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Antisense palmitoyl protein thioesterase 1 (PPT1) treatment inhibits PPT1 activity and increases cell death in LA‐N‐5 neuroblastoma cells
Author(s) -
Cho Seongeun,
Dawson Philip E.,
Dawson Glyn
Publication year - 2000
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/1097-4547(20001015)62:2<234::aid-jnr8>3.0.co;2-8
Subject(s) - apoptosis , programmed cell death , neuroblastoma , biology , microbiology and biotechnology , ceramide , chemistry , cell culture , biochemistry , genetics
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 μM) 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. J. Neurosci. Res. 62:234–240, 2000. © 2000 Wiley‐Liss, Inc.