Cultivation and treatment of insulinoma INS-1 cells overexpressing HNF-1alpha in an inducible system
Rat INS-1 insulinoma cells overexpressing the gene encoding wild-type (WT) HNF-1alpha or a DN mutant of HNF-1alpha (DN-HNF-1alpha) (SM6) under control of a doxycycline-dependent transcriptional activator have been described previously . The SM6 mutant contains a substitution of 83 amino acids in the HNF-1alpha DNA-binding domain, resulting in the formation of non-functional heterodimers with endogenous HNF-1alpha . The level of HNF-1alpha production can be tightly controlled by culturing the cells over defined time periods with doxycycline, with maximal induction at a concentration of 500 ng/ml [9, 22]. INS-1 cells conditionally overproducing WT- or DN-HNF-1alpha were cultured in RPMI 1,640 medium (Life Technologies, Grand Island, NY, USA) supplemented with 6 mmol/l glucose, 2 mmol/l l-glutamine, 1 mmol/l pyruvate, penicillin (100 U/ml), streptomycin (100 μg/ml), 10% FCS (PAA, Cölbe, Germany), 10 mmol/l HEPES (pH 7.4) and 50 μmol/l 2-mercaptoethanol. Cells were plated at a density of 5×104 cells/cm2. After 36 h, culture medium was supplemented with 500 ng/ml doxycycline (Sigma, Munich, Germany) for 12–48 h to induce the production of HNF-1alpha.
Cells were rinsed with ice-cold PBS and lysed in Tris-buffered saline containing SDS, glycerin and protease inhibitors. Protein content was determined using the Pierce (Rockford, IL, USA) BCA Micro Protein Assay kit. Samples were supplemented with 2-mercaptoethanol and denatured at 95°C for 5 min. An equal amount of protein (20–50 μg) was separated with 5–15% SDS-PAGE and blotted to nitrocellulose membranes (Protean BA 85; Schleicher & Schuell, Dassel, Germany). The blots were blocked with 5% non-fat milk in blocking solution (15 mmol/l Tris–HCl pH 7.5, 200 mmol/l NaCl and 0.1% Tween-20) for 2 h at room temperature. Membranes were incubated overnight at 4°C with the following primary antibodies: rabbit polyclonal anti-HNF-1alpha antibody diluted 1:5,000 (kindly provided by Dr R. Cortese, IRBM, Rome, Italy), mouse monoclonal anti-alpha-tubulin antibody (clone DM 1A, 1:10,000; Sigma), and a rabbit monoclonal AKT antibody (diluted 1:1,000 in 5% BSA; Cell Signaling Technology, Beverly, MA, USA). Phosphorylation of AKT1 was investigated using specific antibodies raised against the active, phosphorylated form of AKT1 (Ser473) (1:1,000 in 5% BSA, clone 193H12; Cell Signaling Technology). Membranes were washed and incubated with anti-mouse or anti-rabbit IgG-horseradish peroxidase conjugate (1:1,000–1:5,000; Promega, Mannheim, Germany). Antibody-conjugated peroxidase activity was visualised using the SuperSignal chemiluminescence reagent (Pierce). Membranes were stripped in standard stripping buffer (2% SDS, 62.5 mmol/l Tris–HCl, 100 mmol/l 2-mercaptoethanol, pH 6.8) at 60°C for 30 min, washed twice and reprobed. Densitometric analysis of immunoblots was performed using Metamorph software (Molecular Devices, Downingtown, PA, USA).
