Overexpression of Cysteine Sulfinic Acid Decarboxylase Stimulated by Hepatocarcinogenesis Results in Autoantibody Production in Rats'

Home , Alanine, Cysteine sulfinic acid, Histidine

ICANCERRESEARCH56. 5230-5237, November 15, 19961 Overexpression of Cysteine Sulfinic Acid Decarboxylase Stimulated by Hepatocarcinogenesis Results in Autoantibody Production in Rats'

Toshihiko Kishimoto, Kenji Kokura, Tomoyoshi Nakadai, Yaeko Miyazawa, Toshifumi Wakamatsu, Yasutaka Makino, Takeshi Nakamura, Eiji Hara, Kinichiro Oda, Masami Muramatsu, and Taka-aki Tamura2

Department of Biology, Faculty of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263 fT K., K. K., T N., Y. M., T W.. Y. M., T TI: Biomedical Research and Development Department. Sumitomo Electric Industries, Limited., Taya-cho, Sakae-ku, Yokohama 254 IT K., T NI; Department of Biological Science and Technology, Science University of Tokyo, Yamazaki, Noda 278 fE. H.. K. 0.1; and Department of Biochemistry, Faculty of Medicine. Saitama Medical School, Moroyama. Iruma-gun, Saitama 350-04 (M. MI. Japan

ABSTRACT taneously, and rats bearing various types of chemically induced HCCs, have been developed (13, 14). The Soft-Farber procedure (15) We developed a novel and efficient cDNA subtraction method to isolate is one of the reliable methods to produce HCCs reproducibly in rats by rat hepatocellular carcinoma (HCC)-related genes. cDNAs from Solt chemical carcinogens, and each step during the hepatocarcinogenesis Farber procedure-driven HCCs were synthesized on Latex beads. The subtraction was accomplished by a simple centrifugation, PCR amplifi has been well characterized. In the early stage of the Solt-Farber cation, and dot blot screening. Among 2000 clones from the subtracted procedure, hyperplastic nodules are generated in the precancerous cDNA library, one clone with a full-length 11CC-relatedcDNA was even liver, and several genes such as glutathione S-transferase P (14, 16) tually obtained. Sequence analysis ofthis clone showed it to exhibit 90 and and epoxide hydrolase genes (17) are induced in a carcinogenesis 60%similarity with the rat cysteinesulfinicaciddecarboxylase(CSAD) dependent manner. At the final stage, i.e., around 7 months, the and mammalian glutamic acid decarboxylases (GAD), respectively. Dif treated rats display typical hepatic cancer. ferences between our sequence data on CSAD and those reported previ It is known that carcinogenesis is linked to abnormal expression of ously were observed at two positions, which arose from a single amino acid genes that cause cancers (as oncogenes) or to proteins (as tumor substitution and frame shift mutation. The CSAD expression was re markers) whose genes are activated by carcinogenesis. There are stricted to the liver and kidney of rats. During hepatocarcinogenesis, several approaches to detect alterations in gene expressions. Among expression of the CSAD mRNA and its protein was stimulated in the them, the subtraction technique is frequently used. This technique is precancerous liver and maintained its high expression afterward. Inter estingly, a high level of anti-CSAD autoantibody was detected in the based on depletion of commonly expressed genes from a specific HCC-bearing rats. The titer of anti-CSAD autoantibodies in these rats gene-including gene population. Subtraction is, however, considered was 30â€”200timeshigher than that in normal rats. The anti-CSAD au to be complicated and inconvenient because it uses physically unsta toantibody appeared in the precancerous state and was maintained after ble mRNAs for nucleic acid hybridization; and it requires hydroxy ward, and its pattern of appearance was similar to that of CSAD mRNAs apatite (18) or enzyme reactions to remove commonly expressed and proteins. Thus, we propose that the high-titer CSAD autoantibody genes, as well as the preparation of a cDNA library (19â€”22). Thus, it resulted from increased CSAD gene expression in the liver due to stimu is necessary to develop a better subtraction technique. lation by the 11CC. These results remind us of human autoimmune To study the HCC-associated genes, we used HCCs produced by diseases including insulin-dependent diabetes mellitus and stiff-man syn the Solt-Farber procedure and developed a novel subtraction tech drome, which are caused by autoantibodies against GAD. nique using oligo(dT)-Latex particles (23, 24). By using these proce dures, we identified a gene for CSAD (25, 26). Moreover, we further INTRODUCTION demonstrated the presence of autoantibodies against CSAD in tumor Since a bona fide HCC3-related oncogene has not been identified, bearing rats. There was a good correlation between HCC progression although several external agents such as hepatitis B virus (1â€”4), and the appearance of autoantibody. These results have implications hepatitis C virus (5â€”7),and aflatoxin B (8, 9) are described to be for the possibility of conventional human autoimmune diseases in involved in hepatocarcinogenesis, there is a need for studies on patients with IDDM (27â€”29)and SMS (28, 30), both of which are hepatocarcinogenesis-dependent gene expression. In general, for caused by autoantibodies against GAD, an enzyme having significant studying such expression, the use of experimental animals is one structural and functional similarities with CSAD. suitable approach for the following reasons: (a) genetic background, exposure to carcinogens, diet conditions, and environmental factors can be well controlled; (b) it is easy to obtain a quantity of homolo MATERIALS AND METHODS gous materials; and (c) by using intact tissues, we can identify genes Preparation of Rat Hepatocellular Carcinoma and Extirpation of Rat that promote or prevent carcinogenesis as a body response in addition Liver. Male Wistar rats (5 weeks of age) were used. The Solt-Farber proce to cancer-priming genes. dure (15) was initiated by an i.p. injection of 200 mg/kg DEN. After the rats For investigation of hepatocarcinogenesis, LEC (Long-Evans with had been fed a basal diet for 2 weeks, oral administration of 0.02% a Cinnamon-like coat color) rats (10â€”12),which develop HCC spon 2-acetylaminofluorene was initiated. Three weeks after DEN injection, 70% partial hepatectomy was performed according to the standard protocol. Livers were extirpated in 1, 3, 5, and 7 months after DEN injection under anesthesia Received 4/26/96; accepted 9/16/96. The costs of publication of this article were defrayed in part by the payment of page and stored in liquid nitrogen. In most cases, rats die of HCC by 8 months after charges. This article must therefore be hereby marked advertisement in accordance with DEN injection. All treatments for the rats were according to guidelines 18 U.S.C. Section 1734 solely to indicate this fact. published in â€œInterdisciplinary Principles and Guidelines for the Use of Ani I This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas and for Encouragement of Young Scientists from the Japanese Ministry of Edu mals in Research, Marketing and Education.â€• cation, Science, Sports and Culture, and by grants from the Asahi Glass Foundation, the Synthesis of Subtracted eDNA Library. The subtraction method used in Naito Foundation, and the Ciba-Geigy Foundation (Japan) for the Promotion of Science. this study can be described as follows. First-strand cDNAs from normal and 2 To whom requests for reprints should be addressed. Phone: 81-43-290-2823; Fax: HCC livers are synthesized on oligo(dT)-Latex with dT3O primer, Oligotex 81-43-290-2824. (dT)30 Super (Takara Shuzo), and reverse transcriptase. Then HCC sense 3 The abbreviations used are: HCC, hepatocellular carcinoma; CSAD, cysteine sulfinic acid decarboxylase; GAD, glutamic acid decarboxylase; IDDM, insulin-dependent dia (second)-strand cDNAs are synthesized. Next, the HCC sense cDNAs are betes mellitus; SMS, stiff-man syndrome; DEN, diethylnitrosamine. hybridized with the Latex bead-immobilized first-strand cDNAs of the normal 5230

