The use of an in vitro submerged keratinocyte model to predict induction of squamous metaplasia

An in vitro submerged keratinocyte model of squamous metaplasia (SQ) in epi thelia is being developed to assess the risk associated with exposure to ce rtain environmental agents. Tracheobronchial epithelium (TBE) in vivo can r espond to airborne environmental insult by becoming squamous. Epidemiologic al evidence suggests that cigarette smoke is capable of inducing this chang e. Retinoic acid has been shown to maintain cells in the mucociliary state. SQ is considered protective and adaptive but potentially preneoplastic if unrelenting and is used histologically in the diagnosis of squamous cell ca rcinoma. SQ is characterised by upregulation of the expression of transglut aminase I (TGI), TGI activity leading to the formation of isopeptide cross- linked envelopes and replacement of the mucociliary cell type with non-pola r squamous cells out of contact with the basal lamina. The ability of the i n vitro keratinocyte submerged model to predict the squamous metaplastic re sponse in vivo has been investigated in vitro using TG catalysed fluorescei n cadaverine incorporation as a measure of cross-linked envelope formation, Alamar blue(TM) conversion to measure viability and Coomassie blue incorpo ration to measure total cellular protein. The modulation of the squamous co ndition by retinoic acid (RA), cigarette smoke condensate (CSC) and nicotin e has been assessed in keratinocytes cultured in Green's medium. RA inhibit ed FC incorporation by 95% at 1 x 10(-5) M and simultaneously increased cel l viability providing evidence to support its role in the regulation of the non-differentiated state. Nicotine (0-1 mg/ml) induced a dose-dependent in crease in viability at 6 days, a response that was accompanied by an increa se in FC incorporation at 12 days. CSC (0-5 mug/ml) increased FC incorporat ion after 12 days. Hence, nicotine modulated the squamous condition by up-r egulating TGI activity following a period of hyperactivity. CSC induced a g radual change to the differentiated state and RA served to maintain the cel ls in an undifferentiated state. (C) 2001 Elsevier Science Ltd. All rights reserved.