| Human Molecular Genetics | Pages |
CSR, a scavenger receptor-like protein with a protective role against cellular damage causedby UV irradiation and oxidative stress
Introduction
Results
Isolation of CSR cDNAs
Structure of CSR
CSR expression in response to cellular stress
Cellular localization of CSR
Role of CSR in protection from cell death
Discussion
Materials And Methods
Isolation and sequencing of cDNA
Experimental cell damage and cell treatment
Flow cytometry
Northern analysis
Immunocytochemical staining
Acknowledgements
References
CSR, a scavenger receptor-like protein with a protective role against cellular damage causedby UV irradiation and oxidative stress
DDBJ/EMBL/GenBank accession nos AB007829, AB007830
INTRODUCTION
To adapt to environmental changes and protect themselves from death, cells must increase or induce expression of stress-responsive genes. For example, in response to various stimuli such as reactive oxygen species (ROS), heavy metals, heat shock, radiation, hormones or viral and bacterial infections, normal cells increase the expression of genes that encode proteins related to signalling, proliferative or apoptotic pathways. Among several agents of cellular stress, ROS such as hydrogen peroxide, superoxide (O2-) and hydroxyl radicals (OH-) have attracted attention recently because they can cause severe cell damage and are implicated in aging, cancer and various other diseases (1). The brain in particular is highly sensitive to oxidative damage generated by ROS following ischaemia and reperfusion (2), or as a consequence of neuronal degenerative diseases (3). Therefore, a better understanding of the cellular responses affected by endogenous or exogenous ROS, and of the genes responding to the presence of oxidative agents, is important from a biological and a clinical point of view.
Intracellular levels of ROS become unphysiologically low in the absence of oxygen (hypoxia); in tumours, hypoxia-mediated selective cell growth confers resistance against irradiation and cytotoxic therapy (4). On the other hand, above normal levels of ROS, referred to as oxidative stress, occur frequently in cells exposed to UV light, [gamma]-rays, low concentrations of hydrogen peroxide or certain exogenous chemical reagents such as diethylmaleate (DEM) or 4[beta]-phorbol 12-myristate 13-acetate (PMA), and in cells stimulated with cytokines and other natural ligands for cell surface receptors (5). Normally, ROS are catabolized by means of antioxidant enzymes (superoxide dismutases, microsomal peroxidases, catalase, etc.) and free-radical scavengers (reduced glutathione, reduced thiol, etc.). However, ROS can also lead to events such as proliferation (6) or apoptosis (7) through cellular signalling pathways. An increase in ROS stimulates the activity of mitogen-activated protein kinases (MAPKs) ERK and JNK as well as expression of proto-oncogenes c-fos and c-jun (8). Tyrosine kinases of the Src family are also implicated in the adhesion-dependent activation of neutrophil functions (9). Two redox-related transcription factors, AP-1 and NF-[kappa]B, are known to be induced in hypoxia and reoxygenation (10). To maintain redox balance, oxidative stress-response transcription factors also induce enzymes with antioxidant activities (1). However, cells under severe oxidative stress fail to control their growth cycles, due to oxidation and consequent loss of function of growth-controlling proteins. Oxidants also provoke a decrease in the DNA-binding efficiency of several transcription factors (11,12). However, the mechanisms and factors regulating oxidant and antioxidant responses in eukaryotic cells are not well understood. It is also unclear how many genes are actually involved in redox-related functions.
Here we report the isolation and characterization of a novel gene, CSR (cellular stress response), whose expression is induced mainly by oxidative stress. We demonstrate here that the CSR product has a crucial role in protecting cells from the consequences of oxidative stress, as a scavenger of ROS and their by-products.
RESULTS
Isolation of CSR cDNAs
The entire genomic DNA sequences of a cosmid clone c2, which mapped on chromosome 8p21 (data not shown), were determined and searched for possibly transcribed exons using two exon prediction computer programs, GRAIL 2 and HEXON. Subsequently, using as probes the nucleotide sequences of candidate exons predicted by the programs, we screened a fetal brain cDNA library and isolated four cDNA clones.