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1996 American Association for Cancer Research. HCC-ACTIVATEDCSAD AND ITS AUTOANTIBODY liver. After brief centrifugation, the supernatant is collected, and cDNAs in it particles. After the hybridization and centrifugation, the cDNA pop are amplified by PCR. The PCR products are then introduced into a pBlue ulation enriched for HCC-related cDNA was recovered in the super scriptll vector to prepare a subtracted library. natant, and cDNAs in the supematant were amplified by PCR. Am Dot Blot Screening. Two thousandsclones of plasmidDNAs containing plified cDNAs were introduced into the pBluescript vector, and E. coli HCC-related cDNA from the subtracted library were blotted on Immobilon N was transformed by these cDNAs. From comparison of clones ob (Millipore). To prepare probes for the dot blot hybridization, we synthesized tamed before and after subtraction, we found that the HCC-related mixed cDNAs from mRNAs of each tissue by primer extension with oh go(dT)15 primer (Boehringer Mannheim). Hybridization and washing were clones had been concentrated over 200-fold (data not shown), mdi carried out as recommended by the supplier. cating that we could perform cDNA subtraction efficiently. Preparation of RNA and cDNA Library. Methods to prepare total and We used 2000 colonies to screen for HCC-related cDNAs, using a poly(A) RNAs from normal rat liver and rat HCCs were described previously differential dot blot hybridization technique for this purpose. Plasmid (3 1). The eDNA library was prepared by the Gubler-Hoffman method (32) DNAs from each clone were blotted at corresponding positions on two using a TimeSaver cDNA synthesis kit (Pharmacia). Multiplicity of the library nylon membranes. Hybridization was performed by use of 32P-labeled was 1 X 106plaque-forming units per mg of phage arm DNA. mixed cDNA derived from either normal liver or HCC (Fig. 2). From Northern Blot Hybridization. Three hundreds nanograms of poly(A) comparison of the two signal intensities yielded by each probe, we RNAs were used for Northern hybridization. The standard hybridization mix found that 101 clones exhibited more intense signals with the HCC ture contained 1 X 106cpmlml cDNA probes, 5X SSC [20X SSC: 3 MNaC1, probe than with the normal one (data not shown). Thus, the content of 0.3 M sodium citrate (pH 7.0)], 5X Denhaldt's, 0.5% SDS, 100 mr@isodium PP1 (pH 6.8), 100 @g/mltRNA,100 @.tg/mldenaturedsalmon sperm DNA, and HCC-related clones in the subtracted library was 5%. We sequenced 50% formamide; and hybridization was carried out at 42Â°Cfor 16 h. Mem the inserts of all of these clones and identified with 31 independent branes for multiple tissue Northern blotting, i.e., membranes blotted with genes. The 70 other clones each contained sequences homologous to poly(A) RNAs from eight different tissues (heart, brain, spleen, lung, liver, either of those 3 1 genes. Homology search for the cDNAs indicated skeletal muscle, kidney, and testis), were purchased from Clontech. Hybrid that the above 3 1 clones included 6 known and 13 novel genes. The ization and washing of the multiple tissue Northern blots were carried out as remaining 12 clones were not able to be evaluated because of their recommended by the supplier. short inserts (Table 1). Of the six known genes, we found kininogen Expression of and Generation of Antibody for Recombinant CSAD. and haptoglobin genes whose expressions are enhanced in hepatitis or Recombinant CSAD with a histidine tag [Met-(His)6] at the amino terminus HCC (35â€”38). was overexpressed in Escherichia coli by use of a T7 expression system (33) Isolation of the CSAD Gene. We analyzed the gene expression of and purified with Ni-NTA agarose (Quiagen). The Ni-agarose-puritled CSAD the I3 novel gene-containing clones. RNAs from normal liver and was subjected to preparative SDS-PAGE, and the protein was further purified by electroelution with a Model 422 electroeluter (Bio-Rad). This purified HCC cells were examined by Northern blot analysis using the insert CSAD was used for generation of the antibody. The anti-CSAD antibody was DNA from each novel gene-containing clone as a probe. We found produced by the following method. At the first time, 100 @tgof CSAD was that HCC RNAs contained more transcripts than normal liver RNA injected in complete adjuvant. Two weeks later, 100 @gofCSAD in incom for all of the novel 13 clones and that the sizes of transcripts varied plete adjuvant was injected s.c. once a week for 6 weeks. depending on the clone (data not shown). Of the 13 clones, we found Detection of Autoantibody against CSAD. The recombinant CSAD (100 that one clone, no. 73, exhibited a characteristic and HCC-pronounced ng) was subjected to SDS-PAGE and detected by immunoblotting as described expression pattern (Fig. 3A). The no. 73 probe detected various sizes previously (27, 34). About 1 ml of blood from each rat was collected from a of RNAs ranging from 0.5 to 4.5 kb. We detected transcripts with 0.5, vein. Sera from normal and HCC-bearing rats were prepared by the standard 2.5, 3.0, and 4.0 kb predominantly, whereas those RNAs were poorly method. Anti-CSAD autoantibody in the sera was detected by immunoblotting of the purifiedrecombinantCSAD proteinwith alkalinephosphatase-conju transcribed in the normal liver. From these results, we decided to gated rabbit antirat IgG, followed by the alkaline phosphatase reaction with investigate no. 73 clone intensively. Since the insert of clone no. 73 5-bromo-4-chloro-3-indolyl-1-phosphate as substrate and nitro blue tetrazo was short (â€”200 bp), we isolated the full-length cDNA including hum for color development. no.73 insert from a terminal HCC-derived cDNA library. Finally, we obtained 10 distinct clones. These clones had different genes, and RESULTS each of them exhibited significant homology with no. 73 (data not shown). Subtraction and Screening of HCC-related Clones. To isolate We analyzed the expression of these 10 genes. Among them, novel HCC-associated genes, we used rat HCCs induced by the Northern blot analysis showed that clone no. 73-8 exhibited the most Solt-Farber procedure. The Solt-Farber procedure can reproducibly pronounced HCC specificity (over 8-fold enrichment), and the size of produce terminal HCCs in 7 months. We prepared poly(A) RNAs the no. 73-8 transcript was about 3 kb (Fig. 3B). We isolated several from rats with terminal HCC and synthesized cDNAs by following the cDNAs related to no. 73-8 sequences and connected them. Sequence new subtraction protocol using oligo(dT)-Latex particles, as shown in analysis of no. 73-8 cDNA revealed that it contained one long open Fig. 1. Improvements in our subtraction method may be summarized reading frame that could encode a protein of 506 amino acids (Fig. as the following three points: (a) subtraction was not performed 4A). Homology search of this clone demonstrated that the open between cDNAs and mRNAs but between cDNAs from each tissue; reading frame of the no. 73-8 gene had 90% identity with that of the (b) hybridization for commonly expressed genes was carried out on rat CSAD (25) and about 60% identity with that of various mammalian the surface of Latex particles; thus, common genes can be removed by GAD genes (39â€”44).Furthermore, amino acids from 298 to 319 were a simple centrifugation; and (c) selection of specific genes was done a perfect match to the consensus binding sequence for pyridoxal by mass-screening using cDNA probes representing a native mRNA 5'-phosphate (a coenzyme of the decarboxylases), a sequence con population. served in all known decarboxylases. Our CSAD sequence data were Antisense cDNAs for the normal liver and HCC pohy(A) RNAs different from previously identified rat CSAD in two points (Fig. 4B). were synthesized on oligo(dT)-Latex particles. HCC antisense cDNAs Alanine at position 16 in the previously reported CSAD appeared as on the particles were then linked with a poly(dC) tail by terminal prohine in our sequence. Moreover, the sequence downstream from nucleotide transferase. HCC sense cDNAs were synthesized by primer amino acid 458 of the previously reported CSAD was completely extension using EcoRI-(dG)15 primer and then hybridized with a replaced by a distinct sequence that resulted from deletion of a 10-fold excess of the normal liver-derived antisense cDNAs on the cytidine at 1432 (Fig. 4B). 5231

