Exposure of neuroblastoma cell lines to imatinib results in the upregulation of the CDK inhibitor p27(KIP1) 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 (≥ 10 μM) are substantially higher than those required for inhibition of ligand-induced phosphorylation of wild type c-Kit and PDGFR (≤ 1 μM), 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 1-15 μM 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 p27(KIP1) in the nuclear compartment as a result of increased p27(KIP1) protein stability. We demonstrate that the mechanism of p27(KIP1) stabilization relied on inhibition of p27(KIP1) 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.

Keywords: 5-bromo-2′-deoxyuridine (PubChem CID: 6035); CDK2; Cycloheximide (PubChem CID: 6197); Hoechst 33258 (PubChem CID: 16218619); Imatinib; Imatinib (PubChem CID: 123596); Neuroblastoma; c-Abl; p27(KIP1); pRb.