BK channels contribute to spike repolarization and fast afterhyperpolarzation (fAHP) in cultured CA1 pyramidal cells.

<p>(<b>a</b>) Whole-cell recording from CA1 pyramidal cell in WT organotypic slice culture. A 100 ms depolarizing current pulse was injected, evoking a train of action potentials (<b>a2</b>). Bath application of IbTX (100 nM) slowed repolarization of first action potential in the spike train and abolished the fAHP (<b>a1</b>). (<b>b</b>) Time course of IbTX effect on 1st action potential decay time (90%-10%) in a WT cell (same as in (a)). (<b>c</b>) Same test as in (a) applied to a BK^−/− slice culture (<b>c2</b>). 1st action potential was broader than in WT cells and lacked fAHP. IbTX had no detectable effect (<b>c1</b>). (<b>d</b>) Time course of IbTX effect on 1st action potential decay time (90%-10%) in a BK^−/− cell (same as in (c)). (<b>e</b>) Summary of 1st action potential decay time ± IbTX, in CA1 pyramidal cells from WT and BK^−/−. Decay time of WT cells under control condition was significantly shorter than for the other three groups (n = 4 in each group, <i>p</i><0.01). (<b>f</b>) Summary of fAHP amplitude (measured at the time point corresponding to the peak of the fAHP in WT cells). The fAHP amplitude was close to zero (not measurable) in BK^−/− and after application of IbTX in WT and BK^−/− cells.</p

Cerebral blood flow and NMDA-induced excitotoxicity in WT and BK^−/− mice.

<p>(<b>a</b>) Cerebral blood flow (given as blood perfusion units [BPU], see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015601#s2" target="_blank"><i>Methods</i></a>) was measured with laser Doppler flowmetry during anaesthesia (pre-ischemia), 10 min after onset of occlusion (intra-ischemia) and 10 min after reperfusion onset (post-ischemia), and is presented as mean ± SEM from 7 WT and 5 BK^−/− mice; **<i>P</i><0.01 (One-way ANOVA, followed by Tukey's post hoc analysis). (<b>b</b>) The neurological deficit score was evaluated 24 h after an intra-cerebral microinjection of 50 nmol of a NMDA solution in a volume of 500 nl. (<b>c</b>) <i>Left:</i> Representative WT and BK^−/− brain sections stained with TTC show the lesion area 24 h after NMDA-microinjection; bar: 4 mm. <i>Right:</i> The corresponding statistic based on the analysis of 3 WT and 3 BK^−/− mice. Data are given as mean ± SEM; *<i>P</i><0.05.</p

Lack of BK channels aggravates cell death in CA1, CA3 and DG after <i>in vitro</i> ischemia (IVI).

<p>(<b>a,b</b>) Representative pseudocolour images showing PI fluorescence intensity as indicator of cell death measured 4 h, 8 h, 24 h, 48 h and 72 h after IVI. (<b>a</b>) BK^−/− and WT cultures without IVI (control cultures) maintained for 72 h. Regions of interests (CA1, CA3, dentate gyrus (DG)) are outlined. (<b>b</b>) BK^−/− and WT cultures at different time points after exposure to IVI. Scale bar  = 1 mm. (<b>c</b>) Time course of cell death measured as PI fluorescence in CA1 (<i>upper</i>), CA3 (<i>middle</i>) and DG (<i>lower</i>). Data are given as mean ± SEM; WT IVI: <i>n</i> = 44, BK^−/− IVI: <i>n</i> = 50, WT control: <i>n</i> = 25, BK^−/− control: <i>n</i> = 23; ***<i>P</i><0.005. Slice cultures were from a minimum of two pups per experimental set.</p

Targeted deletion of BK channel increases mortality and severity of brain infarction after transient middle cerebral artery occlusion.

<p>(<b>a</b>) Mortality within 7 h after reperfusion (in 8 WT vs. 8 BK^−/− mice tested), and the neurological deficit score (<b>b</b>) evaluated 7 h after reperfusion from seven WT and five BK^−/− mice. (<b>c</b>) Representative photomicrographs from coronal WT and BK^−/− brain sections (1 mm thick, from rostral (top) towards caudal (bottom)) stained with TTC 7 h after reperfusion. Lack of red staining indicates infarction. The infarction area of every brain section (<b>d</b>) and the resulting infarction volume of the ipsilateral hemisphere (<b>e</b>) are plotted. All data are given as mean ± SEM; WT: <i>n</i> = 7, BK^−/−: <i>n</i> = 5; *<i>P</i><0.05, **<i>P</i><0.01.</p

Table2_Decitabine-induced DNA methylation-mediated transcriptomic reprogramming in human breast cancer cell lines; the impact of DCK overexpression.XLSX

Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors. Although epigenome modulation provides a promising avenue in treating resistant cancer types, more studies are required to evaluate its safety and ability to normalize the aberrant transcriptional profiles. As deoxycytidine kinase (DCK)-mediated phosphorylation is a rate-limiting step in DAC metabolic activation, we hypothesized that its intracellular overexpression could potentiate DAC’s effect on cell methylome and thus increase its therapeutic efficacy. Therefore, two breast cancer cell lines, JIMT-1 and T-47D, differing in their molecular characteristics, were transfected with a DCK expression vector and exposed to low-dose DAC (approximately IC20). Although transfection resulted in a significant DCK expression increase, further enhanced by DAC exposure, no transfection-induced changes were found at the global DNA methylation level or in cell viability. In parallel, an integrative approach was applied to decipher DAC-induced, methylation-mediated, transcriptomic reprogramming. Besides large-scale hypomethylation, accompanied by up-regulation of gene expression across the entire genome, DAC also induced hypermethylation and down-regulation of numerous genes in both cell lines. Interestingly, TET1 and TET2 expression halved in JIMT-1 cells after DAC exposure, while DNMTs’ changes were not significant. The protein digestion and absorption pathway, containing numerous collagen and solute carrier genes, ranking second among membrane transport proteins, was the top enriched pathway in both cell lines when hypomethylated and up-regulated genes were considered. Moreover, the calcium signaling pathway, playing a significant role in drug resistance, was among the top enriched in JIMT-1 cells. Although low-dose DAC demonstrated its ability to normalize the expression of tumor suppressors, several oncogenes were also up-regulated, a finding, that supports previously raised concerns regarding its broad reprogramming potential. Importantly, our research provides evidence about the involvement of active demethylation in DAC-mediated transcriptional reprogramming.</p

Image1_Decitabine-induced DNA methylation-mediated transcriptomic reprogramming in human breast cancer cell lines; the impact of DCK overexpression.tif

Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors. Although epigenome modulation provides a promising avenue in treating resistant cancer types, more studies are required to evaluate its safety and ability to normalize the aberrant transcriptional profiles. As deoxycytidine kinase (DCK)-mediated phosphorylation is a rate-limiting step in DAC metabolic activation, we hypothesized that its intracellular overexpression could potentiate DAC’s effect on cell methylome and thus increase its therapeutic efficacy. Therefore, two breast cancer cell lines, JIMT-1 and T-47D, differing in their molecular characteristics, were transfected with a DCK expression vector and exposed to low-dose DAC (approximately IC20). Although transfection resulted in a significant DCK expression increase, further enhanced by DAC exposure, no transfection-induced changes were found at the global DNA methylation level or in cell viability. In parallel, an integrative approach was applied to decipher DAC-induced, methylation-mediated, transcriptomic reprogramming. Besides large-scale hypomethylation, accompanied by up-regulation of gene expression across the entire genome, DAC also induced hypermethylation and down-regulation of numerous genes in both cell lines. Interestingly, TET1 and TET2 expression halved in JIMT-1 cells after DAC exposure, while DNMTs’ changes were not significant. The protein digestion and absorption pathway, containing numerous collagen and solute carrier genes, ranking second among membrane transport proteins, was the top enriched pathway in both cell lines when hypomethylated and up-regulated genes were considered. Moreover, the calcium signaling pathway, playing a significant role in drug resistance, was among the top enriched in JIMT-1 cells. Although low-dose DAC demonstrated its ability to normalize the expression of tumor suppressors, several oncogenes were also up-regulated, a finding, that supports previously raised concerns regarding its broad reprogramming potential. Importantly, our research provides evidence about the involvement of active demethylation in DAC-mediated transcriptional reprogramming.</p

Table1_Decitabine-induced DNA methylation-mediated transcriptomic reprogramming in human breast cancer cell lines; the impact of DCK overexpression.XLSX

Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors. Although epigenome modulation provides a promising avenue in treating resistant cancer types, more studies are required to evaluate its safety and ability to normalize the aberrant transcriptional profiles. As deoxycytidine kinase (DCK)-mediated phosphorylation is a rate-limiting step in DAC metabolic activation, we hypothesized that its intracellular overexpression could potentiate DAC’s effect on cell methylome and thus increase its therapeutic efficacy. Therefore, two breast cancer cell lines, JIMT-1 and T-47D, differing in their molecular characteristics, were transfected with a DCK expression vector and exposed to low-dose DAC (approximately IC20). Although transfection resulted in a significant DCK expression increase, further enhanced by DAC exposure, no transfection-induced changes were found at the global DNA methylation level or in cell viability. In parallel, an integrative approach was applied to decipher DAC-induced, methylation-mediated, transcriptomic reprogramming. Besides large-scale hypomethylation, accompanied by up-regulation of gene expression across the entire genome, DAC also induced hypermethylation and down-regulation of numerous genes in both cell lines. Interestingly, TET1 and TET2 expression halved in JIMT-1 cells after DAC exposure, while DNMTs’ changes were not significant. The protein digestion and absorption pathway, containing numerous collagen and solute carrier genes, ranking second among membrane transport proteins, was the top enriched pathway in both cell lines when hypomethylated and up-regulated genes were considered. Moreover, the calcium signaling pathway, playing a significant role in drug resistance, was among the top enriched in JIMT-1 cells. Although low-dose DAC demonstrated its ability to normalize the expression of tumor suppressors, several oncogenes were also up-regulated, a finding, that supports previously raised concerns regarding its broad reprogramming potential. Importantly, our research provides evidence about the involvement of active demethylation in DAC-mediated transcriptional reprogramming.</p

