Background Regions within solid tumours often experience oxygen deprivation, which is associated with resistance to chemotherapy and irradiation. The aim of this study was to evaluate the radiosensitising effect of gemcitabine and its main metabolite dFdU under normoxia versus hypoxia and to determine whether hypoxia-inducible factor 1 (HIF-1) is involved in the radiosensitising mechanism. Methods Stable expression of dominant negative HIF-1α (dnHIF) in MDA-MB-231 breast cancer cells, that ablated endogenous HIF-1 transcriptional activity, was validated by western blot and functionality was assessed by HIF-1α activity assay. Cells were exposed to varying oxygen environments and treated with gemcitabine or dFdU for 24 h, followed by irradiation. Clonogenicity was then assessed. Using radiosensitising conditions, cells were collected for cell cycle analysis. Results HIF-1 activity was significantly inhibited in cells stably expressing dnHIF. A clear radiosensitising effect under normoxia and hypoxia was observed for both gemcitabine and dFdU. No significant difference in radiobiological parameters between HIF-1 proficient and HIF-1 deficient MDA-MB-231 cells was demonstrated. Conclusions For the first time, radiosensitisation by dFdU, the main metabolite of gemcitabine, was demonstrated under low oxygen conditions. No major role for functional HIF-1 protein in radiosensitisation by gemcitabine or dFdU could be shown.

Abstract Introduction: Regions within solid tumors often experience mild to severe oxygen deprivation, associated with resistance to chemotherapy and irradiation. The aim of this study was to evaluate the radiosensitizing effect of gemcitabine and its main metabolite dFdU under normal versus reduced oxygen conditions and to determine whether hypoxia-inducible factor 1 (HIF-1) is involved in the radiosensitizing mechanism. Materials & methods: The clonogenic assay was performed in three isogenic MDA-MB-231 breast cancer cell lines differing in HIF-1alpha proficiency (24h 0-8 nM gemcitabine or 0-4 microM dFdU, 0-8 Gy irradiation). Validation of the transfection with dominant negative HIF-1alpha was done by western blot and by assessment of HIF-1alpha activity. The relative expression of 84 genes related to the hypoxia signaling pathway was characterized by human hypoxia signaling pathway PCR array. Using radiosensitizing conditions, cells were collected for cell cycle analysis. Results: HIF-1 activity was significantly inhibited after transfection with a dominant negative protein. Furthermore, anoxia-induced VEGF secretion was significantly lower (p<0.05) in MDA-MB-231 cells stably expressing dnHIF (dominant negative HIF-1alpha) than in MDA-MB-231 EV (empty vector) cells. PCR array indicated that hypoxic conditions significantly influenced the expression of HIF-1alpha and ANGPTL4 (angiopoetin-like 4). A clear radiosensitizing effect under normoxic and anoxic conditions was observed for both gemcitabine and dFdU. The radiosensitizing effect was similar under normoxic and anoxic conditions (p=0.48 for gemcitabine, p=0.56 for dFdU) and the dose enhancement factor (DEF) was not significantly influenced by the cell line used. Under anoxia, DEFs for gemcitabine ranged from 1.26 to 1.59, from 1.11 to 1.77, and from 1.34 to 2.04 in MDA-MB-231 wt, EV and dnHIF cells, respectively. Statistical analysis revealed no significant differences in radiobiological parameters between HIF-1 proficient and HIF-1 deficient cells. Cell cycle analysis showed that, in contrast to previous findings in other cell lines, exposure of MDA-MB-231 cells to low oxygen conditions did not induce a significant increase in the percentage of G0/1 cells (p=0.21). Gemcitabine and dFdU caused a block of cells in the S phase of the cell cycle under both normoxic and anoxic conditions. Conclusion: For the first time, radiosensitization by dFdU, the main metabolite of gemcitabine, was demonstrated under low oxygen conditions. As dFdU has a prolonged half-life, the sustained presence of dFdU in the blood might induce radiosensitization despite the short half-life of the parent drug, gemcitabine. This might be highly relevant, especially considering delivery of the drug to hypoxic tumor regions. No major role for HIF-1 in radiosensitization by gemcitabine or dFdU could be shown. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5722. doi:1538-7445.AM2012-5722

