kn-62 and Lung-Neoplasms

Because changes in intracellular Ca2+ affect progression through the mitotic cell cycle, we investigated the role of Ca2+-binding proteins in regulating cell cycle progression. Evidence was found demonstrating that the activation of Ca2+/calmodulin-dependent protein kinase (CaM kinase) inhibits cell cycle progression in small cell lung carcinoma (SCLC) cells. We also demonstrated that SCLC cells express both CaM kinase type II (CaMKII) and CaM kinase type IV (CaMKIV). Five independent SCLC cell lines expressed proteins reactive with antibody to the CaMKII beta subunit, but none expressed detectable proteins reactive with antibody to the CaMKII alpha subunit. All SCLC cell lines tested expressed both the alpha and beta isoforms of CaMKIV. Immunoprecipitation of CaMKII from SCLC cells yielded multiple proteins that autophosphorylated in the presence of Ca2+ / calmodulin. Autophosphorylation was inhibited by the CaMKII(281-302) peptide, which corresponds to the CaMKII autoinhibitory domain, and by 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine (KN-62), a specific CaM kinase antagonist. Influx of Ca2+ through voltage-gated Ca2+ channels stimulated phosphorylation of CaMKII in SCLC cells, and this was inhibited by KN-62. Incubation of SCLC cells of KN-62 potently inhibited DNA synthesis, and slowed progression through S phase. Similar anti-proliferative effects of KN-62 occurred in SK-N-SH human neuroblastoma cells, which express both CaMKII and CaMKIV, and in K562 human chronic myelogenous leukemia cells, which express CaMKII but not CaMKIV. The expression of both CaMKII and CaMKIV by SCLC cells, and the sensitivity of these cells to the anti-proliferative effects of KN-62, suggest a role for CaM kinase in regulating SCLC proliferation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinase Type 4; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Small Cell; Cell Division; DNA; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Isoquinolines; Lung Neoplasms; Piperazines; Tumor Cells, Cultured

Although small cell lung carcinoma (SCLC) cells express both voltage-gated Ca2+ channels (VGCC) and second messenger-operated Ca2+ channels (SMOCC), little is known about the factors that regulate the activity of these channels in SCLC cells. Ca2+/calmodulin-dependent protein kinase (CaM kinase) type II has been implicated recently in regulating Ca2+ channel activity in other cell types. Because of this, we investigated the effects of the specific CaM kinase antagonist 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tryosyl] -4-phenylpiperazine [sequence: see text] (KN-62) on Ca2+ channel activity in SCLC cells. Incubation with 10 microM KN-62 for 20 min inhibited depolarization-dependent 45Ca2+ influx by 96.1 +/- 2.1% in four independent SCLC cell lines, and by 42.2 +/- 6.8% in the NCI-H146 SCLC cell line. Similar inhibitory effects of KN-62 were observed when Fura-2 was used to measure depolarization-dependent Ca2+ influx. These results indicate that KN-62 potently inhibits VGCC activity in SCLC cells. In contrast, KN-62 (10 microM, 20 min) did not inhibit significantly Ca2+ mobilization induced by muscarinic acetylcholine receptor (mAChR) activation in SCLC cells. This indicates that SMOCC are less susceptible than VGCC to inhibition by KN-62 in SCLC cells. Because mAChR activation also inhibits VGCC activity in SCLC cells, we examined the effects of KN-62 on the mAChR-mediated inhibition of VGCC activity. To do this, we measured depolarization-dependent 45Ca2+ influx in SCLC cells incubated with submaximal concentrations of KN-62 and the mAChR agonist carbachol. Treatment of cells with both drugs resulted in almost twice as much inhibition of VGCC activity as in cells treated with only one of the drugs. This indicates that inactivation of CaM kinase with KN-62 does not suppress the ability of mAChR agonists to inhibit VGCC activity.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Small Cell; Enzyme Inhibitors; Humans; Isoquinolines; Lung Neoplasms; Piperazines; Receptors, Muscarinic; Tumor Cells, Cultured