HCC

Fig. 1. Scheme of the subtraction technique used in this study. Antisense cDNAs were synthesized on Latex beads. Sense cDNAs were synthesized by oligo(dT) primers immobilized with Latex beads. HCC-enriched CDNA Antisense cDNAs of the normal liver were hybrid ized with sense cDNAs of HCC. After centrifuga tion, cDNAs in the supematant were amplified by PCR. PCR products were introduced into the plas @;1 PCR mid vector and analyzed by dot blot hybridization.

Differential @ dot blot

oâ€¢oooo1 0000 OOOO@ 0000@:-0 OO@@@O 000000 @@@QOOO G0000@: 11CCprobe Normal liver probe

Expression of the CSA.D Gene. Next, we examined the tissue during hepatocarcinogenesis (Fig. 6). Although CSAD was expressed distribution of CSAD transcripts. For control of quantification of at a basal level in the normal liver (Fig. 6, Lane 1), its transcripts CSAD mRNA, we measured RNA preparation correctly by several disappeared completely for 48 h upon DEN injection (Fig. 6, Lanes ways (data not shown), because usual ubiquitous probes such as 2â€”4).These results suggest that the CSAD gene expression is down @3-actinand glyceraldehyde 3'-phosphate dehydrogenase are thought regulated by DEN. However, CSAD transcripts were expressed again to change theirexpressionsby oncogenesis andcell growth.Northern in accordance with the appearance of hyperplastic nodules during the blot analysis showed that 3-kb CSAD RNAs were expressed specif precancerous state. Its expression was then five times higher than that ically in liver and kidney (Fig. 5, Lanes 5 and 7). In lung (Fig. 5, Lane in normal liver, and the maximal level was maintained afterward 4), there was a small but significant amountof CSAD RNA whose during the cancerous state (Fig. 6, Lanes 5â€”8).Therefore, the CSAD size was larger (6 kb and greater) than that of the transcripts in liver expression is suggested to be highly induced during hepatocarcino and kidney. No signal was observed in other tissues tested (Fig. 5, genesis. CSAD is thus a candidate of a novel genetic marker for Lanes 1, 2, 3, 6, and 8). These results indicate that CSAD is expressed hepatocarcinogenesis of the rat. In the regenerating liver, which tissue specifically. Moreover, we used Northern blot analysis again to mimics normal hepatocyte growth, CSAD expression increased grad investigate the temporal expression of the CSAD gene in rat liver ually (Fig. 6, Lanes 9â€”11).At 12 h after the hepatectomy, 30% of the 5232

@ Fig. 2. Differential dot blot hybridization to a.. 3 detect HCC-related clones. The same volume of mini-preparation samples of each plasmid DNA 0@..â€¢ was blotted at corresponding positions on two â€¢1 I 0 * membranes and hybridized with HCC-derived eDNA probes (A) or normal liver-derived eDNA probes (B). Arrows, 11CC-relatedclones. [email protected]â€¢ I.. S. S. I

Table I Summary of subtraction to obtain HCC-related cDNAs during hepatocarcinogenesis might yield anti-CSAD autoantibody in cloneNumberOriginalSubtraction step/Content of rats. examined2000HCC-relatedclones As a test for this possibility, recombinant CSAD was blotted on a screening101Independentclones detected by dot blot membrane, and rat sera were used to detect anti-CSAD autoantibodies clones31Novel in the sera. We detected a Mr 55,000 band corresponding to CSAD gene13Known geneâ€• with sera from HCC-bearing rats, whereas no signal was observed 12a Unevaluated geneâ€•6 when serum from normal rats was used (Fig. 8A). These results Includes two clones containing kininogen and haptoglobin genes. suggest that sera from HCC-bearing rats contain significant amounts b These clones could not be evaluated because of their short inserts. of anti-CSAD antibodies. Since normal and HCC-bearing rat sera were prepared from animals of similar age, the rapid increase in the transcripts for the normal liver was observed (Fig. 6, compare Lanes autoantibody titer during hepatocarcinogenesis is most likely age 1 and 9); then CSAD expressionreachedthe normal level by 24 h independent. Similar results were reproducibly obtained regardless of (Fig. 6, compare Lanes 1 and 10). rat individuals (Fig. 8A). The amount of anti-CSAD antibodies in Expression of CSAD during Hepatocarcinogenesis. Antiserum HCC-bearing rats was 30â€”200times greater than that in normal rats against the purified recombinant CSAD was produced by immuniza (Fig. 8B). The appearance of anti-CSAD antibodies in the rat sera tion with rabbits. The titer of anti-CSAD antibody reached its maxi during hepatocarcinogenesis was investigated (Fig. 8C). All sera from mal level (> 10,000-fold increase) after only three injections of the