DNA sequencing of these four cDNA clones yielded a full-length cDNA and defined two alternatively spliced transcripts of 4.0 and 1.9 kb. The larger cDNA encoded a protein of 606 amino acids and the other encoded a protein of 466 amino acids, the transcripts of which are very few in mammalian cells (Fig. 1). These two novel proteins, which share the 457 N-terminal amino acids, were designated CSR1 (606 amino acids) and CSR2 (466 amino acids) since their expression was induced by a cellular stress response as described below. Northern analysis using an MTN blot (Clontech) revealed that the CSR1 and CSR2 transcripts were expressed ubiquitously (data not shown), although expression of both transcripts was relatively low in liver and peripheral blood leukocyte.
Figure 1. Predicted protein sequences encoded by the CSR1 (A) and CSR2 (B) cDNAs. CSR2 is alternatively spliced at codon 457. A predicted transmembrane domain at the N-terminus of the protein is boxed, and a leucine zipper site is double-underlined, with repeated leucine residues represented as larger and bolder characters. Possible N-linked glycosylation sites are enclosed in ovals, and suspected phosphorylation sites conserved for protein kinase C (S/T-X-K/R) (open triangle), for casein kinase 2 (S/T-X-X-D/E) (closed circle) and for tyrosine kinase (R/K-X-X-X-D/E-X-X-X-Y) (asterisk) are marked by symbols under the single-letter code. A possible heme-bindng site (open circle) and microbodies C-terminal targeting signal (open inverted triangle) are also presented. Shading indicates a suspected [alpha]-helical coiled-coil regions, and a series of G-X-Y repeats at the C-terminus of the CSR1 protein is indicated with a surrounding open box. The secondary structure of CSR protein is predicted by using programs such as PROSITE, COIL and PredictProtein.
Structure of CSR
We screened the public databases using the FASTA and BLAST programs to look for proteins with similarities to the CSRs, but no amino acid sequences revealed significant homologies. Subsequently we examined proteins structurally similar to CSRs using the PROPSEARCH program in which proteins sharing structural features are matched on the basis of amino acid composition, average charge and molecular weight. The program predicted that CSR1 is structurally similar to macrophage scavenger receptor type II (13; GenBank accession no. S08278) and that CSR2 resembles a sensor protein (14; GenBank accession no. P30844) with a reliability of >80%. The suspected structure of the CSR protein is shown in Figure 2. CSR1 has three distinct peptide domains, a possible membrane-spanning region (I), an [alpha]-helical coiled-coil domain (II) and a collagen-like domain (III). CSR2 protein lacks the C-terminal collagen-like domain. Region I overlaps with a leucine zipper-like domain in which leucine is repeated four times (Fig. 1), suggesting that the CSR protein may function as a polymer. However, due to the hydrophobicity of its leucine-rich structure, it is uncertain whether region I is actually a membrane-spanning domain. The multiplicity of phosphorylation and glycosylation sites predicted by the PROSITE program (Fig. 1), suggests that CSR proteins would be modified post-translationally.
Figure 2. Schematic representation of the structures of the CSR1 and CSR2 genes and proteins. The CSR gene consists of six exons; exons are depicted as hatched boxes and lines represent 5[prime]- and 3[prime]-untranslated regions. Exon numbers are indicated above each box. CSR1 is spliced to exon 6[prime] and CSR2 is alternatively spliced to exon 6[prime]. The predicted model of the putative CSR protein shows three distinctive domains: I, transmembrane (residues 56-80); II, two [alpha]-helical coiled-coils (residues 128-161 and 295-331); and III, collagen-like (CSR1, residues 457-603). The collagen-like domain is replaced by 10 other residues in CSR2.
