Antisense palmitoyl protein thioesterase 1 (PPT1) treatment inhibits PPT1 activity and increases cell death in LA-N-5 neuroblastoma cells

Infantile neuronal ceroid lipofuscinosis (INCL) is a childhood neurodegener ative disease caused by the selective death of cortical neurons and retinal degeneration, as the result of a palmitoyl protein thioesterase 1 (PPT1) d eficiency. Recently, we showed that overexpression of PPT1 protects LA-N-5 human neuroblastoma cells against apoptotic death (Cho and Dawson [2000a] J . Neurochem, 74:1478-1488) and we now show that inhibition of PPT1 increase s the susceptibility of these cells to apoptotic cell death. Transient tran sfection of LA-N-5 neuroblastoma cells with PPT1-FLAG resulted in a strong expression of PPT-FLAG-tagged protein as evidenced by Western blot analysis and immunofluorescence, Co-transfection of a reverse-oriented (antisense) PPT1 (AS-PPT1) decreased the expression of PPT-FLAG to almost zero, reduced PPT1 enzyme activity (as measured by an in vitro assay) and increased the susceptibility to apoptosis induced by C-2 ceramide. Similarly, inhibition of PPT1 with a synthetic inhibitor (AcG-palmitoyl diaminoproprionate-VKIKK) (DAPI) (100 mu M) increased the susceptibility of the cells to apoptosis i nduced by either C-2-ceramide or etoposide, a common chemotherapeutic agent used in the treatment of neuroblastoma, Cells stably overexpressing PPT1 w ere resistant to apoptosis induced by DAP1 suggesting that the inhibitor ha s a specific action and confirming that low levels of protein palmitoylatio n block the death pathway. Drugs that raise the level of protein palmitoyla tion are pro-apoptotic and PPT1 inhibition may enhance the killing efficacy of chemotherapeutic agents used to kill neuroblastoma-derived cells. (C) 2 000 Wiley-Liss,Inc.