The urokinase plasminogen activator (uPA) system is one of the serine protease systems involved in extracellular matrix degradation, which will facilitate tumor cell invasion and intravasation. Recently, the Medicinal Chemistry group of the University of Antwerp (UAMC) developed some very selective covalently binding uPA inhibitors. Besides its function as possible therapeutic target, uPA and its endogenous inhibitor PAI-1 are described as prognostic biomarkers for breast cancer with the highest level of evidence. Commercial ELISA and PCR kits are available for the screening of uPA in breast tumors. These ex vivo assays measure or predict the total uPA content (active and inactive). We present here a more advanced technique, which will be able to monitor the actual enzymatic uPA activity in a non-invasive manner. The technology is based on the modification of the UAMC inhibitors towards activity based probes containing a fluorescent label. Currently, we are evaluating these probes as imaging tools for uPA monitoring. The developed imaging probes will be profoundly tested in an orthotopic model of human MDA-MB-231 and MCF7 breast cancer cells since these cell lines show a high and low uPA expression respectively. We will use these models to study the potential beneficial characteristics of activity based probes in the field of bio-imaging applications targeted to uPA as a validated biomarker. The current protocol, which is still under evaluation, can be described as followed: Cell suspensions (2.106 cells/100μl) of each cell line were implanted in the mammary fat pads at both flanks of 7 mice, alternatively with and without Matrigel. Tumor size was monitored using bioluminescent imaging (BLI) and caliper measurements. When the tumor volume reached 200 - 400mm3, the rhodamine labeled uPA-inhibitor (uPA-probe) was administered (0.3 mg / kg i.v.). Fluorescent images were taken at 1, 2, 4, 6, 8, 12, 24 and 48 hours after injection. At 48 hours, the mice were sacrificed and the tumor, liver and lung were excised for ex vivo bioluminescent or fluorescent imaging and stored for histological examination. In this presentation, we describe the evaluation of the model and the first promising results. Considering the role of uPA in migration and invasion, the uPA-probe will also be tested in the future for the detection of micrometastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5250. doi:1538-7445.AM2012-5250

Abstract Background Elucidation of biological mechanisms underlying deregulated proliferation and motility of cancer cells and identification of potential therapeutic targets require accurate cell-based monitoring. The xCELLigence Real Time Cell Analysis (RTCA) technology (Roche Applied Science) constitutes a non-invasive and label-free approach to assess cell viability and motility in real time, hereby countering key features of classic label-based endpoint detection methods. Here we show results of in vitro detection of cell viability and migration on 2 cancer cell lines using xCELLigence RTCA DP and correlate these with data obtained from parallel experiments carried out using established assessment methods for each process. Methods and results Kinetic viability and migration measurements were carried out on the MDA-MB-231 (breast cancer) and A549 (lung cancer) cell lines using the xCELLigence RTCA DP instrument. Cell viability was assessed during 10 days of incubation using modified 16-well plates (E-plate) containing microelectrodes at the well bottoms for impedance-based detection of attachment, spreading and proliferation, expressed as a Cell Index (CI) value. Cell migration was measured during 38 hours using 16-well plates (CIM16-plate) consisting of an upper and a lower chamber separated by a microporous membrane equipped with a similar detection system at the bottom side. The Sulforhodamine B (SRB) assay and a 24-well Transwell system served as reference tools to assess viability and migratory kinetics. Viability was estimated by optical density (OD) reading (540 nm) of solubilized cells that were fixed and stained with SRB at a rate of 1 plate per day. Good correlations were observed between SRB and RTCA CI for 5x103 and 104 MDA-MB-231 cells/mL (Spearman's ≤ = 0.79 and 0.84 resp) and similar doubling times (p = 0.459). The experimental Transwell design allowed dynamic quantitation of cancer cell migration by fixing and staining of the insert membranes in methanol and crystal violet in duplicates at 10 time points during a 24 hour-incubation. Pixel area quantitation showed strong correlation with xCELLigence CI (Spearman's ≤ = 0.90 for both cell lines). However, OD measurements (590 nm) correlated even stronger with CI (Spearman's ≤ = 0.96 and 1.00 for MDA-MB-231 and A549). Moreover, analysis of random migration indicated a significant difference between RTCA CI and area / OD (p < 0.001) implying reduced detection limits of the xCELLigence system. Conclusions The similarity between observations as performed with conventional approaches and xCELLigence makes both methods interchangeable. Added with results indicating reduced detection limits, xCELLigence provides an accurate detection platform for high-throughput kinetic screenings and for determination of time-dependent cell proliferation and motility dynamics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4300. doi:1538-7445.AM2012-4300

