Children suffering from neurologic cancers undergoing chemotherapy and radiotherapy are at high risk of reduced neurocognitive abilities likely via damage to proliferating neural stem cells (NSC). Therefore, strategies to protect NSCs are needed. We argue that induced cell-cycle arrest/quiescence in NSCs during cancer treatment can represent such a strategy. Here, we show that hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels are dynamically expressed over the cell cycle in NSCs, depolarize the membrane potential, underlie spontaneous calcium oscillations and are required to maintain NSCs in the actively proliferating pool. Hyperpolarizing pharmacologic inhibition of HCN channels during exposure to ionizing radiation protects NSCs cells in neurogenic brain regions of young mice. In contrast, brain tumor-initiating cells, which also express HCN channels, remain proliferative during HCN inhibition. IMPLICATIONS: Our finding that NSCs can be selectively rescued while cancer cells remain sensitive to the treatment, provide a foundation for reduction of cognitive impairment in children with neurologic cancers.

• MeSH
• hyperpolarizační iontové kanály řízené cyklickými nukleotidy metabolismus   MeSH
• lidé MeSH
• myši MeSH
• nádory farmakoterapie   MeSH
• nervové kmenové buňky metabolismus   MeSH
• proliferace buněk MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The cell cycle coordinates core functions such as replication and cell division. However, cell-cycle-regulated transcription in the control of non-core functions, such as cell identity maintenance through specific transcription factors (TFs) and signalling pathways remains unclear. Here, we provide a resource consisting of mapped transcriptomes in unsynchronized HeLa and U2OS cancer cells sorted for cell cycle phase by Fucci reporter expression. We developed a novel algorithm for data analysis that enables efficient visualization and data comparisons and identified cell cycle synchronization of Notch signalling and TFs associated with development. Furthermore, the cell cycle synchronizes with the circadian clock, providing a possible link between developmental transcriptional networks and the cell cycle. In conclusion we find that cell cycle synchronized transcriptional patterns are temporally compartmentalized and more complex than previously anticipated, involving genes, which control cell identity and development.

• MeSH
• algoritmy MeSH
• buněčný cyklus genetika   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory genetika   metabolismus   patologie   MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• transkripční faktory metabolismus   MeSH
• transkriptom * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• validační studie MeSH

The Oct4 gene codes for a transcription factor that plays a critical role in the maintenance of pluripotency in embryonic and cancer stem cells. Its expression thus has to be tightly regulated. We performed biophysical characterization of the promoter region using a combination of UV absorption, CD, and NMR spectroscopies, native PAGE and chemical probing, which was followed by functional studies involving luciferase reporter assays performed in osteosarcoma and human embryonic stem cell lines. We have shown that the evolutionarily conserved G-rich region close to the Oct4 transcription start site in the non-template strand forms a parallel G-quadruplex structure. We characterized its structure and stability upon point mutations in its primary structure. Functional studies then revealed that whereas the wild type quadruplex sequence ensures high reporter gene expression, the expression of mutated variants is significantly decreased proportionally to the destabilizing effect of the mutations on the quadruplex. A ligand, N-methyl mesoporphyrin IX that increases the stability of formed quadruplex rescued the reporter expression of single-mutated variants to the level of wild-type, but it has no effect on a mutated variant that cannot form quadruplex. These data indicate that the quadruplex acts as a strong, positive regulator of Oct4 expression and as such it might serve as a potential target for therapeutic intervention.

• MeSH
• cirkulární dichroismus metody   MeSH
• embryonální kmenové buňky účinky léků   metabolismus   MeSH
• G-kvadruplexy účinky léků   MeSH
• lidé MeSH
• magnetická rezonanční tomografie metody   MeSH
• mesoporfyriny farmakologie   MeSH
• mutace genetika   MeSH
• nádorové buněčné linie MeSH
• oktamerní transkripční faktor 3 genetika   MeSH
• osteosarkom genetika   MeSH
• počátek transkripce účinky léků   fyziologie   MeSH
• promotorové oblasti (genetika) účinky léků   genetika   MeSH
• reportérové geny genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Glioma-initiating cells (GIC) are considered the underlying cause of recurrences of aggressive glioblastomas, replenishing the tumor population and undermining the efficacy of conventional chemotherapy. Here we report the discovery that inhibiting T-type voltage-gated Ca(2+) and KCa channels can effectively induce selective cell death of GIC and increase host survival in an orthotopic mouse model of human glioma. At present, the precise cellular pathways affected by the drugs affecting these channels are unknown. However, using cell-based assays and integrated proteomics, phosphoproteomics, and transcriptomics analyses, we identified the downstream signaling events these drugs affect. Changes in plasma membrane depolarization and elevated intracellular Na(+), which compromised Na(+)-dependent nutrient transport, were documented. Deficits in nutrient deficit acted in turn to trigger the unfolded protein response and the amino acid response, leading ultimately to nutrient starvation and GIC cell death. Our results suggest new therapeutic targets to attack aggressive gliomas. Cancer Res; 77(7); 1741-52. ©2017 AACR.

