Caveolin-1 (Cav-1) is an integral transmembrane protein and a critical component in interactions of integrin receptors with cytoskeleton-associated and signaling molecules. Since integrin-mediated cell adhesion generates signals conferring radiation resistance, we examined the effects of small interfering RNA-mediated knockdown of Cav-1 alone or in combination with β1-integrin or focal adhesion kinase (FAK) on radiation survival and proliferation of pancreatic carcinoma cell lines. Irradiation induced Cav-1 expression in PATU8902, MiaPaCa2 and Panc1 cell lines. The cell lines showed significant radiosensitization after knockdown of Cav-1, β1-integrin or FAK and cholesterol depletion by β-cyclodextrin relative to nonspecific controls. Under knockdown conditions, proliferation of non-irradiated and irradiated cells was significantly attenuated relative to controls. These findings correlated with changes in expression or phosphorylation of Akt, glycogen synthase kinase 3β, Paxillin, Src, c-Jun N-terminal kinase and mitogen-activated protein kinase. Analysis of DNA microarray data revealed a Cav-1 overexpression in a subset of pancreatic ductal adenocarcinoma samples. The data presented show, for the first time, that disruption of interactions of Cav-1 with β1-integrin or FAK affects radiation survival and proliferation of pancreatic carcinoma cells and suggest that Cav-1 is critical to these processes. These results indicate that strategies targeting Cav-1 may be useful as an approach to improve conventional therapies, including radiotherapy, for pancreatic cancer.
Caveolin-1 (Cav-1) plays a major role in caveolae formation, endo- and exocytosis, lipid homeostasis and tumorigenesis. A scaffolding domain within Cav-1, which consists of 20 amino acids, enables the inactivation of diverse signaling proteins, thus resulting in the regulation of a large variety of cell functions (Carver and Schnitzer, 2003; Shatz and Liscovitch, 2004; Williams and Lisanti, 2005). Interactions and complex formation of Cav-1 with a number of signal-transduction molecules including non-receptor bound tyrosine kinases, Ras and insulin-like growth factor are mediated by accumulation of these molecules in caveolae. Moreover, Cav-1 is an integral transmembrane protein critically acting in crosslinking cell adhesion molecules of the integrin family, cytoskeleton-associated proteins and various signaling molecules (Wary et al., 1998; Razani et al., 2002). Because cell–matrix interactions (Sethi et al., 1999; Cordes and Meineke, 2003; Buttery et al., 2004; Hodkinson et al., 2006; Hehlgans et al., 2007) as well as other focal adhesion proteins such as β1-integrins (Cordes et al., 2006; Estrugo et al., 2007), focal adhesion kinase (FAK) (Kasahara et al., 2002) and integrin-linked kinase (ILK) (Cordes, 2004; Eke et al., 2006; Hess et al., 2007) have been shown to modulate the cellular sensitivity to radiation-induced genotoxic injury, Cav-1 might also contribute to radiation survival regulation.
Cav-1 has been implicated in tumor suppression, differentiation and in promoting oncogenic transformation (Koleske et al., 1995; Galbiati et al., 1998). In the context of our studies, it is interesting that elevated expression of Cav-1 is accompanied by acquisition of multi-drug resistance in various chemotherapy-resistant tumor cell lines (Belanger et al., 2004). The effects mediated by Cav-1 may be cell-type specific since Cav-1 exerted tumor suppressor activity in small-cell lung cancer while promoting survival and proliferation in non-small-cell lung cancer (Sunaga et al., 2004). In pancreatic carcinoma, increased Cav-1 expression has been associated with progression and poor prognosis for patients following surgical resection (Suzuoki et al., 2002). In human breast cancer cell lines, cell proliferation and anchorage-independent growth were impaired by reconstituted expression of Cav-1 (Lee et al., 1998; Fiucci et al., 2002). Conversely, the dominant-negative mutation P132L in the Cav-1 gene has been associated with an invasive form of breast cancer (Hayashi et al., 2001). Expression of Cav-1 is also dysregulated in normal cells adjacent to tumors. In endothelial cells isolated from glioblastomas, Cav-1 is downregulated, and expression returns to the levels observed in normal brain endothelial cells after irradiation (Regina et al., 2004).