CSR expression in response to cellular stress
To investigate the physiological function of CSR, we examined the effect of cellular damage on the expression of CSR in normal human fibroblasts (NHDF). A total of 3×104 NHDF cells were seeded in complete medium in a series of 100 mm culture dishes. One or two days later, cells were washed twice with phosphate-buffered saline (PBS) and irradiated with 5-120 J/m2 UV light, and then RNAs were extracted from the cells 24 h later. As shown in Figure 3A, UV radiation significantly enhanced expression of the CSR gene in normal fibroblasts; the maximum effect was observed when the cells were irradiated at 15 J/m2. We then treated the cells with 5-400 µM hydrogen peroxide for 1 h followed by further incubation in complete medium for 24 h, and extracted RNA for northern analysis. CSR transcripts were significantly elevated by treatment with 10 µM hydrogen peroxide, a relatively low concentration to cause significant DNA damage to cells (Fig. 3B). Under conditions of high UV dose (120 J/m2) and high concentration of hydrogen peroxide (400 µM), microscopic analysis revealed that surviving cells were significantly decreased.
Figure 3. Increased CSR transcription in response to cellular stresses such as UV radiation (A) and hydrogen peroxide (B). A DNA-damaging agent, adriamycin (C), did not induce CSR transcription. CSR transcription induced by UV radiation or exogenous hydrogen peroxide is blocked by the antioxidant N-acetylcysteine (NAC) (D). [beta]-Actin served as a quantity control in each experiment (lower panels). Poly(A)+ RNAs from cells treated in each condition were isolated and analyzed by northern blot as described in Materials and Methods. To investigate further whether reagents other than UV radiation and hydrogen peroxide, which induce DNA damage, could affect induction of the CSR gene, we treated normal human fibroblasts with adriamycin (a DNA-intercalating drug) or bleomycin (which produces DNA single-stranded breakage), as described in Materials and methods. Since neither of the drugs caused a significant increase in CSR transcript (Fig. 3C), we assumed that DNA-damaging agents such as adriamycin and bleomycin could not affect the expression of CSR. As both UV radiation and hydrogen peroxide produce reactive oxygen and free radicals in vivo (8,9,15), we assumed that CSR expression is likely to be related to oxidative stress on the cell. To investigate this possibility, we examined the effect of N-acetylcysteine (NAC), a thiocompound with antioxidant activities that counteracts oxidative stress. We first confirmed that pre-incubation with 3 mM NAC itself was not toxic to normal fibroblasts and did not itself influence CSR expression. However, incubation with NAC before UV irradiation or hydrogen peroxide treatment did suppress the enhancement of CSR expression normally induced by these cellular stresses (Fig. 3D), strong evidence to support our hypothesis that oxidative stress in a cell is important for CSR transactivation.
Cellular localization of CSR
To determine the subcellular localization of CSR proteins, a mammalian expression plasmid (pcDNA3.1) was designed to place a hemagglutinin (HA) epitope tag at the N- or C-termini of the CSR proteins. At 24 h after transfection of the plasmid into the HeLa cell line, lysates were prepared and followed by SDS-PAGE to confirm stable expression of HA-tagged CSR proteins by western analysis (data not shown). The transfectant that expressed CSR most stably and strongly was cultured on a slide chamber, and immunocytochemically stained using anti-HA antibody. This procedure revealed that CSR proteins are located in the cytoplasm (Fig. 4B-D); no staining was observed in negative control cells (Fig. 4A) and the subcellular localization of CSR proteins was the same whether the HA tag was at the N- or the C-terminus. The signal was the strongest around the nuclear membrane, a suspected endoplasmic reticulum (ER)-Golgi zone. To define its location in detail, we used an anti-Golgi-58kDa protein monoclonal antibody as a counterstain. As shown in Figure 4C, the subcellular location of CSR protein overlaps with that of Golgi-58kDa protein, indicating that CSR proteins are likely to be located in the ER-Golgi apparatus. We repeated this experiment after oxidative damage, by treating the transfected cells on the slide with 10 µM hydrogen peroxide for 1 h and incubating them in complete medium for 24 h before immunostaining with anti-HA antibody. As shown in Figure 4D, the CSR proteins diffused widely into the cytoplasm, suggesting that they were secreted into this region to meet the challenge of oxidative damage.
