Specimens
Primary RCC tissue samples were obtained under sterile conditions from 71 patients (Table 1) who underwent nephrectomy at the Department of Urology, University Medical Center Mainz [17]. The study was performed in agreement with the Declaration of Helsinki and approved by local ethics committee (No. 837.005.09, Landesärztekammer Rheinland-Pfalz, Mainz, Germany). Each patient provided informed consent. Samples of tumor tissue and renal cortex, obtained from the opposite kidney pole at a minimum distance of 3 cm from the tumor, were shock frozen in liquid nitrogen and stored at − 80 °C. The RCC diagnosis and tumor grade was verified on hematoxylin and eosin sections.
RNA extraction
RNA was extracted from the frozen tissue samples by using the PeqGold Total RNA Kit (Peqlab). The tissue was first split into pieces of 40–50 mg and then transferred in Precellys ceramic tubes. Afterwards the samples were homogenized by adding 400 μl of RNA lysis buffer T (Peqlab) followed by incubation in a Precellys homogenizer on a frequence of 5000 rpm for 3 × 45 seconds. In contrast cultivated cell lines were lysed by adding 600 μl of RNA lysis buffer T and detached by using a scraper. Afterwards the samples were processed according to the kit-provider. To ensure a completely DNA free RNA extract an additional enzymatic digestion of DNA with DNase (Peqlab) was performed after the first washing step for 15 min at room temperature (RT). The amount of RNA in each sample was photometrically determined, using the Nanodrop1000 (Thermo Scientific).
cDNA synthesis
RNA was transformed into cDNA with M-MLV reverse transcriptase (Invitrogen). Therefore 2–3 μg of isolated RNA was diluted in 20 μl RNase free water and denatured at 70 °C for 10 min. Next a reaction mix was added to every sample, each containing 1 μl oligo-dT primer (0.5 μg/μl), 2 μl RNase inhibitor, 3.5 μl dNTP mix (each 2.5 mM), 4 μl DTT (0.1 M), 8 μl of First strand buffer (0.1 M) and finally 1.5 μl M-MLV reverse transcriptase. Subsequently, the samples were incubated at 37 °C for 90 min and finally the reverse transcriptase was inactivated by raising the temperature on 95 °C for further 10 min.
Real-time PCR
Quantification of gene expression was performed with the LightCycler 480 II (Roche). Specific primers were used to secure a selective amplification. These include the PTEN-specific primers 5‘-GTTTACCGGCAGCATCAAAT-3′ (forward) and 5‘-CCCCCACTTTAGTGCACAGT-3′ (reverse), as well as the PTEN-∆ specific primers 5‘-TCCACAAACAGAACAAGATGC-3′ (forward) and 5‘-ACACACATCACATACATACAAG-3′ (reverse). The primers were added (10 μM each) to a total mixture of 10 μl, containing 5 μl Kapa SYBR Fast reagent (Kapa Biosystems), 3 μl distilled water and 1 μl of the cDNA sample. Each reaction was performed in duplicate and determined by the following program: initial denaturation (3 min; 95 °C), followed by 45 repetitive cycles, including denaturation (5 s; 95 °C), annealing (5 sec; 62–66 °C) and elongation (1 s; 72 °C). A final calculation of a melting curve concluded this analytic procedure. For analysis the geometrical average was calculated referring to the expression level of the house keeping genes TBP (TATA-box binding protein), ATP5J (ATP synthase, mitochondrial F0 complex subunit F6) and PPIA (peptidylprolyl isomerase A) [18], which were measured simultaneously.
Cells and cell culture
The human RCC cell lines A498, 786-O, Caki-1 and Caki-2 were obtained from LGC Promochem and CCF-RCI and CCF-RCII were kindly provided by the establisher, Cleveland Clinic Foundation [19]. Caki-1 and Caki-2 cell culture was maintained in Iscoves medium (Biochrom), supplemented with 10% fetal calf serum, 1% GlutaMax (Sigma) and 1× penicillin/streptomycin (Life Technology). All other cell lines were maintained in RPMI1640 (Gibco) supplemented with 10% fetal calf serum, 2.5% HEPES buffer (Sigma) and 1× penicillin/streptomycin (Life Technology). All cell lines were incubated in a moistened atmosphere at 5% CO2 at 37 °C.