BACKGROUND:Cell viability and motility comprise ubiquitous mechanisms involved in a variety of (patho)biological processes including cancer. We report a technical comparative analysis of the novel impedance-based xCELLigence Real-Time Cell Analysis detection platform, with conventional label-based endpoint methods, hereby indicating performance characteristics and correlating dynamic observations of cell proliferation, cytotoxicity, migration and invasion on cancer cells in highly standardized experimental conditions. METHODOLOGY/PRINCIPAL FINDINGS:Dynamic high-resolution assessments of proliferation, cytotoxicity and migration were performed using xCELLigence technology on the MDA-MB-231 (breast cancer) and A549 (lung cancer) cell lines. Proliferation kinetics were compared with the Sulforhodamine B (SRB) assay in a series of four cell concentrations, yielding fair to good correlations (Spearman's Rho 0.688 to 0.964). Cytotoxic action by paclitaxel (0-100 nM) correlated well with SRB (Rho>0.95) with similar IC(50) values. Reference cell migration experiments were performed using Transwell plates and correlated by pixel area calculation of crystal violet-stained membranes (Rho 0.90) and optical density (OD) measurement of extracted dye (Rho>0.95). Invasion was observed on MDA-MB-231 cells alone using Matrigel-coated Transwells as standard reference method and correlated by OD reading for two Matrigel densities (Rho>0.95). Variance component analysis revealed increased variances associated with impedance-based detection of migration and invasion, potentially caused by the sensitive nature of this method. CONCLUSIONS/SIGNIFICANCE:The xCELLigence RTCA technology provides an accurate platform for non-invasive detection of cell viability and motility. The strong correlations with conventional methods imply a similar observation of cell behavior and interchangeability with other systems, illustrated by the highly correlating kinetic invasion profiles on different platforms applying only adapted matrix surface densities. The increased sensitivity however implies standardized experimental conditions to minimize technical-induced variance.

Abstract Introduction: It has been well documented that the efficacy of both chemotherapy and radiotherapy is directly linked with an adequate oxygen tension, and that hypoxic regions in solid tumors often contain viable cells that are intrinsically more resistant to anticancer treatment. Recently, it has been demonstrated that the cytotoxic drug gemcitabine retains its radiosensitizing potential under low oxygen conditions in lung adenocarcinoma cells. As the transcription factor ‘hypoxia inducible factor 1’ (HIF-1) plays a crucial role in regulating the adaptive responses of tumor cells to survive under hypoxic conditions, the present study investigated the potential influence of HIF-1 status on radiosensitization by gemcitabine. Materials & methods: Three isogenic human breast adenocarcinoma cell lines with different HIF-1 status were included in this study: MDA-MB-231 (breast adenocarcinoma cell line, wt HIF-1), MDA-MB-231 DN-HIF (transfected with dominant-negative HIF-1alpha, abrogating HIF-1 function) and MDA-MB-231 EV (empty vector transfected control, functional HIF-1). Anoxic conditions (<0.1% O2) were achieved in a Bactron IV anaerobic chamber. To analyze the radiosensitizing effect of gemcitabine under normoxic versus anoxic conditions, the clonogenic assay was performed. Cells were treated with 0-8 nM gemcitabine for 24h directly before radiation (0-8 Gy). Using radiosensitizing conditions, cell cycle distribution was monitored flow cytometrically according to the Vindelov method. Results: A clear concentration-dependent radiosensitizing effect of gemcitabine was observed under both normoxic and anoxic conditions (dose enhancement factor (DEF) under normoxia: 1.02-1.70; DEF under anoxia: 1.11-2.04). Combination index (CI) analysis showed an additive interaction between gemcitabine and radiation under normal oxygen conditions (CI 0.902-1.148), and a synergistic interaction under reduced oxygen conditions (CI 0.455-0.889). Statistical analysis using two-way ANOVA revealed no significant differences in radiobiological parameters (DEF, ID10, ID50, mean inactivation dose, surviving fraction at 2 Gy, oxygen enhancement ratio) between MDA-MB-231 EV and MDA-MB-231 DN-HIF cells, suggesting no influence of HIF-1 functionality on radiosensitivity. Considering the cell cycle distribution after treatment with gemcitabine, 24h treatment with 4 or 8 nM gemcitabine established a significant S-phase block in both normoxic and anoxic MDA-MB-231 wt and DN-HIF cells. Conclusion: This study showed that the retained radiosensitizing effect of gemcitabine under anoxic conditions was not tumor tissue specific and could be observed in MDA-MB-231 breast cancer cells. No major role for functional HIF-1 protein in radiosensitization by gemcitabine could be demonstrated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2671. doi:10.1158/1538-7445.AM2011-2671