Quantitative real-time PCR
Expression of Ins1 (insulin) and Akt1 mRNA was examined using relative quantitative real-time PCR. In relative quantification the expression of Ins1 and Akt1 mRNA was measured with respect to the reference gene Actb (β-actin), which was expressed constitutively and at the same level in all the samples analysed. INS-1 cells were harvested from culture treatments at the appropriate time-points. Total RNA was extracted using the RNeasy mini Kit (Qiagen, Hilden, Germany). First-strand cDNA synthesis was performed using 2 μg total RNA as template and M-MLV reverse transcriptase (Invitrogen, Paisley, UK) primed with 50 pmol random hexamers (New England Biolabs, Ipswich, MA, USA). Quantitative real-time PCR was performed using the LightCycler (Roche Diagnostics, Indianapolis, IN, USA) and the QuantiTech SYBR Green PCR kit (Qiagen) as per manufacturers’ protocols and 25 pmol of primer pair concentration. In order to obtain the highest efficiency in real-time PCR using SYBR Green I, the targets were designed to be 150–200 bp in length. Specific primers for each gene analysed were designed using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Each primer pair was tested with a logarithmic dilution of a cDNA mix to generate a linear standard curve which was used to calculate the PCR efficiency. The PCR reactions were performed in a 20-μl volume. The PCR was performed as follows: there was an activation step of HotStartTaq polymerase at 95°C for 15 min, followed by 35 cycles of denaturation at 95°C for 15 s, annealing at 65°C for 20 s, and extension at 72°C for 20 s. After the PCR reaction, the temperature was decreased to 65°C and then gradually raised to 95°C at a rate of 0.2°C/s. The fluorescence signal was continuously monitored during this process for melting curve analysis. The results were judged positive or negative by the presence or absence of the melting temperature peak of 88°C and by the effectiveness of the quantitative score. The data were analysed using Lightcycler Software 4.0 with all samples normalised to Actb.
Transient transfection experiments and evaluation of apoptosis
For transfections, INS-1 cells were plated onto 24-well tissue culture plates. One day later cells were cotransfected with plasmids pEGFP-C1 (Clontech, Palo Alto, CA, USA) and plasmids encoding a constitutively active mutant of Akt1 (CA-AKT1) (myr-AKT1; Upstate Biotechnology, Lake Placid, NY, USA), a DN form of Akt1 (DN-AKT1) (AKT1K179M; Upstate Biotechnology), or with an empty vector (pCMV) using the F2 transfection reagent (Targeting Systems, Santee, CA, USA). Cells were transfected with pEGFP-C1 (20 ng) and a 14-fold excess of the respective kinase expression vector or the empty vector (280 ng). For transfections, 300 ng of the total plasmid DNA mix and 0.3 μl F2 reagent were diluted in 200 μl RPMI medium under serum-free conditions and preincubated at room temperature for 20 min. Cultures were incubated with the DNA-F2-transfection mixture at 37°C for 1 h. After 24 h, the culture medium was supplemented with 500 ng/ml doxycycline to induce DN-HNF-1alpha production. After a further 48 h, nuclei were stained live with Hoechst 33258. Apoptotic nuclei and expression of green fluorescent protein (GFP) were observed and quantified by epifluorescence microscopy as described previously .
Measurement of executioner caspase (DEVDase) activity
Cells were lysed in 200 μl lysis buffer (10 mmol/l HEPES, pH 7.4, 42 mmol/l KCl, 5 mmol/l MgCl2, 1 mmol/l phenylmethylsulphonyl fluoride, 0.1 mmol/l EDTA, 0.1 mmol/l EGTA, 1 mmol/l dithiothreitol, 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 5 μg/ml aprotinin, 0.5% CHAPS). Fifty microlitres of this lysate were added to 150 μl reaction buffer (25 mmol/l HEPES, 1 mmol/l EDTA, 0.1% CHAPS, 10% sucrose, 3 mmol/l dithiothreitol, pH 7.5 and 10 μmol/l of the caspase substrate Ac-DEVD-AMC). The substrate is efficiently cleaved by the apoptotic executioner caspases 3, 6 and 7 . Accumulation of fluorescent AMC fluorescence was monitored over 120 min using an HTS fluorescence plate reader (Perkin Elmer, Langen, Germany) (excitation 380 nm, emission 465 nm). Fluorescence of blanks containing no cell lysate were subtracted from the values. Protein content was determined using the Pierce Coomassie Plus Protein Assay reagent. Caspase activity was expressed as change in fluorescent units per microgram protein and per hour.
Detection of cytochrome-c release
The release of cytochrome-c from mitochondria into the cytosol was analysed by selective permeabilisation of the plasma membrane . Briefly, cells were permeabilised with 100 μg/ml digitonin at 4°C for 10 min. The supernatant representing the cytosol and the mitochondria-containing pellet fraction were separated by centrifugation and denaturated. SDS-PAGE and western blot analysis were performed as described. Cytochrome-c was detected with a monoclonal cytochrome-c antibody (7H8.2C12; Pharmingen, Franklin Lakes, NJ, USA), diluted 1:1,000.
Data are given as means±SEM. For statistical comparison, ANOVA and subsequent Tukey’s test were employed. A p value of p<0.05 was considered to be statistically significant.