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 neurodegenerative disease caused by the selective death of cortical neurons and retinal degeneration, as the result of a palmitoyl protein thioesterase 1 (PPT1) deficiency. 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 increases the susceptibility of these cells to apoptotic cell death. Transient transfection 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) (DAP1) (100 microM) increased the susceptibility of the cells to apoptosis induced by either C(2)-ceramide or etoposide, a common chemotherapeutic agent used in the treatment of neuroblastoma. Cells stably overexpressing PPT1 were resistant to apoptosis induced by DAP1 suggesting that the inhibitor has a specific action and confirming that low levels of protein palmitoylation block the death pathway. Drugs that raise the level of protein palmitoylation are pro-apoptotic and PPT1 inhibition may enhance the killing efficacy of chemotherapeutic agents used to kill neuroblastoma-derived cells.
Cho
S
S
Departments of Pediatrics, Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.
Dawson
P E
PE
Dawson
G
G
D000970:Antineoplastic Agents / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D016373:DNA, Antisense / Q000494:pharmacology*; D006801:Humans; D009447:Neuroblastoma / Q000378:metabolism; D009472:Neuronal Ceroid-Lipofuscinoses / Q000378:metabolism; D009944:Organoplatinum Compounds / Q000494:pharmacology*; D010170:Palmitoyl-CoA Hydrolase / Q000096:biosynthesis / Q000187:drug effects* / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000970:Antineoplastic Agents; D016373:DNA, Antisense; D009944:Organoplatinum Compounds; C042496:ARK 62-62; D010170:Palmitoyl-CoA Hydrolase