Summary To assess the role of oestrogen regulation in the growth of ovarian cancer, we examined the effects of an oestrogen, 17 P-oestradiol, and an anti-oestrogen, tamoxifen, on oestrogen receptor (ER) -positive and -negative human ovarian carcinoma cell lines. As measured by a dextran-coated charcoal adsorption assay, cell lines PEOI, PEO4 (Schwartz et al., 1982;Pagel et al., 1983;Hamerlynck et al., 1985;Campbell et al., 1984), although others have failed to demonstrate such benefits Slevin et al., 1986;Shirey et al., 1984;Landoni et al., 1983).We have recently derived and characterized a series of human ovarian carcinoma cell lines (Langdon et al., 1988). In the present study, we measured the levels of ER in these lines and examined the effects of 17 P-oestradiol and the anti-oestrogen tamoxifen on the growth of two of these lines -the ER +ve line PE04 and the ER -ve line, PE014. We have also assessed the effects of high doses of tamoxifen on 8 of the lines of the series. Materials and methods Cell lines and drugsThe characterization of the cell lines has been described previously (Langdon et al., 1988) but brief details are recorded in Table I. Cell lines were routinely cultured at 37°C, 90% humidity and 5% CO2 in RPMI 1640 + fetal calf serum (FCS) (9:1) with streptomycin (100 gg ml-'), glutamine (2 mM), pyruvate (2 mM), penicillin (100 IU ml-'), and 3-[morpholino] propane sulphonic acid (12.5 mM, pH 7.4). 17 P-oestradiol was obtained from Sigma Ltd., Dorset, UK and tamoxifen was a gift from ICI Pharmaceuticals Ltd., Macclesfield, UK. In the cell growth experiments, both were initially dissolved in ethanol. The final concentration of ethanol in cell growth experiments was always less than 0.01 % v/v, a concentration previously shown to have no effect on these cell lines.Oestrogen receptor assays Assays were performed on cells which were in early plateau phase of their growth using the dextran-coated charcoal adsorption method described previously (Hawkins et al., 1975;Hawkins et al., 1981

The ZR-75-1 ER positive breast cancer cell line, xenografted in female nude mice, has been used to determine the effect of tamoxifen on cell proliferation (as measured by mitosis) and cell death (as evidenced by apoptosis and necrosis). After 2 days treatment, there was a significant rise in apoptosis (p < 0.05), whereas a fall in mitosis was not apparent until 7 days (p < 0.05). Furthermore there was an increase in the apoptotic:mitotic ratio on day 7 (p < 0.05). These changes antedated tumour regression, which did not reach not significance until day 14. Tamoxifen did not increase necrosis (which significantly decreased in treated tumours once they had regressed (p < 0.01). In contrast tamoxifen treatment of xenografted MDA-MB-231 ER-negative breast cancer cells produced no significant effects on growth, apoptosis, or mitosis. This study presents clear evidence for tamoxifen inducing apoptosis in ZR-75-1 xenografts (but not MDA-MB-231 tumours). Since changes in apoptosis and mitosis antedate tumour regression, their assessment may provide the potential by which to predict tumour response to tamoxifen therapy.

Summary The extracellular matrix protein tenascin (TN) is overexpressed in a number of solid tumours. This, however, is the first study to examine TN expression in ovarian tumours. TN protein was examined in frozen sections of 50 human ovarian tumours by immunohistochemistry. Malignant and borderline tumours showed a significantly greater incidence and intensity of stromal staining than benign tumours (P<0.0001 and P=0.038 respectively). Seven omental metastases were also examined and showed a strikingly similar protein distribution to their primary tumour counterparts. The expression pattern of different RNA isoforms, created by alternative splicing of the primary transcript, was identified using reverse transcription-polymerase chain reactions (RT-PCR). The smallest TN RNA splice variant (284 bp) was found in all tumours examined, while the appearance of larger molecular weight transcripts (-490 and 556 bp), as major forms, was predominantly limited to malignant tumours, with 9/12 malignant tumours showing this pattern compared with 1/6 benign tumours. These data suggest that malignant ovarian tumours have increased expression of TN compared with benign tumours and this may be associated with induction of specific isoforms.

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Summary Although EGF receptor expression is generally elevated in human lung squamous carcinoma, the biological significance of this phenomenon and the role of EGF and TGF-a in this disease are poorly understood. We have investigated three human lung squamous carcinoma cell lines (NX002, CX140 and CX143) and have shown, using an antibody (EGFRI) directed against the EGF receptor, that the majority of cells in all three lines express the EGF receptor. Using a ligand binding assay, Scatchard analysis indicated high concentrations (1,300-2,700 fmol mg-' protein) of a single low affinity binding site (Kd = 3-5 nM) within these lines. Addition of EGF or TGF-a at concentrations greater than 0.1 nm resulted in growth inhibition of all three lines and this was associated with an accumulation of cells in the G2/M phase of the cell cycle. Growth inhibitory effects were not explained by an enhancement of cellular differentiation as monitored by involucrin expression and the ability to form cornified envelopes. While the presence of EGF could not be detected in medium conditioned by the NX002 cell line, mRNA for TGF-a was detected in all three lines suggesting the possibility of an autocrine loop. These results together with reports of growth inhibition by EGF and TGF-a in other systems suggest that EGF and similar molecules might have a growth regulatory role in lung cancer cells and modulation of such may have therapeutic potential.The related molecules, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-ax), modulate the growth of a wide variety of normal and malignant cells (reviewed in Burgess et al., 1989). Both factors operate through the EGF receptor and in many malignant diseases, for example lung squamous carcinoma, levels of the EGF receptor are markedly overexpressed relative to normal tissue (Hendler & Ozanne, 1984;Cerny et al., 1986;Berger et al., 1987;Veale et al., 1987;Sobol et al., 1987;Dazzi et al., 1989). The biological significance of this overexpression of receptors in lung squamous carcinoma is not known. Cell lines derived from human lung squamous carcinoma cell lines have been shown to possess high concentrations of EGF receptors (Haeder et al., 1988) and represent useful models to study growth factor modulation. Little is known of the role of EGF and TGF-x in this disease, however, several reports have proposed that TGF-o may act in an autocrine manner in this cell type (Lee et al., 1990;Putnam et al., 1991) while another recent study has suggested that either EGF or TGFa might stimulate squamous differentiation (Levitt et al., 1991).In order to investigate further the role of EGF-like factors in lung squamous carcinoma cells, we have developed cell line models and have examined them for the presence of EGF receptors. Two approaches have been used to detect and measure the levels of this receptor: immunocytochemical staining using an antibody directed against the receptor and a competitive binding assay employing radiolabelled EGF. We have also studied the effects of EGF and T...