Figure 4. Subcellular localization of the CSR protein. The HA epitope-tagged CSR gene was overexpressed in HeLa cells. Anti-HA polyclonal antibodies were used for immunocytochemical staining (left panels). Nuclei were stained with DAPI (right panels). (A) Parental HeLa cells transfected with vector only served as a negative control for background. (B) HeLa cells transfected with an HA-tagged CSR expression vector show a strong signal in the cytoplasmic region near the nucleus. (C) Anti-Golgi-58K protein counterstain defines the ER-Golgi region in the cell. The signal overlaps with that of HA-tagged CSR protein. (D) After exposure to hydrogen peroxide, CSR protein is still present in the ER-Golgi region, but the signals spread wider in the cytoplasm.
Role of CSR in protection from cell death
Genes that respond to cellular stress contribute to determining whether a cell lives or dies. To investigate CSR function further with respect to cellular damage, we performed a test of survival under UV irradiation using cell line H1299. We transfected H1299 cells with the vector only (H1299-vector), with CSR1 (H1299-CSR1), with CSR2 (H1299-CSR2) or with CSR1 and CSR2 together (H1299-CSR1/CSR2). Stable transfectants were selected through exposure to geneticin for 10 days. Each was examined for expression of CSR1 or CSR2 by RT-PCR, and the colonies that showed the highest level of expression were chosen for further analysis. Figure 5 summarizes the average survival rate of each line in three independent experiments. Cells designed to overexpress both CSR1 and CSR2 seemed to be significantly more resistant to UV damage than their parental cells or cells transfected with vector only. The amount of CSR1 and CSR2 expressed in the transfected cells was examined by RT-PCR. It shows that the stronger effect of co-expression of both CSR1 and CSR2, compared with expression of each by itself, is not due to a quantitative combined higher amount of expressed proteins (data not shown). The results suggest that CSR1, which is structurally similar to macrophage scavenger receptor, may play a significant role in response to cellular stress, probably mainly oxidative stress, and that it contributes to cell survival together with CSR2. To investigate the mechanism of oxidative stress resistance further, we measured the amount of ROS in CSR-overexpressing cells and their parental cells (H1299-vector) using 2[prime],7[prime]-dichlorofluorescein diacetate (DCFH-DA) and flow cytometry after exposure to additive hydrogen peroxide. This method is based on the increase of fluorescence activity according to oxidation of DCFH-DA in cells (16). The results shown in Figure 6A clearly indicate that ROS were depleted in CSR1/CSR2-overexpressing cells, while ROS were significantly increased in cell line H1299-vector after 24 h; partial effects were observed in cells overexpressing CSR1 or CSR2 only. It is likely that CSR proteins play an important role in protecting cells by scavenging the ROS produced by oxidative stress. The effect of detoxification by CSR proteins was also seen as a change of cell morphology after oxidative damage. Figure 6B shows differences in the shape of cells exposed to 20 µM hydrogen peroxide for 1 h. CSR-overexpressing cells reverted more rapidly to their normal morphology in comparison with parental cells or H1299-vector transfectants. This result further supported our hypothesis that CSR proteins protect cells by removing cell death-inducing products generated by oxidative stress.
Figure 5. The protective role of induced CSR gene products against cellular damage by UV radiation. Histogram bars representing parental cell, vector-transfected cells, CSR1- or CSR2-overexpressing cells, and CSR1/CSR2-overexpressing cells are identified in the key. After 10 days, surviving cells were measured by MTT assay. Three independent experiments were performed and averaged. Data are presented as means ± SD. Figure 6. Scavenging activity of CSR gene products. Cells were exposed to 20 µM hydrogen peroxide for 1 h and cultured further in fresh medium. (A) Fluorescence distribution of DCFH oxidation by FACS analysis reveals that the level of intracellular ROS is unchanged in CSR1/CSR2-overexpressing cells, significantly increased in cells transfected only with vector and increased only slightly in cells overexpressing either CSR1 or CSR2 separately. Cells were harvested for analysis at 30 min (line) or 24 h (bold line) after hydrogen peroxide treatment, or without hydrogen peroxide treatment (dotted line). (B) Morphological changes after hydrogen peroxide treatment in CSR1/CSR2-overexpressing cells and H1299-vector cells. Photographs were taken before treatment and at 0, 1 and 3 h after treatment.
