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Peripheral Benzodiazepine Receptor: Characterization in Human T-Lymphoma Jurkat Cells

Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Pisa, Italy (F.S., B.C., E.D.P., C.M.); and Department of Human Morphology and Applied Biology, University of Pisa, Pisa, Italy (B.C., A.S., L.R., A.L., M.R., V.G.)

Peripheral benzodiazepine receptor (PBR) has been considered a promising drug target for cancer therapy, and several ligands have been developed for this purpose. Human T-lymphoma Jurkat cells have been considered as lacking PBR and are often used as negative control to prove the specificity of PBR ligands effects. It is surprising that we evidenced PBR protein expression in this cell line by means of Western blotting and immunocytochemistry assays using specific anti-PBR antibodies. PBR intracellular localization was evidenced in mitochondria and nuclei, as demonstrated by confocal and electron microscopy. The binding of the [³H]4'-chloro derivative of diazepam [³H]7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one (Ro5-4864) and the isoquinoline carboxamide derivative [³H]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3 isoquinolinecarboxamide (PK11195) evidenced a single class of binding sites with an unusual affinity constant (K_d) of 1.77 ± 0.30 and 2.20 ± 0.20 µM, respectively. The pharmacological profile of the classic ligands showed that PK11195 was the most potent inhibitor in the radioligand binding assays followed by Ro5-4864 and diazepam, whereas clonazepam, a specific ligand for the central-type receptor, showed a K_i >1.0 x 10^–4 M. By a combined strategy of reverse transcriptase-polymerase chain reaction and Southern blot experiments, we succeeded in isolating and cloning the full-length Jurkat PBR cDNA, called JuPBR. The JuPBR gene showed two single-nucleotide polymorphisms resulting in the two substitutions, Ala147 threonine and His162 arginine, of PBR amino acidic sequence. In conclusion, for the first time, we demonstrated PBR expression in Jurkat cells: the protein bound classic PBR ligands with micromolar affinity constants and presented a modified amino acidic sequence consequent to the detection of two gene polymorphisms.

Address correspondence to: Dr. Martini Claudia, Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, via Bonanno, 6-56126 Pisa, Italy. E-mail: cmartini{at}farm.unipi.it