Chemicals
Gemcitabine (Gemzar®; 2',2'-difluoro- 2'-deoxycytidine; dFdC) was a generous gift from Eli Lilly and Company (Indianapolis, IN) and dissolved in sterile distilled water. Paclitaxel was purchased from Sigma-Aldrich Company (St. Louis, MO) and dissolved in 0.1% acetic acid in methanol. Radiolabeled chlorodeoxyadenosine (8-3H-CdA, 7.8 Ci/mmol) was purchased from Moravek (Brea, CA). All other chemicals were of analytical grade.
Cell culture
The NSC large cell lung carcinoma H520 cell line (mutant-p53) was provided by Dr. William T. Beck (University of Illinois, Chicago, Illinois, USA). The NSC H460 squamous carcinoma cell line (wild-type p53) and H838 adenocarcinoma cell line (wild-type p53) were obtained from the American Type Culture Collection (Manassas, Virginia, USA). The cells were grown in monolayers and maintained in exponential growth in RPMI-1640 medium containing 2 mM L-glutamine supplemented with 10% fetal bovine serum (FBS) and 1% penicillin (10,000 U penicllin per ml)-streptomycin (10 mg of streptomycin per ml) at 37°C at 5% CO2. The medium was further supplemented with insulin (Gibco Life Technologies, Grand Island, New York, USA) for H520 cells.
Growth inhibition assay
Growth inhibition was determined using a dye exclusion assay with trypan blue staining followed by a cell count using a hemocytometer [18]. Briefly, ~3.5 × 105 cells were seeded in duplicate in 6-well flat bottom plates. After 24 hours, the cells were treated with vehicle-control, gemcitabine (ranged from 1 to 15,000 nM) or paclitaxel (ranged from 1 to 3,000 nM) for 24 hours. The fraction of affected cells and unaffected cells for the individual drugs was calculated compared to cells exposed to vehicle-control. The IC50 values were determined using linear regression analysis with the aide of CalcuSyn software (v. 2, Biosoft, Cambridge, UK).
A multiple drug effect analysis was completed to predict the likely drug-drug interaction based on the principles of Chou and Talalay [19]. The combination index (CI) for each fraction affected was simulated and for the final evaluation, the averaged CI at 0.50, 0.75, 0.90 and 0.95 fraction affected was determined [20]. Briefly, ~1 × 106 cells were seeded in duplicate in 60 mm dishes. After 24 hours, the cells were treated with sequential gemcitabine → paclitaxel and paclitaxel → gemcitabine at five different concentrations of a constant ratio based on the ratio of the observed IC50 values of each cell line. The cells were exposed to each drug for 24 hours; the medium containing the first drug was removed, the cells were washed with phosphate buffered saline, then medium containing the second drug was added to the cells. The total culture time was 72 hours. A CI < 0.3, 0.3–0.7, 0.7–0.9, 0.9–1.1, 1.1–1.45, 1.45–3.3 and >3.3 indicates highly synergistic, synergistic, moderate to slight synergistic, nearly additive, slight to moderate antagonistic, antagonistic; strong antagonistic, respectively (CalcuSyn software, v. 2, Biosoft, Cambridge, UK).
Flow cytometry
Flow cytometric measurements were performed after staining the cellular DNA content with propidium iodide to determine the cell cycle distribution and apoptosis following treatment with sequential gemcitabine → paclitaxel or paclitaxel → gemcitabine. Briefly, ~1 × 106 cells were plated in 60 mm dishes and allowed to attach overnight. After treatment with sequential gemcitabine → paclitaxel or paclitaxel → gemcitabine as described for the determination of the CI, the cells were harvested and suspended in a propidium iodide solution (Sigma-Aldrich Co.) as described previously [21] and filtered in 5 ml round bottom tube with cell-strainer cap (BD Falcon). The cell cycle analysis was performed on a Beckman-Coulter EPICS Elite ESP flow cytometer (Hialeah, Florida, USA) using the Multicycle AV program (v. 3, Phoenix Flow Systems, San Diego, Calfornia, USA).
dCK and CDA enzyme specific activity
The effect of paclitaxel on dCK and CDA enzyme specific activity was measured after exposing ~20–30 × 106 cells (seeded in duplicate in 100 mm dishes) to either vehicle-control or paclitaxel at the observed IC50 value for 24 hours. Cells were manually harvested and counted. Total protein was quantified using BCA protein kit (Pierce Biotechnology, Rockford, Illinois, USA)
dCK activity was analyzed using radiolabeled chlorodeoxyadenosine (CdA) as previously described [22, 23]. Briefly, the crude cellular extract was suspended in Tris-HCl buffer and mixed with CdA 256.5 μM plus [8-3H]-CdA (128 μM, specific activity 0.19 μCi/nmol) as substrate. The enzymatic reaction was incubated for 1 hour at 37°C. Enzyme activities were expressed as nmol product formed per hour per mg protein or 106 cells.
