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 g/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: GC55%;
Obs/Exp65 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
elements

(www.itba.mi.cnr.it/webgene/). CpG islands containing repetitive

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 95C for 5 min, b) 21-34

cycles of 95C for 1 min, 55-62C for 1 min, 72C for 1 min. c) An extension of
10 min at 72C. 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 95C and 1 min at 60C.
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 m
sections from each FFPE block from 36 NSCLC-primary specimens and nonneoplasic 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 (1g) 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 37C and then modified by hydroquinone and
sodium bisulfite treatment at 50C 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 95C for 5 min, 56-63C

for 1 min, 72C for 1 min; b) 34-38 cycles of 95C for 1 min, 55-64C for 1 min,
72C for 1 min. c) An extension of 10 min at 72C. 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 Dulbecos modified eagles medium
nutrient misture F-12/Ham media (Sigma Aldrich) with antibiotics. The enzyme
disaggregation was carried out for 20 min at 37C with gentle stirring. Then,
cells were washed twice and resuspended in the same media supplemented
with fetal calf serum. Aliquots (50l) of this single cell suspension were
dispensed into 96-well microtiter plates, and then incubated at 37C 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 g/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 manufactures protocol (Biosource
Europe, Nivelles, Belgium).

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