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[Cancer Research 60, 1914-1920, April 1, 2000]
© 2000 American Association for Cancer Research

Immunology

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

Akiko Maeda2, Hiroshi Ohguro, Tadao Maeda, Ikuo Wada, Noriyuki Sato, Yoshio Kuroki and Takashi Nakagawa

Departments 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 Medical University School of Medicine, Sapporo 060-8543, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cancer-associated retinopathy (CAR) is an ocular manifestation of a paraneoplastic syndrome whereby immunological reactions to retinal antigens aberrantly expressed in tumor cells lead to the degeneration of retinal photoreceptor cells. In our previous study (H. Ohguro et al., Invest. Ophthalmol. Vis. Sci., 40: 82–89, 1999), recoverin, a retina-specific calcium-binding protein, and heat shock cognate protein 70 (hsc 70) were identified as autoantigens recognized by sera from patients with CAR. Therefore, we suggested that autoimmune reactions against both recoverin and hsc 70 might be involved in the pathogenesis of CAR. To elucidate the initial step of the molecular pathology of CAR, we examined the expression of recoverin and hsc 70 by reverse transcription-PCR and Western blot using cell lines of several kinds of cancers, including lung small cell carcinoma, lung adenocarcinoma, gastric cancer, pancreatic cancer, breast cancer, uterine cervical cancer, endometrial cancer, and leukemia. Recoverin was expressed in 21 of the 31 cancer cell lines. The expression levels of hsc 70 were significantly higher in cancer cell lines than in noncancerous cell lines. However, no difference in the expression levels of hsc 70 was observed between recoverin-positive and -negative cell lines. Immunofluorescence labeling by the affinity-purified recoverin antibody revealed the immunoreactivity to recoverin as a granular pattern within the cancer cells. Lung adenocarcinoma A549 cells, which did not express recoverin, exhibited a significant reduction in cell proliferation upon transfection with human recoverin cDNA. Taken together, our present data suggest that the retina-specific calcium-binding protein recoverin is expressed in more than 50% of a variety of cancer cells and may play a significant role in the cell proliferation of these tumor cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A variety of neurological disorders called paraneoplastic syndromes are known to be associated with malignant tumors, although the tumor or its metastases have not invaded the nervous system. This so called "remote effect" of cancer is considered to be mediated on an autoimmune basis. That is, the expression of a tumor antigen presumably triggers immunological responses which in turn recognize the same antigen or shared epitope in the nervous system, resulting in neuronal cell damage. In the peripheral nervous system, Lambert-Eaton myasthenic syndrome is known to be associated with autoantibodies to the calcium channel of the neuromuscular junction, which interfere with the release of acetylcholine and cause proximal muscle weakness (1) . In the central nervous system, paraneoplastic cerebellar degeneration has been identified to be caused by autoantibodies against Purkinje cell antigen (called Yo antigen) detected in some individuals with gynecological tumors (2) . CAR3 has been identified as a paraneoplastic syndrome of the visual system (3, 4, 5) . CAR is found in patients with small cell carcinoma of lung and other malignant tumors and is clinically characterized by photopsia, progressive visual loss with a ring scotoma, attenuated retinal arterioles, and abnormalities of the a- and b-waves of electroretinogram. Histopathology revealed that loss of photoreceptor cells occurs primarily in the retinas of CAR patients (5 , 6) . It was found that CAR is caused by an autoimmune reaction against a photoreceptor-specific Mr 23,000 calcium-binding protein called recoverin (7 , 8) . Functionally, recoverin was found to play a major role in light and dark adaptation by regulating rhodopsin phosphorylation and dephosphorylation in a calcium-dependent manner (9 , 10) . In terms of the generation of autoantibody to recoverin, it was identified that recoverin is aberrantly expressed in the cancer cells or cell lines obtained from CAR patients, and this may trigger the autoimmune reaction (11, 12, 13) . However, preliminary studies have revealed that such aberrant expression of retinal-specific recoverin is not seen in cancer cells without retinopathy. These observations suggested that aberrant expression of recoverin in cancer cells is an initial and critical step in the cause of retinopathy. We still do not know the molecular mechanisms that cause the aberrant expression of recoverin in cancer cells. This knowledge is a key to understanding the molecular pathology of CAR and to designing an effective treatment for retinopathy.