Plasmid construction and stable transfection of RCC cell lines
ORFs of the PTEN and PTEN-Δ isoforms were PCR amplified, inserted into a pcDNA3 vector (Invitrogen) and in frame tagged with a V5 encoding domain. Chemocompetent DH5 E. coli cells were transfected with the expression plasmids. Individual clones were selected according to their resistance to ampicillin and verified by complete sequencing of the plasmid insert. For stable transfection 2 × 105 A498 or 786-O cells were seeded in a 6-well-plate. Next day 5 μg plasmid DNA was mixed with 10 μl P3000 (Thermo Scientific) and 125 μl Opti-Mem (Gibco). At the same time 7.5 μl L3000 (Thermo Scientific) was diluted with 125 μl Opti-Mem. Both mixtures were combined and after 10 min incubation the cells were transfected by adding the mixture dropwise. After 24 h G418 (400 mg/ml) was added to the cell medium. Cells were cultured and resistant clones were selected.
In vitro translation
For cell free in vitro translation (IVT) the 1-step human coupled IVT (Thermo Scientific) kit system was used. cDNA containing the ORF of the PTEN isoforms was inserted into the provided pT7CFE1 vector. For IVT 12.5 μl HeLa lysate was mixed with 2.5 μl accessory proteins. After incubation on ice for 10 min, 5 μl reaction mix, 2 μl of plasmid (0.5 μg/μl) and 3 μl nuclease free water were added. IVT was performed for 3 h at 30 °C.
Knockdown
PTEN-∆ was selectively knocked down in Caki-1 cells, using the synthesized ds-siRNA targeting PTEN-∆: 5’-AAAUUUUAAGGUCAGUUAA-3‘ (sense) and 5’-UUAACUGACCUUAAAAUUU-3′ (antisense) from Sigma Aldrich. Lipofectamine RNAiMAX (Thermo Scientific) was used as transfection reagent. 2 × 105 cells were plated in a 6-well-plate and cultured in 2.5 ml Iscoves medium lacking antibiotics. Each well received either PTEN-∆ siRNA or scrambled locus siRNA (negative control) treatment as reference. In the following PTEN-∆ siRNA, or scrambled locus siRNA and Lipofectamine RNAiMAX (7.5 μl) were each mixed with Opti-Mem complete medium (Gibco) to a total concentration of 150 pmol siRNA per well and in a total volume of 250 μl per well. The diluted transfection reagent was then mixed with the diluted siRNA solution and filled dropwise in the correspondent wells. After incubation at 5% CO2 and 37 °C for 24 h, medium was changed to Iscoves medium without additives and cells were incubated for another 24 h. Cells from each well were then detached, counted and prepared for a cell migration assay. The remaining cells of each sample were centrifuged, each suspended in 600 μl RNA lysis buffer T and frozen at − 80 °C for later RNA extraction.
Cell adhesion assay
For cell adhesion assay amine-binding, maleic anhydride activated clear 96-well-plates (Pierce #15110, Thermo Scientific) were used as described previously [20]. Extracellular matrix components used were fibronectin (10 μg/ml), vitronectin (5 μg/ml), laminin (50 μg/ml), collagen I (10 μg/ml), collagen IV (10 μg/ml) and as control BSA (10 μg/ml) (all Thermo Scientific). Concentrations were selected according to the cell migration assay. Each well was coated with extracellular matrix components at a volume of 100 μl overnight on a rocking shaker at RT and washed twice with 100 μl washing buffer (DPBS with 0.05% Tween 20, ICI Amenic Inc.). Unspecific binding sites were blocked with 200 μl blocking solution (DPBS with 0.5% BSA) per well and incubated for 1 h in a moistened atmosphere at 5% CO2 at 37 °C in air. Meanwhile cells were washed in DPBS, medium was then replaced with Trypsin-EDTA and cells were resuspended in serum-free culture medium. Blocking solution was removed from the wells coated with ECM components and 50 μl of tumor cell suspension were added (4 × 105 cells/ml) per well. After 1 h incubation in a moistened atmosphere at 5% CO2 at 37 °C in air, non-adherent cells were removed with 2 × 200 μl washing buffer (DPBS with 0.05% Tween 20) per well. Adherent cells were fixed with 100 μl 4% paraformaldehyde (Histofix 4%, Roth) for 15 min at RT and stained using 100 μl crystal violet solution (5 mg/ml in 2% ethanol) for 10 min at RT. Afterwards the staining solution was washed out with 3 × 100 μl washing buffer (DPBS with 0.05% Tween 20) per well and the plate was dried on air. For resolving the colorant wells were incubated with 100 μl 2% SDS (Roth) for 30 min. The adsorption was measured at 550 nm (reference value at 650 nm) with GloMax®-Multi detection system (Promega). Experiments were performed in quadruplicates and repeated three times.
