Peripheral benzodiazepine receptor antisense knockout increases tumorigenicity

ABSTRACT: Peripheral benzodiazepine receptors (PBR), first described more than 20 years ago, have been attributed with many putative functions including ones in cellular proliferation and cellular respiration. Hence, it is quite conceivable that deregulation of this receptor could lead to pathology. We and others have reported the existence of PBR overexpression in different human and nonhuman malignancies, but it has never been made clear whether this aberrant malignant PBR expression is a cause or consequence of the cancer. In the current study we induced PBR underexpression by downregulating one critical subunit of the PBR complex, the isoquinoline-binding protein (IBP), using the stable antisense knockout approach, in the MA-10 Leydig cell line. Resultant clones, showing PBR deregulation, also demonstrated increased tumorigenicity, using both in vitro (loss of contact inhibition and growth in soft agar) and in vivo (increased mortality on grafting back into isogenic mice) assays. We suggest that this type of deregulation could be a later event in natural tumor progression. Consequently, PBR deregulation should be more closely studied in human malignancy. Peripheral benzodiazepine receptors (PBR), 1 while widely expressed throughout the body, exhibit different patterns of tissue-specific expression (1-5). Although the PBR were first described more than 20 years ago (6) and a broad spectrum of putative functions has been suggested for them The PBR is an intracellular multisubunit protein receptor, located mainly on the outer mitochondrial membrane (26) and composed of three subunits: the isoquinoline-binding protein (IBP), 1 which is 18 kDa in mass, the voltagedependent anion channel (32 kDa), and the adenine nucleotide transporter (30 kDa) MATERIALS AND METHODS Cells. The MA-10 cell line, originally cloned from the solid M5480P mouse tumor Leydig cell, was a kind gift of Dr. Mario Ascoli (Department of Pharmacology, University of Iowa College of Medicine, Iowa City, IA). MA-10 cell cultures were grown as previously described (30). Briefly, cells were maintained in RPMI 1640 medium with Lglutamine, containing 20 mM HEPES buffer, 15% horse serum, 300 mg/L glutamine, and 50 µg/mL gentamicin sulfate (all purchased from Biological Industries, Beit HaEmek, Israel). MA-10 transfected cells were grown in similar medium, except for the use of 7.5% horse serum and 7.5% fetal calf serum in place of 15% horse serum