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 Costa, Barbara
A1 Salvetti, Alessandra
A1 Rossi, Leonardo
A1 Spinetti, Francesca
A1 Lena, Annalisa
A1 Chelli, Beatrice
A1 Rechichi, Mariarosa
A1 Da Pozzo, Eleonora
A1 Gremigni, Vittorio
A1 Martini, Claudia
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.