RT Journal Article SR Electronic T1 Peripheral Benzodiazepine Receptor: Characterization in Human T-Lymphoma Jurkat Cells JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 37 OP 44 DO 10.1124/mol.105.015289 VO 69 IS 1 A1 Barbara Costa A1 Alessandra Salvetti A1 Leonardo Rossi A1 Francesca Spinetti A1 Annalisa Lena A1 Beatrice Chelli A1 Mariarosa Rechichi A1 Eleonora Da Pozzo A1 Vittorio Gremigni A1 Claudia Martini YR 2006 UL http://molpharm.aspetjournals.org/content/69/1/37.abstract AB 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 (Kd) 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 Ki >1.0 × 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.