On the basis of these findings, we evaluated radiation-dependent expression of Cav-1 as well as the role of Cav-1 in the radiation survival and proliferation response in a number of human pancreatic tumor cell lines. We used Cav-1 small interfering RNA (siRNA)-mediated knockdown or cholesterol depletion by β-cyclodextrin to inhibit Cav-1 function. Cav-1 directly binds cholesterol, an important cofactor for caveolae formation (Hailstones et al., 1998). In order to test for possible synergistic, prosurvival signaling between Cav-1 and β1-integrin or FAK, double knockdown experiments were performed before irradiation. Additionally, the selected pancreatic tumor cell lines were stably transfected with Cav-1-overexpression vectors. Gene expression of Cav-1, β1-integrin, FAK and a variety of downstream target molecules was analysed by DNA microarrays in pancreatic cancer cell lines and in microdissected tissue biopsies from normal pancreas and patients with ductal adenocarcinomas of the pancreas.
Cav-1 expression is induced by irradiation
We first assessed the effect of ionizing radiation on Cav-1 expression. In pancreatic tumor cells, irradiation strongly induced both Cav-1 (Figure 1a) and the number of Cav-1-positive caveolae (Figure 1b) evaluated by western blotting or immunofluorescence staining, respectively.
Pancreas carcinoma cells are sensitized to ionizing radiation by Cav-1 knockdown and cholesterol depletion
Human pancreatic tumor cell lines were transfected using siRNA against Cav-1 before irradiation. PATU8902, MiaPaCa2 and Panc1 cells were significantly (P<0.01) radiosensitized under Cav-1 downregulation using two different Cav-1 siRNAs (nos. 1, 2) relative to nonspecific Duplex XII (Figure 2a). Similar to Cav-1, knockdown of β1-integrin or FAK using two different siRNAs significantly (P<0.01) reduced radiation survival in 6-Gy irradiated pancreatic cancer cell lines (Figure 2b; siRNA no. 2 not shown).
Since Cav-1 interacts with focal adhesion proteins, knockdown of Cav-1 was combined with silencing of β1-integrin or FAK, which resulted in enhanced radiation sensitivity of pancreatic carcinoma cells relative to single knockdown and Duplex XII controls (Figure 2c). A second set of siRNAs with different target sequences within the same genes gave similar results (data not shown). To compare Cav-1 knockdown, which might impact on caveolae function, with another function-disrupting approach against caveolae, cells were depleted of cholesterol by β-cyclodextrin. Exposure of cells to β-cyclodextrin also caused radiosensitization in pancreatic carcinoma cells (Figure 2d). A reduction in the number of caveolae, Cav-1 staining intensity and expression of total Cav-1 after β-cyclodextrin exposure were detectable (Figure 2e and f).
siRNA-mediated knockdown of Cav-1 alters expression of β1-integrin and FAK
Single Cav-1, β1-integrin or FAK knockdown in PATU8902, MiaPaCa2 and Panc1 cells was between 80% and 95% and double knockdown was between 80% and 98% according to densitometric analysis of protein bands (Figure 3a and b). Nonspecific Duplex XII did not reduce the expression of any gene studied. There was minimal change in non-target protein levels (Figure 3a and b). The toxicity of single or double knockdowns was monitored by trypan blue exclusion revealing 82–96% cell viability relative to untransfected controls (Figure 3a). As a second survival parameter, plating efficiencies of knockdown cultures showed a range from 88% to 100%, indicating no significant difference relative to untransfected or Duplex XII controls. Protein silencing after transfection with siRNA no. 2 (data not shown) resulted in similar protein and cell viability data as shown for siRNA no. 1 (Figure 3a).
