1. ¹Faculty of Veterinary Medicine, Institute of Pharmacology, Pharmacy and Toxicology, University of Leipzig, Leipzig, Germany
2. ²Department of Molecular Cell Therapy, Center for Biotechnology and Biomedicine, Faculty of Medicine, University of Leipzig, Leipzig, Germany

Correspondence: Dr S Halwachs, Faculty of Veterinary Medicine, Institute of Pharmacology, Pharmacy and Toxicology, University of Leipzig, An den Tierkliniken 15, 04103 Leipzig, Germany. E-mail: halwachs@vetmed.uni-leipzig.de

Received 27 August 2008; Revised 11 December 2008; Accepted 7 January 2009; Published online 12 February 2009.

Abstract

Concurrent treatment with methotrexate (MTX) and antiepileptic drugs, such as phenobarbital (PB), reduces the efficacy of MTX chemotherapy in childhood acute lymphoblastic leukemia (ALL). This can result from defective Reduced folate carrier (Rfc1)-dependent cellular uptake of MTX. Indeed, we have shown that functional Rfc1 activity is significantly reduced by clinically relevant concentrations of the anticonvulsant drugs PB or carbamazepine in an adequate in vitro model. As PB is known to regulate carrier-associated transport by the nuclear receptor constitutive androstane receptor (CAR), we investigated the involvement of the CAR signaling cascade and the mode of PB-induced downregulation of Rfc1 activity. CAR activation by PB or the CAR agonist 1,4-bis[2-(3,5-dichloro- pyridyloxy)]-benzene resulted in translocation of Ca²⁺-dependent protein kinase C (cPKC) to the plasma membrane related to significantly elevated PKC activities. In contrast, subcellular localization of Ca²⁺-independent PKC was only marginally altered. Studies on intracellular distribution of the Rfc1 protein indicated that PB-induced activation of cPKC was associated with carrier internalization from the plasma membrane into the cytosol independent of the Rfc1 phosphorylation status. In conclusion, we identified for the first time the molecular mechanism of this clinically relevant drug resistance in patients with ALL concurrently receiving MTX chemotherapy and antiepileptic drugs.