Cell lines and culture conditions
Human breast cancer cell line MCF7 was obtained from Karolinska Institute (Stockholm, Sweden). Cells were maintained in IMDM medium supplemented with 2 mM L-glutamine, 100 units penicillin 100 μg/ml streptomycin and 10% (v/v) fetal bovine serum at 37°C in a humidified 5% CO2. For analysis cells were harvested by standard protocol using trypsin/EDTA (Sigma, USA).
Human D-glucuronyl C5-epimerase cloning
Human D-glucuronyl C5-epimerase (GLCE) (NM_015554) was cloned into episomal vector pCEP4 (Invitrogen, USA). Full-length sequence of epimerase cDNA was amplified by PCR with Pfu-DNA-polymerase (Stratagene) and epi-oligo-F 5'-CTAAGATCTAGATATGCGTTGCTTGGCAGCTCGGGTCAACTATAAGACTTTGATTATTA-3' and epi-R 5'-TACAGCGGCCGCTGAAGTGCAGTTTTGGT-3' primers from KIAA0836 clone (AB_020643) coding 5'-truncated sequence of the GLCE (Kazusa DNA Research Institute, Japan). Amplified full-length fragment was cloned into pCEP4 vector and obtained plasmid was defined as epi-pCEP4. GLCE sequence was verified by sequencing (Dye Terminator Cycle sequencing Kit and ABI PRISM 3700 genetic analyzer).
Transfection and selection of stable cell clones
To obtain stable cell clones expressing D-glucuronyl C5-epimerase, MCF7 cells were transfected with epi-pCEP4 or pCEP4 plasmid DNA (0.5 μg DNA/well) using Lipofectamine-ReagentPlus combination (Invitrogen, USA) according to the manufacturer's protocol. Stable cell clones were selected for 2-3 weeks in IMDM medium containing 200 μg/ml Hygromycin (Sigma, USA).
Colony formation assay
pCEP4- or epi-pCEP4-transfected MCF7 cells were stripped 24-48 h after transfection and plated on 100 mm cell culture dishes at 500-1000 cells per plate. After selection by 200 μg/ml Hygromycin for 2 weeks, giemsa-stained colonies were photographed and counted by Quantitione software, Version 4.4.0 (Bio-Rad, USA).
RT-PCR analysis of GLCE expression
Total RNA was isolated from the cells using PureLink Total RNA Purification System (Invitrogen, Carlsbad, CA) according to manufacturer's protocol. Synthesis of cDNA was performed from 1 μg of total mRNA using First Strand cDNA Synthesis kit (Fermentas, Hanover, MD) according to manufacturer's instruction. Multiplex PCR was performed in 20 μl volume (200 ng cDNA, 2 μl 10*PCR buffer 10 mM Tris-HCl, 1,5 mM MgCl2, 50 mM KCl, pH 8,3, 5 pmole of each primer, 0,2 mmole of each dNTP and 1 unit Taq-Polymerase) in Tercik PCR machine (DNA Technology, Russia). PCR conditions: 95°C 10 min, 95°C 15 s, 59°C 15 s and 72°C 1 min with final elongation at 72°C 10 min; 32 cycles for GAPDH and 20 cycles for GLCE. Following primers were used:
GLCE-F, 5'-AAGGGAGACGAGAGGGGAACGAA-3'; GLCE-R, 5'GCCACCTTTCTCATCCTGGTTC-3'; GAPDH-F, 5'-GGGCGCCTGGTCACAA-3'; GAPDH-R, 5'-AACATGGGGGCATCAGCAGA-3'.
Products of amplification were analyzed by electrophoresis in 1.2% agarose gel and visualized using "DNA Analyzer" system (Moscow, Russia). GLCE expression level was estimated as a ratio of GLCE DNA fragment intensity to GAPDH DNA fragment intensity (TotalLab Рrogramme, Nonlinear Dynamics, UK).
Cell proliferation assay in vitro
Cell proliferation rate was estimated using the CY QUANT NF Cell Proliferation Assay (Invitrogen, USA) according to manufacturer's instruction. Cells were plated at densities 100-500 cells/well to 8-12 wells in a 96-well microplate. DNA content in the wells was measured every 24 hours - the medium was removed and 50 μl fluorescent dye was added followed by incubation for 30 min at 37°C. Fluorescent intensity of each sample was measured at 485/530 nm with Fluorescence Microplate Reader (SPECTRA max, "Molecular Devices", UK).
Cancer-related genes expression profiling
To identify possible molecular mechanisms of antiproliferative effect of GLCE, Cancer PathFinder RT2 Profiler™ PCR Array (SABioscience, Frederick, MD) was used. Total RNA was isolated using RNAqueous Micro Kit (Applied Biosystems, Foster City, CA) according to manufacturer's protocol. RNA concentration was measured with Quant-iT Assay Kit and Qubit instrument (Invitrogen, USA), RNA quality was evaluated by electrophoresis. cDNA was synthesized from 1-2 μg total RNA using First Strand cDNA Synthesis kit (Fermentas, Hanover, MD). Real-time PCR was performed with SYBR Green PCR Master Mix (Fermentas, Hanover, MD) and iCycler iQ5 Multicolor Detection System (Bio-Rad, USA) according to manufacturer's instructions. Obtained data were analyzed with Excel-based PCR Array Data Analysis Software (SABioscience, Frederick, MD).