immunogen (data not shown). Because at least five injections is .@ usually required to obtain the maximum antibody titer for ordinary E antigens, CSAD was found to be exceptionally antigenic. By using A I-)t@ this anti-CSAD antiserum, we detected CSAD in liver extracts from z0 ID 00 normal and HCC-bearing rats. This antiserum detected only a Mr z____ 55,000 polypeptide in either extract (Fig. 7), and its apparent molec ular weight were well coincided with that (Mr 55,700) deduced from the cloned CSAD. Although CSAD exhibited the highest similarity to 8 Sâ€” the GAD among the known decarboxylases, we could not detect GAD polypeptide with the anti-CSAD antiserum in brain extracts where 28Sâ€” GAD is abundant (data not shown). These results suggested that the anti-CSAD antiserum probably reacts specifically with CSAD and does not recognize GAD. A comparison between Lanes 1 and 2 of 18Sâ€” Fig. 7A indicates that CSAD polypeptides were present in the HCC liver at about five times their level in the normal liver. Moreover, the expression pattern of CSAD polypeptide stimulated by hepatocarci nogenesis resembled that of the mRNA (Fig. 7B). Thus, it is clear that there is a good coordination between expression patterns of CSAD

mRNA and its polypeptide. .@ . Anti-CSAD Autoantibodies Detected in HCC-bearing Rats. CSAD is structurally similar to GAD (about 60% similarity). More over, we detected a significant GAD enzyme activity with the recom 1 2 12 binant CSAD (data not shown). GAD expression in the human pan probe : No.73 probe : No.73-8 creas and central nervous system has been implicated as an autoantigen in patients with IDDM and SMS, respectively. Moreover, Fig. 3. Northern blot analyses of HCC-related DNAs. A. 2 @zgofpoly(A) RNAs from normal liver and HCC were analyzed with the no. 73 probe. B, 300 ng of poly(A) RNAs CSAD polypeptide was extremely antigenic in rabbits as stated above. from normal liver and HCC cells were analyzed with no. 73-8 probe. Positions of 185 and All these results led us to hypothesize that overexpression of CSAD 285 rRNAs are indicated. 5233

