Nick Brown2, Ken-ichi Arai2, Takashi Yokota2,. Hiro Wakasugi3 and Junji ..... region of the IL-2R/Tac gene (Inoue et al., 1985; Sabe et al., 1988). In this study ...
The EMBO Journal vol.8 no.3 pp.757 - 764, 1989
ATL-derived factor (ADF), an IL-2 receptor/Tac inducer homologous to thioredoxin; possible involvement of dithiol-reduction in the IL-2 receptor induction Yutaka Tagaya, Yasuhiro Maeda, Akira Mitsui', Nobuo Kondo', Hiroshi Matsui', Junji Hamurol, Nick Brown2, Ken-ichi Arai2, Takashi Yokota2, Hiro Wakasugi3 and Junji Yodoi Institute for Immunology, Faculty of Medicine, Kyoto University, Yoshida-Konoe, Sakyo, Kyoto 606, Japan, 'Central Research Laboratory, Aji-no-mo-to Co. Ltd., Kawasaki, Kanagawa, Japan, 2DNAX Research Institute of Molecular and Cellular Biology, Inc., 901 California Avenue, Palo Alto, CA 94304-1104, USA and 3Laboratoire d'immunobiologie des Tumeurs, Laboratoire Associe 1156, Centre de la Recherche Scientifique, Institut Gustave-Roussy, 94805 Villejuif, France Communicated by P.Reichard
HTLV-I transformed T cells not only express a large number of interleukin-2 receptors [IL-2R/p55(Tac)], but also produce a factor named ATL-derived factor (ADF) that augments the expression of IL-2R/p55(Tac). Based on a partial N-terminal amino acid sequence, complementary DNA (cDNA) clones for human and mouse ADF were isolated and sequenced. Recombinant ADF produced by COS-7 monkey kidney cells showed IL-2R/Tac inducing activity on YT cells, which are sensitive for ADF. ADF mRNA was strongly expressed in HTLV-I(+) T cells lines, but not in inactivated cells (THP-1, unstimulated PBMC). Furthermore, in normal human peripheral blood mononuclear cells, the expression of ADF mRNA was enhanced by mitogens or phorbol myristate acetate, suggesting a possible involvement of ADF in the lymphocyte activation. Homology analysis revealed an unexpected relationship between ADF and dithiol-reducing enzyme, thioredoxin, involved in many important biological reactions such as the conversion of ribonucleotides into deoxyribonucleotides, or the stabilization of glucocorticoid receptors. The biological significance of the generation of a redox potential in lymphocyte activation, and the possible involvement of dithiol reduction in the induction of IL-2R/Tac are discussed. Key words: adult-T cell leukemia (ATL)/adult-T cell leukemia derived factor (ATL-derived factor; ADF)/dithiolreduction/IL-2 receptor/Tac antigen/thioredoxin
Introduction Interleukin-2 (IL-2) (Morgan et al., 1976) has a number of biological activities including growth promotion and activation of various lymphoid cells such as T, B and large granular lymphocytes (LGL) with natural killer (NK) activity. High-affinity receptors for IL-2 (IL-2R) are composed of at least two distinct molecules. IL-2R/p55(Tac) (Uchiyama et al., 1981; Leonard et al., 1982) and the recently described IL-2R/p70, an IL-2 binding molecule different from the Tac protein (Sharon et al., 1986; Tsudo et al., 1986; Robb et al., 1987; Teshigawara et al., 1987). (IRL Press
IL-2 signal transduction is tuned by altering the number of high-affinity receptors through the regulation of IL-2R/Tac. Adult T cell leukemia (ATL) (Yodoi et al., 1974; Uchiyama et al., 1977), closely associated with the human T lymphotropic virus-I (HTLV-I) (Poiesz et al., 1980; Hinuma et al., 1981), is an endemic leukemia originally described in Japan. Abnormal high expression of IL-2R/p55, the Tac antigen, is a characteristic feature of ATL leukemic cells (Uchiyama et al., 1985; Yodoi and Uchiyama, 1986) despite the lack of significant IL-2 production by these cells (Yodoi et al., 1983; Arya et al., 1984). It has been suggested that the unregulated expression of IL-2R/Tac plays important roles in the leukemogenesis of ATL. Using a human NK-like cell line YT (Yodoi et al., 1985) bearing a regulable IL-2R/Tac, we have reported a variety of IL-2R/Tac inducers. These include lymphokines such as IL-Is (Shirakawa et al., 1986; Lubinski et al., 1988) and IL-2 (Tagaya et al., 1987), phorbol esters, forskolin, an activator of adenylate cyclase (Narumiya et al., 1987), and the p40x gene of HTLV-I (Sabe et al., 1988). Besides these IL-2R/Tac inducers, we have reported an