Oligonucleotide Array Hybridization and Gene Selection

Supplementary materials and methods

Tittle: IGFBP-3 hypermethylation-derived deficiency mediates cisplatin resistance in non-small cell lung cancer.

Running Tittle: IGFBP-3-methylation as a chemo-resistance biomarker

Cell culture and viability to CDDP

Due to the lack of commercialized cisplatin-resistant NSCLC cell lines, we first established two CDDP-resistant cell lines, derived from the sensitive H23 and H460 NSCLC cell lines. Resistant cell lines were established by in vitro exposure to an initial concentration of 0,05 µg/ml CDDP that was increased periodically during 12 and 7 months respectively. After a high mortality rate, 30% cells survived showing a temporal enlarged and flattened morphology, probably as a survival mechanism to the drug (Levina et al., 2008; Roninson, 2003). Resting periods without drug exposure was intercalated between treatments. Then, cell lines were maintained in the absence of CDDP for a further 20 days before 5Aza-dC and TSA treatment, to avoid making note of transcriptional changes caused by the insult of drug itself and to ensure that the resistance remained stable. Viability comparing sensitive vs. resistant cell lines was determined using the staining method described previously (Chattopadhyay et al., 2006).

Oligonucleotide array hybridization and gene selection Sample amplification and labeling procedures were performed according to the Agilent microarray protocol (www.chem.agilent.com/scripts/generic.asp). Scanning was executed with the Agilent G2565AA microarray scanner using settings recommended by Agilent. Readings from the scanning were processed with Feature Extraction Software that uses replicate population and spike-in statistics to monitor array processing and array analysis to report confident results. All calculations were performed using the gene expression analysis software GeneSpring. We first selected those genes down-regulated in the resistant cell lines in comparison with the parental sensitive cell lines; and which expression was re-established after 5Aza-dC and TSA reactivation treatment, in order to identify specifically those genes silenced by epigenetic mechanisms as a result of the acquired resistant to CDDP. All microarray analyses were done in duplicates starting from the same independently treated population. (Further statistical details are described in Statistical analysis). Gene selection was followed by testing for no evidence of expression in normal cells according to the Cancer Genome Anatomy Project (CGAP) Serial Analysis Gene Expression (SAGE) database, as well as those located in known imprinted regions, were excluded. Then, we analyzed the promoter region using GeneCard website (http://bioinfo.weizmann.ac.il/cards/index.html) to obtain the information regarding to chromosome location, transcripts, and gene, cDNA and upstream sequences from the upregulated genes. We searched all the 5´ CpG islands located in the transcriptional site and at least 1000 bp upstream using different CpG island revealing programs; First we used the CpG Island Searcher, (http://cpgislands.usc.edu/) designed by Daiya Takai D and Jones PA with the parameters the authors described for a CpG island: GC55%; Obs/Exp65 and length 200 bp, because these situations excludes most of the Alu- repetitive elements (Takai and Jones, 2002; Takai and Jones, 2003). In order to confirm the CpG islands position we used the WebGene Home Page website (www.itba.mi.cnr.it/webgene/). CpG islands containing repetitive elements were detected using RepeatMasker Web Server (ftp.genome.washington.edu/cgi-bin/RepeatMasker) and then excluded from the study. The next step was to select genes whose actions involve the inhibition of cell proliferation, apoptosis-related actions and transcription factor regulation using a gene ontology analysis (http://bioinfo.vanderbilt.edu/gotm/).

Reverse transcription and Q-RT-PCR To validate the differential gene expression observed in the oligoarray analysis, semiquantitative and real-time RT-PCR assays were performed in sensitive, resistant and resistant-reactivated cell lines. The Forward and Reverse primers were chosen from different exons in order to avoid genomic DNA contamination (Supplementary Table 1). The PCR reactions were performed under the following conditions: a) 1cycle of 95C for 5 min, b) 21-34 cycles of 95C for 1 min, 55-62C for 1 min, 72C for 1 min. c) An extension of 10 min at 72C. PCR amplification was unaffected by the amplification of GAPDH as an internal control in the same mixture reaction. For quantitative real-time RT-PCR analysis, the following conditions were applied: 10 min at 95 °C and 40 cycles of 15 sec at 95°C and 1 min at 60°C.

