J.H.H. Thijssen

Effects of seven low-dose combined oral contraceptives on sex hormone binding globulin, corticosteroid binding globulin, total and free testosterone

The effect of seven low-dose oral contraceptive preparations on sex hormone binding globulin (SHBG), cortisol binding globulin (CBG), total and absolute free testosterone were investigated in groups of 10 healthy volunteers. All preparations contained about the same amount of ethinylestradiol but they differed in type and/or dose of progestagen. The progestagens studied were: levonorgestrel (LNG; in mono- and triphasic preparations), norethisterone (NET; in monophasic preparation), desogestrel (DSG; in mono- and biphasic preparations) and gestodene (GSD; in triphasic preparation), all 19-nortestosterone derivatives, and the anti-androgen cyproterone acetate (CPA) in a monophasic preparation. Differences observed in SHBG level, which reflect the estrogen-androgen balance, can be attributed to the intrinsic androgenic (or anti-androgenic) properties of the progestagens, and were in agreement with the results of published receptor binding studies, performed in vitro. Based on our results the following ranking (high to low) can be made with respect to the androgenicity of the preparations: monophasic LNG greater than or equal to monophasic NET = triphasic LNG greater than or equal to triphasic GSD = biphasic DSG = monophasic DSG greater than monophasic CPA. An anti-estrogenic effect of the 19-nortestosterone derived progestagens can be excluded by the effect on CBG, a marker for estrogenic activity. All preparations containing a 19-nortestosterone derived progestagen, independent of their type and dose, induce a similar rise in CBG, whereas the preparation with cyproterone acetate induced an even higher CBG level. Irrespective of the effect on total testosterone, which varies between the preparations, the absolute free testosterone level decreased to a comparable degree for all preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Aromatase and COX-2 expression in human breast cancers☆

We have investigated aromatase and the inducible cyclooxygenase COX-2 expression using immunocytochemistry in tumors of a series of patients with advanced breast cancer treated with aromatase inhibitors. Aromatase was expressed in 58/102 breast cancers. This is similar to the percentage previously reported for aromatase activity. Interestingly, aromatase was expressed in a variety of cell types, including tumor, stromal, adipose, and endothelial cells. Since prostaglandin E2 is known to regulate aromatase gene expression and is the product of COX-2, an enzyme frequently overexpressed in tumors, immunocytochemistry was performed on the tissue sections using a polyclonal antibody to COX-2. Aromatase was strongly correlated (P<0.001) with COX-2 expression. These results suggest that PGE2 produced by COX-2 in the tumor may be important in stimulating estrogen synthesis in the tumor and surrounding tissue. No correlation was observed between aromatase or COX-2 expression and the response of the patients to aromatase inhibitor treatment. However, only 13 patients responded. Nine of these patients were aromatase positive. Although similar to responses in other studies, this low response rate to second line treatment suggests that tumors of most patients were no longer sensitive to the effects of estrogen. Recent clinical studies suggest that greater responses occur when aromatase inhibitors are used as first line treatment. In the intratumoral aromatase mouse model, expression of aromatase in tumors is highly correlated with increased tumor growth. First line treatment with letrozole was effective in all animals treated and was more effective than tamoxifen in suppressing tumor growth. Letrozole was also effective in tumors failing to respond to tamoxifen, consistent with clinical findings. In addition, the duration of response was significantly longer with the aromatase inhibitor than with tamoxifen, suggesting that aromatase inhibitors may offer better control of tumor growth than this antiestrogen.

Occurrence, regulation, and significance of progesterone receptors in human meningioma.

The abundant expression of progesterone receptors (PR) in human meningiomas is well established. It is unknown, however, how PR expression is regulated, especially since estrogen receptors (ER) are virtually absent in these tumors. At the mRNA level, ER splice variants occur in meningioma but these appear not to be involved in the apparently autonomous PR expression. In an earlier study, because other ER-inducible proteins were either not expressed at all (pS2) or were expressed at a very low level compared to their expression in breast cancer (Cathepsin-D), the authors have postulated that the autonomous PR expression in meningioma is PR promoter-related rather than ER-related and have studied PR expression in cultured meningioma cells. PR levels appeared to decrease rapidly in vitro in monolayer as well as in three dimensional spheroid cultures. Culture conditions thus are not yet sufficient for the quantitative evaluation of PR expression. To evaluate whether PR deterioration is associated with cell turnover (meningiomas grow much faster in vitro than in vivo), the relationship between expression of the apoptotic proteins Bcl-2 and Bax and PR expression was investigated. Bcl-2 expression was found to be highest in meningioma with low PR levels, and in breast cancer tissue with high PR levels. Bax expression was not related to PR expression in any of the two tissues. Given the potential benefit of antiprogestin treatment and the occurrence in meningiomas of a protein capable of binding to the estrogen-responsive element, the expression of PR in meningioma remains a fascinating phenomenon which requires further investigation.