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1996 American Association for Cancer Research. 11CC-ACTIVATEDCSAD AND ITS AUTOANTIBODY A 1 GCGCTCTGAACCCGTCGTCTGAACCCTCTCTGAACCTTCCTGAAG(TGGAAGATTTCACC60 * 61 CTGATGGCTGACTCAAAACCACTCAGAACCCTGGATGGGGACCCTGTGCCTGTGGAGG(T120 1 MADSKPLRTLDGDPVPVEA 19 121 TTGCTCCGGGACGTGT1TGGGATTGTCGTAGATGAGGCCATTCGGMGGGGACCMTGCC180 20LIRDVFGIVVDEAIRKGINA 39 181 TCTGAGAAGGTCTGCGMTGGAAGGAGCCTGAAGAGCTCAAGCAGCTGCTGGACUGGAG240 4OSEKVCEWKEPEELKQL LOLE 59 241 CTGCAGAGCCAGGGCGAGTCTAGGGAGCGGATCCTGGAGCGCTGCCGGGCTGTGATTCAT300 60LQSQGESRERIIERCRAVIH 79 301 TACAGTGTCAAGACTGGTCACCCCCGGTTCTTCAACCAGCTCTTCTCAGGA1TAGATCCC360 80YSVKIGHPRFFNQLFSGLDP 99 361 CATGCTCTGGCCGGGCGCATCATFACGGAGAGCCTCAATACCAGCCAGTACACATATGAG420 100 H A I A G R I I I E S L N I S Q Y T Y E 119 421 A1TGCCCCCGTG1TTGTGCTCATGGAAGAGGAGGTGCTGAAGAAACTCCGTGCCCTTGTG480 120 I A P V F V I M E E E V L K K L R A L V 139 481 GG@GGAACACTGGGGATGGGGTCUCTGTC@GGTGGUCCATCTCTAACATGTACGCC 540 140 G W N I G D G V F C P G G S I S N M V A 159 541 ATAAACCTGGCCCGCTTFCAGCGCTACCCAGACTGCAAGCAGAGGGGCCTCCGGGCCCTG600 160 I N L A R F Q R Y P D C K Q R G L R A L 179 601 CCACCCTTGGCCCTCTTCACTTCAAAGGAGTGCCACTACTCCATCACCAAGGGAGCTGCT660 180 P P L A I F I S K E C H Y S I I K G A A 199 661 @CTGGGACTrGGCACCGACAGTGTCCGAGTGGTCAAGGCTGATGAGAGAGGGAAGATG720 200 F I G L G I D S V R V V K A D E R G K M 219 721 ATCCCTGAGGATCTGGAGAGGCAGATCAGTCTGGCAGAGGCTGAGGGCTCGGTGCCA1TT780 220 I P E D I E R Q I S L A E A E G S V P F 239 781 CTGGTCAGTGCCACCTCTGGTACCACCGTGCTAGGGGCCUTGACCCCCTGGATGCAATT840 240 L V S A I S G I I V L G A F D P L D A I 259 841 GCCGATG1TTGCCAGCGTCACGGGCTGTGGTTACACGTGGATGCCGCCTGGGGTGGGAGC900 260 A D V C Q R H G I W L H V D A A W G G S 279 901 GTC@GCTGTCCCGGACACACAGGCATCTCCTGGATGGGATCCAGAGGGCTGACTCCGTG 960 280 V I L S R I H R H L L D G I Q R A D S V 299 961 GCCTGGAACCCTCACAAGCTTCTCGCCGCGGGGCTGCAGTGCTCTGCTCTTCTTCTCCGG1020 300 A W N P H K L I A A G L Q C S A L L L R 319 Fig. 4. Structure of the rat CSAD. A. an open reading frame in the 1021 GACACCTCGAACCTGCTCAAGCGCTGCCACGGGTCCCAGGCCAGCTACCTCTTCCAGCAA1080 CSAD eDNA consisting of 1518bp encodes a protein with 506 amino 320 D I S N I I K R C H G S Q A S Y I F Q Q 339 acids. Asterisks under the nucleotide sequence are in-frame stop 1081 GAC@GUCTAC@CGTGG@CTGGACACCGGAGA@GGTGGTGCAGTGTGGCCGCCGC1140 