Non Small Cell Lung Cancer (NSCLC) causes the premature death of over 1 million people worldwide each year, but remains inadequately understood at the molecular level. To provide new insights for NSCLC treatment we performed a molecular characterisation of wild type and platinum drugs resistance in A549 cells. Transcriptome profiling revealed contrasting patterns of gene expression in sensitive and resistant cells and identified genes whose expression was highly correlated with the platinum drugs. Our results revealed a gene set of 15 transcripts whose expression was highly correlated with platinum-resistance in NSCLC A549 cell lines.

Global  profiling of platinum drugs resistant genes in A549 cell lines

Affymetrix GeneChip HT Human Genome U133A HT_HG-U133A

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Non Small Cell Lung Cancer (NSCLC) causes the premature death of over 1 million people worldwide each year, but remains inadequately understood at the molecular level. To provide new insights for NSCLC treatment we performed a molecular characterisation of wild type and platinum drugs resistance in A549 cells. Transcriptome profiling revealed contrasting patterns of gene expression in sensitive and resistant cells and identified genes whose expression was highly correlated with the platinum drugs. Our results revealed a gene set of 15 transcripts whose expression was highly correlated with platinum-resistance in NSCLC A549 cell lines.


Affymetrix:Protocol:Hybridization-EukGE-WS2v4[]

Title: Affymetrix EukGE-WS2v4 Hybridization. Description:

Affymetrix:Protocol:MAS4:ExpressionCall

Title: Affymetrix CHP Analysis (ExpressionCall). Description:

P-AFFY-2

Labeling of samples was carried out according to the manufacturer’s standard protocol

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MTAB-19607

For development of the platinum resistant lines: 1. human NSCLC cell line A549wt and colon cancer cell line H630wt were cultured in F12-K and RPMI 1640. 2. Platinum resistant cells were generated by continuously culturing the drug sensitive parental cell lines in medium containing increasing concentrations of cisplatin, carboplatin or oxaliplatin in a stepwise fashion over 10 months. The lowest cisplatin concentration was 1nM, this was slowly increased to a final concentration of 10µM. The cells were exposed to each drug concentration for at least a 4 week period. After being left in this concentration for 4 weeks they were allowed to recover in drug free medium for a minimum period of 2 weeks.  3. The sensitivity (IC50) to carboplatin, cisplatin and oxaliplatin was determined at each step by MTT assay. The A549 cisplatin (Cis) resistant cell was derived from the A549WT line by maintaining the cells in increasing cisplatin concentrations. 

P-MTAB-19608

For development of the platinum resistant lines: 1.The human NSCLC cell line A549wt and colon cancer cell line H630wt were cultured in F12-K and RPMI 1640 at 37°C, 5% CO2 in incubator. 2. Platinum resistant cells were generated by continuously culturing the drug sensitive parental cell lines in medium containing increasing concentrations of cisplatin, carboplatin or oxaliplatin in a stepwise fashion over 10 months. 3. The lowest cisplatin concentration was 1nM, this was slowly increased to a final concentration of 10µM. The cells were exposed to each drug concentration for at least a 4 week period. After being left in this concentration for 4 weeks they were allowed to recover in drug free medium for a minimum period of 2 weeks. 4. The sensitivity (IC50) to carboplatin, cisplatin and oxaliplatin was determined at each step by MTT assay. 5. Cells were frozen in liquid nitrogen or prepared as cell pellets for further experiments. The same method was performed to develop resistance to carboplatin (Car), and oxaliplatin (Oxa) in A549 and oxaliplatin in H630 cell lines. 6. Resulting cell lines resistant to carboplatin, cisplatin, and oxaliplatin were named according to the cell name, drugs and final concentration, for example A549cis1.0 means to treat A549 cells with medium containing 1.0uM cisplatin at least 4 weeks. 7. For maintaining the phenotype, the A549cis7.5, A549oxa7.5 were kept in 1 µM Cisplatin or oxaliplatin and A549car15.0 was kept in 5 µM carboplatin during culture.