Chemotactic cell migration assay
For chemotactic cell migration analysis a modified Boyden chamber was used (Costar), as described previously [21]. The chamber consists of an upper and a lower department separated by a porous polycarbonate membrane with 8 μm pore diameter (Neuro Probe). Before analysis cells were cultivated in serum-free culture medium for 24 h. According to the instruction of the manufacturer, the lower chemotaxis compartment was filled with 29 μl solution of extracellular matrix components (according to the optimal conditions [22]: fibronectin 10 μg/ml, vitronectin 5 μg/ml, laminin 50 μg/ml, collagen I and IV 10 μg/ml (Thermo Scientific)) diluted in serum-free medium. The lower part was covered by the polycarbonate membrane, having been equilibrated in DPBS for 2 min. Then the upper part was fixed. The wells of the upper part were filled with 50 μl of tumor cell suspension (3 × 105 cells/ml). The chamber was then incubated for 16 h in a moistened atmosphere with 5% CO2 at 37 °C. Afterwards not-migrated cells were removed from the upper membrane side by washing it in buffer solution according to Weise (Merck) and by mechanical detachment using a rubber scraper. Migrated cells were fixed in methanol for 1 min and dyed with hemacolor (Merck). The dyed membrane was transferred onto a microscope slide and covered with immersion oil. The migrated cells were counted on an area of 2.5 mm2 of the porous membrane. The experiment was performed in quadruplicates and repeated three times.
Cell death detection assay
For apoptosis analysis in transfected RCC cells a cell death detection assay (Roche) was performed. 5 × 104 cells were seeded in a 6-well-plate. Next day medium was discarded; cells were scraped off and transferred in 500 μl incubation buffer. After 30 min at RT a centrifugation at 20000 g for 10 min followed. The supernatant was used for cell death detection assay. For the assay the 96-well-plate was incubated with 100 μl coating solution per well over night at 4 °C. Next day the solution was discarded, 200 μl incubation buffer was added per well and incubated for 30 min at RT. A washing step with 300 μl washing solution per well followed for three times. 100 μl of the prepared supernatant was then filled in the wells. After 90 min the solutions were discarded and wells were washed again (3 × 300 μl/well washing solution). Incubation with 100 μl per well coating solution for 90 min at RT followed. After another washing step apoptosis was detected with 100 μl substrate solution per well. The adsorption of individual wells was measured at 405 nm with GloMax®-Multi detection system (Promega). Experiments were performed in quadruplicates and repeated three times. Mean value and standard error rate were calculated.
Cell proliferation assay
To study the effect of transfected RCC cells on proliferation, a colorimetric BrdU incorporation assay (Roche) was performed as previously published [23]. The cells were seeded in quadruplicates into a 96-well-plate (5 × 103 cells/well) and cultured for 48 h. BrdU solution (10 μM) was added to the cells and incubated for 2 h. The cells were fixed and the DNA was denatured in one step by adding 200 μl per well fixDenat solution for 30 min. Incorporated BrdU was detected by an anti-BrdU-POD antibody (100 μl antibody solution per well) within 60 min. Fixed cells were washed three times with 200 μl washing solution per well. The BrdU-antibody complex was detected by 100 μl substrate solution per well. After incubation of 15 min reaction was stopped by adding 25 μl H2SO4 (1 M) per well and proliferation was quantified by measuring the absorbance at 450 nm (reference 690 nm) with GloMax®-Multi detection system (Promega).
Flow cytometric analysis of integrin subunits in renal carcinoma cell lines
The stably transfected 786-O and A498 cells were analyzed for the protein levels of integrin subunits α1, α2, α3, α5, αV, α6, β1 and β3. For this, cells were detached, centrifuged and washed with DPBS. Tumor cells (0.5 × 106 cells) were resuspended in 100 μl DPBS + 1% BSA and treated with labeled anti-integrin subunit antibodies (integrin α1/CD49a PE-conjugated, integrin α3/CD49c Fluorescein-conjugated, integrin α6/CD49f Alexa Fluor 488-conjugated, integrin αV/CD51 PE-conjugated (all purchased from R&D Systems) and integrin α2/CD49b FITC-conjugated, integrin α5/CD49e PE-conjugated, integrin β1/CD29 PE-conjugated, integrin β3/CD61 FITC-conjugated (all purchased from Becton, Dickinson & Company) for 30 min on ice in darkness. Cells were washed by using DPBS and resuspended in 500 μl DPBS + 1% BSA for analysis. Fifteen-thousand counts were used for analysis (BD Calibur, Becton, Dickinson & Company). Protein expression analyses were performed three times.
