Expression profile of HER2-overexpressing CHO cells treated with trastuzumab and pertuzumab

CHO-K1 (wildtype) and CHO-K6 (stablely overexpressing human HER2) cells were terated with 10 ug/ml transtuzumab or pertuzumab and their combination for 24 h and then the whole gene expression was analyzed by cDNA array. CHO-K1 and CHO-K6 were plated in 60 mm dishes in RPMI-1640 medium containing 1% FBS and cultured for 24 h. Afterwards, the old medium discarded and fresh RPMI-1640 supplemented with 10% FBS and containing 10 ug/ml of trastuzumab or pertuzumab or their combination added to the cells and the cell were cultured for 24 h. After the treatment period, the total RNA was isolated using TRIzol reagent (Invitrogen, Waltham, USA) according to standard protocol. Samples for microarray hybridization were prepared according to the Affymetrix Manual Target Preparation for GeneChip® Whole Transcript (WT) Expression Arrays (#902130, Affymetrix Inc., Santa Clara, CA, USA). Amount 100 ng of total RNA were used to make double-stranded cDNA following cRNA synthesis. After purification of cRNA, 15 ug of each cRNA was reverse transcribed into sense-strand (ss) cDNA when unnatural dUTP residues were incorporated. ss cDNA were purified and 5.5 ug of each ss cDNA was fragmented using uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1) at the unnatural dUTP which breaks the DNA strand. Fragmented ss cDNA were terminal labeled with biotin and 3ug of each sample was hybridized to Affymetrix GeneChip® CHO Gene 2.0 ST array (Format 100) for 16 hours at 45C with rotation at 60 rpm in an Affymetrix GeneChip® Hybridization Oven 645. After hybridization, the arrays were washed and stained in an Affymetrix GeneChip® Fluidics Station FS450 and the fluorescent signals were measured with an Affymetrix GeneChip® Scanner 3000 7G. Row data was analyzed by Affymetrix Transcriptome Analysis Console (TAC) 3.0 software (Affymetrix Inc., Santa Clara, CA, USA)