No abstract

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

A human ovarian adenocarcinoma cell line (PE04) has been established as a xenograft in nude mice. In vitro, this cell line is estrogen receptor (ER)-positive and its growth is stimulated by 17 beta-estradiol at concentrations between 10(-12) and 10(-6) M. When xenografted, PE04 cells remain ER-positive and also possess progesterone receptors (PR); treatment with 17 beta-estradiol reduces the concentration of ER and increases levels of PR. Growth of the xenograft is reduced in ovariectomized animals while implantation of estrogen pellets also results in growth inhibition. Similar treatment with estrogen does not inhibit the ER-negative HOX 60 ovarian xenograft, and stimulates growth of the ER-positive ZR-75-I breast carcinoma xenograft. Serum measurements of 17 beta-estradiol confirm that ovariectomy reduces the level of 17 beta-estradiol while implantation of estrogen pellets results in raised levels of the hormone. Tamoxifen inhibits growth of the PE04 xenograft but not that of the HOX 60 xenograft, consistent with ER status. These results indicate that ER-positive PE04 ovarian cancer cells are sensitive to 17 beta-estradiol in vivo but that the response may be of a different type from the in vitro response. This lends further support to the concept that ovarian cancer may be hormone-sensitive and potentially responsive to endocrine therapy.

(Osborne, 1992) and different studies of prognosis have yielded widely differing results (Hawkins, 1993). We have selected four putative biochemical indices of prognosis and evaluated them in a prospective study. The putative markers chosen were the oestrogen receptor (ER), the epidermal growth factor receptor (EGFR), cathepsin D (cath D) and cyclic AMP-binding protein(s) (c-AMP-b).The oestrogen receptor, a nuclear protein (Mr 66 kDa) has been shown to relate to both endocrine sensitivity (Jensen, 1975) and prognosis in multiple studies, including our own previous work (Humeniuk et al., 1982;Hawkins et al., 1987aHawkins et al., , 1991, although some consider that it is only of prognostic significance in the early years after treatment (e.g. Saez et al., 1983). The receptor for epidermal growth factor is a membrane protein (Mr 180 kDa), which has been reported to be a sign of bad prognosis, again in multiple studies (Sainsbury et al., 1987;Gasparini et al., 1992). The proteolytic enzyme, cathepsin D (Mr 52 kDa), has been suggested to be involved in metastasis formation and reported in several studies also to be a sign of bad prognosis when present in high levels (for a review see Rochefort, 1990). Lastly, work in our own laboratories and elsewhere has demonstrated that high levels of cyclic AMP-binding protein(s), the regulatory subunits of the enzyme protein kinase A, represent another sign of poor prognosis, in both retrospective (Miller et al., 1990) and prospective (Miller et al., 1993)

Summary The effect of systemic therapy on tumour oestrogen receptor (ER) concentration has been studied in 88 patients with large, operable, primary tumours (total 89) of the breast. In 26 patients, tumour was not available for study on one occasion (usually post-treatment). Forty-five patients were treated initially by endocrine therapy but, of these, 13 who had failed to respond went on to receive chemotherapy also. Seventeen patients with low concentrations of ER (<20 fmol mg-' protein) were treated directly by chemotherapy. Patients underwent an incisional biopsy for confirmation of diagnosis and determination of pre-treatment ER by radioligand binding assay, followed by systemic therapy for 3 months (or 6 months for both endocrine and cytotoxic therapies). Following 3 months of systemic therapy (6 months when patients received both endocrine and cytotoxic therapies), patients proceeded on to mastectomy and axillary lymphnode clearance. When residual tumour was present within the mastectomy specimen, a portion was selected for ER assay by the pathologist.In both pre-and post-treatment specimens, a section was cut from the face of the tissue portion used for receptor analysis, fixed in formol-saline and stained with haematoxylin and eosin to permit histopathological confirmation of the presence of tumour. Twenty-six patients in whom either the pre-or post-treatment specimen contained <10% tumour, as assessed by the pathologist, have been excluded from the Correspondence: R.A. Hawkins.