Exposure of neuroblastoma cell lines to imatinib results in the upregulation of the CDK inhibitor p27KIP1 as a consequence of c-Abl inhibition

Abstract

Imatinib mesylate is a tyrosine kinase inhibitor with selectivity for abelson tyrosine-protein kinase 1 (c-Abl), breakpoint cluster region (Bcr)-Abl fusion protein (Bcr-Abl), mast/stem cell growth factor receptor Kit (c-Kit), and platelet-derived growth factor receptor (PDGFR). Previous studies demonstrated that imatinib in the low micromolar range exerted antiproliferative effects on neuroblastoma cell lines. However, although neuroblastoma cells express c-Kit and PDGFR, the imatinib concentrations required to achieve significant growth inhibitory effects (≥10μM) are substantially higher than those required for inhibition of ligand-induced phosphorylation of wild type c-Kit and PDGFR (≤1μM), suggesting that additional mechanisms are responsible for the antitumor activity of imatinib on these cells. In this study, we show that treatment of neuroblastoma cell lines with 1–15μM imatinib resulted in a dose dependent inhibition of 5-bromo-2′-deoxyuridine (BrdU) incorporation into newly synthesized DNA. The antiproliferative effect of imatinib was dependent on the upregulation of the cyclin-dependent kinase (CDK) inhibitor p27KIP1 in the nuclear compartment as a result of increased p27KIP1 protein stability. We demonstrate that the mechanism of p27KIP1 stabilization relied on inhibition of p27KIP1 phosphorylation on tyrosine residues by c-Abl. We provide evidence that in neuroblastoma cell lines a significant fraction of cellular c-Abl is phosphorylated on Tyr-245, consistent with an open and active conformation. Notably, exposure to imatinib did not affect Tyr-245 phosphorylation. Given the low affinity of active c-Abl for imatinib, these data provide a molecular explanation for the relatively high imatinib concentrations required to inhibit neuroblastoma cell proliferation.