Bea Pauwels

Comparative Analysis of Dynamic Cell Viability, Migration and Invasion Assessments by Novel Real-Time Technology and Classic Endpoint Assays

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 (Spearmans Rho 0.688 to 0.964). Cytotoxic action by paclitaxel (0–100 nM) correlated well with SRB (Rho>0.95) with similar IC50 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.

Cell cycle effect of gemcitabine and its role in the radiosensitizing mechanism in vitro

PURPOSE The mechanism of radiosensitization by gemcitabine is still unclear. It has been hypothesized that the accumulation of cells in early S phase may play a role in enhancing radiosensitivity. METHODS AND MATERIALS The schedule dependency of the radiosensitizing effect was studied in ECV304, human bladder cancer cells, and H292, human lung cancer cells, by varying the incubation time and time interval between gemcitabine and radiation treatment. To determine the role of cell cycle perturbations in the radiosensitization, the influence of gemcitabine on the cell cycle at the moment of radiation was investigated by flow cytometry. RESULTS The radiosensitizing effect increased with a longer incubation period: Dose enhancement factors varied from 1.30 to 2.82 in ECV304 and from 1.04 to 1.78 in H292 after treatment during 8-32 h, respectively. Radiosensitization decreased with an increasing interval: Dose enhancement factors varied from 2.26 to 1.49 in ECV304 and from 1.45 to 1.11 in H292 after an interval 0-24 h, respectively. Cells were blocked in the early S phase of the cell cycle by gemcitabine. The highest percentage S-phase cells was observed after treatment with the schedules that resulted in the highest radiosensitizing effect. CONCLUSIONS We observed a clear schedule-dependent radiosensitization by gemcitabine. Our findings demonstrated a correlation between gemcitabine-induced early S-phase block and the radiosensitizing effect.

Unraveling the Mechanism of Radiosensitization by Gemcitabine: The Role of TP53

Abstract Pauwels, B., Korst, A. E. C., Andriessen, V., Baay, M. F. D., Pattyn, G. G. O., Lambrechts, H. A. J., De Pooter, C. M. J., Lardon, F. and Vermorken, J. B. Unraveling the Mechanism of Radiosensitization by Gemcitabine: The Role of TP53. Radiat. Res. 164, 642–650 (2005). Gemcitabine has excellent radiosensitizing properties, as shown in both preclinical and clinical studies. Radiosensitization correlated with the early S-phase block of gemcitabine. In the present study, we investigated the role of TP53 in the radiosensitizing effect of gemcitabine. Isogenic A549 cells differing in TP53 status were treated with gemcitabine during the 24 h prior to irradiation. Cell survival was determined 7 days after irradiation by the sulforhodamine B test. In addition, cell cycle perturbation was determined by flow cytometry and TP53 expression by Western blot analysis. Gemcitabine caused a concentration-dependent radiosensitizing effect in all cell lines. Transformed A549 cells were less sensitive to the cytotoxic effect of gemcitabine. The cell cycle arrest early in the S phase was dependent on the drug dose but was comparable in the different cell lines and was not related to functional TP53. Using isogenic cell lines, we have shown that neither TP53 status nor the transfection procedure influenced the radiosensitizing effect of gemcitabine. Since both the radiosensitizing effect at equitoxic concentrations and the cell cycle effect of gemcitabine were independent of TP53 expression, it is likely that TP53 protein does not play a crucial role in the radiosensitizing mechanism of gemcitabine.

The radiosensitising effect of gemcitabine and the influence of the rescue agent amifostine in vitro