The effect of these siRNA treatments on downstream signaling proteins was also documented. Total expression of Akt, glycogen synthase kinase 3β (GSK3β) and c-Jun N-terminal kinase (JNK) remained unaltered after siRNA transfection (Figure 3c and d). Src was induced by Cav-1, Cav-1/β1 or Cav-1/FAK knockdown (Figure 3c and d). p42 mitogen-activated protein kinase (MAPK) and p44 MAPK were both reduced after FAK or Cav-1/FAK silencing and Paxillin increased after Cav-1/β1-integrin knockdown (Figure 3c and d). Phosphorylation of these proteins, which regulates their activity, was examined next. Phosphorylation of Akt-S473 was strongly induced by Cav-1, β1-integrin, Cav-1/β1-integrin and FAK knockdown (Figure 3c and d). GSK3β-S9 phosphorylation was elevated following Cav-1 or Cav-1/β1-integrin silencing. A fourfold or greater increase in Y416-Src phosphorylation was detectable after silencing of β1-integrin or FAK (Figure 3c and d). Phosphorylation of MAPK was induced by Cav-1, β1-integrin, Cav-1/β1-integrin or FAK knockdown and repressed by Cav-1/FAK downregulation. The phosphorylated form of JNK increased under FAK or Cav-1/FAK silencing and was reduced by 50% after ILK knockdown. Phosphorylation of Paxillin-Y118 declined by 70% after FAK knockdown.
Overexpression of Cav-1 reduces the radiosensitivity of pancreatic cancer cells
To confirm that Cav-1 is involved in regulating radiation survival, cells were transfected with a Cav-1 expression vector. Most notably in the PATU8902 and MiaPaCa2 cell lines, overexpressed Cav-1 significantly (P<0.01) protected the cells against radiation-induced cell death as compared to empty vector control transfectants (Figure 4).
Cell cycle redistribution does not account for radiosensitivity changes
To evaluate whether enhancement of radiosensitivity was based on cell cycle alterations such as increased accumulation of cells in the radiation-sensitive G2 phase after protein silencing, we examined the cell cycle distribution at 48 h after siRNA treatment. Knockdown of β1-integrin or FAK significantly (P<0.01) increased the S-phase fraction of PATU8902 cells relative to Duplex XII controls (Figure 5a). Similar data were obtained in MiaPaCa2 and Panc1 cells (Figure 5a).
Silencing of Cav-1, β1-integrin or FAK reduces proliferation
To determine the impact of single and combined knockdown of the proteins of interest on cell proliferation, cell numbers were measured at the time point of termination of colony formation assays. Silencing of Cav-1, β1-integrin or FAK significantly (P<0.01) repressed proliferation of the pancreatic carcinoma cell lines tested relative to controls (Figure 5b). Two Gy irradiation of single-knockdown cell cultures resulted in a two- to threefold reduction in cell proliferation. Combined downregulation of Cav-1/FAK significantly (P<0.01) reduced basal cell proliferation with an additional reduction in cell numbers after 2 Gy (Figure 5b). The specific knockdown of Cav-1, β1-integrin or FAK mediated thus enhanced radiosensitivity and increased cell inactivation through potent growth inhibition.
Gene expression profiling of Cav-1 and related signaling molecules
Gene expression analysis was performed to assess Cav-1 expression in pancreatic cancer tumor specimens and to test whether the cell lines we studied reflect the range of Cav-1 expression as detectable in normal and tumor tissue. In contrast to previous studies analysing whole tissue samples from pancreatic tumors (Iacobuzio-Donahue et al., 2003; Grutzmann et al., 2005), we analysed expression of Cav-1 and related signaling molecules in microdissected tissues of normal pancreatic epithelia versus ductal pancreatic adenocarcinoma (Grutzmann et al., 2004). Expression of the genes of interest was measured in eight pancreatic cancer cell lines, seven normal pancreatic epithelia and 11 ductal adenocarcinoma. Expression was visualized using a heatmap generated by dChip. All analysed genes were highly expressed in subsets of pancreatic carcinoma samples and in a number of pancreatic cancer cell lines (Figure 6a). However, analysis of the overall expression of examined genes failed to assign a differential expression to any gene. Interestingly, Cav-1 expression in MiaPaCa2 and Panc1 cells differed by a factor of 4.91 indicating that the observed effects of Cav-1 knockdown are independent of pretreatment Cav-1 RNA content. Moreover, the Cav-1 expression level detected in MiaPaCa2 cells resembles the gene expression level in primary pancreatic tissues, whereas in Panc1, the expression level of Cav-1 reflected Cav-1 mRNA levels present in highly overexpressing primary adenocarcinoma (Figure 6b).