In addition to recoverin, other retinal antigens including Mr 65,000 protein, enolase (Mr 46,000 protein), and neurofilament (Mr 58,000–62,000, Mr 145,000, and Mr 205,000 proteins) are also recognized by the sera of some CAR patients (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) . Among these retinal autoantigens, recoverin alone or a combination of recoverin and Mr 65,000 protein has most frequently been reported as the immunoreactive band by Western blot analysis. We have recently identified the Mr 65,000 protein as hsc 70 and have suggested that both anti-recoverin and anti-hsc 70 antibodies are involved in the pathogenesis of CAR (28) . These observations allowed us to speculate that hsc 70 may be involved in the aberrant expression of recoverin.

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


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines.
The 33 cell lines used in this study are summarized in Table 1Citation . 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 lines were provided by Dr. M. Hirasawa (Department of Internal Medicine, Section 3, Sapporo Medical University School of Medicine, Sapporo, Japan), cervical and endometrial cancer cell lines were provided by Dr. M. Koizumi (Department of Gynecology, Sapporo Medical University School of Medicine), and C1R cells provided by Dr. M. Takiguchi (Kumamoto University, Kumamoto, Japan) were basically established as described previously (29 , 30) . None of the donors of the tumor cells described above had episodes of visual symptoms of CAR. The other cell lines were either provided by the Japanese Collection of Research Bioresources (Tokyo, Japan) or purchased from American Type Culture Collection. These cell lines were maintained in RPMI 1640 containing 10% fetal bovine serum and antibiotics.


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  		Table 1 Summary of recoverin expression in various cancer cell lines

 
Normal Tissues and Neonatal Thymuses.
Normal tissues (stomach, small intestine, colon, spleen, liver, lung, kidney, prostate, pancreas, and heart) and six newborn thymuses were generously donated by Drs. M. Imamura and S. Yokoyama of the Hokkaido Children’s Hospital and Medical Center (Hokkaido, Japan) who gave consent for participation after the study procedures, which are in accordance with the tenets of the Declaration of Helsinki, were explained to them.

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

Western Blot.
Western blot analysis was carried out as described previously (32) . Briefly, the protein fraction isolated by ISOGEN reagent according to the manufacturer’s procedure (Nippon Gene, Tokyo, Japan) was analyzed by SDS-PAGE using a 12.5% polyacrylamide gel. Separated proteins in a gel were electrotransferred to polyvinylidene difluoride membranes in 10 mM bis-Tris phosphate buffer (pH 8.4) containing 10% methanol. After blocking with 5% skim milk in PBS, the membrane was probed successively with anti-recoverin antibody and horseradish peroxidase-labeled anti-rabbit IgG (Funakoshi Co., Tokyo, Japan). Immunoreactive bands were visualized by an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) according to the method described by the manufacturer.

RT-PCR Analysis.
Total RNA from cell lines was isolated using ISOGEN reagent according to the procedure described by the manufacturer (Nippon Gene) and reverse-transcribed by using Superscript II with oligo(dT) primer (Life Technologies, Inc., Rockville, MD). The incubation was carried out at 42°C for 50 min and then at 70°C for 15 min. The PCR amplifications were performed using 4.4 µl for recoverin or 2.2 µl for hsc)(70 and ß-actin from the RT reaction mixture in 50 µl of PCR mixture containing 50 pmol of sense and antisense primers. After the initial incubation at 94°C for 4 min, 30 cycles of amplification were conducted with denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 2 min. The following primer pairs were used for RT-PCR analysis: (a) sense primer 5'-TGTGTTCCGCAGCTTCGATT-3' and antisense primer 5'-TGAGGCTCAACTAACTGGATCAG-3' for recoverin (expected PCR product, 369 bp); (b) sense primer 5'-TGTGGCTTCCTTCGTTATTGG-3' and antisense primer 5'-GCCAGCATCATTCACCACCAT-3' for hsc70 (expected PCR product, 342 bp); (c) sense primer 5'-CTGTCTGGCGGCACCACCAT-3' and antisense primer 5'-GCAACTAAGTCATA-GTCCGC-3' for ß-actin (expected PCR product, 254 bp). The amplified PCR products were electrophoresed on a 1.5% agarose gel containing ethidium bromide. The densitometric analysis of the bands was performed using Epi-Light UVF500 (Aisin Cosmos R&D Co., Ltd., Tokyo, Japan).

