Genomic aberrations of
Cyclin D1 (CCND1), CDK4, and CDK6 in
neuroblastoma indicate that dysregulation of the G(1) entry checkpoint is an important cell cycle aberration in this pediatric
tumor. Here, we report that analysis of Affymetrix expression data of primary neuroblastic
tumors shows an extensive overexpression of
Cyclin D1, which correlates with histologic subgroups. Immunohistochemical analysis showed overexpression of
Cyclin D1 in neuroblasts and low
Cyclin D1 expression in all cell types in
ganglioneuroma. This suggests an involvement of G(1)-regulating genes in neuronal differentiation processes which we further evaluated using RNA interference against
Cyclin D1 and its
kinase partners CDK4 and CDK6 in several
neuroblastoma cell lines. The
Cyclin D1 and CDK4 knockdown resulted in pRb pathway inhibition as shown by an almost complete disappearance of CDK4/CDK6-specific pRb phosphorylation, reduction of E2F transcriptional activity, and a decrease of
Cyclin A protein levels. Phenotype analysis showed a significant reduction in cell proliferation, a G(1)-specific cell cycle arrest, and, moreover, an extensive neuronal differentiation. Affymetrix microarray profiling of
small interfering RNA-treated cells revealed a shift in expression profile toward a neuronal phenotype. Several new potential downstream players are identified. We conclude that
neuroblastoma functionally depend on overexpression of G(1)-regulating genes to maintain their undifferentiated phenotype.