codons. The underline indicates the conserved pyridoxal 5-phosphate binding site. A putative polyadenylation signal is shown in italics. B, 340 D K F Y N V A L D I G D K V V Q C G R R 359 comparison between our CSAD amino acid sequence (CSAD) and a 1141 GTGGACTGTCTGAAGCTGTGGCTCATGTGGAAGGCGCAGGGTGGGCAAGGGCTGGAGTGG1200 previously identified CSAD sequence (CSAD#; Ref. 26). Asterisks 360 V D C I K L W I M W K A Q G G Q G I E W 379 indicate matching amino acids between the two sequences. 1201 CGCATCGACCAGGCCTITGCTCTCACTCGGTACUGGTGGAGGAGATAAAAAAGCGGGAA1260 380 R I D Q A F A I I R Y L V E E I K K R E 399 1261 GGA1TrGAGTTGGTCATGGAGCCCGAGTTCGTCMCGTGTGC1TCTGG1TFGTGCCTCCC1320 400 G F E I V N E P E F V N V C F W F V P P 419 1321AGCCTGCGGGGGAAGAAGGAGAGCCCAGAUACAGCCAGAGGGGTCTCAGGTGGCCCCT1380 420 S L R G K K E S P D Y S Q R L S Q V A P 439 1381 GTGCTCAAGGAGCGCATGGTGAAGMGGGAACCATGATGATCGGCTACCAGCCCCATGGG1440 4.4Ã˜VLKERMVKKGTMMIGYQPHG 459 1441 ACCCGGGCCAACTTCTTCCGAATGGTGGTGGCCAACCCCATACTGGTCCAGGCCGATATA1500 46Ã˜TRANFFRMVVANPILVQADI 479 1501 GACTTCC1TCTGGGCGAGGCTGGAGCGTCTGGGCCAGGACCTGTGAGCTGCTTCCTCTCT1560 480 D F I L G E A G A S G P G P V S C F I S 499 1561 CTGCCCCACCCAAGCTCTGCATAAGCTCCTGGGTTCCCAMAGCGACCTTTCTAGGAAAC1620 500 1 P H P S S A * 506 1621 AGTGGCCUGACTGTGTGAGCCCCCACACACTAACTCTCCTAGCTMGTA1TGGCTGCCA1680 1681 GACGGTCTCTAAGCACACTACAGTCTG1TCTTACGA&ATGTGCTFCTITFAAGTCGGTCA1740 1741TAGTGGTACACACCGUAATACCAGCACTGGGGAGGCAGAGGCAGAUCAAGCAGATCTC1800 1801 1TGAG1TrGAGGCCAGCCTGGTCTACAGAGCTGGCCTACACAGAAAAA&A.ACCTGTCTCA1860 1861AAAAMAAGAMGGAAGGAAGA.&AGAAAGGAAAAGAAAGAAATATTFTTCA1TAAGA1TA1920 1921 TGTCTATAAAMATrG1TATrAATATGAGAGATATGGTACGATGTATFAAGAAAGCTAGA1980 1981 TATGGGGGTTGGGGATTTAGCTCAGTGGTAGAGCCC1TGCTAGGAAGCGCAAGGCCTGGG2040 2041 TTCAGTCCCCAGCTCCGAAAAAAAGAACCACAAAAAAAAAAAAAAAAA&A.AAAAAAGAAA2100 2101 GCTAGATATGAG11TATATATCATGGTATCTGAG1TAAACTAAAAMAAAAAATACATAG2160 2161 GAAAAGGCGGTGAGTGGAACTGTGCCAAAGGTCAGCAG11TFCCCTGGAAGAGGATAACA2220 2221 GGCTGTTCCTMGTCAGCCTCTCAGACC1TCCCTGCTrCCCCACTTrATrATGTMCCAC 2280 2281 ATCACCTACTFCTGAGATATAACAATA4AGC1TrGTCACTATAAAAAAAAAAAA&AAAAA2340 2341 AMAAAAAAAAAA 2353 B CSAD 1 MADSKPLRTLDGDPVPVEALL 21 *************** ***** CSAD# 1 MADSKPLRTLDGDPVAVEALL 21

CSAD 444 RMVKKGTMMIGYQPHGTRANFFRMVVANPI LVQADIDF LLGEAGASGPG 492 ************** * * CSAD# 444 RMVKKGTMNIGYQPMGPGPTSSEWWWWPTPYWSRPI 478