This study shows, for the first time, that siRNA-mediated knockdown of Cav-1 expression results in an enhanced radiosensitization of pancreatic cancer cells and that this effect might be in part due to the interaction of Cav-1 with focal adhesion proteins. This suggests that Cav-1 acts as prosurvival factor within the cellular response to ionizing radiation. The successful implementation of siRNA-based methods enabled us to uncover the role of the caveolae/lipid raft-associated, regulatory protein Cav-1 in clonogenic survival, proliferation and cytoplasmic signal transduction of pancreatic carcinoma cells.
These findings add new insight into Cav-1's role in promoting cellular resistance against genotoxic agents in human cancer cells. The function of Cav-1 as an integral transmembrane protein critically acting in crosslinking cell adhesion molecules of the integrin family, cytoskeleton-associated proteins and various signaling molecules further supports the important regulatory role of integrin-mediated cell–matrix interactions in the phenomenon termed cell adhesion-mediated radiation resistance (Cordes and Meineke, 2003; Eke et al., 2006; Estrugo et al., 2007). The importance of Cav-1 as linker protein between different focal adhesion proteins in cell adhesion-mediated radiation resistance was demonstrated by the significant reduction in clonogenic radiation survival after siRNA-mediated silencing of Cav-1 expression. Our data are in accord with results obtained in Cav-1-deficient mice irradiated with 15 Gy γ-radiation. Cav-1-deficient mice exhibited reduced survival rates relative to wild-type mice as well as increased apoptosis in Cav-1 null crypt stem cells (Li et al., 2005). Although these data reflect the radiation response of normal tissue following knockout of Cav-1, they also support the hypothesis that Cav-1 is critical to cellular radiation sensitivity. The effects of double-knockdown regimes targeting Cav-1 in combination with either β1-integrin or FAK on radiation survival were only marginal, but neither additive nor synergistic. These findings imply that Cav-1 effects occur via partial overlapping pathways with those used by β1-integrin or FAK (Cherubini et al., 2005). With regard to the additive effects on radiation survival at 2 Gy by double-knockdown regimes, Cav-1 silencing, on the one hand, might biochemically alter caveolar function and perturb downstream signaling by changes in the composition of signaling complexes located at the cell membrane. On the other hand, silencing of β1-integrin or FAK might trigger the cellular sensitivity to ionizing radiation by reducing integrin-mediated cell–matrix interactions in terms of anoikis (cell detachment-induced apoptosis). However, the morphological changes observed upon β1-itegrin or FAK knockdown such as cell rounding and cell detachment were negligible. Thus, this dual modification of prosurvival effects might lead to the significant decrease of radiation survival at 2 Gy. Whether these promising results of this first evaluation of Cav-1 and some of its interactors in the cellular radiosensitivity have implications for radiotherapy have to be assessed in more complex cell culture models or in animals.
In addition to a second set of siRNAs, cholesterol depletion using β-cyclodextrin underscored a critical function of Cav-1 in the cellular radiation survival and proliferation response. However, the effects of cholesterol depletion on modifying the cellular sensitivity to radiation or cytotoxic drugs remain controversial (Miller et al., 1993; Fritz et al., 2003; Yacoub et al., 2007). It can be hypothesized that the degree of dependence of a cell on Cav-1 and cholesterol is correlative to the cellular radio- and chemosensitivity mediated by a reduction of Cav-1 and/or cholesterol. Further studies on tumor-specific targeting of Cav-1 are warranted to evaluate the therapeutic effectiveness from such an approach in combination with conventional anticancer therapies administered in pancreatic carcinoma.
The results of microarray analysis of microdissected tissue showed that the mean Cav-1 mRNA expression showed no significant difference between pancreatic carcinomas and normal epithelium. The usage of MiaPaCa2 and Panc1 cells reflects a high-to-low range in the Cav-1 expression spectrum in pancreatic tumors. Intriguingly, knockdown of Cav-1 in both cell lines enhanced the radiosensitivity, thus, indicating this approach promising and not due to RNA expression levels.