Abstract Introduction: Cellular stress responses are essential for normal cellular growth and development while deregulation of one of these processes can play a role in tumorigenesis. Mammalian target of rapamycin (mTOR) is regulated by a lot of these stress responses and regulates cell growth and cell cycle progression. REDD1 is a response gene that can alter the mTOR status in response to DNA damage, hypoxia and glucose deprivation. Hypoxia has become a big issue in solid tumors due to its adverse effects on tumor behavior. mTOR will be rapidly inhibited by activation of REDD1 under anoxia. REDD1 silencing could therefore establish a continuous translation under anoxic conditions and might resensitize anoxic cells to certain cytostatic drugs and/or radiotherapy that target cellular growth and proliferation. Material & Methods: Human A549 lung carcinoma cells were transfected with short hairpin RNA targeted against REDD1 to constitutively silence REDD1 translation (A549shREDD1). After puromycin selection, REDD1 protein expression was determined by western blot analysis. A549 and A549shREDD1 cells were incubated under anoxic conditions (<0.1% O2) in a Bactron anaerobic IV chamber and harvested at different time points (24-96h). Vindelov staining was used to analyze cell cycle distribution by flow cytometry. The IC50 value of gemcitabine under normoxic and anoxic conditions was determined using the sulforhodamine B test. Cells were treated with gemcitabine (0 – 100nM) for 24h. 2h prior to treatment, cells were incubated under anoxic conditions to induce REDD1 upregulation. Results: An increase of REDD1 protein in A549 cells could already be detected after 2h incubation under anoxia. REDD1 downregulation was confirmed in the A549shREDD1 cells. After a 72h incubation period under anoxia, cell cycle analysis showed an increase of the percentage G0/1 cells in A549 cells compared to A549shREDD1 cells (A549: 80.68 ± 15.06%; A549shREDD1: 65.28 ± 11.23%). Interestingly, the cell fraction in the S phase turned out to be larger in anoxic A549shREDD1 compared to anoxic A549 cells (A549: 14.33 ± 12.96% A549shREDD1: 23.34 ± 6.23%). REDD1 might play a potential role in anoxia-mediated cell cycle arrest. The difference in REDD1 expression had no influence on the IC50 values of gemcitabine of the different cell lines (IC50 normoxia: A549: 17.34 ± 9.21; A549shREDD1: 19.49 ± 2.97; IC50 anoxia: A549: 17.47 ± 5.79; A549shREDD1: 23.83 ± 1.03). Moreover, REDD1 inhibition did not cause a sensitization of anoxic cells. Conclusion: Inhibiting REDD1 translation seems to overcome anoxia-induced G0/1 arrest. This could be an interesting approach to resensitize anoxic cells to conventional cancer treatments that target dividing cells. Although gemcitabine typically interacts with the cell cycle, no difference in IC50 values could be observed between the wild type A549 and REDD1 silenced A549 cells, neither under normoxia nor under anoxia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1662. doi:10.1158/1538-7445.AM2011-1662

