The current study was undertaken to analyse growth and differentiation-related functions of normal keratinocytes (NOK) and an SV40T-immortalized keratinocyte line (SVpgC2a) from buccal mucosa, viewing the latter cell line as a model of a dysplastic epithelium. Morphological and immunohistochemical assessments of organotypic epithelia generated from 10 or 17 d of culture showed three- to five-fold higher apoptotic and proliferative activity in SVpgC2a relative to NOK. Conditions with or without serum (up to 10%) did not significantly influence these parameters in NOK whereas serum supported proliferation of SVpgC2a. Both cell types showed basal expression of collagen IV and laminin 1, indicating basal lamina, as well as vimentin, indicating an activated, proliferative state. Reduced expression of keratin, including the non-keratinizing marker K13, was seen in SVpgC2a. Assessment of proliferative monolayer cultures by microarray showed that NOK transcribed tissue-specific keratins, but also the epidermal keratin K2a, several simple epithelial keratins and low levels of hair keratins. SVpgC2a transcribed keratins seen in epithelial dysplasia, and K2a and hair keratins, albeit at low level. Overall, the results implied aberrant apoptosis, proliferation and keratin expression in the immortalized state of SVpgC2a. Comparison of NOK and SVpgC2a under identical culture conditions may serve to model the progression from a normal to a pre-neoplastic state of buccal epithelium.

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 antigenimmortalized 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. © 2002 Wiley-Liss, Inc. Key words: oral epithelium; biotransformation; phase I and II metabolizing enzymes; mRNA expressionSeveral enzyme systems including cytochrome P450s (CYPs), conjugation enzymes and enzymes involved in detoxification of reactive oxygen species (ROS) cooperate to protect the organism against reactive agents that are potentially carcinogenic and stress inducing. 1 Oxidative metabolism (so called phase I metabolism), carried out by the CYP family of enzymes, initiate biotransformation of compounds, which do not possess functional groups suitable for conjugation and can thereby generate toxic and mutagenic/ carcinogenic intermediates. 2 Following CYP dependent oxidative activation, conjugation enzymes (involved in so called phase II metabolism) including transferases for glutathione, sulfate, acetyl moieties and glucuronic acid generally decrease the reactivity of ...

Enzymes involved in various protective and metabolic processes of carbonyl compounds were analysed utilising a micro-array method in a three-stage in vitro model for oral carcinogenesis involving cultured normal, immortalised and malignant human oral keratinocytes. A complete transcript profiling of identified carbonyl-metabolising enzymes belonging to the ADH, ALDH, SDR and AKR families is presented. Expression of 17 transcripts was detected in normal, 14 in immortalized and 19 in malignant keratinocytes of a total of 12,500 genes spotted on the micro-array chip. For the detected transcripts, about half were changed by cell transformation, and for the various enzyme families, differences in expression patterns were observed. The detected AKR transcripts displayed a conserved pattern of expression, indicating a requirement for the keratinocyte phenotype, while most of the detected SDRs displayed changed expression at the various stages of malignancy. The importance of multiple experiments in using a microarray technique for reliable results is underlined and, finally, the strength of the method in detecting co-expressed enzymes in metabolic pathways is exemplified by the detection of the formaldehyde-scavenging pathway enzymes and the polyol pathway enzymes.

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.

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