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38), suggesting that our subtraction strategy functions properly. These results suggest that cDNA subtraction using ohigo(dT)-Latex is an excellent method to isolate genes specifically expressed in a certain cell type and that the several improvements applied in this procedure I functioned efficiently. We should mention that by further analyses we @ (kb) found our subtraction protocol to be much more sensitive than two I.. dimensional electrophoresis (14) to detect changes in specific proteins 9.5â€” i@ (data not shown). In conclusion, our subtraction technique is benefi 7.5â€” :@:@ cial for isolating with a wide spectrum of genes. In this study, we finally isolated the HCC-rehated clone 73-8. A protein encoded by the 73-8 insert had 90 and 60% sequence simi 4.9 â€” larities with CSAD and GAD (actually two GAD isoforms, GAD65 and GAD67), respectively, and also contained a consensus sequence @ for the pyridoxal 5'-phosphate attachment site (45) characteristics of 2.4 â€” ye the decarboxyhases (Fig. 3B). These results demonstrate that the 73-8 gene encodes CSAD. Although Kaisakia et a!. (25) also reported a CSAD sequence from rat liver, there were two differences between their base sequence data and ours (Fig. 4C). In their data, cytidines at 1.35 positions I 19 and 1433 were changed to guanine and deleted, respec tively (Fig. 4A), which resulted in amino acid substitution from alanine to proline at position 16 and a frame-shift mutation down stream from position 458. We cannot rule out the possibility of a polymorphism dependent on differences in individual rats or alterna 1 23 45 6 78 tive splicing. However, our CSAD sequence was much more similar to the GAD one than theirs, and the apparent molecular weight of the Fig. 5. CSAD gene expression in rat tissues. Two jsg of poly(A) RNA from each tissue native CSAD on SDS-PAGE was consistent with that deduced from was analyzed by Northern blotting using a probe of CSAD DNA from 683 to 1434 including the CSAD-coding region. Arrowhewl. position of the major CSAD mRNA. our sequence data (Fig. 7). Thus, the CSAD molecule that we de scribed here is suggested to be the dominant form in rat liver. CSAD synthesizes a precursor of taurine that becomes a major com HCC-bearing rats at 1, 3, 5, and 7 months after DEN injection also ponent of bile in the liver and a neurotransmitter in the brain. Since contained a high level of anti-CSAD antibodies, and the appearance of CSAD enzyme activity was detected in the normal liver and its gene anti-CSAD antibodies was synchronized with CSAD gene expression expression was stimulated during liver regeneration, CSAD may be during hepatocarcinogenesis (Figs. 6 and 7). Equivalent data were required for both maintenance and growth of hepatocytes. If CSAD reproducibly obtained irrespective of the lot of sera. The background bands of this figure were thought to come from a reaction between E. coli-contaminating proteins and natural antibodies in rats. Because Regenenerating these backgrounds were also faintly visible in normal rat sera as DEN HCC liver shown in Fig. 8C, rats may have several types of natural antibodies, and these antibodies are supposed to be weakly and nonspecifically @ $(w74@â€˜câ€•o000 induced by HCC. These results demonstrate that CSAD is highly \@S@@c@c@cc@ antigenic and that overexpression of CSAD in rat liver stimulated by carcinogenesis yields the autoantibody. (kb) 9.5 DISCUSSION 7.5â€” In human hepatocarcinogenesis, investigation of carcinogenesis dependent gene expression is important, since no apparent cellular I oncogene has been identified thus far. We chose rat hepatocarcino 4.9â€” genesis as a model system for this purpose. In this study, by using a new and efficient method, we carried out the isolation and character ization of rat genes that are expressed in a hepatocarcinogenesis 2.4 â€” â€¢â€¢ @eIM@. a@ dependent manner. We used the Solt-Farber procedure because of its high efficiency and reproducibility to generate HCCs in the rat. Since proteins expressed specifically in naturally occurring rat HCCs were similar to those in other kinds of chemical hepatocarcinogenesis in 1.35â€” spite of the use of carcinogens (14), this model system must be a proper approach for investigating naturally occurring rat HCCs. By using a new subtraction method, we obtained 3 1 independent HCC-related genes from 2000 original clones. Eventually, the cDNA 1 2 3 4 5 6 7 8 91011 subtraction was able to afford at least a 200-fold concentration of Fig. 6. CSAD gene expression during rat hepatocarcinogenesis. Three hundred nano these genes. Among the 3 1 clones, there were six known genes, grams of poly(A) RNAs from normal liver. DEN-treated livers ( I2, 24, and 48 h after DEN injection). HCC livers ( I, 3. 5, and 7 months after DEN injection), and regenerating including those of kininogen and haptoglobin, whose expressions livers (12, 24, and 48 h after partial hepatectomy) were analyzed with the CSAD-coding have been demonstrated to be stimulated by hepatocyte injury (35â€” sequence probe as mentioned in Fig. 5. Arrowhead. the position of CSAD mRNA. 5235