DataSheet1_Decitabine-induced DNA methylation-mediated transcriptomic reprogramming in human breast cancer cell lines; the impact of DCK overexpression.docx

Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors. Although epigenome modulation provides a promising avenue in treating resistant cancer types, more studies are required to evaluate its safety and ability to normalize the aberrant transcriptional profiles. As deoxycytidine kinase (DCK)-mediated phosphorylation is a rate-limiting step in DAC metabolic activation, we hypothesized that its intracellular overexpression could potentiate DAC’s effect on cell methylome and thus increase its therapeutic efficacy. Therefore, two breast cancer cell lines, JIMT-1 and T-47D, differing in their molecular characteristics, were transfected with a DCK expression vector and exposed to low-dose DAC (approximately IC20). Although transfection resulted in a significant DCK expression increase, further enhanced by DAC exposure, no transfection-induced changes were found at the global DNA methylation level or in cell viability. In parallel, an integrative approach was applied to decipher DAC-induced, methylation-mediated, transcriptomic reprogramming. Besides large-scale hypomethylation, accompanied by up-regulation of gene expression across the entire genome, DAC also induced hypermethylation and down-regulation of numerous genes in both cell lines. Interestingly, TET1 and TET2 expression halved in JIMT-1 cells after DAC exposure, while DNMTs’ changes were not significant. The protein digestion and absorption pathway, containing numerous collagen and solute carrier genes, ranking second among membrane transport proteins, was the top enriched pathway in both cell lines when hypomethylated and up-regulated genes were considered. Moreover, the calcium signaling pathway, playing a significant role in drug resistance, was among the top enriched in JIMT-1 cells. Although low-dose DAC demonstrated its ability to normalize the expression of tumor suppressors, several oncogenes were also up-regulated, a finding, that supports previously raised concerns regarding its broad reprogramming potential. Importantly, our research provides evidence about the involvement of active demethylation in DAC-mediated transcriptional reprogramming.</p

Image2_Decitabine-induced DNA methylation-mediated transcriptomic reprogramming in human breast cancer cell lines; the impact of DCK overexpression.tif

Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors. Although epigenome modulation provides a promising avenue in treating resistant cancer types, more studies are required to evaluate its safety and ability to normalize the aberrant transcriptional profiles. As deoxycytidine kinase (DCK)-mediated phosphorylation is a rate-limiting step in DAC metabolic activation, we hypothesized that its intracellular overexpression could potentiate DAC’s effect on cell methylome and thus increase its therapeutic efficacy. Therefore, two breast cancer cell lines, JIMT-1 and T-47D, differing in their molecular characteristics, were transfected with a DCK expression vector and exposed to low-dose DAC (approximately IC20). Although transfection resulted in a significant DCK expression increase, further enhanced by DAC exposure, no transfection-induced changes were found at the global DNA methylation level or in cell viability. In parallel, an integrative approach was applied to decipher DAC-induced, methylation-mediated, transcriptomic reprogramming. Besides large-scale hypomethylation, accompanied by up-regulation of gene expression across the entire genome, DAC also induced hypermethylation and down-regulation of numerous genes in both cell lines. Interestingly, TET1 and TET2 expression halved in JIMT-1 cells after DAC exposure, while DNMTs’ changes were not significant. The protein digestion and absorption pathway, containing numerous collagen and solute carrier genes, ranking second among membrane transport proteins, was the top enriched pathway in both cell lines when hypomethylated and up-regulated genes were considered. Moreover, the calcium signaling pathway, playing a significant role in drug resistance, was among the top enriched in JIMT-1 cells. Although low-dose DAC demonstrated its ability to normalize the expression of tumor suppressors, several oncogenes were also up-regulated, a finding, that supports previously raised concerns regarding its broad reprogramming potential. Importantly, our research provides evidence about the involvement of active demethylation in DAC-mediated transcriptional reprogramming.</p