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Expression of the Sperm Fibrous Sheath Protein CABYR in Human Cancers and Identification of ·-Enolase As an Interacting Partner of CABYR-A

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Expression of the Sperm Fibrous Sheath Protein CABYR in Human Cancers and Identification of ·-Enolase As an Interacting Partner of CABYR-A 1169-1175.qxd 13/7/2022 01:01 Ì ™ÂÏ›‰·1169 ONCOLOGY REPORTS 25: 1169-1175, 2011 Expression of the sperm fibrous sheath protein CABYR in human cancers and identification of ·-enolase as an interacting partner of CABYR-a YEN-TZU TSENG1, JIUN-YI HSIA1,2, CHUN-YU CHEN1, NIEN-TSUNG LIN3, PELE CHOI-SING CHONG4 and CHIOU-YING YANG1 1Institute of Molecular biology, National Chung Hsing University, Taichung 402; 2Division of Thoracic Surgery, Department of Surgery, Taichung Veterans General Hospital, Taichung 407; 3Institute of Microbiology, Immunology and Molecular Medicine, Tzu Chi University, Hualien 970; 4Vaccine Research and Development Center, National Health Research Institutes, Miaoli 350, Taiwan, R.O.C. Received November 3, 2010; Accepted December 6, 2010 DOI: 10.3892/or.2011.1165 Abstract. Calcium-binding tyrosine phosphorylation regu- Introduction lated protein (CABYR), a family of isoforms resulting from alternative splicing, has been identified as a cancer/testis anti- Calcium-binding tyrosine phosphorylation regulated protein gen (CT88) in lung cancer and hypothesized to be a promising (CABYR), encoded by the CABYR gene located at chromo- target for immunotherapy. Here, we report the expression some 18q11.2 and chromosome 18 locus A1 of human of CABYR in various cancer tissues/cell lines. Expression and mouse, respectively, is a family of highly polymorphic profiles of individual isoforms were different among cancers. proteins (1,2). Six alternatively spliced transcripts involving Furthermore, protein and mRNA levels did not correlate two coding regions (CR-A and CR-B) have been shown to for individual isoforms. While CABYR-c/d were the most encode five protein isoforms (CABYR-a - CABYR-e) (Fig. 1) abundant splicing variants, CABYR-a was the predominant with extensive charge and mass polymorphisms in human protein isoform. Finally, CABYR-a, but not CABYR-c, spermatozoa (1). While proteins encoded by both CR-A was found to interact with ·-enolase in vivo. Collectively, the and CR-B are produced during spermatogenesis, only data indicate that CABYR is a CT antigen widely expressed CR-A-encoded acidic isoforms bind calcium. These isoforms in diverse cancer cells. However, individual protein isoforms appear to have a much higher molecular weight than the may be differentially regulated by post-transcriptional and predicted 50-53 kDa when assessed by SDS-PAGE (1,3). post-translational mechanisms and may have a unique role Although CABYR was originally reported as a testis-specific in carcinogenesis. The protein expression pattern of various protein localized exclusively in the sperm flagellum, which CABYR isoforms is important with regard to the consideration gains calcium-binding capacity when phosphorylated during of using CABYR as a target antigen for the development of capacitation (1), CABYR-c and CABYR-d are also detected vaccines for cancer therapy. in the normal human brain and also in the fetal brain and brain tumors in which the expression is more distinct than in the adult brain (4). This evidence, together with the detection _________________________________________ of aberrant expression of CABYR-a/b and CABYR-c in lung cancers and the presence of specific IgG antibodies in Correspondence to: Dr Chiou-Ying Yang, Institute of Molecular some sera from lung cancer patients but not from the healthy Biology, National Chung Hsing University, 250 Kuo Kuang Road, donors, has defined CABYR as a novel cancer/testis (CT) Taichung 402, Taiwan, R.O.C. antigen and a new potential target for the use in development E-mail: [email protected] of therapeutics for human cancer (5,6). Recently, CABYR was found in motile cilia of fallopian Abbreviations: CABYR, calcium-binding tyrosine phospho- tubes and human bronchi in addition to the sperm fibrous rylation-regulated protein; CT, cancer/testis; DMEM, Dulbecco's sheath suggesting that it may have a role in calcium signaling modified Eagle's medium; FCS, fetal bovine serum; SDS-PAGE, pathways that regulate motility of cilia and flagella (7). sodium dodecyle sulfate-polyacrylamide gel electrophoresis CABYR-c and CABYR-d, which lack calcium binding capacity, have been demonstrated to interact with GSK3ß Key words: calcium-binding tyrosine phosphorylation regulated in vitro (4). Both CABYR and GSK3ß genes were found to protein, cancer/testis antigen, liver cancer, esophageal cancer, be up-regulated in smokers, suggesting a role of these proteins ·-enolase in carcinogenesis (8). The present study was undertaken after we identified a recombinant phage displaying