• MeSH
• aminokyseliny metabolismus   MeSH
• biologický transport MeSH
• blokátory kalciových kanálů farmakologie   MeSH
• buněčná smrt MeSH
• dihydropyridiny farmakologie   MeSH
• draslíkové kanály aktivované vápníkem antagonisté a inhibitory   MeSH
• gliom farmakoterapie   metabolismus   patologie   MeSH
• lidé MeSH
• mykotoxiny farmakologie   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádorové kmenové buňky patologie   MeSH
• nádory mozku farmakoterapie   metabolismus   patologie   MeSH
• proteomika MeSH
• signální dráha UPR účinky léků   MeSH
• sodík metabolismus   MeSH
• vápníkové kanály - typ T fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Pluripotent stem cells are the starting cell type of choice for the development of many cell-based regenerative therapies due to their rapid and unlimited proliferation and broad differentiation potential. The unique pluripotent cell cycle underlies both these properties. Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) family channels have previously been reported to modulate mouse embryonic stem cell (ESC) proliferation and here we characterize the effects of HCN inhibitor ZD7288 on ESC proliferation and stem cell identity. The doubling time of cells treated with the HCN blocker increased by ~30 % due to longer G1 and S phases, resulting in a nearly twofold reduction in ESC numbers after 4 day serum-free culture. Slower progression through S phase was not accompanied by H2AX phosphorylation or cell stalling at transition points, although EdU incorporation in treated cells was reduced. Despite the drastic cell cycle perturbations, the pluripotent status of the cells was not compromised by treatment. Cultures treated with the HCN blocker in maintenance conditions maintained pluripotency marker expression on both RNA and protein level, although we observed a reversible effect on morphology and colony formation frequency. Addition of ZD7288 in differentiating media improved FBS-driven differentiation, but not directed differentiation to neuroectoderm, further indicating that altered cell cycle structure does not necessarily compromise pluripotency and drive ESCs to differentiation. The categorically different outcomes of ZD7288 use during differentiation indicate that cell culture context can be determinative for effects of ion-modulatory molecules and underscores the need for exploring their action in serum-free conditions demanded by potential clinical use.

• Publikační typ
• časopisecké články MeSH

Radiotherapy in children causes debilitating cognitive decline, partly linked to impaired neurogenesis. Irradiation targets primarily cancer cells but also endogenous neural stem/progenitor cells (NSPCs) leading to cell death or cell cycle arrest. Here we evaluated the effects of lithium on proliferation, cell cycle and DNA damage after irradiation of young NSPCs in vitro.NSPCs were treated with 1 or 3 mM LiCl and we investigated proliferation capacity (neurosphere volume and bromodeoxyuridine (BrdU) incorporation). Using flow cytometry, we analysed apoptosis (annexin V), cell cycle (propidium iodide) and DNA damage (γH2AX) after irradiation (3.5 Gy) of lithium-treated NSPCs.Lithium increased BrdU incorporation and, dose-dependently, the number of cells in replicative phase as well as neurosphere growth. Irradiation induced cell cycle arrest in G1 and G2/M phases. Treatment with 3 mM LiCl was sufficient to increase NSPCs in S phase, boost neurosphere growth and reduce DNA damage. Lithium did not affect the levels of apoptosis, suggesting that it does not rescue NSPCs committed to apoptosis due to accumulated DNA damage.Lithium is a very promising candidate for protection of the juvenile brain from radiotherapy and for its potential to thereby improve the quality of life for those children who survive their cancer.

• MeSH
• apoptóza účinky léků   účinky záření   MeSH
• chlorid lithný aplikace a dávkování   farmakologie   MeSH
• hipokampus cytologie   účinky léků   účinky záření   MeSH
• kontrolní body buněčného cyklu účinky léků   účinky záření   MeSH
• kultivované buňky MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nervové kmenové buňky cytologie   účinky léků   účinky záření   MeSH
• neurogeneze účinky léků   fyziologie   účinky záření   MeSH
• novorozená zvířata MeSH
• poškození DNA účinky léků   účinky záření   MeSH
• proliferace buněk účinky léků   účinky záření   MeSH
• průtoková cytometrie MeSH
• radioizotopy kobaltu MeSH
• techniky in vitro MeSH
• záření gama MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH