The inducibility of human cytochrome P450 1A by environmental-relevant xenobiotics in the human hepatoma derived cell line HepG2.

Overexpression of the CYP1 family, independent of gender, is focal to the evaluation of the risk of human cancer. We have analysed the ability of 17 anthropogenic environmental xenobiotics widely used in Europe within households and agriculture to induce the human cytochrome P450 1A (CYP1A) in the human hepatoma derived cell line HepG2. The xenobiotics were potent to concomitantly induce both CYP1A mRNA and CYP1A activity in a dose-response relationship. Exceptions were shown by the organophosphate insecticide chlorpyrifos and the imidazole fungicide prochloraz in high concentrations which were capable of both inhibiting the basal or abolishing the initially induced CYP1A activity, respectively. A CYP1A induction has been shown for the first time by the aromatic xenobiotics irgasan, permethrin and azoxystrobin, the nonaromatic tributyltinoxide and for humans by the piperonylbutoxide. The xenobiotics additionally differed by their induced CYP1A isoenzyme pattern. A pronounced CYP1A1 and CYP1A2 mRNA induction was given by the phenyl urea herbicide diuron and benzodiazole insecticide piperonylbutoxide, respectively. In conclusion, out of the environmental xenobiotics, we described new members of human CYP1A inducers which extend chemical structures of biotransformation activators.

Diversity of common alternative splicing variants of human cytochrome P450 1A1 and their association to carcinogenesis.

Cytochrome P450 1A1 (CYP1A1) belongs to the enzymes of biotransformation of phase I. CYP1A1 performs the catalytic activation of exogenous and endogenous substrates to more carcinogenic metabolites. Overexpression of the wild-type and a recently described splice variant (CYP1A1v, ovarian cancer) are attributed to neoplastic transformation. Here we describe novel CYP1A1 splicing variants commonly and frequently transcribed in leucocytes of healthy volunteers, separated from variants exclusively expressed in tumour cell lines. Interestingly, all the novel splicing variants in leukocytes are generated by employing of two nested splice site pairs, one outer canonical and one inner non-canonical splice site pair, within the exon 2 of the human CYP1A1. In general, the frequent presence of common splicing variants in healthy volunteers has to be consider as a physiological feature of human CYP1A1 transcription process, rather than a signature of carcinogenesis.

N-glycosylation of CD97 within the EGF domains is crucial for epitope accessibility in normal and malignant cells as well as CD55 ligand binding.

CD97 is an EGF-TM7 receptor found on various carcinomas where expression levels correlate with dedifferentiation and tumor stage, smooth muscle cells and leukocytes. CD97 acts as an adhesion molecule by binding to its cellular ligand, CD55. In this study, we demonstrate that 2 immunodominant CD97 epitopes are not equally present in the various cell types. Differences were apparent in gastrointestinal tumors and smooth muscle cells where monoclonal antibodies (mAbs) to the first epidermal growth factor (EGF) domain (CD97(EGF)) showed a more restricted staining pattern than mAbs to the stalk region (CD97(stalk)). This discrepancy was not detectable in cultured gastrointestinal tumor cell lines. In fact, the selection of the CD97 mAb influences the result of clinical studies. Thus, we clarified the reason(s) for these differences in CD97 mAb staining on various cell types. We provide evidence that epitope accessibility for CD97(EGF) mAbs depends on N-glycosylation. Immunoprecipitation of CD97 from the Colo 205 tumor cell line revealed the established 78 and 83 kDa products, while a 52 and 57 kDa band were obtained from smooth muscle cells. N-glycosidase F reduced the size of CD97 in Colo 205 cells to 52-57 kDa. Culturing these cells with tunicamycin resulted in the same decrease in size and impaired CD97(EGF) mAb binding. As shown by site-directed mutagenesis, deletion of the N-glycosylation sites located within the EGF domains efficiently disturbed CD97(EGF) mAb immunoreactivity and, importantly, binding of CD55. In conclusion, CD97(EGF) epitope accessibility for mAbs and ligand binding is influenced by cell type-specific N-glycosylation.