DISCUSSION
We have demonstrated here that oxidative stress plays a significant role in up-regulation of CSR transcription and that CSR proteins protect cells from cellular damage by scavenging ROS. When cells are UV irradiated, UV light absorbed directly into the plasma membrane generates intracellular free radicals; UV also affects many types of cellular macromolecules, leading to formation of photoproducts (15). The UV light-dependent signal cascade generated on the plasma membrane subsequently is propagated by phosphorylated proteins. Genes activated in this way by UV light include all those for members of the Jun family, p53, c-Myc, NF-[kappa]B, IL-1, Ras, Raf-1, PKC and many others. Some of the genes activated in this signal pathway may be protective to cells, while others may be part of the cell-damaging machinery (17). Hydrogen peroxide also increases the expression of genes such as c-fos and c-jun; transcription of these early genes is enhanced through various signalling pathways involving MAPKs (8,18), and oxidative stress modulates this response. Eukaryotic cells continuously produce ROS as side products of the electron transfer reaction, and normally control the intracellular redox balance. However, UV radiation and stimulation with exogenous hydrogen peroxide undoubtedly generate ROS above normal levels, the condition referred to as oxidative stress.
Although in our experiments oxygen radicals that are well known as initiators of cell damage, including DNA breakage, were found to enhance CSR expression, anti-cancer drugs (such as adriamycin and bleomycin) damaging to DNA had no influence on this. Our data suggested that CSR expression correlates mainly with oxidative stress, not with DNA damage. The effect of pre-treatment with the antioxidant NAC that is taken up by cells and rapidly converted to glutathione, a scavenger of intracellular free radicals, supported the hypothesis that oxidative stress is likely to be the major factor inducing CSR expression.
Stress genes expressed under cell-damaging conditions are considered to function either to protect cells from death or to induce apoptosis. Our cell survival experiment provided evidence that the role of UV-inducible CSR gene expression is to protect against cell death (Figs 5 and 6). Cells that were overexpressing CSR1 became resistant to UV irradiation in comparison with parental or vector-transfected cells. In addition, we demonstrated that the CSR gene products depleted potentially harmful oxygen derivatives (Fig. 6). However, cells designed to overexpress CSR2 alone had only a small effect although CSR2 significantly enhanced the protective function of CSR1.
Although the mechanism involved in their protective function is still unclear, it is possible that CSR proteins remove intracellular free radicals or side products of cellular damage by transporting them from the ER to lysosomes in the same manner as a structural homologue, macrophage scavenger receptor. A collagen-like triple-helix domain (Gly-X-Y), a characteristic feature of macrophage scavenger receptor, is present only in CSR1 (Fig. 2). Collagenous domains are known to bind to a wide variety of molecules including lipids, macromolecules including plasma components (fibronectin, laminin, collagen, fibrinogen, etc.) and various negatively charged molecules (19). The DNASIS computer program predicted that the 49 uninterrupted Gly-X-Y triple repeats present in CSR1 protein would impart a positive charge to the molecule at physiological pH, because ~50% of its side chains are expected to be charged (Glu, Arg, Lys or Asp). The distribution of charges on the surface of the predicted superhelix could affect its binding to other molecules. Post-translational modification (11 possible N-glycosylation sites and many possible phosphorylation sites, Fig. 1) could also influence the behavior of CSR protein in response to cellular damage as well as its assembly, stability, intracellular sorting and transport. The physiological functions of macrophage scavenger receptors are not well understood, but they may participate in atherosclerosis, adhesion and host defence because of their broad binding specificities (20). We suspect that like the macrophage scavenger receptors, CSR protein might scavenge by-products generated inside damaged cells, harmful oxidative products in particular. This is a reasonable speculation in view of the location of CSR in the cytoplasmic region (Golgi-ER zone, Fig. 4), while macrophage scavenger receptors are in the plasma membrane (21), and its wide secretion into the cytoplasm under oxidative stress.
