cell 0.546 0.08

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ever, in the present study, we found that recoverin was aberrantlyexpressed in cell lines from various cancer patients at a high incidence(21 of 31 cell lines from various cancers). This observation allowed usto speculate that because CAR is a very rare disease, some unknownmechanisms must be required for the generation of autoantibodies toaberrantly expressed recoverin in cancer cells. It was revealed thatself-reactive T cells can be physically deleted within the thymus gland(38). If the expression of recoverin is detected in neonatal thymus,immunocompetent T cells to recoverin can underlie negative selectionand become tolerant. Therefore, immunological tolerance to recoverincan be induced in individuals who express recoverin in the thymus atbirth. In addition, Charukamnoetkanoket al. (39) reported that notranscripts of uveitogenic peptides such as S-antigen and interphoto-receptor retinoid-binding protein were detected in the thymus of thehighly susceptible Lewis rats, whereas the thymus of mice resistant touveitis did express transcripts of these antigens. These facts mayexplain why CAR developed in only a few patients.

Why retina-specific recoverin is expressed in cancer cells is stillunknown. As a possible mechanism, we speculated that the molecularchaperone functions of hsc 70 and the autoimmune reaction to themmay be related to anti-recoverin antibody generation because autoim-mune reaction to hsc 70 was also recognized in most CAR patients.Nevertheless, in the present study, stress-induced expression of hsc 70was significantly increased in cancer cell lines, confirming the reportof Hattori et al. (40), but no statistical difference was observedbetween recoverin-positive and -negative cancer cell lines.

Another important question is what the physiological roles ofrecoverin are in cancer cells. Our current study revealed that: (a)immunofluorescence labeling of recoverin produced a granular pat-tern within the cancer cells, suggesting that recoverin may be associ-ated with endomembranes and may have some specific functions; and(b) transfection of recoverin in A549 cells caused their proliferation toslow down. Functionally, recoverin is believed to be involved in theindispensable role of adaptation to dark and light by regulating rho-dopsin phosphorylation in a calcium-dependent manner in photore-ceptor cells (41). In addition, it was found that calcium-bindingproteins belong to the neuronal calcium sensor gene family, whichincludes S-modulin, neurocalcin hippocalcin frequenin, vilip1, vilip2,vilip3, visinin, HLP2 and neuronal calcium sensor 1, which sharefunctional and structural homologies with recoverin and are widelydistributed within the nervous system. These family members wereshown to regulate rhodopsin phosphorylation in a calcium-dependent

manner, suggesting that they may function in the regulation of thephosphorylation of G-protein-coupled receptors (42). Taken together,our data allow us to speculate that recoverin may be a calcium sensorin cancer cells and may have significant roles in the cellular metab-olism and proliferation of tumors. Therefore, further study to elucidatethe function of recoverin in cancer cells is our next project.

ACKNOWLEDGMENTS

We thank Prof. S. Kawamura, Dr. H. Sahara (Department of Pathology,Section 1, Sapporo Medical University School of Medicine, Sapporo, Japan),Drs. M. Imamura and S. Yokoyama for providing human cDNAs and Drs. M.Hirasawa, M. Koizumi, and M. Takiguchi for donating cell lines. We are verygrateful to Prof. Y. Niitsu (Department of Internal Medicine, Section 4,Sapporo Medical University School of Medicine) and Dr. H. Sano (Depart-ment of Biochemistry, Section 1, Sapporo Medical University School ofMedicine) for valuable discussion on the present study.

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Fig. 9. WST-1 assay of human recoverin and GFP transfectants. Human recoverin orGFP cDNA was transfected into A549 lung adenocarcinoma cells, and cell proliferationof each was compared by WST-1 assay. The ratios of recoverin transfectants:control (GFPtransfectants) after 24- and 48-h incubations were plotted. Experiments were performed in12 replicates. The details of the proliferation assay are described in “Materials andMethods.” Data were expressed as the mean6 SE of each experiment.

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2000;60:1914-1920. Cancer Res Akiko Maeda, Hiroshi Ohguro, Tadao Maeda, et al. Calcium-binding Protein (Recoverin) in Cancer Cell LinesAberrant Expression of Photoreceptor-specific

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