Endogenous oestrogens and androgens in normal and malignant endometrial and mammary tissues

Because of a well-established mechanism of action, tissue concentrations of steroid hormones are thought to be more closely related than blood levels to the biological effects exerted by these hormones. The results of studies on oestrogen and androgen concentrations in malignant and normal breast tissues are presented. Normal fatty and epithelial breast tissues and malignant tumour samples which had been obtained from pre- and postmenopausal women of two countries (Poland and The Netherlands) differing in the incidence of this malignancy were studied. In both countries highly comparable oestradiol concentrations in the breast were found. The median hormone levels in tumour tissue of 0.65 pmol/g tissue did not change with age. They were significantly higher than in normal epithelial (0.48 and 0.25 pmol/g in pre- and postmenopausal women) and fatty tissues (0.54 and 0.19 pmol/g respectively). Particularly in postmenopausal women, hormone levels in tumour tissue were much higher than plasma concentrations, which are comparable in both populations. Oestrone levels decreased with age in normal and malignant breast tissues. In both countries median levels in normal and fatty tissues of premenopausal women were similar (1.10 pmol/g tissue) but higher than those in postmenopausal patients (0.45 pmol/g tissue. Significantly lower levels were found in the malignant tissue samples of Polish premenopausal women (0.70 pmol/g) than in Dutch women (1.05 pmol/g); similarly, after menopause the tissue concentrations were higher in Dutch (0.55 pmol/g) than in Polish (0.31 pmol/g) patients. Thus lower oestrone tissue levels were observed in tumours from the country with the lower incidence for breast cancer. In a comparable study of uterine tissues, obtained from pre- and postmenopausal women, higher oestradiol concentrations than in the breast were found, whereas estrone levels were very similar. The levels in the uterus did not correlate with those in the plasma; no relation with histology was observed. The results of androgen measurements in breast tissues were in agreement with the concept that, particularly, androstenedione and testosterone could play a role as substrates for local aromatization. Lower concentrations were observed in the tumours than in the normal and fatty tissues. More extensive investigations will be needed to clarify the role of local formation (aromatization, hydrolysis by sulphatase) of oestrogens in tissues and of the interconversion of less active (oestrone) to more active (oestradiol) oestrogens.

Intratumoral levels of estrogens in breast cancer

Breast cancer tissue is an endocrine organ and particularly the estrogen biosynthetic properties of this tissue have been well studied. The concentration of estradiol in breast cancer tissue from postmenopausal patients is considerably higher than that in the circulation and appears to depend largely on local production. Androgenic precursor steroids are abundantly present, but estrogen storage pools like fatty acid derivatives appear to be less important than initially thought. New, potent and highly specific aromatase inhibitors effectively inhibit peripheral conversion of androgens to estrogens (Cancer Res. 53: 4563, 1993) as well as intratumour aromatase, median aromatase activity being 89% lower in the tissue from patients pretreated with aromatase inhibitor 7 days prior to surgery (P < 0.001). Also the intratissue concentrations of estrogens were decreased (64% and 80% reduction, respectively for estrone and estradiol; P = 0.001 and <0.05; Cancer Res. 57: 2109, 1997). These results illustrate that intratissue estrogen biosynthesis is effectively inhibited by the new generation of aromatase inhibitors. The pathophysiological consequences of this finding are currently under study.

Functional analysis of an alternatively spliced estrogen receptor lacking exon 4 isolated from MCF-7 breast cancer cells and meningioma tissue

Abstract An alternatively spliced mRNA coding for a variant estrogen receptor (ER) missing exon 4 (ER Δ4) was detected in the breast tumor cell line MCF7 and meningioma tissue by using the reversed transcriptase PCR technique. The trans-activational properties of this mutant ER were assessed in embryo carcinoma P19EC and human choriocarcinoma JEG3 cells by co-transfection of the ER Δ4 expression vector with an oxytocin promoter construct containing an estrogen-responsive element. ER Δ4 did not trans-activate the oxytocin promoter in either a hormone-dependent or -independent manner. Co-transfection of ER Δ4 together with the wtER did not show any interference of ER /gDA4 on the stimulation of the oxytocin promoter by the wtER. ER Δ4 was translated in vitro. Its capacity to bind estradiol, and the binding of the variant to a synthetic estrogen-responsive element were compared to those of the wild-type receptor. ER Δ4 did not bind to a synthetic estrogen-responsive element, nor did it bind estradiol. Hence, ER Δ4 appears to be a silent variant and we speculate that it is without any role in tumor progression.