The presence of potential sites for heme binding and a C-terminal targetting signal for microbodies including microsomes and other organelles (identified in CSR2 protein), that is also present in many redox-related or carrier proteins including cytochrome c, acyl-co-enzyme A oxidase and sterol carrier protein, supports our suggested mechanism of cell protection. Others have reported that overexpression of Raf-1, Ras or Src renders cells more resistant to UV radiation (9). All of these gene products are activated by oxidative stress. Other redox-related genes such as heme oxygenase, ferritin, superoxide dismutase and glutathione reductase also protect cells from oxidative stress, through either their ability to scavenge free radicals or their enzymatic activity (22,23).
Accumulated cell damage contributes to development of cancer or other diseases. The evidence shown in this study of CSR and its ROS scavenging activity open interesting avenues to pursue with regard to the mechanisms of signal pathways elicited by cellular oxidative stress, and prompt us to investigate the regulatory factor for CSR expression. Moreover, the protective function of CSR protein against UV irradiation is of considerable significance because harmful UV radiation is gradually increasing worldwide. Considering the recognized physiological roles of oxidative products in several diseases and in the aging process, further studies of CSR function may yield clues to the cellular mechanisms involved in such conditions. In addition to the protective function against cellular oxidative stress, other important biological roles of CSR should be investigated further.
MATERIALS AND METHODS
Isolation and sequencing of cDNA
The cosmid clone c2 was obtained from human genomic DNA as a part of the human genome analysis of chromosome 8p in our laboratory. The entire genomic DNA sequence contained in cosmid clone c2 was determined by the `shot-gun' method using an ABI377 autosequencer (Perkin Elmer). This sequence was analyzed with the GRAIL 2 (24) and HEXON (25) computer programs to predict probable exons. Each candidate exonic sequence was used as a hybridization probe to screen 5×105 plaques of a random-primed fetal brain cDNA library (Stratagene). Four clones were obtained; their nucleotide sequences were analyzed by searching for structural homologies, secondary protein structure and motifs in the GenBank and EMBL databases by means of the BLAST (26), FASTA (27), COIL (28), PredictProtein (29), PROPSEARCH (30) or PROSITE (31) computer programs. The archived data are updated daily at the Human Genome Center, Institute of Medical Science, University of Tokyo.
Experimental cell damage and cell treatment
The normal human fibroblast cell line NHDF4042, derived from neonatal skin, was purchased from Iwaki (Japan). The immortal tumour cell lines including HeLa subclone (cervical carcinoma) and H1299 (lung carcinoma) were obtained from the ATCC (Rockville, MD). All cultures were maintained in recommended media in 5% CO2 at 37°C. Subconfluent (50-70%) cultures were treated 24-48 h after seeding as described previously (32,33), with radiation or with one of the agents described below.
UV irradiation from a 40 W UV lamp (Toshiba, Japan) was performed at a 50 cm distance for 5-120 s. Before irradiation, the medium was removed and cells were washed twice with PBS. New medium was added immediately after UV treatment. The UV dose was estimated with a Radiometer model 254 (Atto, Japan). Hydrogen peroxide (30% solution; Wako, Japan) was added to medium containing 1% fetal bovine serum (FBS) after the cells were washed twice with PBS. For other experiments, adriamycin (Kyowa, Japan) and bleomycin (Nippon Kayaku, Japan) were diluted with PBS to the appropriate concentrations. For adriamycin treatment, cells were exposed for 24 h at various concentrations. For bleomycin, 70 µg/ml was added to the medium; after 4 h of exposure to this reagent, the cells were washed with PBS and fresh complete medium was added.
