Drugs and Chemicals
RPMI 1640, fetal calf serum (0.45 µm membrane filtered and heat-inactivated), penicillin (10,000 U/ml), streptomycin (10 mg/ml), L-glutamine (200 mM) and Trypan blue solution (0.05%) were obtained from Biological Industries, Israel. Trizol reagent was obtained from Life Technologies. Moloney Murine Leukemia Reverse-Transcriptase, Taq DNA polymerase, RNAse inhibitor, DNA size marker (100-3000 bp) were obtained from Fermentas, USA. The set of deoxynucleotide (dNTP), Isopropanol, Agarose, MOPS formamide and MTT were obtained from Sigma, USA. PBS was obtained from Oxoid, England. Diethylepyrocarbonate (DEP-C) was obtained from Applichem, Germany. Formaldehyde (37%) was obtained from Merck, Germany. OligodT was obtained from Integrated DNA Technology, USA. Chloroform was obtained from Lab Scan Analytical Sciences, Ireland. Twenty-five centimeter square tissue culture flasks were obtained from Corning, USA. Seventy-five centimeter square tissue culture flasks were obtained from TPP, Switzerland. VCR was a gift from Dr. Ali Uur Ural, Gülhane Military Medical School, Ankara, Turkey.
HL 60 Cell Line
HL60 cells were obtained from Gülhane Military Medical
School, Department of Haematology, Ankara, Turkey.
HL-60/VCR, a vincristine-resistant line was developed by
stepwise exposure of HL-60 to increasing concentrations
of vincristine. The cells were grown in RPMI 1640 medium
containing 10% heat-inactivated fetal calf serum (FCS),
500 U/ml penicillin and 0.5 mg/ml streptomycin in 25 cm2
tissue culture flasks. The cells were incubated in CO2
incubator at 37ºC in the presence of 5 % CO2. The medium
was refreshed every five days.
Cell Survival (MTT) Assay
2x104 cells were plated in 100 ml of the growth medium in the absence or presence of increasing concentrations of VCR into 96-well plates at 37ºC in 5% CO2 for 24 h and
48 h. The cells were then incubated with 3-[4, 5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) (5 mg/ml) at 37ºC for 4 h. Then the medium was removed and the converted dye was solubilized with the addition of acidic isopropanol (0.1 N). Plates were examined by using a microplate reader at 570 nm and the concentration of drug that inhibited cell survival by 50% (IC50) was determined from cell survival plots .
Isolation of RNA
Total RNA was isolated from 1x107 sensitive HL60 and HL60/VCR cells by using Trizol reagent (consist of guanidium thiocyanate, phenol and sodium citrate). Quantification of RNA was conducted by spectrophotometer. Measuring the absorbance at wavelengths of 260 nm and 280 nm will provide information about protein contamination of RNA. Reading at 260 nm was used to calculate the concentration of nucleic acid in a sample . The intactness of total RNA was confirmed by two sharp bands 28S rRNA and 18S rRNA . Total RNA was stored at -80ºC.
Reverse transcription was carried out for 60 min at 42ºC in a total volume of 20 µl, containing ribonuclease inhibitor (20 U), 0.5 mg oligo-dT, 2.5 mM of each dNTP and 200 units of Moloney Murine Leukemia Virus-reverse transcriptase and a sample of 3 µg of total cellular RNA. Once the cDNA copy created using the mRNA template, the PCR was conducted immediately or the cDNA was stored at -20ºC until required for analyses.
Polymerase Chain Reaction
Two sets of primers were used in all reactions to obtain amplification of an endogenous control, β-2-microglobulin (120 bases) and a specific target gene of interest, MDR1 gene (258 bases). Following an initial denaturation step of 94 ºC for 5 minutes, 35 cycles of PCR amplification were performed, each consisting of a denaturation step of 94 ºC for 30 seconds, annealing at 62 ºC for 45 seconds and extension at 72 ºC for 1 min. At the end of the 35 cycles, a 5-minute extension phase at 72 ºC was included to provide complete synthesis. The amplified fragments were visualized by running on 2% agarose gel at 90 V for 1 hour and then by ethidium bromide staining.
Primers were obtained from IDT, USA. The PCR primers used to amplify MDR1 gene were 21/20 nucleotide long oligonucleotides. The sequence of sense strand primer was 5'TACAGTGGAATTGGTGCTGGG and the sequence a n t i s e n s e s t r a n d p r i m e r w a s 5'CCCAGTGAAAAAATGTTGCCA. Nucleotides were chosen to avoid any co-amplification of MDR3 gene . For the amplification of the β-2-microglobulin 20-mer oligonucleotides were used. The sequence sense strand primer was 5'CTTACTGAAGAATGGAGAGAGA and the sequence antisense strand primer was 5'CTTACATGTCTCTATCCCACTT .