John O. White

Tamoxifen inhibits growth of oestrogen receptor-negative A549 cells

The non-steroidal anti-oestrogen tamoxifen inhibits proliferation of the A549 human lung adenocarcinoma cell line (EC50 congruent to 10 nM) yet there was no evidence of oestrogen receptor expression as determined by ligand binding assay and northern blotting. 17-beta-Oestradiol had no effect on A549 cell proliferation (1 pM-1 microM) and moreover a 100-fold excess failed to reverse the effect of 10 nM tamoxifen as did a 100-fold excess of the steroidal anti-oestrogens ICI 164384 and ICI 182780. However, 4-hydroxytamoxifen which had no significant effect on A549 cell growth (1 pM-1 microM) completely antagonized the effect of 10 nM tamoxifen when used at a 100-fold excess. In the presence of oleic acid and stearic acid (10 microM) the growth inhibitory effect of tamoxifen in A549 cells was greatly enhanced, unlike effects mediated by the anti-oestrogen binding protein described in other cells where these fatty acids had no effect. These results indicate the presence of a unique and highly sensitive mechanism in A549 cells whereby concentrations of tamoxifen relevant to classical receptor binding can inhibit cell growth in the absence of the oestrogen receptor.

The inhibitory effects of danazol, danazol metabolites, gestrinone, and testosterone on the growth of human endometrial cells in vitro

Danazol and gestrinone are effective drugs in the treatment of endometriosis. Their mechanism of action remains uncertain, but may be related to their androgenic activity. The authors examined the effect of danazol on human endometrial cells cultured in vitro, its two major metabolites, ethisterone and 2 hydroxymethyl ethisterone, gestrinone, and testosterone (T) at 1X and 10X expected plasma concentrations. Danazol and T suppressed growth by 20.8 and 25.0% (P less than 0.01), respectively, at the lower dose, and by 26.9 and 35.5% (P less than 0.01), respectively, at the 10-fold higher dose. No significant suppression of growth occurred with gestrinone, ethisterone, or 2 hydroxymethyl ethisterone. The results provide further evidence that danazol and T (but not gestrinone) may act by a direct effect on endometrial tissue.

The biochemistry of human endometrium after two regimens of postcoital contraception: a dl-norgestrel/ethinylestradiol combination or danazol.

A combination of 0.5 mg levonorgestrel (in 1 mg dl-norgestrel) and 0.1 mg ethinylestradiol was administered to eight volunteers 48 hours after the start of the luteinizing hormone surge. A second dose was given 12 hours later. Endometrial samples were obtained 24 hours after the first dose was given. The steroid receptor concentration was compared with ovulatory spontaneous cycles. The dl-norgestrel/ethinylestradiol combination caused a significant reduction in receptor concentration. Isocitrate dehydrogenase (a progestin-sensitive enzyme) was also altered, suggesting an effect on endometrial metabolism. Danazol was used in a similar fashion, with two doses each of 400 mg. Nine volunteers were studied. A similar pattern of alteration of endometrial biochemistry was demonstrated but did not reach statistical significance. The relevance to the postcoital use of hormones is discussed.

Hormonal control of proliferation in the Ishikawa endometrial adenocarcinoma cell line.

Steroid hormone regulation of proliferation of the Ishikawa human endometrial adenocarcinoma cell line was investigated in defined tissue culture medium. Oestradiol increased cell number following treatment for greater than 8 days; 4-OH tamoxifen, used alone, induced growth in a similar manner to oestradiol and was not antagonistic when used in combination with oestradiol. Progesterone decreased cell number 4 days after treatment but thereafter the effect was lost; the effect of progesterone was abolished in the presence of Phenol Red, consistent with the oestrogenic properties of this indicator. Oestradiol together with progesterone for greater than 8 days resulted in maximal growth and was preceded by an apparent increase in synthesis of a protein of molecular weight 36 kDa pI 8.

Adhesion Molecules and the normal Endometrium

The factors contributing to a fertile endometrium receptive to the presenting embryo remain unclear despite continual advances in the field of reproductive medicine. In most animals, it is thought that the endometrium undergoes a series of changes in response to ovarian steroids leading to a period called the window of implantation. Prior to and subsequent to this phase, the uterus is refractory to embryo attachment. In humans, the window of implantation starts approximately six days after ovulation (Day 20) and usually lasts for four days. Associated with the onset of receptivity is the appearance of transient (over 24–48 hours) apical epithelial cellular protrusions called pinopodes. These coincide with an increase in epithelial secretory activity and dilatation of the glands, prominence of epithelial subnuclear vacuoles and cessation of mitotic activity in the epithelial cells. Coincident with the conversion of the endometrial surface from a non-receptive to a receptive state are alterations in the expression of cell surface molecules. The MUC1 mucin is thought to have a pivotal role in embryo attachment and implantation, and is likely to have an impact upon the expression and function of other adhesion molecules, such as the integrins, cadherins (CADs) and CD44.

Isolation of microsomal poly(A)-RNA from rat brain directing the synthesis of the myelin encephalitogenic protein in Xenopus oocytes.