In addition to cell survival, cell proliferation is prominently influenced by irradiation (Iliakis, 1997). Cav-1−/− mice models have demonstrated that Cav-1 expression suppresses mitogenesis and avoids constitutive activation of MAPK, overexpression of cyclin D1 and proliferating cell nuclear antigen (PCNA) and decrease in p21Cip/WAF−1 (Williams et al., 2004). Experiments with primary mouse embryonic fibroblasts from Cav-1 transgenic mice demonstrated that Cav-1 promotes exit of cells from the S phase of the cell cycle (Galbiati et al., 2001). As shown here, PATU8902, MiaPaCa2 and Panc1 cells were potently growth inhibited following Cav-1, β1-integrin or FAK knockdown, which reflects the alterations observed in clonogenic survival. However, at maximum siRNA-mediated knockdown of targeted proteins, PATU8902, MiaPaCa2 and Panc1 cells revealed a significant increase in either S or G1 phase cells, coincident with a reduction in G2. As the G1 and S phases are considered to represent more radioresistant cell cycle phases, our data do not support cell cycle redistribution because of siRNA-mediated knockdown as determinants of clonogenic survival in these studies.
Changes in signal transduction, in particular in the phosphoinositide 3-kinase/Akt and Ras/Raf/MAPK cascades, were examined after Cav-1 downregulation. In endothelial cells, Cav-1 knockdown using siRNA has been demonstrated to alter Akt and MAPK signaling independent from Cav-1's role in guiding specific signaling molecules to lipid rafts (Gonzalez et al., 2004). Deletion of the Cav-1 scaffolding domain significantly diminished Akt phosphorylation and cell viability of prostate cells (Li et al., 2003). Cav-1-dependent activation of MAPK gave controversy results. Galbiati et al. (1998) observed an inhibitory role of Cav-1 on MAPK cascade activation in Cav-1−/− mouse embryonic fibroblasts while Cohen et al. (2003) found no substantive difference in MAPK activation in Cav-1 wild-type versus Cav-1−/− fibroblasts. Interactions of Cav-1 with integrins and the tyrosine kinase Fyn revealed that Src is a connector between integrins, Grb2 and the Ras/MAPK cascade to control cell cycling. Our data demonstrate that Cav-1 knockdown induces Akt, GSK3β and MAPK phosphorylation while phosphorylation of Src, JNK and Paxillin remain stable. Src-Y416 phosphorylation, however, showed strong increase after silencing of β1-integrin or FAK. Downregulation of β1-integrin mediated changes in GSK3β, Src and MAPK phosphorylation and Paxillin expression; an effect overruled by simultaneous Cav-1 knockdown. Under combined knockdown, Cav-1/β1 stimulated GSK3β phosphorylation. Thus, integrin signaling seems strongly affected by Cav-1. However, the impact of these changes on the radiation sensitivity of the human pancreatic carcinoma cells tested has not yet been determined.
Taken together, our findings not only support the notion of a regulatory function of Cav-1 in signaling via β1-integrin or FAK but also uncover Cav-1 as modifier of radiation cell survival and proliferation in vitro. It is tempting to speculate that novel targeting therapeutics against Cav-1 might improve the effectiveness of conventional radio- and chemotherapeutic regimes in pancreatic carcinoma. Additional studies will be required to address this issue. These results further contribute to the understanding of cell adhesion-mediated radiation resistance and to the development of targeted therapies that are urgently needed to improve patient outcome after diagnosis with highly lethal carcinoma of the pancreas.
Materials and methods
Pancreatic carcinoma cell lines PATU8902, MiaPaCa2 and Panc1 were purchased from DMSZ (Braunschweig, Germany) or ATCC (Manassas, VA, USA), respectively. Cells were cultured in DMEM GlutaMAX 1 medium (Gibco, Karlsruhe, Germany) supplemented with 10% fetal calf serum (PAA, Linz, Austria) and 1% non-essential amino acids (Gibco) at 37°C in a humidified atmosphere containing 10% CO2. For all experiments, asynchronous growing cell cultures were used. To evaluate viability, cells were mixed with an equal volume of 0.4% trypan blue solution and immediately scored to determine exclusion of dye by light microscopy.
