Down-regulation of protein kinase Cα and γ and enhanced TPA-induced neurite formation in DAN-transfected neuroblastoma cells

Abstract

DAN gene was first isolated by differential screening between rat 3Y1 and v- src-transformed 3Y1 cells and showed a tumor-suppressive activity toward v- src-transformed 3Y1 cells. When DAN-transfected neuroblastoma cells were treated with a tumor promoter phorbol ester, TPA, neurite-like processes appeared within 2 h whereas no apparent change was observed in the parent and vector-transfected cells up to 8 h. This suggests some difference in TPA-receptor, protein kinase C (PKC), between DAN-transfectants and the control cells. DAN-transfected SH-SY5Y cells showed complete loss in PKCα and a large decrease in PKCγ. Similar down-regulation in PKCα and PKCγ was also observed in DAN-transfected Ha- ras-transformed NIH 3T3 cells. The decreased level of PKCα was partially recovered after treatment with a calpain inhibitor, ZLLH. A 150-kDa proteolytic product of a calpain-specific substrate, non-erythroid α-spectrin, was detectable in DAN-transfected SH-SY5Y cells but not in the parent or vector-transfected control cells. This suggests that DAN-transfected cells contain activated calpain which may cause down-regulation of PKC and hence induce the altered TPA response.