ATL-derived factor (Yodoi et al., 1984; Okada et al., 1985; Teshigawara et al., 1985; Tagaya et al., 1987, 1988) produced by many HTLV-1 transformed T cells. ADF enhances the expression of IL-2R/Tac not only on YT cells, but also on some HTLV-I transformed T cells (Okada et al., 1985) through enhanced transcription of the IL-2R/Tac gene (Tagaya et al., 1987). It was implicated that ADF is an autocrine factor involved in the abnormal expression of IL-2R/Tac in HTLV-I transformed cells. Some lymphokines, such as IL-lot and 1, were recently reported to have IL-2R/Tac inducing activity (Shirakawa et al., 1985; Lubinski et al., 1988). Although IL-la was produced by some HTLV-I transformed T cell lines, there was no apparent correlation between the IL-2R/Tac inducing activity and the quantity of IL-la (Noma et al., 1986). Our previous report (Tagaya et al., 1988) showed the differences between ADF and IL-la in target cell specificity as well as in antigenicity. Subsequently, we purified the ADF protein, and determined the partial N-terminal amino acid sequence, which had no significant homology with many cytokines including all the interleukins whose cDNA had been cloned (Tagaya et al., 1988). Wakasugi and co-workers independently reported an IL-l like factor produced by an EBV transformed B cell line 3B6 (Rimsky et al., 1986; Wakasugi et al., 1987). This factor, named 3B6-IL-1, was different from known IL-1 species (IL-la and ,B). The 20 N-terminal amino acid sequence of 3B6-IL-1 was identical to that of ADF, indicating a close relationship of the two factors. We cloned both human and mouse ADF cDNA clones using oligonucleotide probes based on the amino acid sequence of purified human ADF. ADF proved to have an unexpected homology with thioredoxin, an enzyme catalyzing reduction of proteins and ribonucleotides in many 757
Y.Tagaya et al.
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IrAMAACCI T90% on the amino acid level. Expression of ADF product in mammalian cells Two ADF clones in vector pCDSRcr named pADF-2 and pADF-3 were expanded and transfected to monkey COS cells using the DEAE-dextran method (Yokota et al., 1985). After 48 h culture, the supernatant and cell pellets were collected. The supernatant was subjected to YT cell assay. As shown in Figure 2A,the supernatant of transfected cells showed clear IL-2R/p55 (Tac) inducing activity on YT cells (Figure 2a-C, increase in mean fluorescence intensity (MFI); 24.8-97.9). In contrast, the supernatant of control COS cells showed little or marginal ADF activity (Figure 2a-B), increase of MFI; 24.8-26.6). As a positive control, crude ADF was also examined (Figure 2a-A, increase of MFI;
24.8-78.7). Detection of ADF product in the COS cell supernatant To confirm that the IL-2R/p55 (Tac) inducing activity of COS-7 cell supernatant was due to the transfected gene product, the supernatant was subjected to Western blot
analysis using anti-ADF polyclonal antibody raised against the N-terminal 10mer synthetic peptide of ADF conjugated with BSA to check the correlation between IL-2R/Tac inducing activity and the quantity of ADF protein. As shown in Figure 2b, COS-7 cells transfected with ADF cDNA produce a protein recognized by the anti-ADF antibody (Lanes 2 and 6), when the supernatant of mock COS-7 cells showed only a marginal band (Lanes 1 and 5). By the transfection of human ADF clone into COS-7 cells, both intracellular (lysate) and secreted (supematant) forms of ADF were remarkably increased. The distribution of secreted form of ADF is shown to be -50% by the density of the band detected by autoradiography (Figure 2c; Lane 1 versus 3). These data correlate well with the measured ADF activity in the supernatant. Expression of ADF mRNA in lymphoid cells Using the human NK-like cell line YT, we screened the supernatant of several human lymphoid cells for IL-2R/Tac inducing activity. Most HTLV-I( +) T cells such as ATL-2 (Maeda et al., 1985) and Hut 102, showed potent ADF activity in their supernatant. Furthermore, one EB virus transformed B cell line, 3B6 (Wakasugi et al., 1987) producing the IL- 1 like factor also showed ADF-like
760
