12-O-Tetradecanoylphorbol 13-acetate induced differentiation in human lung squamous carcinoma cells

Abstract

Summary Three human lung squamous carcinoma cell lines (NX002, CX140 and CX143) demonstrate features of squamous differentiation including involucrin synthesis and competence to form comified envelopes. 12-O-Tetradecanoylphorbol 13-acetate inhibits growth of these cell lines and this growth inhibition is associated with enhanced differentiation.Squamous cell carcinoma is the most common subtype of non-small cell lung cancer and represents 40% of all lung cancer cases (Minna et al., 1989). The factors that control proliferation and differentiation in this disease are poorly understood and an improved knowledge of these might aid the development of therapeutic strategies such as the use of differentiation induction in this tumour type (Bloch, 1984;Sartorelli, 1985). Lung squamous carcinoma cells undergoing terminal differentiation demonstrate a number of features including expression of involucrin (a protein precursor found beneath the plasma membrane prior to terminal differentiation) and the ability to form cornified envelopes (Miyazaki et al., 1982;Banks-Schlegel et al., 1985;Levitt et al., 1990;Said et al., 1983;Salge et al., 1990 (Broers et al., 1988).In the present study, we describe differentiation features within three new cell lines and show how they relate to the xenografts from which they were derived. The effect of the differentiation inducer 12-O-tetradecanoylphorbol 13-acetate (TPA) on the growth and differentiation of these cell lines was then investigated. Materials and methodsThe three xenografts from which the cell lines were derived were obtained from untreated patients in 1983 and brief details of these xenografts have previously been reported (Fergusson et al., 1986). The pathology of both the NX002 and CX140 xenografts is consistent with that of poorly differentiated lung squamous carcinoma and has remained constant over the last 7 years. The CX143 xenograft was obtained from a patient with adenosquamous carcinoma and although both adeno and squamous components could be observed in early passages of the xenograft, the pathology of later passages was consistent with a poorly differentiated squamous carcinoma.Cell lines were derived from all three xenografts between their 20th and 27th passages and grown in RPMI 1640 media supplemented with 5% foetal calf serum (FCS), streptomycin (100 igml-'), penicillin (1OOIUml-') and glutamine (2g.M) and kept at 37°C in 5% CO2 and 90% humidity. For these experiments, cell lines were in their 6th-20th passage.For experiments wherein the effects of TPA on growth were examined, cells in exponential phase of growth were trypsinised and plated into 24 well trays at a density of 5 x I04 cells/well. Twenty-four hours later, TPA at concentrations ranging from 10`0 to 10-6 M was added to quadruplicate wells. After 4 days, cells were trypsinised and counted using a ZF coulter counter.For detection of antigens, cells were stained by an indirect immunoperoxidase method using avidin-biotinylated horseradish peroxidase complex (Hsu et al., 1981). Mouse monoclonal anti...