Abstract Formalin-fixed paraffin-embedded (FFPE) tissue is the most common tissue specimen widely available. Moreover, long clinical follow-up is on hand. Therefore, FFPE material is a precious source of material for identifying predictive and/or prognostic biomarkers in cancer research on the basis of gene expression. However, the main drawback of FFPE tissue is the significant reduction in quantity and quality of the extracted RNA. The aim of this study is the comparison of different commercially available kits for the RNA isolation in FFPE tissue material. Five commercially available kits were compared and evaluated to identify the most optimal RNA isolation procedure for FFPE material. The FFPE sections used in this study derived from cervical cancer patients treated by surgery. Three paraffin sections (10 μm) per patient were used to isolate RNA, according to the manufacturer's protocol. The following RNA isolation kits were used: Rneasy® FFPE isolation kit (Qiagen), RecoverAllTM Total Nucleic Acid isolation kit (Ambion), ArrayGradeTM FFPE RNA isolation kit (SA Biosciences), NucleoSpin® FFPE RNA isolation kit (Macherey-Nagel) and QuickExtractTM FFPE RNA Extraction Kit (Epicentre® Biotechnologies). RNA extraction was carried out in RNAse free environment. The concentration and purity (A260/A280 ratio and A260/A230 ratio) were measured using a Nanodrop ND-1000 spectrophotometer. The integrity of the isolated RNA was assessed by capillary electrophoresis with an Agilent 2100 Bioanalyzer using Agilent RNA 6000 Series Nano kits, expressed in RNA integrity numbers (RIN). The mean total RNA eluated by the different kits were as follows: Qiagen 25957 ± 19417ng, Ambion 8249 ± 2898ng, SA Biosciences 8070 ± 3700ng and Machery-Nagel 622 ± 394ng. The A260/A280 and A260/A230 ratio was used to evaluate the purity of the samples. The mean A260/A280 ratios were as follows: Qiagen: 1.81 ± 0.23, SA Biosciences: 0.66 ± 0.36, Ambion: 1.03 ± 0.37 and Machery-Nagel: 1.04 ± 0.61 and for the mean A260/A230 ratios: Qiagen: 1.88 ± 0.09, SA Biosciences: 1.61 ± 0.32, Ambion: 1.54 ± 0.30 and Machery-Nagel: 1.88 ± 0.61. The RNA extractions from Epicentre® could not be measured by Nanodrop and, therefore, were excluded from further analysis. The mean RIN values were as follows: Qiagen: 2.19 ± 0,37, SA Biosciences: 2.09 ± 0.38, Ambion: 2.44 ± 0.12 and Macherey-Nagel: 2.30 ± 0.44. Based on the Nanodropdata, the most consistent results were obtained with the Rneasy® FFPE Isolation Kit (Qiagen). However, RNA isolated with RecoverAllTM Total Nucleic Acid isolation kit (Ambion) gave slightly higher RIN values. Taken these results together, the kit from Qiagen appears the most appropriate kit to be used in our further studies that require RNA isolation from FFPE material. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 280. doi:10.1158/1538-7445.AM2011-280

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL It is known that gemcitabine and difluorodeoxyuridine (dFdU) have radiosensitizing properties. It has become evident that solid tumors often contain hypoxic regions and that this is one of the causes of resistance or decreased sensitivity to cancer therapy. In hypoxic conditions, the transcription factor, hypoxia-inducible factor-1 (HIF-1), is upregulated. HIF-1 is responsible for the cellular and adaptive responses of tumors to survive in hypoxic conditions. Recently, it has been demonstrated that gemcitabine retains its radiosensitizing potential under hypoxia. In the present study, the radiosensitizing potential of dFdU under anoxic conditions is investigated as well as the role of HIF-1 protein. In order to explore the role of HIF-1, human tumor cell lines included in this study were MDA-MB-231 (breast adenocarcinoma cell line, wt HIF-1), MDA-MB-231 DN-HIF (transfected with dominant-negative HIF-1α) and MDA-MB-231 EV (empty vector transfected control). Anoxic conditions (<0.1% O2,) were achieved in a Bactron IV anaerobic chamber. To analyze the radiosensitizing effect of dFdU, the clonogenic assay was performed. Cells were exposed to normoxic or anoxic conditions and were simultaneously treated with 0, 2 or 4 microM dFdU for 24h immediately before irradiation (0-8 Gy, room temperature, linear accelerator). Immediately following radiation, anoxic cells were reoxygenated and cells were washed with drug-free medium. Using radiosensitizing conditions, cells were collected for cell cycle analysis by flow cytometry. A clear concentration-dependent radiosensitizing effect of dFdU was observed under both normoxic and anoxic conditions (dose enhancement factor (DEF) under normoxia: 1.74-3.38; under anoxia: 2.08-3.81). Combination index (CI) analysis showed a synergistic to additive interaction between dFdU and radiation under normoxia (CI 0.651 ± 0.175), yet an additive interaction under anoxia (CI 0.784 ± 0.120). Statistical analysis using two-way ANOVA revealed that cell survival was significantly influenced by the concentration of dFdU, the dose of radiation, the oxygen tension and the cell line. Post hoc analysis indicated no significant difference between the three cell lines for DEF, ID50, mean inactivation dose and survival fraction at 2 Gy, suggesting that the functionality of HIF-1 protein has no impact on the radiosensitizing effect of dFdU. Considering the cell cycle distribution after treatment with dFdU, 24h treatment with 2 or 4 microM dFdU established a significant S-phase block in both normoxic and anoxic MDA-MB-231 wt and DN-HIF cells. In conclusion, this study showed that dFdU has a clear concentration-dependent radiosensitizing effect in MDA-MB-231 breast cancer cells under normoxic as well as anoxic conditions. No major role for functional HIF-1 protein in radiosensitization by dFdU could be demonstrated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2661. doi:10.1158/1538-7445.AM2011-2661

