Lars Ekblad

Coomassie Staining as Loading Control in Western Blot Analysis

In Western blotting, immunodetection of housekeeping proteins is routinely performed to detect differences in electrophoresis loading. The present work describes a much faster and simpler protein staining method, which is compatible with ordinary blocking conditions. In addition, the method can be used after immunodetection with superior linearity compared to ordinary staining methods. After immunoblotting and staining, protein bands can be further identified using peptide mass fingerprinting.

Epidermal growth factor receptor status and persistent activation of Akt and p44/42 MAPK pathways correlate with the effect of cetuximab in head and neck and colon cancer cell lines.

ObjectiveThe aim of this study was to investigate the effect of epidermal growth factor receptor (EGFR) blockade on cell survival and on downstream signalling pathways using the monoclonal antibody cetuximab.MethodsWe used three colon cancer cell lines, of which one was EGFR-negative, and two head and neck squamous cell carcinoma (HNSCC) lines. EGFR expression and gene copy number were measured by immunohistochemistry and FISH analysis, respectively. The effect of cetuximab, irradiation or the combination of both on cell growth was estimated by SRB assay. Western blotting was used to determine the phosphorylation of intracellular signalling proteins and cell cycle phase distribution was measured by flow cytometry.ResultsThe addition of cetuximab had only limited effects on cell growth, with a maximum inhibition of approximately 30%, but was correlated with the amount of protein expression and gene copy number of EGFR. When combined with irradiation, the effect of cetuximab was only additive and not dependent on the inherent radio-sensitivity of the cell lines. Persistent phosphorylation of Akt and/or p44/42 MAPK was detected by western blot in all of the cell lines, whereas there was no phosphorylation of Jak2 or STAT3.ConclusionsNone of these factors alone could predict the sensitivity to cetuximab. Rather, the results suggest that it might be necessary to determine the activation status of several intracellular signalling proteins to better predict the sensitivity to cetuximab treatment.

Reduced drug accumulation is more important in acquired resistance against oxaliplatin than against cisplatin in isogenic colon cancer cells.

Preclinical studies have indicated that there is only partial cross-resistance between cisplatin and oxaliplatin. The molecular background for this is incompletely known. To investigate the differences in resistance, we rendered a colon cancer cell line (S1) resistant against cisplatin and oxaliplatin and characterized the subclones with regard to cross-resistance, platinum uptake, and gene expression profiles. Four oxaliplatin and four cisplatin-resistant cell lines were produced from S1 by step-wise increasing the concentrations of the drugs in the growth medium. Cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and platinum accumulation in cell lysates and DNA preparations by inductively coupled plasma mass spectroscopy. Gene expression was investigated by cDNA microarrays. The protein expression of the ATP-binding cassette B1 (ABCB1) was measured by immunohistochemistry. The cisplatin-resistant cell lines were 1.5–6.2-fold resistant against cisplatin and the oxaliplatin-resistant sublines 2.6–17-fold resistant against oxaliplatin. There was a limited degree of cross-resistance. Oxaliplatin resistance could be explained to a larger degree by reduced drug accumulation whereas mechanisms for increased tolerance against platinum incorporation in DNA seemed to be of higher importance for resistance against cisplatin. A greater number of ABC transporters were upregulated in the oxaliplatin-resistant cell lines compared with those selected for cisplatin resistance. ABCB1 was highly overexpressed in the three most oxaliplatin-resistant sublines, but significantly underexpressed in the two most cisplatin-resistant cell lines. This was also confirmed by immunohistochemistry. However, functional tests did not show any increase in ABCB1 transport activity in the oxaliplatin-resistant sub-lines compared with S1.

Autocrine epidermal growth factor receptor ligand production and cetuximab response in head and neck squamous cell carcinoma cell lines

PurposePredictive strategies for the treatment efficacy of cetuximab are currently not available for head and neck cancer. We investigated the correlation between the expression of epidermal growth factor receptor (EGFR) ligands and EGFR expression, and the growth inhibitory activity of cetuximab in a panel of head and neck squamous cell carcinoma (HNSCC) cell lines.MethodsThe growth inhibiting effect of cetuximab was measured for eight HNSCC cell lines and correlated with the autocrine production of five EGFR ligands as measured by ELISA, and the mRNA expression of two ligands, as measured by quantitative RT-PCR. EGFR expression was assessed by western blot analysis.ResultsThere was a good correlation between the expression of four of the EGFR ligands (TGF-α, amphiregulin, epiregulin and epigen) and the growth inhibiting effect of cetuximab. TGF-α had the highest predictive potential but had to be combined with epigen for full prediction. EGFR expression also correlated with cetuximab sensitivity but less clearly.ConclusionsThe results indicate that the expression of several EGFR ligands has to be used to predict sensitivity to cetuximab in HNSCC. This has to be further evaluated in clinical samples.