Abstract Microsomal poly(A)-RNA was isolated from brains of myelinating rats. Both free polysomes and the rough endoplasmic reticulum contain this poly(A)-RNA. The poly(A) segment is 80–100 nucleotides long. Brain microsomal poly(A)-RNA directs the synthesis of the myelin encephalitogenic protein in Xenopus oocytes.

c-myc oncogene expression and clinical outcome in carcinoma of the cervix

The expression of the c-myc oncogene was studied in paraffin-embedded specimens of cervical biopsies using a monoclonal antibody which binds to the 62,000 Dalton protein encoded by the c-myc gene. A range of cervical cancers from intraepithelial neoplasia to advanced grade IV tumours were studied together with normal cervical biopsies; c-myc status was correlated to clinical progress. There was no correlation seen between the clinical stage of the disease at presentation and c-myc expression. The 15 patients with c-myc negative cervical cancers were shown to have better disease free (mean--95.4 mos) and total survival (mean 118.0--mos) compared to the 16 patients that were c-myc positive 28.4 and 48.4 mos respectively). The pattern of recurrence differed between the two groups with c-myc positive tumours more likely to develop extra pelvic metastatic disease. The c-myc status of cervical cancer offers a prognostic indicator that could be useful in guiding treatment decisions.

TAMOXIFEN INHIBITS THE RELEASE OF ARACHIDONIC ACID STIMULATED BY THAPSIGARGIN IN ESTROGEN RECEPTOR-NEGATIVE A549 CELLS

In pre-labelled A549 cells the tumour promoter thapsigargin (50 nM) stimulates the release of [5,6,8,9,11,12,14,15-3H(N)]-arachidonic acid (3H-AA) by ca. 300% above basal levels. A549 cells are estrogen receptor negative (ER-), yet this stimulation by thapsigargin is inhibited in a dose-dependent manner by a 3 h pre-treatment with the anti-estrogen tamoxifen (1-20 microM). Moreover, the presence of excess (100 microM) estradiol does not reverse this effect of tamoxifen. Thapsigargin stimulated 3H-AA release is not inhibited over the same concentration range by 4 hydroxy-tamoxifen nor by the steroidal anti-estrogen ICI 164384. However, the steroidal anti-estrogen ICI 182780 inhibits thapsigargin stimulated 3H-AA release in a similar manner to tamoxifen and this effect is also not reversed by the presence of excess estradiol. Stimulation of 3H-AA release by EGF (10 nM), IL-1beta (1 ng ml-1) and bradykinin (100 nM) was unaffected by these concentrations of tamoxifen. Ionomycin (10 microM) stimulates 3H-AA release by ca. 700% and A23187 (10 microM) by ca. 300% above basal levels. Pre-treatment with tamoxifen (1-20 microM) inhibits 3H-AA release stimulated by both these agents and again the presence of excess estradiol does not reverse this effect. Unlike the effects of glucocorticoids on 3H-AA release in A549 cells the effects of tamoxifen are not reversed by neutralizing anti-bodies to lipocortin 1. Arachidonic acid release is central to cell proliferation in A549 cells and we propose that this action of tamoxifen could explain the anti-proliferative effect seen in these cells and could have important implications for control of cell proliferation of ER- cells in general.

The effects of medroxyprogesterone acetate on enzyme activities in human endometrial carcinoma

Creatine kinase (CK), isocitrate (ICDH) and glucose-6-phosphate dehydrogenase (G-6-PD) activities and cytosol oestrogen (REc) and progestin (RPc) receptors have been measured in twenty post-menopausal subjects with endometrial carcinoma. Diagnostic curettage and hysterectomy specimens were obtained from each patient with intervening medroxyprogesterone acetate therapy (MPA). In RPc rich tumours the activity of creatine kinase was significantly increased following MPA and was attributed to an increase in the BB-isoform. There was a similar though less significant increase in ICDH activity. Neither CK nor ICDH were significantly increased following MPA therapy in progesterone receptor poor specimens. There was a small but significant increase in G-6-PD activity following MPA regardless of RPc content. REc was decreased in some but not all RPc rich tumours following MPA and the trend was significant. The highly significant increase in CK activity confirms the apparent response of this enzyme to progesterone in normal human endometrium during the menstrual cycle.

Activated oestrogen receptors in breast cancer and response to endocrine therapy.

The status of oestrogen and progesterone receptors has been measured in 147 primary breast tumours. In addition to the measurement of cytoplasmic oestrogen receptors, the ability of these receptors to bind to oligo(dT)-cellulose has been assessed. This indicates the capability for activation of cytoplasmic receptors to a form able to bind in the nuclear compartment in vivo and thus be part of a functional receptor pathway. All the receptor concentrations measured were increased in the postmenopausal group of patients. All nuclear oestrogen receptors in this group were available for labelling at 4 degrees C, in contrast to the premenopausal group. The apparent functionality of the oestrogen receptor pathway could be equally assessed either by the co-presence of cytosol progesterone receptor with nuclear oestrogen receptor (30 or 4 degrees C) or with activated cytosol oestrogen receptor. The presence of activated cytosol oestrogen receptor was as reliable (80%) as the presence of either nuclear oestrogen receptor at 30 (83%) or 4 degrees C (81%) in predicting the response of breast tumours to endocrine therapy.