Transfections were performed using the mammalian expression vector pcDNA3.1 (Invitrogen) and TransIT[trade]-LT1 (Mirus) according to the manufacturer's instructions. Another mammalian expression vector, pCMV-[beta] (Clontech), was used each time to permit us to monitor the efficiency of transfection by means of [beta]-galactosidase staining 24 h later. To select stable transfectants, we cultured the cells for 10 days in medium containing Geneticin (G418 sulfate) (Gibco BRL). Isolated single colonies were analyzed by RT-PCR and/or western blotting to confirm stable expression of CSR or HA epitope-tagged CSR genes.
Flow cytometry
Cells (2×105 total) were exposed to 20 µM hydrogen peroxide for 1 h and then cultured further with fresh complete medium. Before analysis by flow cytometry, cells were pre-incubated with 5 µM DCFH-DA (Molecular Probes) for 30 min at 37°C. After pre-incubation, cells were washed with PBS and harvested. Cells were resuspended in PBS and analyzed with FACSCalibur (Becton Dickinson). Equal numbers of cells from the gates were recorded in the same condition.
Northern analysis
Northern blots containing poly(A)+ RNA from normal human tissues were purchased from Clontech. To examine CSR expression after treatment, RNAs were extracted from cells using Trizol (Gibco BRL) according to the manufacturer's protocol. Poly(A)+ RNA was isolated using Oligotex-dT30 [super] (JSR, Japan) according to the manufacturer's suggested protocol. Isolated mRNAs (0.5-1 µg) were separated on 1.5% agarose gels with 20% formaldehyde, transferred to Biodyne B transfer membranes (PALL) and hybridized in 50% (v/v) formamide, 5× SSPE, 2× Denhardt's solution, 2% (w/v) SDS and 1×106 c.p.m./ml of an [[alpha]-32P]dCTP-labeled DNA probe generated by random priming. CSR RNA levels were normalized to the level of actin RNA. Hybridized membranes were washed twice in 2× SSC/0.05% SDS at room temperature for 15 min and once in 0.1× SSC/0.1% SDS at 50°C for 10 min. Northern membranes were exposed to BAS 1000 imaging plates (Fuji, Japan) for 12-24 h. A 0.24-9.5 kb RNA ladder (Gibco BRL) was used as a size marker for electrophoretic bands.
Immunocytochemical staining
Full-length CSR1 and CSR2 were cloned into mammalian expression vector pcDNA3.1 (Invitrogen). Constructed vectors were confirmed by sequencing. An HA epitope tag (YPYDVPDYA) (34) and restriction sites were placed at the N- or C-termini of CSR. Epitope-tagged cDNA was transfected into HeLa cells; cells that transiently expressed CSR were seeded in a two-well slide chamber (Falcon) and cultured for 24 h before immunocytochemical staining. Cultured cells were washed briefly with PBS and fixed using cold methanol. After washing with PBS, cells were incubated at room temperature for 30 min in 2% bovine serum albumin, and subsequently incubated with anti-epitope (HA) polyclonal antibodies (MBL) at 37°C for 1 h. Incubation with fluorescein isothiocyanate (FITC)-conjugated second antibodies (Cappel) at 37°C for 40 min was followed by washing with PBS three times. For counterstaining, anti-Golgi-58K antibody (Sigma) and rhodamine-conjugated second antibodies (Leinco Tech.) were used in the same procedure. Nuclei were stained with 4[prime],6-diamidine-2[prime]-phenylindole dihydrochloride (DAPI) (Boehringer Mannheim) and observed under a fluorescence microscope (Nikon Eclipse E800).
ACKNOWLEDGEMENTS
This work was supported in part by a special grant for Strategic Advanced Research on Cancer from the Ministry of Education, Culture, Sports and Science of Japan, and a `Research for the Future' Program Grant (96L00102) of the Japan Society for the Promotion of Science.
REFERENCES
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