To confirm the identity of the bands, the PCR product for recoverin was cloned into pCRII vector with a TA cloning kit (Invitrogen, Carsbad, CA). The nucleotide sequences of the clones were determined using an ABI Genetic analyzer PRIM 310 and an AmpliCycle sequencing kit (Perkin-Elmer, Foster City, CA).

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

Transfection of Human Recoverin cDNA into A549 Cells.
Human recoverin cDNA was obtained from Dr. S. Kawamura (Department of Biology, Osaka University, Osaka, Japan). Transfection of human cDNA into A549 cells was performed by the method described by Kawamoto et al. (33) , with some modifications. Briefly, 1 µg of human recoverin cDNA inserted in the pIRES puro expression vector or GFP cDNA in the same vector (control) was mixed with 4 µl of LipofectAMINE in a total of 400 µl of RPMI 1640 for 15 min at room temperature. Each mixture was then added to 6-well plates of A549 cells and incubated at 37°C. Twenty-four h after the incubation, 100 µl of FCS and puromycin (final concentration, 20 µM) were added to the mixture, which was incubated for an additional 48 h at 37°C for selection of cells expressing the plasmids. For further incubation, RPMI 1640 containing 10% FCS and 2 µM puromycin was used.

Cell Proliferation Assay.
The cell proliferation of A549 cells transfected with human recoverin cDNA was estimated by using a WST-1 assay according to the manufacturer’s guidelines (Boehringer Mannheim). A549 cells transfected with GFP cDNA were used as a control. This assay is based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenase in viable cells (34) . Briefly, A549 transfectants (2 x 104 cells/well) were incubated with 100 µl of culture medium in 96-multiwell plates. After 24 or 48 h of incubation, 10 µl of solution containing 3.3 mg/ml WST-1 were added to each 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
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In the present study, we examined aberrant recoverin expression and determined the expression levels of hsc 70 mRNA in cancer cells to understand the molecular pathology of the onset of CAR, using cell lines derived from several types of cancers. Fig. 1Citation shows recoverin mRNA expression analyzed by RT-PCR using the 31 cancer cell lines and 2 EBV-transformed B lymphocytes as controls. Twenty-one of 33 cell lines (1 of 4 lung small cell carcinoma cell lines, 3 of 5 lung adenocarcinoma cell lines, 3 of 3 gastric signet cell carcinoma cell lines, 3 of 3 breast cancer cell lines, 5 of 6 cervical/endometrial cancer cell lines, 3 of 3 oral squamous cell cancer cell lines, and 1 of 2 leukemia cell lines) expressed recoverin mRNA (summarized in Table 1Citation ). As shown in Fig. 2Citation and Table 1Citation , this aberrant expression of recoverin in cancer cell lines was also confirmed by Western blot analysis using the affinity-purified anti-recoverin antibody. In concurrence with the findings of Murakami et al. (35) , who reported that recoverin was exclusively expressed within photoreceptor cells and retinal bipolar cells, we also found no expression of recoverin in normal adult tissues (stomach, small intestine, colon, spleen, liver, lung, kidney, prostate, pancreas, and heart; Fig. 3Citation ). However, recoverin was found to be expressed in three of six of newborn thymuses (Fig. 4Citation ). The identity of the bands from cancer cell lines and thymuses was confirmed by sequencing the PCR products (Fig. 5Citation ).



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  		Fig. 1. Expression of mRNA for recoverin in various tumor cell lines. Two µg of RNA from 31 tumor cell lines and from 2 EBV-transformed B lymphocytes (controls) were reverse-transcribed to generate cDNA pools, and then 4.4 µl from a 22-µl cDNA pool were used for PCR, using specific primers as described in "Materials and Methods." PCR products were evaluated by agarose gel electrophoresis and ethidium bromide staining.

 


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  		Fig. 2. Western blot analysis for recoverin. Whole cell lysates containing approximately 20 µg of proteins were loaded on SDS-PAGE gel, followed by electrotransfer to a polyvinylidene difluoride membrane. Western blot analysis was performed using the affinity-purified anti-recoverin polyclonal antibody (1:400 dilution). The details of the Western blot are described in "Materials and Methods." Western blots of selected recoverin-positive and -negative cell lines by PCR are shown in the top and bottom panels, respectively. The results of Western blot analysis of the other tumor cells are summarized in Table 1Citation .