On the significance of in situ production of oestrogens in human breast cancer tissue.

We have previously shown that human breast cancer is autonomous in the regulation of its intra-tissue oestradiol concentration. Breast fatty tissue does not have this capacity, but rather reflects changes in the peripheral oestradiol concentration. To further evaluate the relative contribution of breast cancer and fatty tissue to the maintenance of tumour oestradiol we investigated whether a tumour-directed gradient in aromatase activity and oestrogen levels existed in mastectomy specimens. No such gradient was found, however, for aromatase, oestrone, oestradiol and their sulphates. Aromatase activity (expressed per gram of tissue) and the concentrations of oestradiol, oestradiol sulphate and oestrone sulphate were higher in tumour than in breast fatty tissue. Fatty tissue had a higher oestrone concentration. It is tentatively concluded that breast tumour aromatase activity is more important for the maintenance of tumour oestradiol levels than aromatase in breast fatty tissue.

Wild type and alternatively spliced estrogen receptor messenger RNA in human meningioma tissue and MCF7 breast cancer cells

Human meningiomas are rich in progesterone receptors (PR), which appear to be expressed autonomously. To investigate whether estrogen receptor (ER) variants which do not bind the ligand, but may constitutively induce PR expression, prevail in meningioma, we amplified cDNA by PCR in order to detect mRNA coding for the ER in meningioma which were ER-negative/PR-positive at the protein level. We screened for a portion of the ER which includes the DNA binding domain, the hinge region and the ligand binding domain. For this part of the ER we found a wild type mRNA in all 8 meningiomas tested. No mutations were detected. Apart from this transcript we found two alternatively spliced products missing exons 4 and 7, respectively in 8/8 meningioma specimens. These two products were not exclusive for meningioma, since they were also detected in the MCF7 breast cancer cell line which was used as control. ER deletion mutants missing exon 7 have already been reported [Ref. 1; Molec. Endocr. 5 (1991) 1571-1577]. These are dominant negative. To our knowledge, this is the first report on ER mutants missing exon 4. The presence of ER variants missing exon 4, which is probably not able to bind heat shock protein 90 and therefore may be constitutively active, might explain the autonomous expression of PR in meningioma.

Endogenous steroid hormones and local aromatase activity in the breast

To test the hypothesis of an increased activity of the enzyme aromatase in adipose tissue from affected when compared with non-affected quadrants of patients with breast cancer, the aromatase activity has been measured in tumour and fatty tissues dissected at specific sites from the breasts of 16 patients. Activity was measured after extensive purification of the product formed. Results, expressed in fmol/g of tissue, did not show a higher activity in the affected vs the non-affected quadrants. In the tumours, higher activities were found when expressed per g of tissue. Per mg of DNA, an indicator of the number of cells, tumour enzymatic activity was lower than in fatty tissues. The relations between the products of aromatase, oestrone and oestradiol in the various tissues point to the importance of additional enzymatic processes, especially of the reductive 17 beta-oestradiol dehydrogenase, in the accumulation of high quantities of oestradiol in the malignant tissue.

Plasma oestrone, oestradiol and androstenedione levels in post-menopausal women: relation to body weight and height.

To study the relation between body weight and height and the plasma sex-hormone levels wer measured the plasma levels of oestrone (E1), oestradiol (E2) and androstenedione (A) in a group of healthy post-menopausal women with a wide range of body weight. The sex hormones were measured by radioimmunoassay with highly specific antisera after purification of the plasma extract by column chromatography. Our findings show that there is significant positive correlation between the E1 level and body weight as well as A level and, to a lesser extent, between the E1 level and Quetelet index. For E2 there is no correlation with these parameters. There is also a very slight correlation between A level, body weight and Quetelet index. Calculation of the partial correlation coefficient shows that E1 correlates to the same degree with body weight and A level, whereas the A level does not correlate with body weight at a fixed value for the E1 level. We conclude that variation in the E1 level depends to the same degree on the variation in body weight as well as the variation in A level.