IL-2R/Tac inducing activity in the supernatant (data not shown). By Northern blot analysis using ADF cDNA as a probe, we observed the expression of a single species of mRNA with a size of 0.6 kb in HTLV-I( +) ATL-2 (Figure 3A, lanes 1 and 2), Hut 102 (lane 3) and EBV (+) 3B6 cells (lane 4). Myeloid/monocyte cell lines, THP-l or U937, did not show detectable levels of ADF mRNA expression (lanes 8 and 9), which is consistent with the data that ADF activity was not detected in the supernatant of these cells (Teshigawara et al., 1985). On the contrary, when human peripheral blood mononuclear cells (PBMC) were stimulated with phytohemagglutinin (PHA; 10 ,g/ml) or phorbol myristate acetate (PMA; 20 ng/ml), the induction of ADF mRNA was observed (lanes 5-7). At the same time, significant enhancement of IL-2R/Tac inducing activity was detected in the supernatant (data not shown).
Comparison of genomic DNA structure coding ADF gene between normal and A TL-derived cells Since ADF is produced by most HTLV-I transformed cells, we studied the possible structural changes in the ADF gene between normal and HTLV-I transformed cells by Southern blot analysis. High molecular weight DNA from human normal PBMC and HTLV-I(+) T cell line ATL-2 was digested by restriction endonuclease and hybridized with 32P-labelled ADF cDNA. As shown in Figure 3B, no apparent difference was detected between these cells. Homology between ADF and thioredoxin Based on the obtained sequence, we screened the database of protein primary structure to search for proteins homologous to ADF. As expected from the partial amino acid sequence, there was no significant homology to known cytokines, except for IL-1f3 and the whole ADF sequence. There was a marginal similarity between ADF (iesidues 70-93) and IL-1(3 (residues 178-218) (data not shown). As shown in Figure 4, human and mouse ADF had a significant homology with Escherichia coli derived thioredoxin (Holmgren, 1968). In mammalian cells, the sequence of the active site (Holmgren, 1985) and a partial amino acid sequence of thioredoxin from rat Novikoff hepatoma has been published (Guevera et al., 1983). This partial amino acid sequence was highly homologous to human and mouse ADF, suggesting a close relationship between ADF and thioredoxin. Effect of dithiol-reduction on IL-2R/Tac inducing activity of ADF To clarify the relationship between IL-2R/Tac inducing activity of ADF and dithiol-reducing activity of ADF deduced from the homology to thioredoxin, the effect of 2-mercaptoethanol (2-ME) on the induction of IL-2R/Tac of YT cells was examined. As shown in Table I, crude ADF, COS derived ADF and E. coli thioredoxin have IL-2R/Tac inducing activity, which was further enhanced 3-fold by pretreatment with l0-3 M 2-ME. On the contrary, the IL-2R/Tac inducing activity of IL-lax was not affected by the same treatment (data not shown). In addition, 2-ME alone did not affect the expression of IL-2R/Tac on YT cells [medium versus medium (2-ME)]. These results collectively suggested that dithiol-reduction specifically modulates the IL-2R/Tac inducing activity of ADF, but not that of IL-1. -
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Discussion Previous studies on the mechanism of the abnormal expression of IL-2R/Tac by HTLV-I have collectively suggested that p40%, a trans-activator protein encoded by the pX region of the HTLV-I genome, activates the expression of IL-2R/Tac by interacting with some unknown cellular factors rather than directly binding to the 5' upstream region of the IL-2R/Tac gene (Inoue et al., 1985; Sabe et al., 1988). In this study, we isolated the ADF cDNA clone which directed the production of ADF protein having IL-2R/Tac inducing activity in COS cells. To clarify whether p4ox promotes the production of ADF or not, analysis of the promoter region of ADF gene is required. It is particularly interesting that the ADF protein is homologous to the dithiol reducing enzyme, thioredoxin, which is a potent endogenous hydrogen donating enzyme. The published 41 amino acid sequence of rat Novikoff hepatoma thioredoxin (Guevara et al., 1983) is highly homologous to that of human and mouse ADF protein. Recombinant ADF produced by monkey COS-7 cells and E. coli can catalyze the degradation of insulin (A.Mitsui, unpublished observation), one of the reactions specific for thioredoxin (Holmgren, 1979). It is thus possible that ADF is identical to thioredoxin. However, the existence of other proteins homologous to thioredoxin such as protein-disulfide isomerase (Edman et al., 1985) and some of the phospholipase C isozymes (Benette et al., 1988) has also been reported. It is urgently required to clarify whether ADF