BackgroundFormalin-fixed paraffin-embedded (FFPE) tissue is the most common tissue specimen widely available. Moreover, long clinical follow-up is on hand. Therefore, FFPE material is a precious source of material for identifying predictive and/or prognostic biomarkers in cancer research on the basis of gene expression. However, the main drawback of FFPE tissue is the significant reduction in quantity and quality of the extracted RNA. The aim of this study is the comparison of different commercially available kits for the RNA isolation in FFPE tissue material. MethodsFive commercially available RNA isolation kits were tested and the concentration, purity, integrity, and raw cycle threshold values were determined. ResultsThe mean total RNA concentrations were as follows: Qiagen 25957±19417 ng, Ambion 8249±2898 ng, SA Biosciences 8070±3700 ng, and Macherey-Nagel 622±394 ng. The mean A260/A280 ratios were as follows: Qiagen: 1.81, SA Biosciences: 0.66, Ambion: 1.03, and Macherey-Nagel: 1.04. The mean A260/A230 ratios were as follows: Qiagen: 1.88, SA Biosciences: 1.61, Ambion: 1.54, and Macherey-Nagel: 1.88. The RNA extractions from Epicentre could not be measured by the Nanodrop and, therefore, were excluded from further analysis. The mean RNA integrity number (range, 2.09 to 2.47) and the mean raw cycle threshold values (range, 33.43 to 35.37) were more or less the same for all the tested RNA isolation kits. ConclusionsAltogether, on the basis of the number of adequate isolations, the kit from Qiagen seems to be the most appropriate kit to be used in our further studies that require RNA isolation from FFPE material.

Purpose: Whereas radiosensitization by gemcitabine is well studied under normal oxygen conditions, little is known about its radiosensitizing potential under reduced oxygen conditions. Therefore, the present study evaluated the impact of anoxia on gemcitabine-mediated radiosensitization.Methods and Materials: The clonogenic assay was performed in three isogenic A549 cell lines differing in p53 status (24 h, 0-15 nM gemcitabine, 0-8 Gy irradiation, normoxia vs. anoxia). Using radiosensitizing conditions, cells were collected for cell cycle analysis and apoptosis detection.Results: Whereas wild-type p53 A549-LXSN cells were more sensitive to radiation than p53-deficient A549-E6 cells, both cell lines showed similar radiosensitization by gemcitabine under normoxia and anoxia. Independent of p53 functionality, gemcitabine was able to overcome anoxia-induced G(0/1) arrest and established an (early) S phase block in normoxic and anoxic cells. The percentage early and late apoptotic/necrotic cells increased with the gemcitabine/radiation combination, with a significant difference between A549-LXSN and A549-E6.Conclusions: This study is the first to show that gemcitabine retains its radiosensitizing potential under low oxygen conditions. Although radiosensitization was observed in both p53 wild-type and p53-deficient cells, p53 status might influence induction of apoptosis after gemcitabine/radiation treatment, whereas no effect on cell cycle progression was noticed. (C) 2011 Elsevier Inc.

