Detection of anti-livin antibody in gastrointestinal cancer patients.

Abstract

Livin, a recently described member of the inhibitor of apoptosis protein (IAP) family, contains a single baculovirus IAP repeat and a carboxyl-terminal RING finger (1)(2)(3). Like other proteins in the IAP family, livin binds specifically to a terminal effector cell-death protease, in this instance, caspase-9 (1)(2)(3). The consequences are substantially reduced caspase activity and reduced cell death in response to diverse apoptotic stimuli (1)(2)(3). Semiquantitative reverse transcription-PCR methods have detected human livin mRNA in fetal kidney, heart, and spleen and in adult tissues such as heart, lung, spleen, ovary, and placenta (4). In addition, livin mRNA is overexpressed by some cancer cells, including melanoma, breast cancer, cervical cancer, colon cancer, prostate cancer, leukemia, and lymphoma cells (4). As with survivin overexpression (5)(6)(7), livin overexpression by cancer cells may lead to anti-livin antibody responses and cytotoxic T-lymphocyte responses against the cancer. In the present study, we examined livin mRNA expression in gastrointestinal cancer cell lines and the prevalence of antibody responses against livin in patients with various gastrointestinal cancers. We cultured human pancreatic cancer cell lines (PANC-1, Capan-1, AsPC-1, MIAPaCa-2, and BxPC-3), gastric cancer cell lines (MKN-1, MKN-45, and TMK-1), colon cancer cell lines (HT-29, SW480, SW620, and LSI180), and a hepatoma cell line (HepG2) in RPMI-1640 with 100 mL/L calf serum at 37 °C in 5% CO2. Livin mRNA expression was quantified by a previously reported method (8). The sequence of the forward primer was 5′-TCAGTTCCTGCTCCGGTCA-3′, that the reverse primer was 5′-CGTCTTCCGGTTCTTCCCA-3′, and the sequence of the TaqMan probe was 5′-CCACAGTGTGCAGGAGACTCACTCCC-3′. As an internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA was used and amplified with TaqMan control reagents (Perkin-Elmer Applied Biosystems). The conditions of the one-step reverse transcription-PCR have been described previously …