Housekeeping genes involved in non-malignant breast phenotypes are widely expressed in multiple cancers and provide novel biomarkers of tumor classification

Clinically relevant biomarkers are useful to determine cancer patients' prognosis and treatments. To discover new putative biomarkers, we performed in silico analysis of a 325-gene panel previously associated with breast epithelial cell biology and clinical outcomes. Sixteen public datasets of microarray samples representing 8 cancer types and a total of 3,663 patients' samples were used for the analyses. Feature selection was used to identify the best subsets of the 325 genes for each classification, and linear discriminant analysis was used to quantify the accuracy of the classifications. A subset of 102 of the 325 genes were found to be housekeeping (HK) genes, and the classifications were repeated using only the 102 HK subset. The 325-gene panel and 102 HK subset were able to distinguish colon, gastric, lung, ovarian, pancreatic, and prostate tumors and leukemia from normal adjacent tissue, and classify disease subtypes of breast and lung cancers and leukemia with 70% or higher accuracy. HK genes have been overlooked as potential biomarkers due to their relative stability. This study describes a set of HK genes as putative biomarkers applicable to multiple cancer types worth following in subsequent validation studies.

Effects of Trichostatin A on the Chondrogenesis from Human Mesenchymal Stem Cells

Histone deacetylase inhibitors (HDACi) are a class of compounds that suppress the function of histone deacetylases (HDACs). This study was performed to examine the effects of Trichostatin A (TSA), a typical HDACi, on chondrogenesis of human bone marrow mesenchymal stem cells (hBMMSCs) and related molecular pathways. After evaluating the concentration for cytotoxicity and HDAC activity, hBMMSCs underwent chondrogenic differentiation in pellet culture with or without TSA for 21 days. The weight of TSA-treated pellets was 25% lower than that of untreated pellets. DNA level was not significantly different, but glycosaminoglycan content per DNA level was lower in TSA-treated pellets than that of untreated pellets. Gene expression of the chondrogenic markers (SOX9, Aggrecan, and Col2A1) decreased by by 12.9-fold, 8.9-fold, and 7.6-fold respectively in TSA-treated pellets compared with that in TSA-untreated pellets. TSA-treated pellets had lower cell density and lower proteoglycan staining content compared with those of TSA-untreated pellets. A microarray analysis from TSA-treated pellets showed that 1,467 chondrogenic-related genes were downregulated and 1,524 were upregulated by more than 2-fold compared with TSA-untreated pellets. Col10A1, TGF-β3, and SOX9 decreased significantly by 10-fold, 2.1-fold, and 3.2-fold respectively in TSA-treated pellets compared with those in untreated pellets, whereas expression of BMP4 and FGFR3 increased significantly by 2.1-fold and 5.4-fold respectively. It is concluded that TSAinhibits chondrogenesis and does not seem to be useful for cartilage tissue engineering of hBMMSCs.

Differential Expression Patterns of Leukaemia Associated Genes in Leukaemia Cell Lines Compared to Healthy Controls

