CDR2 antigen and Yo antibodies

Ovarian cancers were obtained from 16 patients aged 41–79 years operated at the Department of Gynaecology, Haukeland University Hospital (Table 1). None of the patients received chemotherapy or immunotherapy before the operation. Two of the patients had PCD. From these two patients only formalin-fixed cancer tissue was available. Colon cancer was obtained from a patient without paraneoplastic neurological syndrome. Normal human cerebrum and cerebellum were obtained from autopsy of a patient without neurological disorder. The following cancer cell lines were obtained from ATCC (www.​lgcstandards-atcc.​org): lung cancer (A549), ovarian cancer (SK-OV-3), neuroblastoma (SK-N-SH), and cervical cancer (HeLa). Tissue and cell lysates were made using total protein extraction kit from Chemicon (www.​chemicon.​com). Lysates from human ovary tumor and human normal ovary tissue were purchased from Prosci Inc (www.​prosci-inc.​com). Recombinant CDR2 protein was produced in a coupled transcription/translation (ITT) system with a T7 RNA polymerase and nuclease-treated rabbit reticulocyte lysate, and CDR2-negative lysate was prepared with the ITT reaction without plasmid [12, 13]. Protein concentrations were determined using a BioRad DC protein assay (www.​biorad.​com). Tissue and lysates were kept at −80°C and serum samples at −20°C. Yo antibodies have previously been found in 3 of 182 sera from patients with ovarian cancer at the Department of Gynaecology, Haukeland University Hospital, and the 14 patients included in this report were part of this study [13]. In addition, we also included two patients with PCD and Yo antibodies.

Total RNA was extracted from 14 ovarian cancers by homogenization in a Trizol reagent (www.​invitrogen.​com) and RTL buffer containing β-mercaptoethanol followed by RNeasy mini-kit (www.​qiagen.​com). We also used a panel of human total RNA from 11 different types of tissue (brain, heart, kidney, liver, lung, trachea, mammary gland, prostate, skeletal muscle, testis and uterus), as well as human reference total RNA (www.​clontech.​com). RNA was stored at −80°C until use. From each of the samples, we reverse transcribed 100 ng of RNA to cDNA using TaqMan RT reagents according to the manufacturer’s instructions (www.​appliedbiosystem​s.​com).

Real-time quantitative polymerase chain reaction (RT-PCR) was used to quantify mRNA expression in the 14 ovarian cancers, the panel of human total RNA and the human reference total RNA. All primers and probes were obtained from Assay-on-Demand gene expression products at Applied Biosystems (www.​appliedbiosystem​s.​com). As endogenous control genes, we used human large ribosomal protein (RPLPO), human transcription factor IID/TATA box-binding protein (TBP) and the housekeeping gene human glyceraldehyde phosphate dehydrogenase (GAPDH), including the test gene CDR2. We performed the RT-PCR in the ABI prism 7900 Sequence Detection System run by standard PCR cycling conditions. The relative quantification of mRNA expression was based on the standard curve method (User Bulletin #2, Applied Biosystems). Human reference total RNA (www.​clontech.​com) was used to generate the standard curve. We included a “no template control” in each run. Each sample was run and analyzed in triplicate.

The expression of CDR2 was normalized to all the three reference genes (PO, TBP, and GAPDH). The data were transferred to Graphpad Prism, version 3.0 (www.​graphpad.​com) and calculated using Mann–Whitney nonparametric U-test. P < 0.05 was considered statistically significant. We assessed the three reference genes to evaluate the validity of the normalization of the CDR2 expression. All the three reference genes were highly significantly correlated (r = 0.50–0.65, P < 0.008–0.0002) according to the Spearman rank correlation test (Graphpad Prism).

CDR2 cDNA was sequenced from the 14 ovarian cancers. The following primers for amplifying CDR2 were used: 293U20 5′-CTA GAA GAC CCA GCC GAG AT-3′, 659L19 5′-TTG CAG GCA TTC AAT CGT T-3′, 293u21 5′-CTA GAA GAC CCA GCC GAG ATG-3′, 975L21 5′-CCT TTA ACA CGA GCC CAT ATT-3′, 874U20 5′-GCC CTG ATG AAG AGG AAA AT-3′, 1370L20 5′-GCT GTA CTG CGT GTC CAC TT-3′, 1265U29 5′-AGG GAG TGA CAT CGT GAA GG-3′, and 1819L20 5′-CCT CGT TGT ATG CAT TTG CT-3′. We used one tenth cDNA as a template for a PCR reaction using Amplitaq Gold polymerase (www.​appliedbiosystem​s.​com). cDNA was denaturated at 95°C for 10 min followed by 35 PCR cycles (95°C for 30 s, 55°C for 30 s, and 72°C for 60 s) and then held at 4°C. The PCR product was purified using Montage PCR Centrifugal Filter Devices according to the manufacturer’s instructions (www.​Millipore.​com). The PCR product was bidirectionally sequenced using a Big Dye terminator 3.1 cycle sequencing kit according to the manufacturer’s recommendation on an ABI 3700 DNA Analyzer (www.​appliedbiosystem​s.​com). ChromasPro was used to analyze the sequence files (www.​softpedia.​com), BLAST for identifying the DNA sequence (www.​ncbi.​nml.​nih.​gov), and Clustal W2 for aligning sequences (www.​ebi.​ac.​uk/​Tools/​clustalw2).

We subjected the various extracts to a 10% SDS-PAGE and performed western blotting. The following concentrations were loaded to the gels: 10 μg of each cancer extract, 20 μg of the ovarian lysates according to the manufacturer (www.​prosci-inc.​com), and 100 μg of the human cerebrum and cerebellum extracts. The membranes were first incubated with a 1:200 dilution of the anti-CDR2 monoclonal antibody (sc-100320) and then with a goat anti-mouse HRP antibody (sc-2005) diluted 1:1000, both antibodies obtained from Santa Cruz Biotechnology, Inc. (www.​scbt.​com). The reactions were developed by the ECL Plus Western Blotting Detection system (RPN2132) (www.​healthcare.​com) and then read using a Fuji-las 1000 luminescent image analyzer (www.​fujifilm.​com).

Intracellular localization of CDR2 was done in the ovarian, lung, and cervical cancer cell lines. The cell lines were grown and treated as previously described [10]. After fixation and permeabilization, the cells were incubated with a 1:200 dilution of anti-CDR2 polyclonal antibody (ab59566) or anti-CDR2 monoclonal antibody (sc-100320) (www.​abcam.​com) and then with Alexa Fluor 488 goat anti-rabbit antibody (A-11008) or with Alexa Fluor 488goat anti-mouse antibody (A-11001) (www.​probes.​invitrogen.​com), respectively, diluted 1:200 in blocking solution. The slides were mounted with Vectashield with or without DAPI (www.​vectorlabs.​com). Indirect immunofluorescence was done using Zeiss LSM 510 Meta. Absorption experiments were performed as previously described [15] by diluting the anti-CDR2 polyclonal antibody to one titer below the highest dilution still producing staining of HeLa cells. The antibody was incubated with equal volume of the recombinant CDR2 protein overnight. The antibody–antigen complex was spun down, and the supernatant was applied to the cells as described and analyzed at a Zeiss Axiovert M200.

Intracellular localization of CDR2 was also done in three ovarian cancer biopsies (from patients 1, 15, and 16 in Table 1) and in one colon cancer biopsy using the anti-CDR2 polyclonal antibody (ab59566) (www.​abcam.​com) diluted 1:400. The immunoperoxidase staining followed standard procedures at the Department of Pathology, Haukeland University Hospital.