Gemcitabine (Gemzar®) was a kind gift from Eli Lilly (Indianapolis, IN, USA). Stock solutions were prepared in distilled water and fresh dilutions were prepared before each experiment. Dilazep, tri-N-octylamine, 1,1,2-trichlorotrifluoroethane, propidium iodide and MTT were purchased from Sigma-Aldrich (St. Louis, MI, USA). Bradford protein assay solution was purchased from Bio-Rad Laboratories (Ontario, Canada). [3H]Gemcitabine (14 Ci/mmol) and [3H]uridine (17.7 Ci/mmol) were purchased from Moravek Biochemicals Inc. (Brea, CA, USA). Ecolite™ was purchased from ICN (Costa Mesa, CA, USA). Antibodies against Bax were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); antibodies against Bcl-2 were purchased from DAKO (Glostrup, Denmark). Peroxidase-conjugated secondary antibodies were purchased from Covalab (Oullins, France). Enhanced chemiluminescence western blot detection reagents (ECL system) were purchased from Amersham (Amersham Corp, Buckinghamshire, UK). Other reagents used were analytical or HPLC grade and commercially available.
The RL-7 cells are derived from a human follicular lymphoma [43
]. The resistant variant RL-G was developed using step-wise increases of concentration of gemcitabine over a 12 month period. The maximum concentration used during selection was 2 μM. All cells were grown on 25 cm2
flasks at 37°C in RPMI containing 10% fetal calf serum, 1% L-glutamine and 2% penicillin-streptomycin in a humidified atmosphere containing 5% CO2
[3H]Gemcitabine transport and accumulation
Cellular uptake assays using suspensed cells have been described previously [2
]. Assays (106
cells/tube) were conducted at room temperature in transport buffer (20 mM Tris/HCl, 3 mM K2
, 1 mM MgCl2
O, 2 mM CaCl2
, 5 mM glucose and 130 mM NaCl, pH 7.4, 300 ± 15 mOsm) at various time intervals. Cell pellets were lysed with 5% Triton X-100 and mixed with Ecolite™ scintillation fluid to measure the cell-associated radioactivity (Beckman LS 6500 scintillation counter; Beckman-Coulter Canada, Mississauga, ON). Initial uptake rates were measured over 2–10 s and drug accumulation was determined after exposure of cells to 10 μM [3
H]gemcitabine for 4 h.
Extraction of nucleic acids for [3H]Gemcitabine scintillation assay
Exponentially growing RL-7 and RL-G cells were exposed to 10 μM [3H]gemcitabine for 4 h at 37°C. Cells were harvested by centrifugation and washed with phosphate-buffered saline. Aliquots (5 × 106 cells) were snap-frozen in liquid nitrogen and stored at -70°C before HPLC analysis and nucleic acid extraction. DNA was isolated using a Wizard genomic DNA kit (Promega Corp., Madison, WI, USA). Total RNA was extracted using an RNA easy kit (Qiagen, Valencia, CA, USA) and treated with RNase-free DNase 1 (Pharmacia Biotech) to remove any DNA contamination. Radioactivity incorporation was determined by scintillation counting. Experiments were performed in duplicate.
Cell pellets (prepared as described above) were extracted with 10% trichloroacetic acid on ice for 15 min and centrifuged to remove cell the protein precipitate. The acidic extract was neutralized with 2 volumes of tri-N
-octylamine/1,1,2-trichlorotrifluoroethane (1:4) and the aqueous layer passed through a 0.22 μm filter before injection onto a Partisil SAX 10 column (250 × 4.6 mm internal diameter). The resolution of the nucleotides was achieved using a gradient (0%A-80%B) and based on a method described by Gandhi [45
]. Buffer A, 10 mM, pH 2.9; buffer B, 750 mM ammonium phosphate, pH 4.5. The flow rate of the eluent was 1.5 mL/min and was monitored by UV absorbance (260 nm). Fractions were also collected for radioactivity measurements on a scintillation counter.
RNA extraction, RT-PCR and quantitative PCR
The level of mRNA expression of metabolic factors involved in gemcitabine resistance was assessed by quantitative real time RT-PCR, performed in a LightCycler detection system (Roche, Mannheim, Germany) as previously described [22
]. Briefly, cDNA (5 μl) was mixed with primers (300 nM each), LightCycler-FastStart DNA Master SYBR Green I (Roche) (hENT1, hENT2, CDD, RNR-M1, RNR-M2 and DNA POL) or LightCycler-FastStart DNA master hybridization probes (Roche) (18S, dCK, cNII, dNT-1 and dNT-2), and probes (130 nM; if necessary) in a total volume of 20 μl. These reactions were prepared in duplicate. Primers and probes sequences for hENT1, dCK, CDD, cNII, dNT-1, RNR-M2 and DNA POL are published elsewhere [22
]. Primers for hENT2 were as follows: for: atgagaacgggattcccagtag ; rev: gctctgattccggctcctt. Primers for RNR-M1 were as follows: for: gcagctgagagaggtgcttt; rev: aatggttgtagaattaagaatagc. Primers and probes for dNT-2 were as follows: for: catcagcatttgggagtcaa; rev: cgacacaatctgctccagaa; probe: 5'-fam-cgtcttcatctgcacaagcccca-tamra. All samples were analyzed in three separate experiments.
