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Technical Briefs |
Departments of
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Clinical Laboratory Medicine,
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Surgery, and
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Pathology, Sapporo Medical University School of Medicine, Sapporo 060-8543, Japan
aaddress correspondence to this author at: Department of Clinical Laboratory Medicine, Sapporo Medical University, South-1, West-16, Chuo-ku, Sapporo 060-8543, Japan; fax 81-11-622-7502, e-mail watanabn{at}sapmed.ac.jp
Survivin, a recently described member of the inhibitor of apoptosis protein family, contains a single baculovirus inhibitor of apoptosis protein repeat and lacks a C-terminal RING finger (1). Like other proteins in the inhibitor of apoptosis protein family, survivin binds specifically to terminal effector cell death proteases, specifically caspase-3 and -7. The consequences are substantially reduced caspase activity and reduced cell death in response to diverse apoptotic stimuli (2). Expression of survivin is detected during fetal development in humans, but is not detected in healthy adult tissues except for the thymus and placenta (1). Survivin is expressed by most cancers, including carcinoma of the lung, stomach, colon, pancreas, breast, and prostate, as well as by neuroblastomas, melanomas, and lymphomas (1)(3)(4)(5)(6)(7)(8)(9)(10).
Overexpression of survivin by cancer cells may lead to anti-survivin antibody responses and cytotoxic T-lymphocyte responses against the cancer (11)(12). In the present study, we examined the occurrence of antibody response against survivin in patients with various gastrointestinal cancers.
Blood samples were collected from 33 healthy blood donors and 63 gastrointestinal cancer patients after histologic diagnosis. After centrifugation, sera were divided into aliquots and stored at -80 °C. The ELISA for anti-survivin antibodies was established. Recombinant His-tagged T7-TAT-myc-survivin protein was prepared by the following procedure. We received pcDNA3-myc-survivin as a generous gift from Dr. J. C. Reed (Burnham Institute, La Jolla, CA) (2). SacI and XhoI were used to digest pcDNA3-myc-survivin for ligation into pET21a. In turn, pET21a-T7-TAT-myc-survivin was digested with NdeI and XhoI and ligated into pET15b. The resulting pET15b-T7-TAT-myc-survivin was transfected into BL21 cells, which then overexpressed recombinant survivin protein. Recombinant protein was purified with a nickel-nitrilotriacetate column (QIAGEN) according to the manufacturer’s instructions. Purity of the recombinant protein was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Coomassie blue staining. As a control, His6-T7-TAT-green fluorescent protein was used (provided by the Department of Pathology at Sapporo Medical University, Sapporo, Japan).
As an antigen for coating wells in the ELISA, purified recombinant His6-T7-TAT-myc-survivin was diluted in 50 mmol/L bicarbonate buffer (pH 9.5) to a final protein concentration of 5 mg/L as determined by the method of Bradford, using a commercially available reagent set (Bio-Rad). Recombinant His6-T7-TAT-green fluorescent protein was used as a control antigen at the same concentration in the same buffer.
Survivin or control antigen solution was placed in wells of 96-well plates (Corning) and incubated overnight at 4 °C. After removing antigen solutions and washing five times with phosphate-buffered saline (PBS) with 0.5 mL/L Tween 20 (T-PBS), plates were blocked with 10 g/L bovine serum albumin in PBS for 2 h at room temperature. After emptying the wells and washing five times with T-PBS, 100 µL of serum sample diluted in PBS (1:100 by volume) was added to each well and incubated for 1 h at room temperature. Samples were then removed and wells were washed five times with T-PBS, after which each well was incubated for 30 min with a 1:2000 dilution of rabbit anti-human IgG F(ab')2 conjugated with horseradish peroxidase (DAKO). After removal of this antibody solution and washing five times with T-PBS, each well was developed by o-phenyldiamine. After a 10-min incubation in darkness, the reaction was stopped with 0.25 mol/L H2SO4, and absorbance was measured at 492 nm.
Anti-p53 antibodies were detected with a commercially available ELISA method (Pharma Cell), using recombinant wild-type p53 according to the manufacturer’s instructions.
The cutoff value for positivity in the anti-survivin ELISA, determined from healthy donor samples as the mean absorbance +2 SD, was 0.936. The cutoff value for anti-p53 positivity, determined from healthy donor samples as the mean absorbance +2 SD, was 0.236. Sera from 25 of 63 gastrointestinal cancer patients (39.7%) were reactive with recombinant survivin protein by ELISA, whereas none of the control sera from healthy donors was reactive. Subgroup results for gastrointestinal cancers were as follows: 13 of 30 positive sera for biliary-tract cancer; 4 of 10 positive for gastric cancer; 3 of 10 positive for colorectal cancer; 1 of 3 positive for hepatoma; 3 of 7 positive for pancreatic cancer; and 1 of 3 positive for esophageal cancer. Sera from 15 of 63 gastrointestinal cancer patients (23.8%) and 2 of 33 healthy donors (6.1%) reacted with recombinant wild-type p53 protein in the anti-p53 ELISA. Overall, sera from 30 of 63 gastrointestinal cancer patients (47.6%) reacted by ELISA with recombinant survivin protein, recombinant wild-type p53 protein, or both (Fig. 1A
).