a peptide derived from CABYR 1169-1175.qxd 13/7/2022 01:01 Ì ™ÂÏ›‰·1170 1170 TSENG et al: CHARACTERIZATION OF A CANCER TESTIS ANTIGEN CABYR Figure 1. Diagram of CABYR genomic structure and transcript variants. Translation start codons (▼) and stop codons (∗) are indicated. Relative positions of the primer pairs used to detect splicing variants (1/2, 3/4, and 5/6) and prepare the biotinylated probe (7/8) are indicated by arrows. that was affinity-selected by a rabbit serum against hepato- Southern hybridization. Southern hybridization was carried cellular carcinoma HepG2 cells. Here, we show that CABYR out essentially as previously described (9) using biotinylated is aberrantly expressed in a wide range of human cancers. CABYR DNA obtained by PCR with primers CABYR-7 and Interestingly, although the CABYR-c/d isoforms are more CABYR-8 (Table I, Fig. 1). PCR amplification conditions readily detected at the mRNA level, CABYR-a is the prevalent were as described above, except that the nucleotide mixtures isoform at the protein level. In addition, we found that contained 1.25 mM biotin-11-dATP (Perkin-Elmer, Waltham, CABYR-a, but not CABYR-c, interacts with ·-enolase, MA, USA) in addition to 2.5 mM each of dTTP, dGTP, and suggesting that CABYR may have a role in modulating dCTP, and 1.25 mM dATP. After hybridization, the membrane ·-enolase function. was incubated with alkaline phosphatase-conjugated strep- tavidin (Invitrogen) and developed using CDP-star chemilu- Materials and methods minescence reagent (Perkin-Elmer). The hybridization signals were then detected by an ImageMaster VDS-CL system (GE Cell culture and tissue specimens. DMEM supplemented with Healthcare, Piscataway, NJ, USA). 10% FBS and 50-μg/ml gentamycin was used to cultivate the hepatocellular carcinoma cell lines, HepG2 and Huh7, the Plasmids. For nucleotide sequencing, PCR products were nasopharyngeal carcinoma cell lines, NPC039, NPC076 and cloned into pGEM-T Easy vector (Promega Co., Madison, BM1, the human embryonic kidney cell line, 293T, the breast WI, USA). To produce recombinant CABYR proteins for cancer cell line Hs578T, the lung adenocarcinoma cell lines immunization, the complete coding regions of CABYR-a CL1-0 and CL1-5, and the human glioblastoma-astrocytoma (GenBank ID: HM594166) and CABYR-c (GenBank ID: cell line U-87 MG. Tissue specimens were obtained from HM594168) were PCR amplified from NPC076 cell cDNA Taichung Veterans General Hospital, Taiwan and Buddhist with primer pairs CABYR-F/CABYR-a-R and CABYR-F/ Tzu Chi General Hospital, Hualien, Taiwan. CABYR-c-R, respectively, and cloned into pET21b (Merck KGaA, Darmstadt, Germany) to generate pET-CABYR-a and RT-PCR. Total RNA was prepared using TRIzol reagent pET-CABYR-c. To express Flag-tagged CABYR in 293T (Invitrogen, Carlsbad, CA, USA) according to the manu- cells, DNA fragments encoding the full length CABYR-a were facturer's instructions. RNA was reverse transcribed in a amplified from pET-CABYR-a using primer pairs CRA-F/ reaction mixture of 20 μl containing 5 μg of total RNA, CRA-R, while CABYR-c was amplified from pET-CABYR-c 2.5 μg of oligo dT15, 1 nmole of dNTP and Superscript with primer pairs CRA-F/CRB-R. Both amplification products II reverse transcriptase (Invitrogen). Normal tissue cDNA were cloned into the EcoRI/NotI site of pcDNA-Flagß to were purchased from Clontech (Mountain View, CA, USA). generate Flag-CABYR-a and Flag-CABYR-c. cDNA was amplified by PCR using Taq DNA Polymerase (Invitrogen) with 1 μl of the cDNA as the template. Thermal Antibodies. Anti-CABYR antibodies were prepared by immu- cycle conditions were as follows: 94˚C for 5 min followed by nization of rabbits with E. coli produced His-tagged CABYR. 30 cycles of 94˚C for 1 min, 60˚C for 2 min and 72˚C for Briefly, pET-CABYR-a and pET-CABYR-c plasmids were 1 min, and then extension at 72˚C for 10 min. Integrity of transformed separately into E. coli BL21(DE3) and the trans- the cDNA was verified by amplification of the ß-actin gene. formants were grown in LB for 3 h, after which expression PCR products were confirmed by nucleotide sequencing or was induced with IPTG (1 mM). His-tagged CABYR-a and Southern hybridization. Primers used in this study are listed CABYR-c proteins were purified using a HisBind purification in Table I. kit (Merck KGaA) following the manufacturer's protocol. 1169-1175.qxd 13/7/2022 01:01 Ì ™ÂÏ›‰·1171 ONCOLOGY REPORTS 25: 1169-1175, 2011 1171 Table I. Primer sequence used in this study. ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Primer Sequencea ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– RT-PCRb CABYR-1 5'-GCTCAGCTTGCTGCTCAGATG-3' CABYR-2 5'-TCCCTGAAATGTTGACCCAG-3' CABYR-3 5'-cgaaTtCCCAGATGGAAAAATCTAC-3' CABYR-4 5'-gcggccgcTcaTTCTGGCAGTTGCTC-3' CABYR-5 5'-agaatTgCCAAAATGATTTCTTCAAAGCCC-3' CABYR-6 5'-TAACATCTGAGCAGCAAGCTG-3' ß-actin-F 5'-GCTTGACTCAGGATTTAAAAACTGGAACGG-3'
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