In this study, the radiosensitising effect of different concentrations of gemcitabine and the combination of gemcitabine/radiotherapy with the rescue agent amifostine was investigated in different human tumour cell lines. The cells were treated with gemcitabine (0-8 nM) for 24 h prior to radiation (0-8 Gy). Amifostine (ami) and alkaline phosphatase (AP) were added 30 min before radiation. Cell survival was determined 7 or 8 days after radiation treatment by the sulforhodamine B (SRB) test. For ECV304 cells, the dose enhancement factor (DEF) varied from 1.39 to 2.98 after treatment with 1-6 nM gemcitabine. FaDu, H292, A549 and CAL-27 seemed to be less sensitive, with DEFs ranging from 1.02 to 2.67. These cells were also less sensitive to the cytotoxic effects of single-agent gemcitabine. Amifostine with AP clearly showed a protective effect in combination with gemcitabine/radiotherapy. In H292 cells, the protection factor (PF) of amifostine after treatment with gemcitabine and radiotherapy varied from 1.64 to 1.86. In ECV304 cells, the PF varied from 2.20 to 2.29. In conclusion, a clear concentration- and cell line-dependent radiosensitising effect of gemcitabine was observed in all cell lines. Amifostine with AP showed protection against the radiosensitising effect of gemcitabine. If the protection in vivo indeed occurs selectively in normal tissues, then amifostine could prevent or strongly minimise the increased toxicity resulting from the radiosensitising effect of the combination of gemcitabine and radiotherapy, without influencing the antitumour effect.

Comparative study of the radiosensitising and cell cycle effects of vinflunine and vinorelbine, in vitro

BackgroundVinca alkaloids are an important class of anticancer agents and semisynthetic vinca alkaloids are developed to improve the therapeutic index of this class of drugs. In the present study, a direct comparison was made between vinflunine and vinorelbine regarding their radiosensitising and cell cycle effects.MethodsFour human tumour cell lines were tested under identical experimental conditions, using equitoxic concentrations of vinflunine and vinorelbine.ResultsVinflunine and vinorelbine induced a comparable radiosensitising effect (p-value never below 0.01) when cells were incubated for 24 h immediately prior to radiation. Regarding the cell cycle effects, a statistically significant concentration-dependent G2/M block was seen after 24 h incubation with vinorelbine in all tested cell lines. Similar results, with small cell line-related differences, were observed with vinflunine.ConclusionThe radiosensitising effects of both semisynthetic vinca alkaloids were comparable (not statistically different) and nearly always cell line-specific and concentration-dependent. The cell cycle effects could be related to the observed radiosensitising effects. Considering the more favourable toxicity profile of vinflunine, this agent might be more promising than vinorelbine for chemoradiation studies in the clinic.

The relation between deoxycytidine kinase activity and the radiosensitising effect of gemcitabine in eight different human tumour cell lines

BackgroundGemcitabine (dFdC) is an active antitumour agent with radiosensitising properties, shown both in preclinical and clinical studies. In the present study, the relation between deoxycytidine kinase (dCK) activity and the radiosensitising effect of gemcitabine was investigated in eight different human tumour cell lines.MethodsTumour cells were treated with dFdC (0–100 nM) for 24 h prior to radiotherapy (RT) (γ-Co60, 0–6 Gy, room temperature). Cell survival was determined 7, 8, or 9 days after RT by the sulforhodamine B test. dCK activity of the cells was determined by an enzyme activity assay.ResultsA clear concentration-dependent radiosensitising effect of dFdC was observed in all cell lines. The degree of radiosensitisation was also cell line dependent and seemed to correlate with the sensitivity of the cell line to the cytotoxic effect of dFdC. The dCK activity of our cell lines varied considerably and differed up to three fold from 5 to 15 pmol/h/mg protein between the tested cell lines. In this range dCK activity was only weakly related to radiosensitisation (correlation coefficient 0.62, p = 0.11).ConclusionGemcitabine needs to be metabolised to the active nucleotide in order to radiosensitise the cells. Since dFdCTP accumulation and incorporation into DNA are concentration dependent, the degree of radiosensitisation seems to be related to the extent of dFdCTP incorporated into DNA required to inhibit DNA repair. The activity of dCK does not seem to be the most important factor, but is clearly a major factor. Other partners of the intracellular metabolism of gemcitabine in relation to the cell cycle effects and DNA repair could be more responsible for the radiosensitising effect than dCK activity.

Role of cell cycle perturbations in the combination therapy of chemotherapeutic agents and radiation

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.

Chemoradiation interactions under reduced oxygen conditions: Cellular characteristics of an in vitro model

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.

Expression analysis on archival material: comparison of 5 commercially available RNA isolation kits for FFPE material.

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.

COUNTING CLONOGENIC ASSAYS FROM NORMOXIC AND ANOXIC IRRADIATION EXPERIMENTS MANUALLY OR BY USING DENSITOMETRIC SOFTWARE

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.