Human anaplastic thyroid carcinoma cells are sensitive to NK cell-mediated lysis via ULBP2/5/6 and chemoattract NK cells.

Purpose: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive forms of cancer with no curative therapies available. To date, strategies to target ATC by immunotherapy have not been evaluated. We investigated whether ATC would be a suitable target for natural killer (NK) cell–based immunotherapy. Experimental Design: We first established seven new cell lines from ATC tumors, three from papillary thyroid carcinoma tumors and analyzed them together with eight additional ATC cell lines. Cells were analyzed for sensitivity to lysis by NK cells and their ability to chemoattract and regulate the activity of NK cells. In addition, fresh tumor samples and peripheral blood from six patients with ATC were analyzed for NK cell infiltration and phenotype. Results: We observed that ATC cell lines are sensitive to lysis by ex vivo expanded NK cells and that the lysis was abrogated upon blockade of NKG2D. Sensitivity of thyroid cancer cell lines to NK cell–mediated lysis correlated with surface expression of UL16-binding protein 2 on tumor cells. Moreover, ATC cell lines produced high levels of CXCL10 and stimulated migration of expanded NK cells and ATC tumors were enriched for NK cells expressing the cognate chemokine receptor CXCR3. However, compared with NK cells in peripheral blood, ATC tumor–derived NK cells displayed a suppressed phenotype with a downregulated expression of NKG2D. In vitro, suppression of NK cell–mediated lysis and NKG2D expression by ATC cells was restored upon neutralization of prostaglandin-E2. Conclusions: ATC cell lines are sensitive to NK cell–mediated lysis via ULBP2/5/6 and chemoattract CXCR3-positive NK cells. Patients with ATC may benefit from NK cell–based immunotherapy. Clin Cancer Res; 20(22); 5733–44. ©2014 AACR.

Early Changes in 2-Deoxy-2-[18F]Fluoro-d-Glucose Metabolism in Squamous-Cell Carcinoma During Chemotherapy in Vivo and In Vitro

AIM The aim of this study was to investigate early changes in uptake of 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) in vivo and in vitro in a squamous-cell carcinoma (SCC) cell line originating from a human head and neck SCC during cytotoxic therapy with respect to metabolism in tumor cells and in surrounding stromal tissue. MATERIALS AND METHODS In 60 nude mice with xenografted SCC, 50 animals were treated with cisplatin. Early changes in the tumor FDG uptake following therapy were evaluated sequentially with phosphor imaging. Using this technique, areas with focal hypermetabolism were detected. The cells creating the focal hypermetabolism were then identified histopathologically on the corresponding sections. In addition, early FDG uptake versus the number of viable tumor cells was measured in vitro following cisplatin treatment. RESULTS An early transient increase in FDG uptake in tumor cells was seen on day 1 in treated tumors, followed by a rapid decrease confirmed by subsequent tumor regression. This metabolic flare was present in all treated tumors but not in the controls. In vitro, an increase in FDG uptake per cell was observed. CONCLUSIONS Our results provide new insights into the early metabolic changes in squamous-cell carcinomas subjected to cytotoxic therapy and thus contribute to the discussion on the feasibility of early predictive PET studies.

Localization of phosphatidylinositol 4-kinase isoenzymes in rat liver plasma membrane domains

The presence of different isoenzymes of phosphatidylinositol 4-kinase in isolated rat liver plasma membranes and their further distribution in plasma membrane domains was examined. Both wortmannin-sensitive and -insensitive PtdIns 4-kinase activities were detected in highly purified plasma membranes obtained by aqueous two-phase affinity partitioning. The wortmannin-sensitive enzyme was identified as the 230 kDa isoform by Western blotting, whereas the 92 kDa isoform was not detected in plasma membranes. The apparent molecular weights of these isoforms were 205 and 105 kDa on SDS polyacrylamide gel electrophoresis, but approximately 500 and 230 kDa respectively on gel filtration, suggesting that both enzymes either are dimers or composed of heterologous subunits. Approximately 25% of the total 230 kDa isoenzyme present in liver, and only ca 5% of the wortmannin-insensitive one, was associated with the plasma membrane fraction. Plasma membrane domains were isolated by a combination of sucrose and Nycodenz gradient centrifugations. The 230 kDa isoform was identified in the blood sinusoidal domain, but not in the bile canalicular one, and was also found in lateral plasma membranes. The wortmannin-insensitive isoenzyme was present only in this latter material. The functional implications of this distribution of PtdIns 4-kinase isoenzymes in plasma membrane regions are discussed.

