ABCG2 is required to control the sonic hedgehog pathway in side population cells with stem-like properties.

The Sonic Hedgehog (Hh) pathway has been implicated in the maintenance of stem or progenitor cells in many adult tissues. Importantly, abnormal Hh pathway activation is also associated with initiation of neoplasia, but its role in tumor growth is still unclear. Here, we demonstrate that cyclopamine, a plant-derived alkaloid product used to inhibit the Hh signaling pathway, reduces the Side Population (SP) obtained by Hoechst 33342 (Ho342) dye measurements. In addition, cyclopamine is able to modulate, along with oxysterols and other products, the ABCG2 transporter by increasing Ho342 and mitoxantrone uptake. Therefore, if the SP is solely measured as a Ho342 dye extruding fraction, this may be significantly modulated by the inhibition of ABCG2 transport fraction, independently from the action of cyclopamine on the Hh pathway. Our results indicate that ABCG2 may act in the upstream regulation of the Hh signaling pathway to protect the stemness of the SP compartment, giving support to the cancer stem cell hypothesis and suggesting that ABCG2 is not only critical for increased resistance to anticancer agents.

Analysis of stemness gene expression and CD133 abnormal methylation in neuroblastoma cell lines.

Neuroblastoma is the most common extracranial solid tumor in children, accounting for up to 10% of all childhood malignancies. Cellular heterogeneity is a hallmark of this embryonal cancer, as distinct neural crest lineages can be found within the same tumor sample. The aim of our study was to investigate the presence of a subpopulation of immature cells with features of cancer-like stem cells in 10 neuroblastoma cell lines. RT-PCR and flow cytometry were performed in order to analyze different kinds of 'stemness genes' such as: NESTIN (NES), CD133, SOX-2, BMI1, c-KIT, MELK1, MUSASHI-1 (MSI1), FAS, CD44 and VIMENTIN (VIM). In addition, glial and neuronal markers such as NCAM1, GFAP and B-TUBULIN III (TUBB3) were analyzed. Epigenetic changes within the CD133 (Prominin-1) gene promoter were also analyzed. Neuroblastoma cell lines showed a particular pattern of expression, suggesting the presence of an immature cancer stem cell-like subpopulation. The CD133 protein, commonly used to enrich putative cancer propagating stem cell-like populations in different kinds of solid tumors, presented a half-methylated DNA state in 7 of the 12 neuroblastoma cell lines analyzed. An increase in RNA and protein levels of CD133 was achieved following demethylation by assays using 5-aza-2'-deoxycytidine (5-Aza-dC). Since cancer stem cells are believed to be responsible for tumor metastasis, escape from anticancer therapies and disease relapse, their therapeutic targeting and analysis is crucial in neuroblastoma. Moreover, the regulation of CD133 by epigenetic changes may provide an innovative mechanism of CD133 expression as its regulation still remains unclear.