HCC-ACflVATED CSAD AND ITS AUTOANTIBODY

the Salt-Farber-treated rats well correlated with that in the content of A CSAD mRNA (Fig. 6) and its polypeptide (Fig. 7B). These results strongly suggest that the production of the autoantibody can be attributed 0 to CSAD overexpression in HCC cells. 0 0 z I Although we detected increased amounts of CSAD polypeptide in the total cell lysates of HCCs (Fig. 7B), the amounts of CSAD in the (kDa) soluble fraction from both normal and HCC livers were almost the 64 â€” same (data not shown). Thus, we can hypothesize that overexpressed CSAD proteins become denatured and insoluble and thereby become transformed into antigenically potent peptides. If this hypothesis is 50â€” correct, CSAD-derived new epitopes would be recognized by the immune system including cytotoxic T cells (48, 49). Alternatively, CSAD-overexpressing cells may die of necrosis, resulting in the 36â€” leakage of intracellular CSAD into the blood. Yet another hypothesis, that CSAD comes from other tissues that are indirectly stimulated by hepatocarcinogenesis, seems unlikely because of the coordinate ex pression of CSAD and its autoantibody (see above) and the lack of apparentabnormalityin other tissues(data not shown). B At presenttime, we cannot completely rule out the possibility that the anti-CSAD autoantibody reacts with other proteins. However, our anti E HCC CSAD antibody did not react with GAD that has the highest homology 0 with CSAD among known decarboxylases. Moreover, we did not find z !i 3 5 7 1

@â€˜- 04 A E00 @oo z II (kDa) @,

@ @d;1@*

Fig. 7. Expression of CSAD polypeptide in rat liver during hepatocarcinogenesis. Ten ,zg of the liver homogenates from normal or HCC-carrying rats were subjected to SDS-PAGE,andCSADwasanalyzedbyuseofthespecificantiserum.A,CSADproteins B in the normal livers and terminal HCCs were analyzed. B. liver homogenates from normal Normal HCC and HCC-bearing rats 1. 3, 5, and 7 months after DEN injection were analyzed. Arrow head, position of CSAD. .4

overexpression could be a cause of hepatocarcinogenesis, CSAD action might result in some actual carcinogenic substances in vivo in addition to I @ taurine. On the other hand, if such abnonnal CSAD expression is a result 4@, * of hepatocarcinogenesis, CSAD expression may be transcnptionally ac tivated by an intrinsic oncogene directly or indirectly. Alternatively, removal of carcinogenic substances from the liver might require more X5000Xl0000 bile,whichdemandwouldleadto greaterCSADactivity. Previous biochemical studies reported that CSAD and GAD can C Co utilize both cysteine sulfinic acid and glutamic acid as substrates (46). E HOC 0 @@ Actually, the recombinant CSAD had a significant GAD activity (data z P1 71 not shown). Mammalian GADs include two isoforms, GAD65 and GAD67, which exhibit 65% sequence identity, and are suggested to have originated from a single gene (47). Moreover, we demonstrated that CSAD has 60% sequence similarity with GAD65. These strongly suggest that CSAD is closely related to GAD. It is well known that GAD expressedin the humanpancreasand central nervous system causes autoimmune diseases known as IDDM and SMS, respectively. This indicates that GAD protein is highly antigenic. Fig. 8. Anti-CSAD autoantibodies in HCC-bearing rats. The anti-CSAD autoantibody As described above, we found that GAD and CSAD are structurally was detected as described in â€œMaterialsandMethodsâ€•byuse of recombinant CSAD. A, scm (X 1000 dilution) from normal and two independent HCC-bearing rats (HCCI and related and that recombinant CSAD exhibited an extremely high antige HCC2) 7 months after DEN injection were analyzed. B, sera from normal and terminal nicity in rabbits. In this study, we demonstrated that HCC-bearing rats HCC-bearing rats were analyzed at the dilutions indicated. C. 1000-fold diluted sera from normalandHCC-bearingrats(1,3, 5,and7 monthsafterDENinjection)wereanalyzed. contained increased amounts of anti-CSAD antibodies (30-200 times Arrowheads and asterisks, positions of the recombinant CSAD and nonspecific back than the amount in normal rats; Fig. 8B). The increase in antibody titer in ground,respectively. 5236

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1996 American Association for Cancer Research. Hcc.AcTIvATED CSAD AND ITS AUTOANTIBODY any significant changes in organs except liver. Especially, brain and degrading subtraction (EDS). Nucleic Acids Res., 22: 4381â€”4385, 1994. pancreas were normal where GAD is produced, and some changes must 23. Hara. E., Kato, T., Nakada, S., Sekiya, S., and Oda. K. Subtractive eDNA cloning using oligo(dT)30-Iatex and PCR: isolation of eDNA clones specific to undifferen occur by related autoimmune diseases (data not shown). These results tiated human embryonal carcinoma cells. Nucleic Acids Res.. /9: 7097â€”7104. 1991. strongly suggest that anti-CSAD autoantibody was derived from CSAD 24. Hara, E., Yamaguchi. T., Tahara, H., Tsuyama, N., Tsurui, H., Ide, T., and Oda, K. overexpression in liver and was specific for CSAD. 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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1996 American Association for Cancer Research. Overexpression of Cysteine Sulfinic Acid Decarboxylase Stimulated by Hepatocarcinogenesis Results in Autoantibody Production in Rats

Toshihiko Kishimoto, Kenji Kokura, Tomoyoshi Nakadai, et al.

Cancer Res 1996;56:5230-5237.

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