Affinity partitioning of biotinylated mixed liposomes : effect of charge on biotin--NeutrAvidin interaction

The partitioning behaviour of biotinylated mixed liposomes in aqueous poly(ethylene glycol)/dextran two-phase systems containing NeutrAvidin-dextran suggests that the biotin-NeutrAvidin affinity interaction is charge dependent. Biotinylated phosphatidylcholine liposomes with a low negative surface charge distributed in the NeutrAvidin-containing bottom phase at neutral pH, but the introduction of additional negative charges by including phosphatidylserine or the surfactant sodium dodecylsulfate in the liposomes caused them to distribute in the poly(ethylene glycol)-rich top phase instead. By gradually lowering the pH of the affinity two-phase system below the isoelectric point (6.3) of NeutrAvidin, negatively charged phosphatidylserine/phosphatidylcholine liposomes increasingly were attracted by NeutrAvidin to the bottom phase. It is suggested that acidic amino acids present at the rim of the biotin-binding pocket of NeutrAvidin may interact electrostatically with charged residues of the closely apposed liposome surface affecting the affinity interaction.

Cell-line-specific stimulation of tumor cell aggressiveness by wound healing factors - a central role for STAT3

BackgroundLocal recurrence is a major factor affecting survival after treatment for head and neck squamous cell carcinoma (HNSCC). It is possible that the normal processes involved in wound healing after surgical removal of a primary tumor can boost the regrowth of residual cancer cells, thereby contributing to the recurrent growth. In this work, we collected human wound fluids and used them to investigate the effect of wound healing factors on HNSCC cell lines in vitro.MethodsWound fluids were collected from thyroidectomized patients diagnosed with benign disease and were included in assays of cell proliferation, migration, cell scattering, and invasion. The involvement of intracellular signaling pathways and membrane receptors were investigated by western blotting and the inclusion of specific inhibitors.ResultsOne out of four cell lines was greatly stimulated in proliferation, migration, cell scattering, and invasion by the addition of wound fluid as compared with addition of fetal bovine or human serum. These effects were accompanied by a sharp increase in activation of signal transducer and activator of transcription 3 (STAT3). Inhibition of STAT3 activation abolished the wound fluid response, showing that STAT3 plays an important role in the wound healing response. Several of the observed phenotypic changes were epithelial-to-mesenchymal transition (EMT)-like, but the appropriate changes were not seen in any of the EMT markers investigated. The involvement of c-Met or epidermal growth factor receptor family members was excluded, while the interleukin-6 receptor was found to be partly responsible for the activation of STAT3.ConclusionsIn conclusion, we found cell-line-specific effects of wound healing factors on HNSCC, setting the stage for therapy development and predictive opportunities.

Regression analysis and modelling of data acquisition for SELDI-TOF mass spectrometry

MOTIVATION Pre-processing of SELDI-TOF mass spectrometry data is currently performed on a largel y ad hoc basis. This makes comparison of results from independent analyses troublesome and does not provide a framework for distinguishing different sources of variation in data. RESULTS In this article, we consider the task of pooling a large number of single-shot spectra, a task commonly performed automatically by the instrument software. By viewing the underlying statistical problem as one of heteroscedastic linear regression, we provide a framework for introducing robust methods and for dealing with missing data resulting from a limited span of recordable intensity values provided by the instrument. Our framework provides an interpretation of currently used methods as a maximum-likelihood estimator and allows theoretical derivation of its variance. We observe that this variance depends crucially on the total number of ionic species, which can vary considerably between different pooled spectra. This variation in variance can potentially invalidate the results from naive methods of discrimination/classification and we outline appropriate data transformations. Introducing methods from robust statistics did not improve the standard errors of the pooled samples. Imputing missing values however-using the EM algorithm-had a notable effect on the result; for our data, the pooled height of peaks which were frequently truncated increased by up to 30%.