P-MTAB-19609

The RNA isolation procedure for the Affymetrix microarray analysis was performed using TRIzol Reagent (GIBCO, Carlsbad, CA) according to the manufacturer's instruction (Affymetrix, Santa clara, CA). 1. 1×107 cells were added to 1ml TRIzol reagent for cell lyses. The cell lysate was passed a 21G needle at least 10 times to homogenize and shearing genomic DNA and then centrifuged for 10 minutes at 12,000G, 2-8C.  2. All supernatant was transferred to a new tube and incubated at RT (room temperature) for 5 minutes. 3. 200µl of chloroform was added and solution was mixed by vigorous shaking for 15s. Following centrifugation for 15 minutes at 12,000G, the aqueous phase was removed.  4. RNA was precipitated by addition of 500µl of isopropanol and incubation at RT for 10 minutes. Then the solution was centrifuged at no more than 12,000G for 10 minutes at 2-8C. 5. The RNA pellet was washed in 1ml 75% (v/v) ethanol and then centrifuged for 5 minutes at 7500G after which the supernatant was removed and RNA pellet air dried at RT.  6. RNA was re-suspended in 30µl RNase free water and incubated on ice for 30 minutes to ensure complete dissolution of RNA.  7. The quantification and quality of total RNA was assessed by ratio of 260/ 280nm (between 1.9~2.1) using spectrophotometer and electrophoresis through a 1% agarose gel. Quality of total RNA was further analysed by a BioAanlyser 2100 (Agilent technologies, Palo, CA). All procedures were carried out on ice. (Parameters: Extracted product = total_RNA, Amplification = none)

P-MTAB-19610

For development of the platinum resistant lines: 1. Human NSCLC cell line A549wt was cultured in F12-K till 80% confluence. 2. Carboplatin resistant cells were generated by continuously culturing the drug sensitive parental cell lines in medium containing increasing concentrations of carboplatin in a stepwise fashion over 10 months. The lowest carboplatin concentration was 1nM, this was slowly increased to a final concentration of 17.5µM. The cells were exposed to each drug concentration for at least a 4 week period. After being left in this concentration for 4 weeks they were allowed to recover in drug free medium for a minimum period of 2 weeks.  3. The sensitivity (IC50) to carboplatin, cisplatin and oxaliplatin was determined at each step by MTT assay. 

P-MTAB-19611

All cells were maintained in F12K medium with penicillin/streptomycin (0.5 ml/100 ml media), 10% FCS (Fetalclone) (Hyclone, Thermo Fisher Scientific, USA) and cultured in a 5% CO2, 37°C incubator. In addition, the cell pellets were harvested with a scraper at 80% confluence, washed three times with PBS and stored at -80C, when required for further analyses (Parameters: time unit = seconds, temperature unit = C, media = F12K)

P-MTAB-19612

All cells were maintained in RPMI1640 medium with penicillin/streptomycin (0.5 ml/100 ml media), 10% FCS (Fetalclone) (Hyclone, Thermo Fisher Scientific, USA) and cultured in a 5% CO2, 37°C incubator. In addition, the cell pellets were harvested with a scraper at 80% confluence, washed three times with PBS and stored at -80C, when required for further analyses (Parameters: time unit = seconds, temperature unit = C, media = RPMI1640)

P-MTAB-19613

For development of the oxaliplatin resistant lines: 1. Human NSCLC cell line A549wt or H630wt was cultured in F12-K/RPMI1640 medium till 80% confluence. 2. oxaliplatin resistant cells were generated by continuously culturing the drug sensitive parental cell lines in medium containing increasing concentrations of oxaliplatin in a stepwise fashion over 10 months. The lowest oxaliplatin concentration was 1nM, this was slowly increased to a final concentration of 7.5µM. The cells were exposed to each drug concentration for at least a 4 week period. After being left in this concentration for 4 weeks they were allowed to recover in drug free medium for a minimum period of 2 weeks. 3. The sensitivity (IC50) to carboplatin, cisplatin and oxaliplatin was determined at each step by MTT assay.

Global profiling of platinum drugs resistant genes in A549 cell lines

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