The transcriptosomal response of human A549 lung cells to a hydrogen peroxide-generating system: relationship to DNA damage, cell cycle arrest, and caspase activation.

Intracellular oxidative stress is a dynamic situation characterized by the accumulation of reactive oxygen metabolites, such as hydrogen peroxide. This is traditionally associated with both macromolecular damage and adaptive changes in gene expression, aimed at preventing cellular demise. However, the overall extent of such genetic changes is not well characterized. Here we present a comprehensive analysis of altered mRNA profiles in human A549 type II lung epithelial cells in response to hydrogen peroxide, at concentrations failing to induce necrotic toxicity. The results of an Affymetrix-based screen of the steady-state levels of mRNAs for several thousand genes revealed a complex pattern of transcriptional and/or posttranscriptional response to oxidative stress, which can be functionally related to both the oxidation and repair of damaged DNA, the induction and permanency of cell cycle arrest, and caspase-3 activation. Many of the genetic events can be related to activation of the p53/p21 pathway, but many other novel inductions and suppressions were detected, revealing the intricacy of the response. The data also disclosed a potential interaction between hydrogen peroxide treatment and increased sensitivity to cell killing by TRAIL, which could be functionally confirmed at the level of induction of caspase-3 activity.

Differentiation of human hepatoma cells during confluence as revealed by gene expression profiling.

Certain human hepatocarcinoma cells undergo differentiation when grown at confluence. In order to understand the basis for this differentiation, we investigated the phenotypic changes occurring during confluent growth of the human hepatoma B16A2 cell line. The global gene expression profile of B16A2 cells grown during confluence for 5 weeks was investigated using microarrays containing complementary sequences corresponding to approximately 10,000 genes, and compared with profiles of adult human liver and HepG2 cells. The major part of gene products detected were shared by all three systems and the hepatoma cell lines expressed surprisingly high levels of liver-enriched transcription factors. During confluence of B16A2 cells, the majority of transcriptional changes monitored were directed towards the phenotype of adult human liver in vivo, although the changes accounted for less than 10% of those necessary to acquire a native hepatic phenotype. Several markers of liver differentiation and regeneration were changed in similar manner as observed in developing liver and during liver regeneration. In conclusion, the data indicate that differentiation in vitro of the B16A2 cell line during confluence partially resembles that of hepatic differentiation and regeneration in vivo, implying a partial normalization of a low differentiated phenotype.

Microarray assessment of fibronectin, collagen and integrin expression and the role of fibronectin-collagen coating in the growth of normal, SV40 T-antigen-immortalised and malignant human oral keratinocytes.

Extracellular matrix proteins affect the growth and survival of epithelial tissues. Accordingly, surface coating with fibronectin and collagen is a common practice for promoting keratinocyte culture. In this study, the expression of fibronectin and collagen-related factors, including integrins, by normal (NOK), SV40 T-antigen-immortalised (SVpgC2a) and malignant (SqCC/Y1) human oral keratinocytes, under standardised, serum-free conditions, was investigated by using microarray analysis. Cell growth was also studied in the presence and absence of a matrix consisting of human fibronectin and bovine collagen type I (FN-COL). Fibronectin transcripts were abundant in all cells, whereas 16 of 29 collagen chains and 14 of 24 integrin subunits were variably detected. With regard to both the expression level and the number of transcripts, higher collagen and lower integrin expression was observed in SVpgC2a cells than in NOKs and SqCC/Y1 cells. The cell types differed with regard to colony-forming efficiency and the rate and kinetics of growth at high cell density. For all cell types, FN-COL coating consistently stimulated cell migration, without influencing growth in mass culture or clonal density. The results demonstrate the transcription of genes associated with the formation and function of fibronectin and collagen in oral epithelium, and variably altered expression patterns in transformed states, and show that keratinocyte lines can be successfully transferred without the stimulus from extracellular FN-COL.

Protein S-glutathionylation correlates to selective stress gene expression and cytoprotection.

