Epidermal growth factor regulates topoisomerase II activity and drug sensitivity in human KB cells.

Because of its unique DNA-cleaving and strand-passing activities, topoisomerase II is involved in many aspects of DNA metabolism, including replication, transcription, recombination, and repair. The cytotoxic potential of topoisomerase II-targeted drugs, such as etoposide, is related to their ability to stabilize covalently linked enzyme-DNA complexes, which are intermediates in the enzyme's catalytic cycle. Epidermal growth factor receptor is expressed on the cell surface of the majority of squamous cell carcinomas, and epidermal growth factor binding is known to stimulate a number of cellular transduction pathways, including tyrosine kinase, protein kinase C, and phospholipase C. Because topoisomerase II is a proliferation-dependent protein and has been shown to be a high-affinity substrate for many of these cellular transduction pathways, the effects of epidermal growth factor on cellular regulation and sensitivity to etoposide were studied with the human oral cavity squamous cell line, KB. Topoisomerase II catalytic activity was rapidly and transiently inhibited after the addition of epidermal growth factor to the cellular growth media. Western blot on nuclear extracts did not demonstrate alterations in topoisomerase II polypeptide levels to account for changes in catalytic activity. Epidermal growth factor treatment also led to the formation of stabilized, covalently linked enzyme-DNA complexes. Furthermore, epidermal growth factor-induced, topoisomerase II-mediated DNA strand breaks were additive to those induced by etoposide. This study indicates that epidermal growth factor specifically regulates the catalytic and DNA-cleaving activities of topoisomerase II in KB cells. This may direct clinical strategies for circumventing the intrinsic cellular resistance to chemotherapy commonly observed in squamous cell carcinomas of the head and neck.

Binding, uptake, and release of nicotine by human gingival fibroblasts.

Previous studies of the effects of nicotine on fibroblasts have reported an altered morphology and attachment of fibroblasts to substrates and disturbances in protein synthesis and secretion. This altered functional and attachment response may be associated with changes in the cell membrane resulting from binding of the nicotine, or to disturbances in cell metabolism as a result of high intracellular levels of nicotine. The purpose of the present study, therefore, was to 1) determine whether gingival fibroblasts bound nicotine and if any binding observed was specific or non-specific in nature; 2) determine whether gingival fibroblasts internalized nicotine, and if so, at what rate; 3) determine whether gingival fibroblasts also released nicotine back into the extracellular environment; and 4) if gingival fibroblasts release nicotine intact or as a metabolite. Cultures of gingival fibroblasts were prepared from gingival connective tissue biopsies. Binding was evaluated at 4 degrees C using a mixture of 3H-nicotine and unlabeled nicotine. Specific binding was calculated as the difference between 3H-nicotine bound in the presence and absence of unlabeled nicotine. The cells bound 1.44 (+/- 0.42) pmols/10(6) cells in the presence of unlabeled nicotine and 1.66 (+/- 0.55) pmols/10(6) cells in the absence of unlabeled nicotine. The difference was not significant. Uptake of nicotine was measured at 37 degrees C after treating cells with 3H-nicotine for time periods up to 4 hours. Uptake in pmols/10(6) cells was 4.90 (+/- 0.34) at 15 minutes, 8.30 (+/- 0.75) at 30 minutes, 12.28 (+/- 2.62) at 1 hour and 26.31 (+/- 1.15) at 4 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding and uptake of N-nitrosonornicotine by oral epithelial cells.

N-nitrosonornicotine (NNN), a significant nitrosamine component of tobacco binds to and is taken up by cells prior to its activation by intracellular enzymes. A variety of factors may affect the binding of substances to a cell including the cell type, medium composition and the cell membrane lipid composition. This study examines the binding and uptake of NNN to various cell types and relates these to the cells' lipid composition. Hamster buccal pouch keratinocytes were exposed to NNN and the amount of bound NNN determined. This was compared with cells pretreated with 12-0-tetradecanoylphorbol-13-acetate (TPA). While the keratinocytes bound significant amounts of NNN, this binding was not specific. Pretreatment of the cells with TPA significantly increased the amount of NNN bound. Cultures of human gingival fibroblasts and a human liver cell line also bound NNN, in quantities greater than that bound to the keratinocytes. The TPA caused changes in long chain unsaturated fatty acids although major changes in lipid metabolism were not detected. The fatty acid composition of the liver cells more closely resembled that of the TPA treated keratinocytes than the untreated ones. The data suggest that NNN binds to the cells non-specifically and the binding may be related to the amount of long-chain, unsaturated fatty acids present.