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761
Y.Tagaya et al. Table I. Effect of dithiol-reduction on the IL-2R/Tac inducing activity of ADF Mean fluorescence intensity
Sample
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23.7 24.3 56.3 83.4 43.0 68.4
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NT, Not tested. Samples were incubated with or without l0-3 M 2-ME overnight at 370C, and were dialyzed against RPMI-1640 at 40C. The IL-2R/Tac inducing activity of each sample was examined by YT cell assay. The 2-ME treated sample are cited as (2-ME). ADF; Purified ADF from the culture supernatant of ATL-2, COS-ADF; recombinant ADF produced by COS cells (as described in Figure 2). Thioredoxin; Ecoli thioredoxin.
is the human thioredoxin itself or a unique member of such a thioredoxin gene family. Thioredoxin contains a redox active disulfide (-Cys Gly - Pro -Cys-) and has a variety of biological activities as a hydrogen donor, including formation of deoxyribo-
nucleotides from ribonucleotides catalyzed by ribonucleotide reductase (Laurent et al., 1964), degradation of insulin (Holmgren, 1979) and activation of the glucocorticoid receptor (Grippo et al., 1983). In phage T7, host E. coli thioredoxin is associated with T7 DNA polymerase (Mark et al., 1976) and essential for phage DNA replication (Tabor et al., 1986). The potentiation of the IL-2R/Tac inducing activity of ADF by 2-ME pretreatment indicates the possible involvement of certain types of reduction/oxidation reactions in the ADF-mediated target cell activation. There is accumulating evidence indicating the crucial role of thiol reducing agents for optimal growth of animal cells. Dithiol-relating reducing agents such as 2-ME (Goodman and Weigle, 1979) or 8-mercaptoguanosine (Goodman and Weigle, 1983) support the growth of lymphoid cells. 2-ME can partly replace the role of macrophages in antigenpresentation reactions (Chen and Hirsch, 1972). A serum factor that acts as a target of these reducing agents (2-ME activated serum factor; MaSF) (Opitz et al., 1978) has been also reported. Unanue et al. also reported a similar factor in the serum as 2ME-FCS (Sidman and Unanue, 1979). Considering the possibility that ADF, as the endogenous dithiol reducing agents, promotes antigen-independent growth of lymphocytes in vivo, the involvement of ADF in the HTLV-I-related transformation is quite likely. Sequence analysis of ADF cDNA showed the lack of a signal peptide. While many secreted proteins have signal peptides in the N-terminal region, some cytokines such as PDGF or IL- la and ,3 lack the signal peptide. Interestingly, metabolic labeling by [35S]Met suggested the active release of ADF product in the supernatant of transfected COS-7 cells. Histochemical analysis using anti-ADF antibody showed the localization of ADF on the cell surface of HTLV-I(+) T cell line cells. Since thioredoxin has been
762
shown to be located at membranes in mammalian cells (Rozell et al., 1985; Stemme et al., 1985), this fact may give an alternative possibility that membrane bound ADF may be released as a soluble ADF. As a soluble factor, ADF may have its receptor on the target cell surface. However, considering the homology between ADF and thioredoxin, which is often coupled with a specific NADPH dependent reductase system (Holmgren, 1985), it is also possible that a specific reductase protein may act as an ADF acceptor. A stoichiometric analysis using radiolabeled recombinant ADF will clarify the nature of the cell surface binding molecules for ADF. The specific activity of purified ADF in the induction of IL-2R/Tac in YT cells was -10-fold lower than that of IL- 1 (Tagaya et al.; Wakasugi et al., unpublished observations), suggesting that the mode of ADF activity is somehow different from that of IL-I, which was shown to act by way