(56) CyPD OVEREXPRESSION IN COS-7 CELLS

N.I. Folarin¹, R.J. Heads², S.L. Hart², C.J. Green¹ and L.V. Andreeva^1
1Northwick Park Institute for Medical Research, The Rayne Institute 
²The Institute of Child Health, London, UK

Cyclophilins were first described as intracellular receptors for cyclosporine A (CsA).  One of the intracellular cyclophilins has been identified as an integral part of the mitochondrial permeability transition pore (MPTP) complex.  Formation of the MPTP is considered to be responsible for mitochondrial transmembrane potential collapse and consequently cellular death in the events of ischaemia-reperfusion injury.  cDNA encoding human cyclophilin-D gene was kindly provided by Dr. Dirk Bergsma and subcloned into the vector pcDNA3.  The hCyp3 (CyPD) gene product contains a consensus mitochondrial targeting sequence and shares identity with the mitochondrial CsA receptor.  In order to study the overexpression of this gene, the pcDNA3-CyPD construct was transiently transfected into COS-7 cells.  After overexpression was confirmed by Western blotting, RT-PCR and PPI-ase activity, COS-7 cells were subjected to severe ischaemia and subsequently reperfused.  Cell viability analysis demonstrated that overexpression of mitochondrial CyPD had no significant effect on the cellular response to ischaemia-reperfusion injury.  We are currently investigating the effects of overexpressing CyPD in stressed cardiomyocytes, upon mitochondrial transmembrane potential, apoptosis and necrotic cell death.

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