kn-93 and Neuroblastoma

We previously demonstrated a loss in Ca(2+)/Calmodulin-dependent protein kinase (CaM kinase) activity in SH-SY5Y undergoing thapsigargin-mediated apoptosis. To extend that finding we report that CaM kinase inhibition potentiates thapsigargin-mediated cell death. CaM kinase inhibitor KN93 on its own exhibits little toxicity up to 10 mM, as measured by release of lactate dehydrogenase (LDH) into the culture medium. In SH-SY5Y cells pretreated with KN93 and the non-selective protein kinase inhibitor k252a and then treated with 2 mM thapsigargin, loss of viability is significantly greater than in cells treated with thapsigargin alone. Pretreatment with the pan-caspase inhibitor Z-D-DCB prevented the thapsigargin-mediated increase in LDH release. Furthermore, thapsigargin-induced caspase-3-like activation, demonstrated by poly(ADP)ribose polymerase cleavage and pro-caspase-3 processing, was elevated in the presence of KN93.

Topics: Apoptosis; Aspartic Acid; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Caspase 3; Caspase Inhibitors; Caspases; Cell Survival; Enzyme Inhibitors; Humans; Indole Alkaloids; Neuroblastoma; Neurons; Protease Inhibitors; Sulfonamides; Thapsigargin; Tumor Cells, Cultured

Since the alpha and beta isoforms of CaM kinase II are known to be expressed almost exclusively in the brain, we compared the effect of overexpression of the beta isoform of CaM kinase II with that of the alpha isoform. The subcellular distribution of the alpha isoform was different from that of the beta isoform, although the catalytic properties of the alpha and beta isoforms expressed in transfected cells were similar to those of brain CaM kinase II. The alpha isoform was found in the soluble fraction more than in the particulate fraction, whereas most of the beta isoform bound to subcellular structures. In the cell overexpressing alpha and beta isoforms of CaM kinase II, neurite extension was promoted when compared with the morphology of neo transfectants. Neurite outgrowth of cells overexpressing CaM kinase II was further stimulated by the treatment of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a selective but not absolutely specific inhibitor of protein kinase C. The morphological change was rapid and observed within 1 h followed by H-7 treatment. Morphological changes, such as the number of cells with neurites and length of neurites were greater in the beta cells than in the alpha cells. Chelerythrine, a specific inhibitor of protein kinase C, also stimulated the neurite outgrowth of these cells. Some substrates of CaM kinase II related to neurite outgrowth were detected in cells overexpressing CaM kinase II stimulated with H-7. These results suggest that CaM kinase H and protein kinase C play an important role in the control of cell change, and that the subcellular distribution of CaM kinase II is important for regulating cellular functions efficiently.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amino Acid Sequence; Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cloning, Molecular; Electrophoresis, Gel, Two-Dimensional; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Immunoblotting; Isoenzymes; Isoquinolines; Mice; Molecular Sequence Data; Neurites; Neuroblastoma; Phosphorylation; Substrate Specificity; Sulfonamides; Transfection; Tumor Cells, Cultured