Thus, additional characterization of these cells via whole genome profiling was merited in order to identify the extent of genomic reprogramming that occurred in the variant cell types as well as to uncover candidate genes responsible for maintaining the hepatic phenotype. In order to identify gene products that may play a key role in initiating and/or maintaining the liver phenotype, we profiled genome-wide expression in hepatoma cells and hepatoma-derived variant cell lines. Our strategy was to compare gene expression profiles of a hepatoma parental cell line with that of a panel of 4 independently derived hepatoma variant cell lines. Our goal was to determine the full extent of reprogramming that occurs AZD8055 in the variant cell types leading to loss of hepatic function as well as to identify candidate genes that are responsible for maintaining regulatory circuitry controlling liver function. All cell lines described are derived from the rat liver tumor line H4IIEC3. Fg14 cells are an adenine phosphoribosyltransferase-positive (APRT+), xanthine–guanine phosphoribosyltransferase-positive (GPT+) cell line derived from the APRT− and hypoxanthine–guanine phosphoribosyltransferase-negative (HPRT−) Fado-2 cells by stable transfection of Gpt and Aprt transgenes driven by the human α1-antitrypisin (α1AT, SERPINA1) gene promoter (− 640 to − 2 bp). Dedifferentiated cell lines, including M29, H11, HS2 and M38 cells, were derived from the Fg14 cells by negative selection against both Aprt and Gpt transgene gene expression using 20 μg/ml 2,6-diaminopurine (DAP) and 30 μg/ml 6-thioxanthine, respectively [19]. M29 and M38 cells have a reversion rate to APRT+ of approximately 10− 5, while the H11 cells revert at a frequency of 10− 3 and HS2 cells at < 10− 8. All cells were maintained in 1:1 Ham's F12/Dulbecco's modified Eagle's medium (FDV) containing 5% fetal bovine serum (FBS) (Gibco) and 5 μg/100 ml penicillin–streptomycin (Gibco) at 37 °C in a humid 5% CO2 chamber. RNA was extracted from nearly-confluent monolayers using a Qiagen RNeasy Mini Kit (Cat #74104) following the kit protocol with the addition of a DNaseI (Cat #79254) digestion step, as per protocol. Briefly, approximately 107 cells were lysed with RLT buffer, samples spun through a Qiagen column shredder to ensure homogenization, and RNA collected on an RNEasy column. Samples were washed and then digested with DNase I for 15 min at RT. RNA was further washed then eluted. RNA integrity was determined by 28S and 18S rRNA visualization following gel electrophoresis in MOPS with 1% agarose-2.2 M formaldehyde gels, as described [20]. RNA purity and concentration were determined by nano-drop spectrophotometry at 260 and 280 nm. Triplicate samples of RNA extracted from independent cultures of each cell line (with the exception of the M29 cells) were used for microarray analysis.