During situations of oxidative stress phenotypic adaptation to altered redox state is achieved by changes in expression of selected genes. The mechanisms regulating this may involve reversible S-glutathionylation of cellular proteins. In this study we compared and contrasted changes in gene expression patterns in human type II lung epithelial A549 cells and human endothelial ECV304 cells in correlation to glutathione oxidation and the formation of glutathione-protein mixed disulphides, after exposure to subtoxic levels of hydrogen peroxide, formed in the medium by addition of glucose oxidase, or the thiol oxidant diamide. Both the number of specific mRNAs and their levels of induction were grossly correlated to the degree of S-glutathionylation of cellular protein. Thus, diamide induced the expression of a variety of protein and DNA chaperones and transcriptional regulators, particularly in ECV304 cells. On the other hand, the peroxide failed to induce many of these species, in association with only minimal disturbances to glutathione homeostasis. The induction of the chaperone responses at the level of mRNA was clearly shown to translate into a more resistant morphological phenotype in response to both heat shock and oxidative stress induced by the DNA-damaging pro-oxidant potassium bromate.

Transcript profiling of enzymes involved in detoxification of xenobiotics and reactive oxygen in human normal and simian virus 40 T antigen-immortalized oral keratinocytes.

The metabolic detoxification capacity may critically regulate the susceptibility of human tissues to cancer development. We used standardized and quantitative, reverse transcription-polymerase chain reaction (StaRT-PCR) and microarray chip techniques to analyze transcript levels of multiple detoxification enzymes in cultured normal human oral keratinocytes (NOK) and the Siman virus 40 T antigen-immortalized oral keratinocyte line SVpgC2a, viewing the latter as a model of a benign tumor state. With good agreement between the 2 methodologies, NOK and SVpgC2a were found to express transcripts for cytochrome P450 enzymes (CYPs), factors related to CYP induction and enzymes involved in conjugation reactions or detoxification of reactive oxygen. The cell types expressed similar levels of CYP 2B6/7, CYP 2E1, P450 oxidoreductase, the aryl hydrocarbon receptor nuclear translocator, sulfotransferase 1A1, sulfotransferase 1A3, epoxide hydrolase, glutathione S-transferase M3, glutathione S-transferase pi and catalase, superoxide dismutase 1, glutathione peroxidase 1 and glutathione peroxidase 3. In contrast, SVpgC2a exhibited comparatively higher levels of CYP1A1, 1B1, aryl hydrocarbon receptor, glutathione S-transferase M1, 2, 4, 5, glutathione S-transferase theta 1 and superoxide dismutase 2 and comparatively lower levels of UDP glycosyltransferase 2 and microsomal glutathione S-transferase 1. Some transcripts, e.g., CYP 2A6/7, were not detected by either standard, non quantitative RT-PCR or the above methods, whereas others were barely quantifiable by StaRT-PCR, i.e., were present at 1-10 molecules/10(6) molecules of actin. Overall, the expression analysis demonstrated presence of mRNA for multiple enzymes involved in foreign compound metabolism and detoxification pathways, including several enzymes not previously reported for oral epithelium. Generally, the comparison of NOK from 2 individuals indicated relatively similar transcript levels of these enzymes. In contrast, differences between NOK and SVpgC2a, e.g., for CYP1B1, may reflect alteration caused by immortalization and aid identification of early stage tumor markers in oral epithelium.

Identification of end points relevant to detection of potentially adverse drug reactions.

Expectations are high that the use of proteomics, gene arrays and metabonomics will improve risk assessment and enable prediction of toxicity early in drug development. These molecular profiling techniques may be used to classify compounds and to identify predictive markers that can be used to screen large numbers of chemicals. One of the challenges for the scientific community is to discriminate between changes in gene/protein expression and metabolic profiles reflecting physiological/adaptive responses, and changes related to pathology and toxicology. In these proceedings we provide a brief overview of the technologies with focus on proteomics and the possible applications to mechanistic and predictive toxicology. The discussion also includes strengths and limitations of molecular profiling technologies.