Introduction: The phenotype and genotype of cancer cells portray hallmarks of cancer which may have clinical value. Cancer cell lines are ideal models to study and confirm these characteristics. We previously established two subtracted cDNA libraries with differentially expressed genes from an acute myeloid leukaemia patient with poor prognosis (PP) and good prognosis (GP). Objective: To compare gene expression of the leukaemia associated genes with selected biological characteristics in leukaemia cell lines and normal controls. Methodology: Expression of 28 PP genes associated with early fetal/embryonic development, HOX-related genes, hematopoiesis and aerobic glycolysis/ hypoxia genes and 36 GP genes involved in oxidative phosphorylation, protein synthesis, chromatin remodelling and cell motility were examined in B-lymphoid (BV173, Reh and RS4;11) and myeloid (HL-60, K562) leukaemia cell lines after 72h in culture as well as peripheral blood mononuclear cells from healthy controls (N=5) using semi-quantitative polymerase chain reaction (PCR) method. Cell cycle profiles were analysed on flow cytometry while MTT cytotoxicity assay was used to determine drug resistance to epirubicin. Results: Genes expressed significantly higher in B-lymphoid leukaemia cell lines compared to healthy controls were mostly of the GP library i.e. oxidative phosphorylation (3/10), protein synthesis (4/11), chromatin remodelling (3/3) and actin cytoskeleton genes (1/5). Only two genes with significant difference were from the PP library. Cancer associated genes, HSPA9 and PSPH (GP library) and BCAP31 (PP library) were significantly higher in the B-lymphoid leukemia cell lines. No significant difference was observed between myeloid cell lines and healthy controls. This may also be due heterogeneity of cell lines studied. PBMC from healthy controls were not in cell cycle. G2/M profiles and growth curves showed B-lymphoid cells just reaching plateau after 72 hour culture while myeloid cells were declining. IC50 values from cytotoxicity assay revealed myeloid cell lines had an average 13-fold higher drug resistance to epirubicin compared to B-lymphoid cell lines. Only CCL1, was expressed at least two-fold higher in myeloid compared to B-lymphoid cell lines. In contrast, MTRNR2, EEF1A1, PTMA, HLA-DR, C6orf115, PBX3, ENPP4, SELL, and IL3Ra were expressed more than 2-fold higher in B-lymphoid compared to myeloid cell lines studied here. Conclusion: Thus, B-lymphoid leukaemia cell lines here exhibited active, proliferating characteristics closer to GP genes. Higher expression of several genes in B-lymphoid compared to myeloid leukaemia cell lines may be useful markers to study biological differences including drug resistance between lineages.

Subtractive Hybridization Identifies Stem Cell-Associated Genes in an Acute Myeloid Leukemia with Poor Prognosis

Introduction: Current prognostic markers have improved survival prediction, however, it has not advanced treatment strategies. Gene expression profiling may identify biological markers suitable as therapeutic targets. Leukaemia stem cell is associated with adverse outcome, however, its biological characteristics are still being investigated. We observed higher in vitro cell viability in acute myeloid leukaemia (AML) samples with poor prognosis, which may be stem cell related. Objective: The objective of this study was to profile highly expressed genes in an AML sample of poor prognosis/high viability and compare with a sample of good prognosis/low viability. Method: Subtractive hybridization was performed on two AML samples with high blast counts (>80%), a poor prognosis, PP (disease free survival, DFS12 months) sample. The PP sample had higher CD34+ counts (73% vs 46%) and higher cell viability than the GP sample. cDNA libraries were subsequently cloned and sequenced. Results: cDNA subtracted from the PP samples was identified as genes active during fetal/embryonic development (LCOR, CNOT1, ORMDL1), HOX- related genes (HOXA3, PBX3, SF3B1), hematopoiesis (SELL, IL-3RA) and aerobic glycolysis/hypoxia (PGK1, HIGD1A) -associated genes. Majority of GP clones isolated contained genes involved in oxidative phosphorylation, OXPHOS (COXs, ATPs, MTND4 and MTRNR2), protein synthesis (including ribosomal proteins, initiating and elongation factors), chromatin remodeling (H2AFZ, PTMA), cell motility (MALAT1, CALM2, TMSB4X), and mitochondria (HSPA9, MPO) genes. Conclusion: Thus, the PP sample exhibited stem cell-like features while the GP sample showed cells at a high level of cell activity. These genes are potential prognostic markers and targets for therapy.

HOX gene analysis in the osteogenic differentiation of human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the capacity to differentiate into osteoblasts during osteogenesis. Several studies attempted to identify osteogenesis-related genes in hMSCs. Although HOX genes are known to play a pivotal role in skeletogenesis, their function in the osteogenesis of hMSCs has not yet been investigated in detail. Our aim was to characterize the expression of 37 HOX genes by multiplex RT-PCR to identify the ones most probably involved in osteogenic differentiation. The results showed that the expression patterns of four HOX genes were altered during this process. In particular, the expression levels of HOXC13 and HOXD13 were dramatically changed. Real-time PCR and Western blot analysis were performed in order to further analyze the expression of HOXC13 and HOXD13. The qRT-PCR results showed that transcription of HOXC13 was up-regulated by up to forty times, whereas that of HOXD13 was down-regulated by approximately five times after osteogenic differentiation. The Western blot results for the HOXC13 and HOXD13 proteins also corresponded well with the real-time PCR result. These findings suggest that HOXC13 and HOXD13 might be involved in the osteogenic differentiation of hMSCs.