of binding to a specific receptor molecule. The fact seems to be consistent with the possibility that the action of ADF may be mediated by certain types of oxidation/reduction reactions. Indeed, our preliminary data showed that ADF in the culture supernatant of ATL-2 cells has a potent reducing activity (T.Inamoto et al., in preparation). Northern and Western blot analysis clearly indicated the inducible nature of ADF in lymphoid cells. Human PBMC stimulated with mitogens or phorbol esters produced markedly larger amounts of ADF than in the resting state, indicating that the generation of ADF with dithiol reducing activity is necessary for the growth and activation of hematopoietic cells. There is evidence showing unexpected relationships between the cytokine system and the metabolic pathway. The receptor for insulin like growth factor II (IGF-II) is identical to mannose-6-phosphate binding protein (Morgan et al., 1987). Chaput et al. (1988) demonstrated that mouse
neuroleukin is 90% homologous with pig muscle phosphohexose isomerase, an enzyme catalyzing the conversion of glucose-6-phosphate to fructose-6-phosphate in glycolysis. These data may imply a possible general relationship between certain cytokine systems and intracellular metabolic enzyme systems, inspiring an attractive model for the evolutionary origin of some cytokine systems. Work is in progress to clarify the role of ADF in the physiological and pathological activation of animal cells including lymphocytes.
Materials and methods Cells and tissue culture YT cells, established from a 14 year old ALL patient, are a human LGL cell line bearing NK-like activity. ATL-2 cells are a HTLV-I transformed T cell line established from an ATL patient. Both cell types were maintained in RPMI 1640 medium (Nissui Co. Ltd., Tokyo, Japan) supplemented with 2 mM L-glutamine/10% heat inactivated fetal calf serum (FCS; MBA Laboratory, Walkersville, MD) in the presence of antibiotics. Human PBMCs were isolated from venous blood of a healthy donor by using Ficoll -Hypaque (Pharmacia, Uppsala, Sweden). Isolated cells were plated in 24-well tissue culture plates (COSTAR, Cambridge, MA) at a concentration of 2 x 106 cell/ml in 10% FCS-RPMI 1640 medium. For the stimulation of these cells, 10 Itg/ml phytohemagglutinin (Difco Laboratories, Detroit, MI) or 20 /g/ml phorbol myristate acetate were added to the culture. Lytl +2-/9 cells were from a cloned T cell line derived from C57BL6 mice and were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 50 /M 2-mercaptoethanol/2 mM L-Gln/10% heat inactivated FCS.
Dithiol-reduction in IL-2R induction by ADF Biological assays for ADF For the purpose of quantitation of the IL-2R/Tac inducing activity of ADF or other materials, cells were cultured with the ADF sample for 24 h at 37°C and stained with FITC-conjugated anti-TAC mouse IgG2a mAB and analyzed by flow cytometry using Ortho Spectrum IIITM (Ortho Pharmaceutical, Raritan, NJ) in the linear fluoresence scale (Channels 0-255). Gains of the green detector were set so that the mean fluorescence intensity of unstimulated YT cells was -25 in the histogram channel.
Isolation of mRNA and Northern blot analysis Total cellular RNA was extracted from the ATL-2 cells by guanidium thiocyanate extraction. mRNAs with poly(A)+ tails were purified using oligo(dT) -cellulose affinity chromatography. Approximately 100 ug of poly(A)+ mRNA was obtained from 1-2 x 10 cells. Polyadenylated RNA (5 jig) or total RNA (20 14g) was denatured using glyoxal and DMSO at 50°C for 1 h and then subjected to 1.2% agarose gel electrophoresis. RNA was blotted onto a nylon membrane (Genescreen plusTM, NEN, Boston, MA) and was hybridized with 32P-labeled ADF cDNA for 24 h at 42°C, washed in 1 x SSC, 0.1 % SDS and autoradiographed. Extraction of cellular high mol. wt DNAs and Southern blot analysis High mol. wt DNAs were extracted from the cells and were cut by the restriction endonuclease EcoRI. A 5 ,ig DNA sample was subjected to 0.7% Agarose electrophoresis, blotted onto a nitrocellulose filter (Schleicher and Schuell, Dassel, FRG) and hybridized with 32P-labeled ADF cDNA for 24 h at 42°C, washed in 2 x SSC and 0.2 x SSC sequentially, and then autoradiographed.