 


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  		Fig. 3. Expression of recoverin in normal adult tissues. cDNA pools derived from normal adult tissues were examined. We used 4.4 µl from a 22-µl cDNA pool for PCR, using specific primers as described in "Materials and Methods." PCR products were evaluated by agarose gel electrophoresis and ethidium bromide staining.

 


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  		Fig. 4. Expression of recoverin in neonatal thymuses. cDNA pools derived from six neonatal thymuses were investigated. We used 4.4 µl from a 22-µl cDNA pool for PCR, using specific primers as described in "Materials and Methods." PCR products were evaluated by agarose gel electrophoresis and ethidium bromide staining.

 


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  		Fig. 5. The identity of the bands was confirmed by sequencing the PCR product. RT-PCR amplification with RNA isolated from the SSTW-2 cell line demonstrated 100% identity with the known human recoverin sequence 189–574 (underlined). Arrows indicate the PCR primers used for amplification. The raw nucleotide sequences (number 100–160) obtained by ABI Genetic analyzer PRIM 310 were in agreement with recoverin sequence 325–385 (shown in the bottom panel). All other PCR products from other tumor cell lines and thymus showed an identical nucleotide sequence.

 
To determine the expression level of hsc 70 mRNA in these cancer cell lines and initially for confirmation of the semiquantitative RT-PCR analysis, the levels of amplification were measured after various numbers of PCR cycles. The intensity of the signal increased linearly up to 18 cycles in both hsc 70 and ß-actin, which was used as an internal control (Fig. 6Citation ). The ratio of intensity of hsc 70:ß-actin at 18 cycles was calculated to estimate the expression of hsc 70. As shown in Table 2Citation , hsc 70 was expressed at significantly higher levels in cancer cells than in control cells (peripheral blood mononuclear cells) from five healthy individuals. However, when we compared the correlation of hsc 70 and recoverin expression levels, we found that there was no significant difference in hsc 70 expression between recoverin-positive cancer cells and recoverin-negative cancer cells.



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  		Fig. 6. Semiquantitative RT-PCR for hsc 70. The levels of amplification were measured after various numbers of PCR cycles (B; Refs. 43 and 44 ). A, the intensity of the signal increased linearly up to 18 cycles in both hsc 70 and ß-actin, which was used as an internal control. The ratio of hsc 70:ß-actin intensity at 18 cycles was calculated to estimate the expression of hsc 70.

 

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  		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 (Table 1)Citation , 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 both hsc 70 and ß-actin as an internal control, the ratio of hsc 70:ß-actin at 18 cycles was calculated to estimate the expression of hsc 70. The details are described in "Materials and Methods." Data represent mean ± SE of each experiment.

 
To elucidate the physiological and pathological aspects of the aberrant expression of recoverin, localization of recoverin within the cancer cells was determined by immunocytochemistry using the affinity-purified anti-recoverin antibody. As shown in Fig. 7Citation , the punctate structure at the perinuclear region of the cells was stained with the antibody, suggesting that recoverin is associated with endomembrane systems such as endosomes/lysosomes.



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  		Fig. 7. Immunocytochemistry for recoverin. Cells were cultured overnight on coverslips coated with 0.1% polylysin, fixed in ice-cold 3.7% formaldehyde, and permeabilized in methanol for 20 min at -20°C. The coverslips were incubated with primary antibody for 30 min at 20°C and incubated with FITC-labeled secondary antibody for 30 min at 20°C. The specific antibody binding was visualized on a laser scanning confocal microscope (Bio-Rad). The details of the immunocytochemistry are described in "Materials and Methods." This figure shows SSTW-2 as a representative result.

 
We next examined whether recoverin may have some particular physiological role in the cancer cells besides that of an autoantigen. Because it has been reported that CAR patients had a preferable prognosis compared with cancer patients without retinopathy (36) , we speculated that aberrant expression of recoverin may affect the cell proliferation process. To test our hypothesis, human recoverin (Fig. 8Citation ) or GFP cDNA was transfected into A549 cells in which recoverin was not expressed, and cell proliferation rates were compared with each other by WST-1 assay. The rates of cell proliferation of recoverin transfectants were reduced to 83.4% and 73.7% after 24- and 48-h incubations, respectively, as compared with GFP-transfectants (Fig. 9Citation ). This result demonstrated that expression of recoverin modulated the cell proliferation of A549 cells.