Introduction: Solid tumors often contain hypoxic regions. In hypoxic conditions, the transcription factor, hypoxia-inducible factor-1 (HIF-1), is upregulated. HIF-1 is responsible for the cellular and adaptive responses of tumours to survive in hypoxic conditions. HIF-1 is also thought to influence genes involved in resistance to therapy. In this study, we examined the role of HIF-1 in radiosensitization of gemcitabine under normoxic and anoxic conditions. Materials & methods: Two isogenic breast cancer cell lines were used that differ in HIF-1 expression: MDA-MB-231 (HIF-1 wt) and MDA-MB-231 DN-HIF (HIF-1 deficient). Anoxic conditions ( Results: The IC50 values of gemcitabine were comparable under normoxic and anoxic conditions: 14.1±2 and 15.0±5.2 nM for MDA-MB-231 and 6.3±1.3 and 7.3±1.1 nM for MDA-MB-231 DN-HIF cells (p=0.569). A concentration dependent radiosensitizing effect of gemcitabine was observed in both cell lines under normoxic and anoxic conditions; DEFs ranged from 1.1 to 2.0. Two-way ANOVA revealed that the radiosensitizing effect was not significantly influenced by the oxygen tension (p=0.698). Gemcitabine was able to overcome the anoxia-induced G0/1 phase block and established a significant S-phase block in both normoxic and anoxic cells. Using qRT-PCR, we showed an increase of VEGF expression in the wt MDA-MB-231 cells in anoxia. An increase of VEGF expression was seen with a longer incubation time under anoxia. Conclusion: From these experiments, we can conclude that HIF-1 protein does not seem to play a role in the cytotoxic and radiosensitizing effect of gemcitabine in MDA-MB-231 breast cancer cells. In addition, HIF-1 protein did not influence the cell cycle effect of gemcitabine, declaring the comparable radiosensitizing effect in the different cell lines. There is no difference between normoxia and anoxia in the different experiments. Similar experiments will be performed with the metabolite of gemcitabine, difluorodeoxyuridine. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5551.

Abstract Introduction: It has been well documented that the efficacy of both chemotherapy and radiotherapy is directly linked with an adequate oxygen tension, and that hypoxic regions in solid tumors often contain viable cells that are intrinsically more resistant to anticancer treatment. As the antimetabolite gemcitabine is a potent radiosensitizer, often used in the clinic, the influence of oxygen deficiency on the radiosensitizing effect of gemcitabine was investigated. Materials & methods: The human tumor cell lines included were A549 (lung carcinoma cell line, wt p53), A549-E6 (transduced with the HPV type 16 E6 gene, abrogating the p53 function) and A549-LXSN (vector transfected control, functional p53). Anoxic conditions (<0.1% O2,) were achieved in a Bactron IV anaerobic chamber. To analyze the radiosensitizing effect of gemcitabine under normoxic versus anoxic conditions, the clonogenic assay was performed. Cells were exposed to normoxic or anoxic conditions and were simultaneously treated with 0-15 nM gemcitabine for 24h directly before radiation (0-8 Gy). Immediately following radiation, anoxic cells were reoxygenated and all cells were washed with drug-free medium. Cell cycle analysis and determination of apoptotic cell death was performed using flow cytometry. P53 expression was studied by Western blot. Results: A clear concentration-dependent radiosensitizing effect of gemcitabine was observed under both normoxia and anoxia (dose enhancement factor 1.27-1.70 and 1.28-2.06 respectively). Combination index (CI) analysis showed an additive to strongly synergistic interaction between gemcitabine and radiation under both normal and reduced oxygen conditions (CI 0.672-1.083 and 0.587-1.090 respectively). Two-way ANOVA revealed that cell survival was significantly influenced by the concentration of gemcitabine, radiation dose, oxygen tension and cell line used. Post hoc analysis indicated a significant difference between A549-LXSN and A549-E6 cells, suggesting a potential role for p53 in radiosensitization by gemcitabine. Independent of the p53 functionality, gemcitabine was able to overcome the anoxia-induced G0/1 phase block and established a significant S-phase block in both normoxic and anoxic cells. The percentage early and late apoptotic/necrotic cells increased significantly with the combination of gemcitabine and radiation, and was significantly influenced by the oxygen concentration. Interestingly, a significant difference between A549-LXSN and A549-E6 was observed. Conclusion: This study demonstrated, for the first time, that gemcitabine retains its radiosensitizing potential under low oxygen conditions. The p53 protein might be involved in this process, not by affecting cell cycle progression, but rather by acting on the induction of apoptotic cell death. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 460.

