Melanoma Cell Culture
Melanoma cell lines were derived from metastatic melanoma lesions from patients treated at the Surgery Branch, National Cancer Institute (NCI), Bethesda, MD and kindly donated by Dr Steven A. Rosenberg. All patients signed an informed consent approved by the Institutional Review Board of the National Cancer Institute. Two cell lines were purchased from ATCC, namely A375 and SKMEL28; one additional cell line, PIG1, immortalized from human melanocytes was kindly donated by Dr. Caroline Le Poole from Northwestern University, Chicago, Illinois, USA. These three cell lines were tested for validation purposes. Cells were cultured at 37˚C, in 5% CO2 with RPMI 1640 medium supplemented with 10% heat-inactivated FBS (Gemini Bioproducts), 0.01% GPS (penicillin, L-glutamine, streptomycin), and 0.01% fungizone (250 mg/L, Invitrogen).
DNA Isolation, BRAF And NRAS Sanger Sequencing
Genomic DNA was isolated using QIAamp DNA Mini Kit (Qiagen, Germantown, MD) according to the standard protocol. DNA quality and quantity was estimated using Nanodrop (ThermoScientific, Pittsburgh, PA).
Each sample was screened for mutations in exons 11 and 15 of BRAF genes and exon 1 and 2 of NRAS gene. PCR was performed in a 20 µl final volume, containing 50 ng of genomic DNA, 10 µl of Qiagen HotStarTaq Master Mix Kit (Valencia, CA) and 500 nM of forward and reverse primers with the following cycling conditions: initial denaturation at 95 °C for 10 min; 35 cycles at 95 °C for 30 sec, 56 °C for 30 sec and 72 °C for 30 sec; final step 72 °C for 10 min. Primers were selected using Primer3 software (http://frodo.wi.mit.edu/):
BRAF_ex11_F: 5’-TCCCTCTCAGGCATAAGGTAA-3’
BRAF_ex11_R: 5’-CGAACAGTGAATATTTCCTTTGAT-3’
BRAF_ex15_F: 5’-TCATAATGCTTGCTCTGATAGGA-3’
BRAF_ex15_R: 5’-GGCCAAAAATTTAATCAGTGGA-3’
NRAS_ex1_F: 5’-CACTAGGGTTTTCATTTCCATTG-3’
NRAS_ex1_R: 5’-TCCTTTAATACAGAATATGGGTAAAGA-3’
NRAS_ex2_F: 5’-ATAGCATTGCATTCCCTGTG-3’
NRAS_ex2_R: 5’-CACAAAGATCATCCTTTCAGAGA-3’
In each PCR reaction distilled water was used as a negative control. PCR products were purified with Exosap-IT (USB Corporation, Cleveland, OH) and labeled using Big Dye terminator kit v3.1 (Applied Biosystems, Foster City, CA). Excess dye terminators were removed using DyeEx 96 Kit columns following the manufacturer’s instructions (Qiagen). Sequencing was performed using Biosystems 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA) and analyzed by Sequencher software (Genecodes, Ann Arbor, MI).
RNA-Seq And Data Analysis
Total RNA was isolated from the cell lines using miRNeasy minikit (Qiagen) according to the manufacturer’s protocol. RNA quality and quantity was estimated using Nanodrop (Thermo Scientific) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). A A260/A280 ratio of ~ 2.0 was considered indicative of RNA of good purity. A RIN (RNA Integrity Value) of 7 was set as cut off for RNA quality. Only samples with RIN > 7 were used for further experiments. Enrichment in mRNA molecules was obtained by using oligo (dT) magnetic beads (Ambion® Poly (A)Purist™ MAG Kit). After the mRNA was fragmented in short fragments (approximately 200 bp), CDNA was synthesized by random hexamer primers (Illumina TruSeq Stranded mRNA Library Prep Kit). The double-stranded cDNA was purified by QiaQuick PCR extraction kit (Qiagen) and went through an end repair process with the addition of a single ‘A’ base, and then ligation of the adapters. These products were then purified by agarose gel electrophoresis and enriched with PCR to create the final cDNA library. The library products were sequenced and 300 bp sequences were generated via the GAIIx Illumina sequencing platform. Raw reads were imported on a commercial data analysis platform CLC Genomics Workbench (CLC bio, MA, USA). An average of 20 million reads were generated for each sample run as per Illumina recommendations. Quality control checks on raw sequence data from each sample were performed using the QC analysis application tool. Adapter trimming was done to remove ligated adapter from 3′ end of the sequenced reads with only one mismatch allowed. After reads have been processed to meet a quality standard, they were aligned to the Human reference genome UCSC-Hg19, using the ultra high-throughput short aligner provided by CLC bio software. A Transcript Discovery analysis was performed to generate a transcript annotation file with an estimation of the relative abundances of each transcript by counting the number of reads that mapped to the genomic location of that transcript. Transcription level assessment has been obtained by the number of fragments per kilobase of transcript per million fragments mapped (RPKM).
