Key role for p27Kip1, retinoblastoma protein Rb, and MYCN in polyamine inhibitor-induced G1 cell cycle arrest in MYCN-amplified human neuroblastoma cells

Alpha-difluoromethylornithine (DFMO) inhibits the protooncogene ornithine decarboxylase (ODC) and is known to induce cell cycle arrest. However, the effect of DFMO on human neuroblastoma (NB) cells and the exact mechanism of DFMO-induced cell death are largely unknown. Treatment with DFMO in combination with SAM486A, an S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor, has been shown to enhance polyamine pool depletion. Therefore, we analysed the mechanism of action of DFMO and/or SAM486A in two established MYCN-amplified human NB cell lines. DFMO and SAM486A caused rapid cell growth inhibition, polyamine depletion, and G(1) cell cycle arrest without apoptosis in cell lines LAN-1 and NMB-7. These effects were enhanced with combined inhibitors and largely prevented by cotreatment with exogenous polyamines. The G(1) cell cycle arrest was concomitant with an increase in cyclin-dependent kinase inhibitor p27(Kip1). In a similar fashion, DFMO and DFMO/SAM486A inhibited the phosphorylation of the G(1)/S transition-regulating retinoblastoma protein Rb at residues Ser795 and Ser807/811. Moreover, we observed a dramatic decrease in MYCN protein levels. Overexpression of MYCN induces an aggressive NB phenotype with malignant behavior. We show for the first time that DFMO and SAM486A induce G(1) cell cycle arrest in NB cells through p27(Kip1) and Rb hypophosphorylation.