Bisulfite sequencing and methylation-specific PCR

DNA was extracted from 27 human cancer cell lines, including H23, H23R, H460 H460R and 41S and 41R cell lines together with series of 5 µm sections from each FFPE block from 36 NSCLC-primary specimens and non- neoplasic lung tissues. The cell lines DNA was used for the analysis of CpG island promoter methylation and epigenetic treatment validation of the selected genes by bisufite sequencing. Bisulfite modified tumor DNA from primary samples was PCR amplified with specific primers for methylated versus unmethylated DNA to study the IGFBP-3 promoter methylation status by methylation specific PCR. Bisulfite modification of DNA allows the identification of CpG sites ethylated. This modified DNA can be amplified and sequenced, providing information regarding the CpG island status. DNA modification was developed as follows; genomic DNA (1g) from mock, resistant and 5-aza and TSA treated cell lines cultures and normal lung tissue and primary tumors was denatured by NaOH (0.2M) for 10 min at 37C and then modified by hydroquinone and sodium bisulfite treatment at 50C for 17 hours under a mineral oil layer. Modified DNA was purified using the Wizard DNA Clean-Up system (Promega, Madison, WI, USA). Modification was completed by NaOH (0.3M) treatment for 5 min at room temperature, followed by precipitation with glycogen, 10M ammonium acetate and ethanol precipitation. DNA fragments of 316-496 bp in size containing the promoter CpG island were PCR amplified from bisulfite-modified cell line and normal tissue DNAs for each gene analyzed. Primers were designed, when possible, to exclude binding to any CpG dinucleotide to ensure amplification of both methylated or unmethylated sequences (Supplementary table 1). PCR reactions were performed under the following conditions: a) 1cycle of 95C for 5 min, 56-63C for 1 min, 72C for 1 min; b) 34-38 cycles of 95C for 1 min, 55-64C for 1 min, 72C for 1 min. c) An extension of 10 min at 72C. The PCR products were run into a 1.5% agarose gel, then cut and cleaned by Qiaquick (MinElute, Qiagen, Valencia, CA, USA) and direct sequencing performed on all genes. In addition, some gene products were cloned into a TOPO vector (Invitrogen) and at least ten colonies from each gene analyzed by sequencing. Bisulfite modified primary NSCLC tumor DNAs were used to analize the IGFBP-3 methylation status by MSP. Specific primers were designed according the results obtained from the cell lines bisulfite sequences, to detect methylated or unmethylated modified DNA specifically, (Supplementary Table 1) that makes MSP a very sensitive technique capable of detecting one methylated allele in a background of 1000 unmethylated. MSP amplification of tumor DNA was performed for 39 cycles at 950C denaturing, 57-590C annealing and 720C extension with a final extension step of 5 minutes. In each set of DNAs modified and PCR amplified, a cell line known to be hypermethylated for IGFBP-3 from the bisulfite sequencing data as a positive control and two normal lung tissue DNA as a negative control were included. Each set of DNAs modified and PCR amplified, includes normal human lymphocyte DNA in vitro methylated with Sss I methylase according to the manufacturers instructions (New England Biolabs, Beverly, MA, USA) used as a positive control, normal DNA as negative control and water with no DNA template as a control for contamination. After PCR, samples were run on a 6% non-denaturing acrylamide gel with appropriate size markers and the presence or absence of a PCR product analyzed.

Culture and cisplatin treatment of human cancer tissues Fresh tumor tissue was minced and passed through a nylon mesh. Cells were put into a sterile flask containing a mixture of enzymes: colagenase type II and hyaluronidase (Sigma Aldrich) in Dulbeco’s modified eagle’s medium nutrient misture F-12/Ham media (Sigma Aldrich) with antibiotics. The enzyme disaggregation was carried out for 20 min at 37ºC with gentle stirring. Then, cells were washed twice and resuspended in the same media supplemented with fetal calf serum. Aliquots (50µl) of this single cell suspension were dispensed into 96-well microtiter plates, and then incubated at 37ºC in a humidified 5% CO2 atmosphere for 7 days in the drug presence. Cisplatin response was tested by quadruplicate at the following concentrations 0, 0.01, 0.1, 1, 10 and 100 µg/ml. In order to measure cell viability, alamar blue was added directly into culture media at a final concentration of 10% and the plate was returned to the incubator. Optical density of the plate was measured at 570 and 600 nm with a standard spectrophotometer at 3h after adding alamar blue. Cell viability was calculated according to manufacture’s protocol (Biosource Europe, Nivelles, Belgium).

Chattopadhyay S, Machado-Pinilla R, Manguan-Garcia C, Belda-Iniesta C, Moratilla C, Cejas P et al (2006). MKP1/CL100 controls tumor growth and sensitivity to cisplatin in non-small-cell lung cancer. Oncogene 25: 3335-45.

Levina V, Marrangoni AM, DeMarco R, Gorelik E, Lokshin AE (2008). Drug-selected human lung cancer stem cells: cytokine network, tumorigenic and metastatic properties. PLoS ONE 3: e3077.

Roninson IB (2003). Tumor cell senescence in cancer treatment. Cancer Res 63: 2705- 15.

Takai D, Jones PA (2002). Comprehensive analysis of CpG islands in human chromosomes 21 and 22. Proc Natl Acad Sci U S A 99: 3740-5.

Takai D, Jones PA (2003). The CpG island searcher: a new WWW resource. In Silico Biol 3: 235-40.