Fumihiko Kugawa

Buprenorphine hydrochloride induces apoptosis in NG108-15 nerve cells

A morphine alkaloid derivative, buprenorphine hydrochloride, induces apoptosis in NG108-15 cells. Apoptosis was detected mainly by apoptosis-specific DNA fragmentation and morphological changes. This apoptosis was dose-dependent and the time-course experiment indicated that DNA fragmentation occurred within 4 h after administration of buprenorphine hydrochloride. Specific inhibitors of the previously characterized apoptotic signal cascade as well as antagonists for opioid receptors were tested. Zn2+, herbimycin A, caspase inhibitors YVAD (Ac-Tyr-Val-Ala-Asp-CHO) and DEVD (Ac-Asp-Glu-Val-Asp-CHO), naloxone and naltrindole had no effect on apoptosis-specific DNA fragmentation. The serine protease inhibitor TPCK (N-tosyl-L-phenylalanyl chloromethyl ketone) specifically inhibited apoptosis-specific DNA fragmentation induced by buprenorphine hydrochloride; however, cell viability measurements revealed that cell death still occurred in NG108-15 cells. Thus TPCK pretreatment before buprenorphine hydrochloride administration induced apoptosis-independent cell death, presumably necrosis, in NG108-15 cells. This suggests that an unidentified serine protease, presumably functioning in the buprenorphine hydrochloride-specific death-signal cascade, could be pivotal for the rapid apoptosis observed in NG108-15 cells upon treatment with buprenorphine hydrochloride.

Expression of the Polyubiquitin Gene Early in the Buprenorphine Hydrochloride-induced Apoptosis of NG108-15 Cells

To clone genes expressed early in the buprenorphine hydrochloride (Bph)-induced apoptosis of NG108-15 nerve cells, we adopted a previously reported rapid and simple differential display (DD) cloning procedure. Complementary DNA was generated from differentially expressed mRNAs by reverse transcription (RT) using a fully degenerate 6-mer oligonucleotide as the primer. PCR amplification was then conducted using a combination of three arbitrary but defined 10-bp nucleotide primers. The differentially generated DNA fragments were detected by agarose gel electrophoresis, and 9 were excised from the gel and subcloned into a sequencing vector. Three DNAs that were specifically expressed upon Bph-induced apoptosis of NG108-15 cells were sequenced. Their specific expression was then confirmed by reverse-transcription PCR. One was identified as the mouse polyubiquitin gene c, and the others remain unidentified. Northern and western blots indicated the transcription and translation of polyubiquitin early in Bph-induced apoptosis. The polyubiquitination of apoptotic cellular proteins was also confirmed.