Human phospho-kinase array
The activity of 46 intracellular signaling kinases was quantified by using a human phospho-kinase array (R&D, Minneapolis, USA). The kinase array was performed according to the instructions in the manual. Protein extracts from RCC cells were prepared by using 200 μl lysis buffer 6 included in the kit. The cells were rinsed twice with ice-cold DPBS and scraped off with a cell scraper in lysis buffer. After 30 min incubation on ice, the extracts were centrifuged at 14000 g, 4 °C for 10 min. Protein concentrations were determined using BCA-reagents (Pierce BCA Protein assay kit, Thermo Scientific). The phospho-kinase array membranes were incubated with array buffer 1 for 1 h on a rocking platform. On each membrane 1 ml of the protein lysates (300 μg) was added and incubated overnight at 4 °C on a rocking platform. The membranes were washed three times with washing buffer and shaken with antibody cocktails for 2 h. After a 30-min treatment with streptavidin-HRP solution, the membranes were exposed to a chemiluminescent reagent. Signals were visualized using a chemiluminescent detector (FluorChemE, Protein Simple). For quantification a computer-based pixel counting system was used (AlphaView, Protein Simple).
Western blot analysis
For preparation of protein extracts from cell culture, tumor cells (7.5 × 105 cells) were seeded on 100 mm2 cell culture plates. For protein extraction, cells were washed with DPBS and mechanically detracted in lysis buffer (2 mM HEPES, 0.02 M NaCl, 0.05 mM MgCl2, 0.04 mM EDTA, 0.1% Triton X-100, 5 μM DTT, 1% Phosphatase Inhibitor Cocktail II (Sigma), 1% Protease Inhibitor Cocktail (Sigma)) with a cell scraper [24]. The solution was transferred into a 2 ml reaction tube and placed on ice. After incubation for 30 min on ice the samples were centrifuged for 10 min at 14000 g. The supernatant was transferred to a new tube and stored at − 20 °C. For evaluating protein concentrations of the extracts, BCA-reagents (Pierce BCA Protein assay kit, Thermo Scientific) were used. For protein precipitation 9-fold volume of acetone was used.
Equal amounts of protein (50 μg per lane) were separated by size using SDS-PAGE with 10% or 7.5% polyacrylamide gels. Afterwards gels were transferred on PVDF membrane by semi-dry blotting. Membranes were blocked according to instruction of antibody manufacturers for 1 h. Next, membranes were incubated with a primary antibody in blocking solution overnight at 4 °C on a roll mixer. The monoclonal antibodies against AKT, phospho-AKT S473 and T308, p38, phospho p38 (T180/Y182), PTEN (138G6), SAPK/JNK, phospho SAPK/JNK (T183/Y185) and V5-tag (all CST) were used at a dilution of 1:1000. β-actin antibody (Sigma) was employed at a dilution of 1:5000. After washing, the membranes were incubated with HRP-linked secondary antibodies (DAKO) at a dilution of 1:1000 for 1 h at RT and after washing bound antibodies were visualized by adding enhanced chemiluminescent solution (Perkin/Elmer) and detected in a chemiluminescent detector (FluorChemE, Protein Simple). For quantification bands were quantified by densitometry evaluation using a computer-based pixel counting system (AlphaView, Protein Simple). These values were referred to β-actin values of the same membrane as loading control. The cell culture experiments were performed three times.
Statistical analysis
For statistical analysis we used IBM-SPSS 22.0 and Microsoft Excel 2013. Expression results were quantified and presented as relative units. Significances of tissue specimen analyses were calculated by using the Log Rank test in relation to lymph node metastasis and overall survival. Regression analyses were performed by using a Pearson correlation. All other results using RCC cell lines were presented in % of control cells. Differences in expression levels, activity levels, apoptosis rates, adhesion and migration potential were performed using the Student’s T-test. Statistical significance was assumed at a p-value of < 0.05.