BRAF Inhibitors Treatment
The three discordant cell lines (MEL-2523, MEL-3025, MEL-3104) and the control WT (MEL-2805) and V600E (MEL-2492) cell lines were treated with BRAF inhibitors prior to running the proliferation assay and DNA and RNA mutational testing.
About 5 × 106 cells were seeded in complete medium the day before treatment and incubated overnight at 37C with 5% CO2. Two BRAF-specific (PLX4720 and Vemurafenib) and one BRAF nonspecific inhibitor (Sorafenib) were purchased from Selleckchem, Houston TX and used for the inhibition treatment. Final concentration of PLX4720, Vemurafenib and Sorafenib were respectively 1uM, 2uM and 5uM. Upon treatment, cells were used for DNA and RNA isolation (see below). Each experiment was done in triplicate. The same experiments were repeated on three additional cell lines for reproducibility, namely A375, SKMEL28 and PIG1. A375 and SKMEL28 are commercially available cell lines carrying the BRAF V600E mutation. PIG1 is a cell line transformed from normal epidermal melanocytes.
MTT Proliferation Assay
The MTT assay is a colorimetric test used to assess the cell metabolic activity[15]. The NAD(P)H-dependent oxidoreductase enzymes constitutively present in the cells are able to reduce the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan (an insoluble form), which is purple in color. The acquisition of color changes is the basis of this colorimetric assay. The cytotoxic effects of each inhibitor were tested using the MTT proliferation assay. About 5 × 103 cells in 100 µl of complete medium were seeded into each well of a 96-well tissue culture plate and incubated overnight at 37C incubator with 5% CO2. Cells were treated with indicated inhibitors in serum-free medium for a period of 24 h, 48 h and 72 h. Each treatment condition was done in triplicate. Then 20 µl of MTT (5 mg/ml) was added to each well and plates were further incubated for 4 h at 37C. The formed crystals were solubilized by addition of 150 µl of DMSO in each well and cells agitated on an orbital shaker for 20 min. The optical density was measured using a spectrophotometer at a wavelength of 570 nm. The BRAF inhibition experiment was repeated at least three times, in which each cell lines was assessed in triplicate.
RNA And DNA Isolation After Inhibition Treatment
Total RNA from the 15 cell lines was extracted using miRNeasy minikit (Qiagen) according to the manufacturer’s protocol. RNA quality and quantity was estimated using Nanodrop (Thermo Scientific) and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). A RIN of 7 was set as cut off for RNA quality. Only samples with RIN > 7 were used for further experiments. First- and second-strand cDNA were synthesized from 300 ng of total RNA according to manufacturer’s instructions (Ambion WT Expression Kit).
Genomic DNA was isolated using QIAamp DNA Mini Kit (Qiagen, Germantown, MD) according to the standard protocol. DNA quality and quantity was estimated using Nanodrop (ThermoScientific, Pittsburgh, PA). Only DNA of A260/A280 of ~ 1.8 were processed for downstream analyses.
BRAF Allele-specific PCR
Allele specific PCR was performed to detect BRAF V600E mutation and wild type BRAF sequence. Two PCR reactions (BRAF wild-type and BRAF V600E specific, respectively) were performed for each sample. For the RNA allele-specific PCR, the reverse primer was used for both the PCR reactions and designed spanning exon 15 and exon 16. The forward primers were designed to possess two bases substitution at 3’-end compared to wild-type sequences. These primers were, respectively:
BRAF15_R: 5’-GATGACTTCTGGTGCCATCC-3’
BRAF15_WT_F: 5’-TAGGTGATTTTGGTCTAGCTACAGT-3’
BRAF15_V600E_F: 5’-GGTGATTTTGGTCTAGCTACAAA-3’
For the DNA PCR, primers were designed as previously reported [16].
The PCR reaction was performed in a 25 µL final volume, containing 200 nM of forward and reverse primers, 2 µL of cDNA, 12.5 µL of 2X GoTaq MasterMix (Promega, Madison, WI, USA), which included BRYT Green as intercalating dye. In each reaction 0.3% HiDi formamide (Life Technologies, Grand Island, USA) was added to increase primer-annealing specificity. Cycling conditions were as following: initial denaturation at 95 °C for 10 min; 40 cycles at 95 °C for 15 sec, 60 °C for 1 min. Reactions were run on 7500 Fast Real-Time machine (Applied Biosystem, Carlsbad, CA, USA). To verify primers specificities, melting curves were generated at the end of PCR reaction. Fluorescent data were acquired during the extension phase. After 40 cycles, a melting curve for each gene was generated by increasing the temperature from 60 °C to 95 °C (1 °C for each step), while the fluorescence was measured. For each experiment a no-template reaction was included as a negative control. The qPCR data was analyzed using the 2^-(delta Ct) method and GAPDH as housekeeping gene.
Statistical Analyses
Paired two-tailed Student’s t-test was used to determine differences of cell lines’ responsiveness to the inhibitors at day 3. ANOVA (analysis of variance) test was used to evaluate the effects of BRAF inhibitors. All the analyses were performed using Statgraphics Centurion (V. 15, StatPoint, Inc.).