Flow cytometric characterization of the DAOY medulloblastoma cell line for the cancer stem-like phenotype.

Side population (SP) analyses and CD133 expression have identified cells with stem-like potential in normal and cancerous tissue. Whether stem-like cells exist in cancer cell lines is hotly debated. We have interrogated the DAOY medulloblastoma cell line with respect to stem-like potential. Vital staining for Hoechst 33342 efflux capacity and CD133 immunophenotyping were performed on DAOY cells to assess the presence of the SP and the CD133 stem cell markers, respectively. SP/non-SP and CD133(+)/CD133(-) DAOY cells were sorted into separate fractions for limiting dilution analysis (tumor sphere assay) and asymmetric division assessment. SP/non-SP cells were also sorted separately for viability (XTT assay), cell size, cell cycle status, and proliferative capacity (carboxyfluorescein succinimidyl ester (CFSE)) evaluation. A minor proportion of cells displayed either the SP or the CD133(+) phenotypes. CD133 expression mapped to both the SP and non-SP compartments, with CD133(+) cells being enriched almost fourfold within the non-SP gate. The SP, non-SP, CD133(+), and CD133(-) fractions were all capable of reconstituting the original parental DAOY population. Slight clonogenic enrichment was observed in only the SP fraction; however, both CD133(+) and CD133(-) cells displayed equivalent stem cell-like frequencies. SP cells were resistant to Hoechst 33342-mediated toxicity relative to the parental population and differed from the non-SP cells with respect to increased cell size, decreased S-phase, and slightly decreased proliferative capacity. The multiparametric strategy described in this study revealed that the SP and CD133(+) subset may be two independent compartments. Our results highlight the need for new reliable specific cancer stem cell marker(s) as Hoechst 33342 efflux and CD133 expression might not be suitable for selectively isolating cancer stem-like cells from cell lines, as shown for the DAOY cells. As such, care must be used in interpreting therapeutic studies targeting the stem cell compartment of cancer cell lines.

The cellular and developmental biology of medulloblastoma: current perspectives on experimental therapeutics.

Dysregulation of various signaling pathways that govern cerebellar development with respect to cell proliferation, growth arrest, apoptosis and differentiation has been postulated to contribute to medulloblastoma tumourigenesis. This review will highlight the unique nature of cerebellar development in terms of its derivation from two germinal matrices and significant postnatal expansion of the granule cell precursor (GCP) compartment resulting in granule cell development and migration to form the mature cerebellar cortex. The molecular signals that are critical for timely cell cycle exit and differentiation may become dysregulated leading to unrestrained cell proliferation and enhanced cell survival; indeed, changes in these molecular markers have been observed in medulloblastoma biopsy specimens. Furthermore, transgenic models that faithfully replicate these changes develop medulloblastoma that, by in large, recapitulates the clinico-histopathological features of these tumours. Cellular and developmental biological approaches have contributed greatly to the current debate on the relevance of the cancer stem cell hypothesis in understanding medulloblastoma initiation and propagation. Penultimately, research findings are being translated into experimental therapeutics that target the aberrant signal transduction machinery in medulloblastoma cells and that will hopefully lead to an improved risk-benefit profile.

Impaired medulloblastoma cell survival following activation of the FOXO1 transcription factor.

Medulloblastoma is the most frequent type of childhood brain tumour. The insulin-like growth factor I receptor (IGF-IR) plays a significant neuroprotective role in medulloblastoma survival through regulation of the downstream effectors of the phosphoinositide-3-kinase-protein kinase-B (PI3K-PKB/c-Akt) pathway. One such target is Forkhead box O1 (FOXO1; FKHR), which is part of the FOXO family of Forkhead transcription factors. Phosphorylation by Akt results in cytoplasmic sequestration of FOXO1 thus inhibiting the expression of genes controlling cell death, cell proliferation, differentiation, cellular metabolism and oxidative stress. Here we show that serum starvation of medulloblastoma cells is accompanied by nuclear translocation of FOXO1. IGF-I stimulation of serum-starved cells resulted in rapid phosphorylation of Akt and FOXO1, and was associated with a significant increase in cell viability. In contrast, expression of a constitutively active form of FOXO1 that cannot be phosphorylated led to a significant reduction in medulloblastoma cell viability, even in the presence of growth factors provided by fetal bovine serum (FBS). These data suggest that the transcription factor FOXO1 may be a critical effector of medulloblastoma growth suppression.