Comparative analysis of DNA repair in stem and nonstem glioma cell cultures.

It has been reported that cancer stem cells may contribute to glioma radioresistance through preferential activation of the DNA damage checkpoint response and an increase in DNA repair capacity. We have examined DNA repair in five stem and nonstem glioma cell lines. The population doubling time was significantly increased in stem compared with nonstem cells, and enhanced activation of Chk1 and Chk2 kinases was observed in untreated CD133(+) compared with CD133(-) cells. Neither DNA base excision or single-strand break repair nor resolution of pH2AX nuclear foci were increased in CD133(+) compared with CD133(-) cells. We conclude that glioma stem cells display elongated cell cycle and enhanced basal activation of checkpoint proteins that might contribute to their radioresistance, whereas enhanced DNA repair is not a common feature of these cells.

NK cells recognize and kill human glioblastoma cells with stem cell-like properties.

In this study, cancer cells were isolated from tumor specimens of nine glioblastoma patients. Glioblastoma cells, cultured under suitable culture conditions, displayed markers typical of neural stem cells, were capable of partial multilineage differentiation in vitro, and gave origin to infiltrating tumors when orthotopically injected in NOD/SCID mice. These cells, although resistant to freshly isolated NK cells, were highly susceptible to lysis mediated by both allogeneic and autologous IL-2 (or IL-15)-activated NK cells. Indeed, all stem cell-cultured glioblastoma cells analyzed did not express protective amounts of HLA class I molecules, while expressing various ligands of activating NK receptors that triggered optimal NK cell cytotoxicity. Importantly, glioblastoma stem cells expressed high levels of PVR and Nectin-2, the ligands of DNAM-1-activating NK receptor.

Different response of human glioma tumor-initiating cells to epidermal growth factor receptor kinase inhibitors.

Because a subpopulation of cancer stem cells (tumor-initiating cells, TICs) is believed to be responsible for the development, progression, and recurrence of many tumors, we evaluated the in vitro sensitivity of human glioma TICs to epidermal growth factor receptor (EGFR) kinase inhibitors (erlotinib and gefitinib) and possible molecular determinants for their effects. Cells isolated from seven glioblastomas (GBM 1-7) and grown using neural stem cell permissive conditions were characterized for in vivo tumorigenicity, expression of tumor stem cell markers (CD133, nestin), and multilineage differentiation properties, confirming that these cultures are enriched in TICs. TIC cultures were challenged with increasing concentrations of erlotinib and gefitinib, and their survival was evaluated after 1-4 days. In most cases, a time- and concentration-dependent cell death was observed, although GBM 2 was completely insensitive to both drugs, and GBM 7 was responsive only to the highest concentrations tested. Using a radioligand binding assay, we show that all GBM TICs express EGFR. Erlotinib and gefitinib inhibited EGFR and ERK1/2 phosphorylation/activation in all GBMs, irrespective of the antiproliferative response observed. However, under basal conditions GBM 2 showed a high Akt phosphorylation that was completely insensitive to both drugs, whereas GBM 7 was completely insensitive to gefitinib, and Akt inactivation occurred only for the highest erlotinib concentration tested, showing a precise relationship with the antiproliferative effects of the drug. Interestingly, in GBM 2, phosphatase and tensin homolog expression was significantly down-regulated, possibly accounting for the insensitivity to the drugs. In conclusion, glioma TICs are responsive to anti-EGFR drugs, but phosphatase and tensin homolog expression and Akt inhibition seem to be necessary for such effect.

SOX2 silencing in glioblastoma tumor-initiating cells causes stop of proliferation and loss of tumorigenicity.

Glioblastoma, the most aggressive cerebral tumor, is invariably lethal. Glioblastoma cells express several genes typical of normal neural stem cells. One of them, SOX2, is a master gene involved in sustaining self-renewal of several stem cells, in particular neural stem cells. To investigate its role in the aberrant growth of glioblastoma, we silenced SOX2 in freshly derived glioblastoma tumor-initiating cells (TICs). Our results indicate that SOX2 silenced glioblastoma TICs, despite the many mutations they have accumulated, stop proliferating and lose tumorigenicity in immunodeficient mice. SOX2 is then also fundamental for maintenance of the self-renewal capacity of neural stem cells when they have acquired cancer properties. SOX2, or its immediate downstream effectors, would then be an ideal target for glioblastoma therapy.

Effects of Emx2 inactivation on the gene expression profile of neural precursors.

Emx2 plays a crucial role in the development of the diencephalon and dorsal telencephalon. Thus, Emx2-null mutants have abnormal cortical lamination and a reduction in size of the caudal and medial areas of the prosencephalon. Emx2 is expressed in neural precursors of the subventricular zone in vivo and in cultured neurospheres in vitro where it controls the size of the transit-amplifying population, affecting proliferation and clonal efficiency of neural stem cells. To identify the cellular processes mastered by Emx2, and possibly the molecular mechanisms by which the gene exerts its action, we compared the expression profile of cultured neurospheres derived from wild-type and Emx2-null mouse embryos. The differential expression of several genes was also confirmed by semiquantitative RT-PCR, real-time PCR and cytofluorimetric analysis in different preparations of neurospheres, and by in situ hybridization. The gene expression profile suggested a role for Emx2 in regulating the differentiation and migration properties of neural precursor cells. This involvement was confirmed in vitro, where the altered clonogenicity and impaired migration of Emx2-null cells were partially corrected by transduction of the Emx2 gene. Taken together, our results indicate that Emx2 is indeed involved in the transition between resident early progenitors (perhaps stem cells) and more mature precursors capable of migrating out of the ventricular zone, becoming postmitotic and differentiating into the appropriate cell type, and help explain the alterations observed in the brains of knock-out mice.

Pituitary adenylate cyclase-activating polypeptide in the brain, spinal cord and sensory organs of the zebrafish, Danio rerio, during development.

The distribution of Pituitary adenylate cyclase-activating polypeptide (PACAP) was investigated in the brain, pituitary and sensory organs of the zebrafish, Danio rerio, during development, in juvenile and adult specimens, using the immunofluorescence method. In 24 h post fertilization (hpf) embryos, PACAP immunoreactive cells appeared in the rostral telencephalon, dorsal diencephalon, caudal and medial rhombencephalon, spinal cord and retina. At 48 hpf stage, positive cells were present in the dorsal diencephalon, medial rhombencephalon, spinal cord, retina and olfactory placode (Op). At 72 hpf stage, additional immunoreactive elements appeared in the medial telencephalon, hypothalamus, mesencephalic tegmentum, retina and otic sensory epithelium (Ose). At day 5, new immunoreactive cells were found in the anterior rhombencephalon and pituitary pars distalis. At day 13, positive cells were mainly concentrated in the mesencephalic tegmentum and spinal cord. In the telencephalon, diencephalon, rhombencephalon and pituitary, the distribution of positive cells was similar to that previously reported. At 1 month stage, positive cells were detected in the hypothalamus, nucleus of the medial longitudinal fascicle (nMlf), rhombencephalic griseum centrale (Gc) and pituitary pars distalis. At 2-3 month stages, immunoreactive elements were found in several hypothalamic nuclei, in the mesencephalic nucleus isthmi, cerebellum and pituitary. In adults, PACAP immunoreactivity was confined to a few brain regions and the pituitary. PACAP immunoreactivity was transiently expressed in several regions suggesting that the peptide may have a role in the control of cells differentiation and proliferation during zebrafish ontogeny. The finding of positive fibers in the pituitary from day 5 onward indicates that PACAP may function from this stage as a hypophysiotropic peptide.