BACKGROUND:Based on their different mechanisms of action, non-overlapping side effects and radiosensitising potential, combining the antimetabolites pemetrexed (multitargeted antifolate, MTA) and gemcitabine (2',2'-difluorodeoxycytidine, dFdC) with irradiation (RT) seems promising. This in vitro study, for the first time, presents the triple combination of MTA, dFdC and irradiation using various treatment schedules.METHODS:The cytotoxicity, radiosensitising potential and cell cycle effect of MTA were investigated in A549 (NSCLC) and CAL-27 (SCCHN) cells. Using simultaneous or sequential exposure schedules, the cytotoxicity and radiosensitising effect of 24 h MTA combined with 1 h or 24 h dFdC were analysed.RESULTS:Including a time interval between MTA exposure and irradiation seemed favourable to MTA immediately preceding or following radiotherapy. MTA induced a significant S phase accumulation that persisted for more than 8 h after drug removal. Among different MTA/dFdC combinations tested, the highest synergistic interaction was produced by 24 h MTA followed by 1 h dFdC. Combined with irradiation, this schedule showed a clear radiosensitising effect.CONCLUSIONS:Results from our in vitro model suggest that the sequence 24 h MTA --> 1 h dFdC --> RT is the most rational design and would, after confirmation in an in vivo setting, possibly provide the greatest benefit in the clinic.

The clonogenic assay is the method of choice to determine cell reproductive death after in vitro irradiation treatment. Traditionally, colony quantification has been performed by manual counting, a very laborious, time-consuming and rather subjective task. In this study, we compared manual counting by two skilled investigators with automated counting using the freely available ClonoCounter program. Five human tumour cell lines were irradiated under normoxia (21% O(2)) or anoxia (<0.1% O(2)), after 24 h or 6 h anoxic preincubation. Colonies were quantified manually or using the ClonoCounter software. A positive correlation between the absolute number of colonies counted manually and automatically was shown. Though there was a general trend of underpredicting the absolute number of cell colonies when counting automatically, survival curves were very similar, and in none of the cell lines were significant differences in radiobiological parameters such as mean inactivation dose, surviving fraction at 2 Gy and oxygen enhancement ratio detected. Our results suggest that the ClonoCounter provides sufficient reliability to be implemented for counting human tumour colonies in in vitro irradiation experiments. In contrast to several previously reported computer-aided colony-counting methods, it is a freely available program, requiring only minimal instrument costs.

The combination of radiotherapy with chemotherapeutic agents that sensitize tumor cells to ionizing radiation has long been regarded as a promising strategy to enhance cancer therapy. Many chemotherapeutic agents interact with radiation and enhance the cytotoxic or anti-tumor effect of radiation through a number of mechanisms. These include an increase in initial radiation damage, inhibition of cellular repair, cell cycle redistribution, enhancement of apoptosis, counteracting hypoxia and overcoming accelerated repopulation. This article focuses on the role of cell cycle perturbations in the radiosensitivity of cancer cells.

Hypoxic tumour regions often contain viable cells that are more resistant to chemotherapy and/or radiotherapy, making it of key importance to analyse new combination treatments under both normoxic and hypoxic conditions. In this study, the impact of moderate hypoxia and anoxia on cellular characteristics was investigated in isogenic A549 cells differing in p53 status. VEGF expression, doubling time, cell cycle distribution, induction of apoptosis and p53 protein expression were evaluated. Radiation survival curves yielded an oxygen enhancement ratio of 1.16-1.67. In conclusion, an in vitro hypoxia model that will be highly useful to analyse chemoradiation interactions is presented.

Introduction: Over the last decades, it has been well documented that poor oxygenation is a characteristic feature of most human solid tumors. In addition, the efficacy of radiotherapy is directly linked with an adequate oxygen tension and hypoxic regions often contain viable tumor cells that are intrinsically more radioresistant. In this study, the effect of severe hypoxia (anoxia, Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 254.