Increased calreticulin stability in differentiated NG-108-15 cells correlates with resistance to apoptosis induced by antisense treatment

Abstract

Since its first identification as a high-affinity calcium-binding protein over two decades ago [T.J. Ostwald and D.H. MacLennan, Isolation of a high-affinity calcium-binding protein from sarcoplasmic reticulum, J. Biol. Chem., 249 (1974) 974–979], calreticulin has become recognized as a multifunctional protein involved in a wide variety of cellular processes. We have previously shown that it has a protective function in Ca 2+-mediated cell death [N. Liu, R.E. Fine, E. Simons and R.J. Johnson, Decreasing calreticulin expression lowers the Ca 2+ response to bradykinin and increases sensitivity to ionomycin in NG-108-15 cells, J. Biol. Chem., 269 (1994) 28635–28639]. We report here that in NG-108-15 neuroblastoma×glioma hybrid cells, calreticulin protein levels increase markedly when these cells are induced to differentiate by treating them with N, N-dibutyryl cAMP (db-cAMP). We demonstrate that the reason for this increase is mostly due to a large increase in the turnover time of calreticulin in differentiated cells. We also show that a calreticulin antisense oligonucleotide, CrtAS1, previously described by Liu and co-workers [N. Liu, R.E. Fine, E. Simons and R.J. Johnson, Decreasing calreticulin expression lowers the Ca 2+ response to bradykinin and increases sensitivity to ionomycin in NG-108-15 cells, J. Biol. Chem., 269 (1994) 28635–28639] causes cell death in undifferentiated NG-108-15 cells when antisense treatment is extended for more than 24 h. This effect is not seen in NG-108-15 cells that have been induced to differentiate with db-cAMP until the cells have been treated with antisense for more than 4 days, due to the increased stability of Crt in these cells. Our results indicate that the mechanism by which these cells die is likely to be apoptosis.