Irradiation was delivered at room temperature using single doses of 240 kV X-rays (Isovolt 320/10; Seifert, Ahrensburg, Germany) filtered with 3 mm beryllium. The absorbed dose was measured using a Duplex dosimeter (PTW, Freiburg, Germany). The dose-rate was approximately 1 Gy/min at 13 mA. Applied doses ranged from 0 to 6 Gy.
The sequences of Cav-1, β1-integrin and FAK siRNA were selected based on a method described previously (Elbashir et al., 2001). Two target sense sequences (nos. 1, 2) that effectively mediated silencing were as followed:
Caveolin-1: no. 1, 5′-IndexTermAAAUACAAGAUCUUCCUUCCU-3′; no. 2, 5′-IndexTermAAUGUGAUUGCAGAACCAGAA-3′.
β1-integrin: no. 1, 5′-IndexTermAATGTAACCAACCGTAGCA-3′; no. 2, 5′-IndexTermGCGCATATCTGGAAATTTG-3′ (Cordes et al., 2006).
FAK: no. 1, 5′-IndexTermAATTTGCCAACCTTAATAGAG-3′; no. 2, 5′-IndexTermAACCTCGCAGTCATTTATCAT-3′ (all from MWG, Ebersberg, Germany). Cells were transfected with the specific or nonspecific (Duplex XII; 5′-GUAUAUAAGCAAGCAUUACUU-3′) siRNAs using oligofectamine (Invitrogen GmbH, Karlsruhe, Germany). Depletion of examined proteins was confirmed by western blotting.
Cav-1 expression vector and transfection
pCav-1 (mouse Cav-1; 97% homology to human) and pcDNA3 vectors were kindly provided by M Liscovitch (Weizmann Institute of Science, Israel; Fiucci et al., 2002). Selection was performed under 2000 μg/ml G418. The expression of Cav-1 in the transfectants was confirmed by western blotting. The stable transfectants were pooled and used as a population designated pCav-1 and pcDNA3.
Immunofluorescence staining and laser confocal scanning microscopy
A total of 2 × 103 cells were plated onto Lab Tek chamber slides (Nunc, Hamburg, Germany). After 24 h, cells were irradiated with 6 Gy or treated with 10 mM β-cyclodextrin for 1 h and fixed using 2% paraformaldehyde/phosphate-buffered saline 24 h thereafter. Immunostaining using anti-caveolin-1 monoclonal antibodies (mAbs) and secondary Cy2-conjugated AffiniPure goat anti-mouse IgG was performed as published (Cordes, 2004). Fluorescence images were obtained using a Leica MRC1024 confocal laser scanning microscope equipped with Leica Confocal Software (Wetzlar, Germany).
Colony formation assay
This assay was used for measurement of clonogenic cell survival following siRNA- or DNA-transfection, β-cyclodextrin exposure (Sigma, Taufkirchen, Germany) in combination with irradiation (Cordes, 2004). At 48 h after siRNA transfection, the time of maximum silencing of protein expression, single cells were irradiated with 0–6 Gy. β-Cyclodextrin was applied to the cells (10 mM, 1 h) before irradiation. Between 8 and 21 days after irradiation, cells were stained with Coomassie blue and colonies of >50 cells were counted. Plating efficiencies were calculated as follows: numbers of colonies formed/numbers of cells plated. Surviving fractions were calculated as follows: numbers of colonies formed/numbers of cells plated (irradiated) × plating efficiency (unirradiated). Each point on survival curves represents the mean surviving fraction from three independent experiments.