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To determine the analytical specificity of the survivin ELISA, all serum samples (100 µL of a 1:100 dilution) were incubated with 30 mg/L recombinant survivin protein or green fluorescent protein control antigen for 1 h at 37 °C and then subjected to the survivin ELISA described above. Reactivity of sera decreased substantially after adsorption to recombinant survivin protein, but not after adsorption to control antigen.
In addition, anti-survivin monoclonal antibodies (mAbs) and sera reacting or not reacting with recombinant survivin protein in the survivin ELISA were tested against recombinant survivin protein by Western analysis. Recombinant survivin protein was separated by 100 mL/L sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane (Milipore). The membrane was blocked with 10 g/L bovine serum albumin solution in PBS and cut into strips. After washing with T-PBS, strips were incubated overnight at 4 °C with serum samples diluted 1:100 by volume or anti-survivin mAbs (1.0 mg/L; Clone 91618.11; Genzyme/Techne). After washing with T-PBS, strips were incubated for 30 min at room temperature with a 1:1000 dilution of rabbit anti-mouse immunoglobulin or a 1: 2000 dilution of rabbit anti-human IgG F(ab')2 conjugated with horseradish peroxidase (DAKO). After washing with T-PBS, strips were developed by use of a diaminobenzidine solution (Sigma). Staining patterns of anti-survivin mAbs (lane A) and sera for two patients with gastric cancer (lanes B and C) and two healthy donors (lanes D and E) are shown in Fig. 1B
. Anti-survivin mAb and sera from gastric cancer patients recognized the 32-kDa recombinant survivin protein (His6-T7-TAT-myc-survivin). No reactivity was found in sera from the two healthy donors.
We demonstrated anti-survivin antibodies in patients with various gastrointestinal cancers. When anti-p53 antibodies were additionally considered, the overall prevalence of antibody reactivity was not increased. However, determination of survivin antibodies may be useful for detecting gastrointestinal cancers.
Survivin expression has been detected in 53.2% of colorectal cancers (6), and mRNA transcripts encoding survivin were detected in 63.5% of recurrent colorectal cancers by a reverse transcriptase-PCR (8). However, Rohayem et al. (11) reported that 4 of 49 colorectal cancer patients (8.2%) and 11 of 51 sera from lung cancer patients (21.6%) reacted with His-tagged recombinant survivin protein in an anti-survivin ELISA. In the present study, antibody responses against survivin were not always apparent in all patients whose cancers expressed survivin. In addition, survivin expression has been detected in 70.7% of breast cancer patients (10), but antibody responses against survivin were 6.3% (1 in 16) of breast cancer patients (unpublished data). Our results and the reported data suggest that the site of tumor origin influences anti-survivin reactivity.
We found anti-survivin reactivity 1 year after operation in a biliary-tract cancer patient who had no anti-survivin antibodies before or shortly after surgery (data not shown). At the time reactivity was detected, a local recurrence of cancer was found. In addition, anti-survivin reactivity in three biliary-tract cancer patients who had high anti-survivin reactivity before their operations did not show a decrease at 1 week after operation (data not shown). In one of three patients, a local recurrence of cancer was found at the time of writing.
References
The following articles in journals at HighWire Press have cited this article:
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  J. T. Chang, F.-H. Wong, C.-T. Liao, I-H. Chen, H.-M. Wang, and A.-J. Cheng Enzyme Immunoassay for Serum Autoantibody to Survivin and Its Findings in Head-and-Neck Cancer Patients Clin. Chem., July 1, 2004; 50(7): 1261 - 1264. [Full Text] [PDF]
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 C. Casati, P. Dalerba, L. Rivoltini, G. Gallino, P. Deho, F. Rini, F. Belli, D. Mezzanzanica, A. Costa, S. Andreola, et al. The Apoptosis Inhibitor Protein Survivin Induces Tumor-specific CD8+ and CD4+ T Cells in Colorectal Cancer Patients Cancer Res., August 1, 2003; 63(15): 4507 - 4515. [Abstract] [Full Text] [PDF]
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A. Yagihashi, K. Asanuma, N. Tsuji, T. Torigoe, N. Sato, K. Hirata, and N. Watanabe Detection of Anti-Livin Antibody in Gastrointestinal Cancer Patients Clin. Chem., July 1, 2003; 49(7): 1206 - 1208. [Full Text] [PDF]
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 Y. Hirohashi, T. Torigoe, A. Maeda, Y. Nabeta, K. Kamiguchi, T. Sato, J. Yoda, H. Ikeda, K. Hirata, N. Yamanaka, et al. An HLA-A24-restricted Cytotoxic T Lymphocyte Epitope of a Tumor-associated Protein, Survivin Clin. Cancer Res., June 1, 2002; 8(6): 1731 - 1739. [Abstract] [Full Text] [PDF]
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