14 research outputs found

    Effect of hypoxia and/or etoposide on the HIF-1α protein level and HIF-1 DNA binding activity

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    <p><b>Copyright information:</b></p><p>Taken from "Differential effects of hypoxia on etoposide-induced apoptosis according to the cancer cell lines"</p><p>http://www.molecular-cancer.com/content/6/1/61</p><p>Molecular Cancer 2007;6():61-61.</p><p>Published online 26 Sep 2007</p><p>PMCID:PMC2099441.</p><p></p> A549, MCF-7 or HepG2 cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours. , HIF-1α was detected in total cell extracts by western blotting. a-tubulin was used to assess the total amount of proteins loaded on the gel. , after the incubation, nuclear extracts were performed from three independent experiments and hybridized in the ELISA well containing specific DNA probes (TransAM assay). Detection was performed using an anti-HIF-1α antibody. Results are expressed in absorbance (means ± 1 SD, n = 3)

    Comparison of the results obtained with real time RT-PCR and DNA microarrays analyses for , , and genes

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    <p><b>Copyright information:</b></p><p>Taken from "Differential effects of hypoxia on etoposide-induced apoptosis according to the cancer cell lines"</p><p>http://www.molecular-cancer.com/content/6/1/61</p><p>Molecular Cancer 2007;6():61-61.</p><p>Published online 26 Sep 2007</p><p>PMCID:PMC2099441.</p><p></p> After the incubation, total RNA was extracted, submitted to reverse transcription and then to amplification in the presence of SYBR Green and specific primers. was used as the house keeping gene for data normalization. For real-time RT-PCR results, data are given in fold-induction. For DNA microarray results, mean ratios indicate a fold-increase or decrease in gene expression. They are highlighted in blue if statistically non significant, in yellow for quantitative data and in green for qualitative data, given with + or - signs (according to the inserted table)

    Gene expression profiling, for genes involved in regulating cell cycle, in A549, MCF-7 and HepG2 cells incubated with or without etoposide under normoxic or hypoxic conditions

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    <p><b>Copyright information:</b></p><p>Taken from "Differential effects of hypoxia on etoposide-induced apoptosis according to the cancer cell lines"</p><p>http://www.molecular-cancer.com/content/6/1/61</p><p>Molecular Cancer 2007;6():61-61.</p><p>Published online 26 Sep 2007</p><p>PMCID:PMC2099441.</p><p></p> Please refer to supplementary data [Additional file ] for results obtained for the 62 genes for which there was a significant variation in expression for at least one of the conditions. Cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours before RNA extraction, reverse-transcription and cDNA hybridization, as described in Materials and Methods. Each value is the average of three ratio values calculated from three independent experiments ± 1 S.D. Mean ratios indicate a fold-increase or decrease in gene expression. Qualitative values are given with + or - signs (according to the inserted table). The red vertical bars correspond to undetected cDNA. Duplicates or unique value are noted with a red 2 or 1 behind the corresponding column

    Gene expression profiling, for genes involved in regulating apoptosis, in A549, MCF-7 and HepG2 cells incubated with or without etoposide under normoxic or hypoxic conditions

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    <p><b>Copyright information:</b></p><p>Taken from "Differential effects of hypoxia on etoposide-induced apoptosis according to the cancer cell lines"</p><p>http://www.molecular-cancer.com/content/6/1/61</p><p>Molecular Cancer 2007;6():61-61.</p><p>Published online 26 Sep 2007</p><p>PMCID:PMC2099441.</p><p></p> Please refer to supplementary data [Additional file ] for results obtained for the 62 genes for which there was a significant variation in expression for at least one of the conditions. Cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours before RNA extraction, reverse-transcription and cDNA hybridization, as described in Materials and Methods. Each value is the average of three ratio values calculated from three independent experiments ± 1 S.D. Mean ratios indicate a fold-increase or decrease in gene expression. Qualitative values are given with + or - signs (according to the inserted table). The red vertical bars correspond to undetected cDNA. Duplicates or unique value are noted with a red 2 or 1 behind the corresponding column

    Effect of p53 silencing on the expression of BIM, NOXA and BAD.

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    <p>HepG2 cells were transfected with 50 nM p53 siRNA (p53) or non-targeting control siRNA (NT) or left untransfected (/) for 24 hours. Minimum 6 hours later, cells were incubated under normoxia (N, 21% O<sub>2</sub>) or hypoxia (H, 1% O<sub>2</sub>) with (ETO) or without (CTL) etoposide (50 µM) for 16 hours. Proteins were detected in total cell extracts by western blotting, using specific antibodies. ß-actin was used as loading control. One experiment representative out of three. Uncropped western blots are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047519#pone.0047519.s001" target="_blank">Figure S1</a>.</p

    Effect of hypoxia, etoposide and paclitaxel on the mRNA expression level of genes involved in the apoptotic pathway.