The CDA activity was measured using a spectrophotometric method as described by Dr. Vincenzetti [24]. The crude cellular extract was suspended in a Tris-HCl buffer and freeze-thawed rapidly three times. The extract was subsequently centrifuged for 15 minutes at 12,000 g and the resulting supernatant was suspended in the Tris-HCl buffer. The enzymatic reaction was performed in a 96 well UV-Vis transparent plate (BD Falcon) and initiated with the addition of the substrate cytidine (167 μM). The mixture was incubated at 37°C and the change in absorbance at 282 nm was measured for 10 minutes using a Synergy HT multidetection microplate reader (Biotek, Winooski, Vermont, USA). Enzyme activities were expressed as mmol substrate consumed per minute per mg protein or 106 cells.
Gene expression
Total RNA and protein was extracted form cells exposed to vehicle-control or paclitaxel at varying concentrations for 24 hours using the PARIS™ kit (Ambion, Austin, Texas, USA) according to manufacturer's instructions. Total RNA was treated with TURBO DNA free (Ambion) to remove DNA contamination and the concentration was measured at 260 nm. The total RNA was reverse transcribed using random primers and the High Capacity cDNA reverse transcription kit (Applied Biosystems) per the manufacturer's product information.
The human hypoxanthine phosphoribosyltransferase (HPRT) gene was selected as an endogenous control after assessing the gene expression of 11 potential controls using the TaqMan human endogenous control plate (Applied Biosystems). HPRT produced ΔCT values that deviated little from zero, indicating relative to other candidate controls, that the expression of HPRT remains relatively consistent across the samples tested regardless of type of cells or treatment. Primers and probes for the dCK and CDA were from Applied Biosystems Assay on-Demand Gene expression products. The cDNA was amplified by quantitative real-time PCR in triplicate using the following thermal profile: an initial incubation at 50°C for 5 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds followed by annealing and extension at 60° for 1 minute with the Applied Biosystems 7900 HT sequence detection system. The quantitation of gene expression was performed relative to the calibrator (vehicle-control cells) using the ΔΔCT calculation for dCK and the relative standard curve calculation for CDA. A validation experiment was performed that demonstrated the efficiencies were 0.08 for dCK and 1.1 for CDA. To use the ΔΔCT calculation, the efficiencies should be less than 0.1.
Western blot
Total protein was separated on a 12% SDS-polyacrylamide gel for dCK or a 14% SDS-polyacrylamide gel for CDA and transferred to a polyvinylidene diflouride (PVDF) membrane [25, 26]. The membrane was probed with the either dCK-pep antibody (obtained from Dr. Hatzis) at a 1:4,000 dilution or CDA antiserum (obtained from Dr. Momparler) at a 1:175 dilution followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG (Pierce, Rockford, Illinois, USA). The membrane was also probed with β-actin (Sigma-Aldrich Co) at 1:12,000 dilution, followed by incubation with horseradish peroxidase-conjugated anti-mouse IgG (Calbiochem, San Diego, California, USA) antibody as an endogenous control. Immuncomplexes were visualized by SuperSignal West Pico chemiluminescent substrate kit (Pierce, Rockford, IL) and the band density was semi-quantitated using ImageJ (v. 1.38×, http://rsb.info.nih.gov/ij/index.html) software.
Analysis of gemcitabine, difluorodeoxyuridine and the phosphorylated metabolites
The cells were treated with vehicle-control or paclitaxel at the observed IC-50 value for 24 hours followed by gemcitabine at the observed IC-50 value for 24 hours. The cell medium and pellet were manually harvested and stored at -80°C until analysis. The phosphorylated metabolites were analyzed by Dr. Hilde Rosing within the Department of Pharmacy and Pharmacology at the Netherlands Cancer Institute/Slotervaart Hospital in Amsterdam, Netherlands using their previously described LC-MS method [27]. The lower limit of quantitation was 26.8 nM for the monophosphate, 27.0 nM for the diphosphate and 2.53 nM for the triphosphate.
Gemcitabine and its deaminated metabolite dFdU were analyzed in our laboratory using our previously published method with hexanes used to wash the culture medium [28]. The lower limit of quantitation was 0.25 μM for both gemcitabine and dFdU.
Statistical analysis
All results are expressed as the mean ± the standard deviation of three independent experiments conducted in at least triplicate. Statistical significance was determined by a two sided paired t test or analysis of variance and the level of significance was set at P < 0.05 a priori. A correlation analysis was conducted to determine the relationship between the ratio of dCK to CDA mRNA levels and combination index.