For each sample (RL-7 or RL-G cell lines) and the calibrator (K562 cell line), the relative amount of a target gene and a reference gene (18S) were determined. Crossing point values (Ct), which are the PCR cycle numbers at which the accumulated fluorescent signal in each reaction crosses a threshold above background, were obtained with the LightCycler software 3.5 (Roche) using the second derivative maximum method. (Ct) values are a function of the amplification efficiency of the respective PCR. These data were then exported into the RelQuant software (Roche) as *.txt files. This software provides efficiency-corrected, calibrator-normalized quantification results. Results are expressed as the target/reference ratio of the sample divided by the target/reference ratio of the calibrator and therefore are corrected for sample inhomogenities and detection-caused variances. The efficiency-corrected quantification performed by Relquant is based on relative standard curves describing the PCR efficiencies of each target and the reference gene. The relative standard curves are determined and are used for each analysis.
Ratio results obtained with the RelQuant software were considered as final relative PCR arbitrary units. Results were then expressed as % of PCR arbitrary units in RL-G cells related to RL-7 cells PCR arbitrary unit expression.
DNA extraction and PCR assay for the dCK gene
Genomic DNA was prepared with a phenol/chloroform extraction method [46
]. The coding sequence of the 7 last exons of the human dCK gene were amplified in a final reaction volume of 25 μl using PCR primers (Table ), Taq
DNA polymerase (Invitrogen) and 250 ng of genomic DNA. The PCR program profile was: 10 min at 94 °C followed by 50 cycles of 30 sec at 94 °C, 30 sec at 60 °C and 30 sec at 72 °C. The PCR products were separated on agarose gel 1,5% using low molecular DNA weight (Invitrogen) as marker of size.
Primer sequences used for semi-quantitative PCR assessment of the deoxycytidine kinase gene.
Flow cytometric detection of cell cycle and apoptosis
For analysis of DNA content and cell cycle distribution, RL-7 and RL-G cells were treated with gemcitabine 0.3 μM for 24 h and 72 h. After drug-exposure, 106 cells/ml were resuspended in 2 ml of propidium iodide solution (50 μl/ml), incubated at 4°C overnight and then analyzed by flow cytometry. For apoptosis determination, we used the Annexin-V-Fluos Staining Kit (Boehringer Mannheim, Gmbh, Germany) as recommended by manufacturers. Flow cytometry was performed on a FACScalibur (Becton Dickinson, San Jose, CA, USA). For determination of the apoptotic fraction, analysis was performed using CellQuest™ software (Becton Dickinson, San Jose, CA, USA). Cell cycle distribution and DNA ploidy status were calculated after exclusion of cell doublets and aggregates on a FL2-area/FL2-width dot plot using Modfit LT 2.0™ software (Verity Software Inc, Topsham, ME, USA).
Protein expression was determined by western blot analysis in untreated RL-7 and RL-G cells and after 24 h incubation with gemcitabine 0.3 μM. Briefly, protein was extracted from cells with cold lysis buffer (20 mM Tris-HCL pH 6.8, 1 mM MgCl2, 2 mM EGTA, 0.5% NP40, STI 1 mg/ml, leupeptin 100 μg/ml, aprotinin 100 μg/ml, benzamidine 30 mg/ml, TPCK 1 mg/ml and PMSK 5 mg/ml). Cell lysates were resolved by 12% SDS-PAGE, and transferred onto a nitro-cellulose membrane (Hybond-ECL, Amersham Corp, Buckinghamshire, UK). The blots were incubated with the appropriate dilution of primary antibody, followed by incubation with peroxidase-conjugated secondary antibody. Protein signals were detected by chemiluminiscence and exposure to Kodak film (Eastman Kodak Company, Rochester, NY, USA). Horizontal scanning densitometry was performed on western blots by utilizing acquisition into Adobe PhotoShop (Apple, Cupertino, CA, USA).
Experiments were performed in 4 week-old female athymic nude mice (IFFA-CREDO, L'Arbresle, France). The animals were kept in conventional housing. Access to food and water was not restricted. RL-7 and RL-G xenografts were developed in groups of five mice. Each animal (average body weight 25 g before injection) received a subcutaneous injection in the right flank area containing 106 RL-7 or RL-G cells in serum-free RPMI 1640 (day 0). After 24 h, gemcitabine was given intraperitoneally at a dose of 200 mg/kg once a week (6 doses; 3 consecutive weeks followed by 1 resting week) in the treated group. Initial drug toxicity studies were performed in non-tumor bearing mice at these doses and no major toxicities were observed (weight loss <10%).
The mice were inspected daily for s.c. tumor development and evaluation of clinical condition, and once tumor development occurred, tumor size was determined by caliper measurements while monitoring changes in animal weight twice a week. Animals were euthanized when their total tumor burden reached approximately 5,000 mg (15% of body weigth) to avoid animal discomfort or if clinical conditions may anticipate the potential for animal suffering.
The endpoints for assessing tumor activity were as follows: (a) tumor weight (mg) = (A × B2)/2, where A and B are the tumor length and width (in mm), respectively; (b) tumor growth inhibition = T/C, where T is the weight of treated tumor and C is the weight of control group, both evaluated at 60 days after injection. All studies involving mice were performed under Institutional Review Board-approved protocol.