Construction of cDNA libraries On a template of 5 jg mRNA, a RNA -DNA heteroduplex was constructed using AMV reverse-transcriptase (Life Science, St Petersburg, FL). The hybrid strand was treated with RNase H, and double stranded cDNA was synthesized by DNA-dependent DNA polymerase. An EcoRI linker was attached to both ends of the cDNA, and digested with restriction enzyme EcoRI. The EcoRI-EcoRl fragments were inserted in the XgtlO vector also treated with EcoRl. The pcDSRca cDNA libraries were constructed with mRNA from ATL-2 cells and Con A stimulated mouse Lyt +2 -/9 cells by using a modified pcDV I plasmid containing a poly-linker at the previous location of the BamHI site and modified pLl plasmid carrying the R-U5 sequence of HTLV-I long terminal repeat downstream to the SV40 early region promoter. Probes were labeled with 32P at the 5' end using T4 polynucleotide kinase (Takara, Otsu, Japan). The isolated clones were digested with EcoRI. DNA sequence analysis The nucleotide sequence of cDNA inserts from XgtlO or pCDSRn libraries were analyzed by the dideoxy chain termination method with denatured supercoiled plasmid DNA.
DNA transfection and biosynthetic labeling with [35S]Met Monkey derived COS cells (- 106) were seeded onto 60 mm plates 1 day before transfection. Transfections were performed with 10 14g plasmid DNA in I ml DMEM containing 50 mM Tris-Cl (pH 7.4) and 400 ug DEAE-dextran (Pharmacia) per ml. After 4 h, the solution was removed and replaced with 2.0 ml DMEM containing 1% FCS and 1 mM chloroquine. After 48 h culture, cells were labeled with 0.5 mCi of [35S]Met in 0.5 ml (per well of 24-well plate) of DMEM for 16 h at 370C. Radiolabeled cells and conditioned medium were collected separately. Cells were lysed in SDS-PAGE buffer, subjected to SDS-PAGE, and then autoradiographed.
Immunoblotting of ADF samples Samples were subjected to 15% SDS -PAGE under reducing or non-reducing conditions, and transferred to a polyvinyllidene difluoride (ImmobilonTM Millipore, Bedford, MA) membrane in 0.1 M Tris, 0.192 M glycine, 20% methanol. The filter was blocked in 2% skim milk (Difco Laboratories, Detroit, MI), 0.05% Tween 20-PBS, pH 7.2, overnight at 40C, and incubated with specific anti-ADF antibodies (Tagaya et al., 1988). After several washes in 0.05% Tween 20-PBS, the filters were hybridized with biotinylated-goat anti-rabbit polyclonal antibody, and developed using a Bectastain kit (Vector Laboratories, Burlingame, CA) according to the supplier's recommendation.
2-ME For the reduction, all samples were incubated with or without 10-3M 2-ME at 37°C overnight and were extensively dialyzed against serum free
RPMI 1640. The reduced and non-reduced samples were then subjected to YT cell assay. ADF was purified from the supernatant of ATL-2 as described elsewhere (Tagaya et al., 1988). E.coli thioredoxin was purchased from IMCO (Stockholm, Sweden).
Acknowledgements We greatly appreciate the valuable advice of Drs Keisuke Teshigawara, Masafumi Okada, Hisataka Sabe, Shigetada Nakanishi and Tasuku Honjo at Kyoto University, and give sincere thanks to Dr Arne Holmgren at Karolinska Institutet for stimulating discussions. We would like to thank Drs S.Tonouchi, and T.Sato at Aji-no-moto Co. Ltd., for their technical assistance in the cloning and sequencing of the human ADF gene, Patricia Meyerson for traiisfection work, Kyoko Yokota for constructing cDNA libraries in vector pCDSRa and to Ms M.Teranishi and I.Sasaki for writing this manuscript. This work was supported by a Grant-in-Aid for Scientific Research and Special Project Research-Cancer Bioscience from the Ministry of Education, Science and Culture of Japan.
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