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    <p>Results were obtained using “TLDA Human Apoptosis Panel” (Applied Biosystems) (TLDA). HepG2, A549, MDA-MB231 and Hep3B cells were incubated 16 hours under normoxia (N, 21% O<sub>2</sub>) or hypoxia (H, 1% O<sub>2</sub>) in the presence or not of etoposide (E, 100 µM in Hep3B cells and 50 µM in the other cell types) or paclitaxel (T, 10 µM) in HepG2 cells. After incubation, total RNA was extracted, submitted to reverse transcription and then to TLDA analysis. 18S was used as housekeeping gene for data normalization. Actual numerical values are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047519#pone.0047519.s005" target="_blank">Table S2</a>. Genes shown in bold are genes whose expression was validated by qRT-PCT.</p

    Effect of hypoxia on chemotherapeutic agents-induced cell death.

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    <p>HepG2, A549, U2OS, MDA-MB231, HT-29, Hep3B and PC-3 cells were incubated under normoxia (21% O<sub>2</sub>) or hypoxia (1% O<sub>2</sub>) in the absence (CTL) or presence of etoposide (ETO), paclitaxel (TAX), cisplatin (CIS) or camptothecin (CPT) for 16 (A, C, E, G, J) or 40 hours (B, D, F, H, I, K). The concentration used for each molecule is indicated in the graphs (in µM). (A, C, E, G, J) Caspase-3/7 activity was assayed by measuring the fluorescence of free AFC released from the cleavage of the caspase-3/7 specific substrate Ac-DEVD-AFC. Results are expressed in relative fluorescent units (RFU) as means ±1 SD (n  = 3). (B, D, F, H, I, K) LDH release was assessed. Results are presented in percentages as means ±1 SD (n  = 3). (A-K) Statistical analysis was carried out with ANOVA 1. ns: non-significant; *: P<0.05; **: P<0.01; ***: P<0.001. Symbols above a normoxic column are a comparison with the normoxic control and symbols above a hypoxic column are a comparison with the corresponding normoxic condition (with the same drug).</p

    Schematic representation of the effects of hypoxia on the etoposide-induced effects on BCL2-family proteins.

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    <p>(A) Schematic representation of the results obtained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047519#pone-0047519-g004" target="_blank">figure 4</a>. Anti-apoptotic proteins are represented outlined in green and pro-apoptotic proteins are outlined in red. The activation arrows and inhibition signs come from the hypothesis of the interactions between BCL-2 family proteins as explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047519#pone.0047519-Adams1" target="_blank">[69]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047519#pone.0047519-Fletcher1" target="_blank">[70]</a>. The proteins whose abundance is increased by etoposide are outlined with a thicker line. A yellow filling represents that, in the presence of etoposide, hypoxia decreased the abundance of the protein (or the death) as compared to normoxia. A pink filling represents that, in the presence of etoposide, hypoxia increased the abundance of the protein as compared to normoxia. (B) Schematic representation of the hypothetic relations between BIM, BAK, MCL-1 and NOXA. These possible mechanisms are represented for normoxic (21% O<sub>2</sub>) or hypoxic (1% O<sub>2</sub>) HepG2 cells incubated with or without etoposide (ETO; 50 µM) and with or without silencing (−) of NOXA and/or BIM. The hypothetic relations between these proteins are explained in details in the text. Anti-apoptotic proteins are represented outlined in green and pro-apoptotic proteins are outlined in red.</p

    Effect of hypoxia on chemotherapeutic agents-induced cell death.

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    <p>HepG2, A549, U2OS, MDA-MB231, HT-29, Hep3B and PC-3 cells were incubated under normoxia (N, 21% O<sub>2</sub>) or hypoxia (H, 1% O<sub>2</sub>) in the absence (control) or presence of etoposide, methotrexate, paclitaxel, cisplatin or camptothecin. Apoptosis was assayed by measuring caspase-3/7 activity (Ap) and overall cell death was assessed by measuring LDH release (CD) after 16 or 40 hour incubation respectively. Caspase-3/7 activity was assayed by measuring the fluorescence of free AFC released from the cleavage of the caspase-3/7 specific substrate Ac-DEVD-AFC. The p53 status of each cell line is specified in the second column: wild type (wt), mutated or null. The concentrations used for each molecule on each cell type are indicated. “weak death” means that some cell death (statistically different from the control) is sometimes observed but not reproducible. A light grey cell indicates that hypoxia decreases drug-induced cell death; a dark grey cell indicates that hypoxia does not modify drug-induced cell death; and a black cell indicates that hypoxia aggravates drug-induced cell death. <u>hypoxia protect against cell death</u>; <i>hypoxia has no effect on cell death</i>; <b>hypoxia aggravates cell death.</b></p

    Effect of hypoxia, etoposide and paclitaxel on the mRNA expression level of genes involved in the apoptotic pathway.

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    <p>HepG2, A549, MDA-MB231 and Hep3B cells were incubated 16 hours under normoxia (N, 21% O<sub>2</sub>) or hypoxia (H, 1% O<sub>2</sub>) in the presence or not of etoposide (E, 100 µM in Hep3B cells and 50 µM in the other cell types) or paclitaxel (T, 10 µM) in HepG2 cells. After incubation, total RNA was extracted, submitted to reverse transcription and then to real-time PCR in the presence of SYBR Green and specific primers. Actual numerical values are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047519#pone.0047519.s006" target="_blank">Table S3</a>. Genes shown in bold are genes whose expression was assessed at the protein level by western blot analyses.</p
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