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

 


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  		Fig. 9. WST-1 assay of human recoverin and GFP transfectants. Human recoverin or GFP cDNA was transfected into A549 lung adenocarcinoma cells, and cell proliferation of each was compared by WST-1 assay. The ratios of recoverin transfectants:control (GFP transfectants) after 24- and 48-h incubations were plotted. Experiments were performed in 12 replicates. The details of the proliferation assay are described in "Materials and Methods." Data were expressed as the mean ± SE of each experiment.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recoverin expressed exclusively within photoreceptor cells and retinal bipolar cells is known to be a highly pathogenic molecule, based on the fact that immunization with purified recoverin induced high serum antibody titers to recoverin, the activation of immunocompetent T cells, and photoreceptor degeneration in rats (37) . In CAR, aberrant expression of retina-specific recoverin in tumor cells is suggested to be a possible mechanism of autoantibody production (11, 12, 13) . Similar to this, a Purkinje cell antigen (Yo antigen) was recognized within the tumors in paraneoplastic cerebellar degeneration of patients with gynecological tumors (2) . Nevertheless, such an antigen was not detected in similar tumors obtained from individuals without neurological symptoms. Therefore, aberrant expressions of neuron-specific molecules in tumor cells seem to be a key causal mechanism in the degeneration of the target neuronal regions. However, in the present study, we found that recoverin was aberrantly expressed in cell lines from various cancer patients at a high incidence (21 of 31 cell lines from various cancers). This observation allowed us to speculate that because CAR is a very rare disease, some unknown mechanisms must be required for the generation of autoantibodies to aberrantly expressed recoverin in cancer cells. It was revealed that self-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 selection and become tolerant. Therefore, immunological tolerance to recoverin can be induced in individuals who express recoverin in the thymus at birth. In addition, Charukamnoetkanok et al. (39) reported that no transcripts of uveitogenic peptides such as S-antigen and interphotoreceptor retinoid-binding protein were detected in the thymus of the highly susceptible Lewis rats, whereas the thymus of mice resistant to uveitis did express transcripts of these antigens. These facts may explain why CAR developed in only a few patients.

Why retina-specific recoverin is expressed in cancer cells is still unknown. As a possible mechanism, we speculated that the molecular chaperone functions of hsc 70 and the autoimmune reaction to them may be related to anti-recoverin antibody generation because autoimmune reaction to hsc 70 was also recognized in most CAR patients. Nevertheless, in the present study, stress-induced expression of hsc 70 was significantly increased in cancer cell lines, confirming the report of Hattori et al. (40) , but no statistical difference was observed between recoverin-positive and -negative cancer cell lines.

Another important question is what the physiological roles of recoverin are in cancer cells. Our current study revealed that: (a) immunofluorescence labeling of recoverin produced a granular pattern within the cancer cells, suggesting that recoverin may be associated with endomembranes and may have some specific functions; and (b) transfection of recoverin in A549 cells caused their proliferation to slow down. Functionally, recoverin is believed to be involved in the indispensable role of adaptation to dark and light by regulating rhodopsin phosphorylation in a calcium-dependent manner in photoreceptor cells (41) . In addition, it was found that calcium-binding proteins belong to the neuronal calcium sensor gene family, which includes S-modulin, neurocalcin hippocalcin frequenin, vilip1, vilip2, vilip3, visinin, HLP2 and neuronal calcium sensor 1, which share functional and structural homologies with recoverin and are widely distributed within the nervous system. These family members were shown to regulate rhodopsin phosphorylation in a calcium-dependent manner, suggesting that they may function in the regulation of the phosphorylation of G-protein-coupled receptors (42) . Taken together, our data allow us to speculate that recoverin may be a calcium sensor in cancer cells and may have significant roles in the cellular metabolism and proliferation of tumors. Therefore, further study to elucidate the 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 very grateful to Prof. Y. Niitsu (Department of Internal Medicine, Section 4, Sapporo Medical University School of Medicine) and Dr. H. Sano (Department of Biochemistry, Section 1, Sapporo Medical University School of Medicine) for valuable discussion on the present study.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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

2 To whom requests for reprints should be addressed, at Department of Ophthalmology, Sapporo Medical School of Medicine, S-1 W-16, Chuo-ku, Sapporo 060-8543, Japan. Fax: 81-11-613-6575; E-mail ooguro@sapmed.ac.jp. Back

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

Received 9/ 7/99. Accepted 2/ 3/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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