Total protein extractions and western blotting
At 1 h after exposure to β-cyclodextrin or 48 h after siRNA transfection, cells were harvested by scraping and lysed using modified radioimmuno precipitation assay buffer (50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, protease inhibitor cocktail complete (Roche, Mannheim, Germany), 5 mM Na3VO4, 5 mM NaF). Amounts of total protein extracts were determined using BCA (bicinchoninic acid) assay (Interchim, Montlucon Cedex, France) and samples were stored at –134°C until use. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Probing and detection of specific proteins was performed with enhanced chemiluminescence (ECL) (Amersham, Freiburg, Germany) after antibody binding. The following antibodies are used: anti-phospho-MAPK, anti-MAPK, anti-JNK, anti-Akt-S473, anti-Paxillin-Y118, anti-Src-Y416, anti-GSK3β, anti-GSK3β-S9 (Cell Signaling, Frankfurt a.M., Germany), anti-FAK, anti-β1-integrin (BD, Heidelberg, Germany), anti-Akt (Acris, Hiddenhausen, Germany), anti-phospho-JNK, anti-caveolin-1, horseradish peroxidase-conjugated goat anti-rabbit and rabbit anti-mouse antibodies (Santa Cruz, Heidelberg, Germany), anti-Paxillin, anti-Src, anti-β-actin (Sigma).
Analysis of cell cycle and proliferation
BrdU (5-bromo-2-deoxyuridine; Serva, Heidelberg, Germany)/propidium iodide (PI) co-staining was performed 48 h after siRNA transfection as published (Cordes et al., 2006). RNase A type III-A was from Sigma, pepsin 0.7 FIP-U from Merck (Darmstadt, Germany), anti-BrdU mAbs from BD. The distribution of cells in the different phases of the cell cycle was analysed from the DNA-dot-blots and -histograms using CELLQuest software (BD). Cell proliferation was determined as published (Cordes et al., 2006).
Freshly frozen tissue samples of pancreatic ductal adenocarcinomas (PDACs; n=11) were obtained from surgical specimens from patients who underwent surgery at the Department of Visceral-, Thoracic- and Vascular-Surgery, University Hospital Carl Gustav Carus, Dresden University of Technology and the Department of General Surgery, University of Kiel, Germany, between 1996 and 2003. Normal ductal specimens were obtained from nine patients who underwent pancreatic resection. Before surgery, all patients had signed an informed consent form that had been approved by the local ethics committee. Specimens were immediately sectioned and microscopically evaluated. Suitable samples of tumor tissue or normal tissue were snap frozen in liquid nitrogen and stored at −80°C until further processing. Frozen tissue specimens were cut into 10 μm sections, fixed in 70% ethanol, stained with hematoxylin and eosin (H&E) and coverslipped. Suitable areas were marked and subjected to microdissection. The tissue blocks were serially cut into 5 μm thin sections, fixed in 70% RNase free ethanol and stained with H&E. Stroma from PDAC and from chronic pancreatitis was dissected manually using a sterile injection needle. Poly A+-RNA was prepared using a PolyATtract 1000 kit (Promega, Heidelberg, Germany), according to the manufacturer's recommendations. For each sample, the cDNA synthesis and repetitive in vitro transcription were performed three times. Microdissection and RNA preparation were performed as published (Grutzmann et al., 2003). The U133 A/B Affymetrix GeneChip set utilized in this study consists of over 44 000 probe sets. The CEL Files obtained from the Affymetrix MAS 5.0 software were used for further analysis. The files were loaded into dChip2006 (www.dchip.org), normalized and expression values were calculated using the PM/MM model (Li and Wong, 2001). To minimize the noise within the gene expression data set, we truncated low expression level to zero. Heatmap generation was performed using dChip.
Mean±s.d. of different end points was calculated with reference to untreated controls defined as 1.0 or in a percentage scale. Student's t-test was performed using the data of three independent experiments and results were considered statistically significant if P-value of less than 0.05 was reached. All experiments were repeated at least three times.
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We thank M Hiber, B Reincke and G Schröder for excellent technical assistance and M Liscovitch (Weizmann Institute of Science, Israel) for pcDNA3-Cav-1 and control vectors. The work was supported in part by grants from the German Ministry of Education and Research (BMBF Contract 03ZIK041 to NC), by the Deutsche Krebshilfe (70-2937-SaI to RG) as well as a MRC fellowship and RO1 CA73820 to EJB.
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