Expression of progesterone induced blocking factor (PIBF) by human cell lines

Abstract

3191 Background: PIBF, synthesized by gamma delta T cells exposed to progesterone, acts as an inhibitor of natural killer cells at the maternal-fetal interface. NK cells are also critical to host antitumor defense. PIBF expression by malignant cells could allow evasion from the immune system. The role of PIBF in human cancer remains unknown. Methods: We examined PIBF expression by human cell lines using immunohistochemistry and RT-PCR. All cell lines were maintained in RPMI 1640 5% fetal calf serum without antibiotics at 37oC in a 5% CO2 incubator. Cells in log phase growth with greater than 95% viability were stained with anti-PIBF antibody and DAKO Rabbit EnVision Plus kit according to their protocol. RNA extracted by Qiagen kit, was treated with DNAse I and RNAse inhibitor, followed by heating. RT step using Superscript II from Invitrogen according to their protocol was followed by PCR amplification of cDNA for 40 cycles using Taq polymerase and primers for PIBF (accession number AF330046 L: 5'- CCT-GCG-TGA-CTT-TGA-GTT-GA-3'; R: 5'-CTG-TCA-GCT-GCT-TCA-GTT-GG-3'). Expected product size was 206 bp. GAPDH served as positive control. The PCR products were separated by agarose gel electrophoresis, visualized by ethidium bromide staining, and photographed. Results: All cell lines tested expressed PIBF mRNA: T cell lines: SRIK-NKL, MOT, HUT 102, HUT 78, HTLV1+transformed CD8+ cell line, SRID-TL, JURKAT, SRIA-TL, SRIP-TL, CEM; Myeloid lineage: THP-1,U937, K562, HL60, SRIS-EOSL, ML-1, HEL; B cell lineage: T-PLL, R-CLL, 729pH6neo, SRIH-B(ATL), U266, 8226; epithelial: MDE, KATO-3, CaCo-2, MeWo, MCF-7; fibroblast: BG9; G-CSF mobilized CD34+ normal cells . PCR without RT step gave no product. In addition, SRIK-NKL, MOT, U937, HL60, R-CLL, MD-B, 729pH6neo, SRIH-B (ATL), T-PLL, and MeWo were stained for protein. Only SRIK-NKL (natural killer), U937 (monoblast), HL60 (myeloid), and T-PLL (B cell) were positive by IHC. Conclusions: PIBF mRNA was widely expressed, and protein was detected in 4/10 leukemic lines tested indicating a role for PIBF in some malignancies and suggesting PIBF as a novel target for therapy. No significant financial relationships to disclose.