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ASTRACT

The effect of two anti-oestrogens, 4-OH tamoxifen and ICI 164,384, on growth and progesterone receptor (PR) concentration was investigated in the endometrial carcinoma cell line, Ishikawa. Growth stimulation in response to 4-OH tamoxifen was antagonized by ICI 164,384, the latter having no agonist effect when used as a single agent. Similarly, ICI 164,384 antagonized oestradiol-stimulated cell growth. PR was significantly increased following treatment with 4-OH tamoxifen, this response being antagonized in the presence of ICI 164,384. Oestradiol increased PR, although to a lesser extent than did 4-OH tamoxifen;the effect of oestradiol on PR was also antagonized by ICI 164,384. Used as a single agent, ICI 164,384 induced a moderate but statistically significant increase in PR, thus demonstrating partial agonist activity. This agonist property of ICI 164,384 may provide a mechanism of maintaining PR, which is down-regulated during conventional progestin therapy, without undesirable mitogenic activity.

ABSTRACT

Expression of the RNA coding for the ACTH—β-lipotrophin precursor, pro-opiomelanocortin (POMC), has been demonstrated in five human small-cell lung cancer (SCLC) cell lines. Using Northern and slot-blot hybridization analysis of RNA and a bovine POMC cDNA as probe, the processed POMC RNA from SCLC cells was found to be approximately 1350 nucleotides in length, which is larger than that found in the normal human pituitary. Expression of the POMC gene was confirmed by measurement of ACTH precursors secreted by the cells, using a novel two-site immunoradiometric assay based on monoclonal antibodies, which directly quantifies both POMC and pro-ACTH but does not recognize ACTH. Levels of POMC in medium accumulated throughout the growth of the cells, in contrast to POMC RNA which showed a relatively constant level of expression. We conclude that human SCLC cell lines are valuable models for studying the aberrant expression and regulation of the human POMC gene.

ABSTRACT

In the normal pituitary, glucocorticoids are the principal negative regulator of the pro-opiomelanocortin (POMC) gene which gives rise to the biologically active peptides ACTH and β-endorphin. In Cushing's syndrome, ACTH-secreting pituitary tumours show a degree of glucocorticoid resistance, whilst ACTH-secreting extra-pituitary tumours have an even greater resistance to glucocorticoid excess. In an attempt to understand the mechanism of this phenomenon, we have compared the effects of glucocorticoids on POMC mRNA and peptide secretion in human and mouse corticotroph adenoma cells and in small cell lung carcinoma (SCLC) cells. ACTH precursor peptides were inhibited within 24 h by 25–50 nm hydrocortisone in primary cultures from a human corticotroph adenoma. In the mouse corticotroph adenoma cell line (AtT20), inhibition of both ACTH precursors and ACTH was not observed after 24 h but, by 10 days, glucocorticoids suppressed peptide levels with a concentration causing 50% inhibition of 50 nm hydrocortisone and maximal inhibition at 500 nm hydrocortisone. In marked contrast, there was no response to 500 nm hydrocortisone in the five SCLC cell lines (COR L103, COR L42, COR L24, COR L31, DMS 79) all of which secrete ACTH precursors. However, two of the five SCLC cell lines (COR L31 and DMS 79) were responsive to 1000 nm hydrocortisone. POMC mRNA, quantitated by slot-blot analysis, gave similar results for the five SCLC cell lines, implying that the abnormality may occur at the level of gene expression. When one of the three resistant cell lines (COR L103) was incubated with 2000 nm hydrocortisone or 2000 nm dexamethasone a clear suppression of precursor peptides and POMC mRNA was observed. This suggests that the resistance to glucocorticoid inhibition is relative rather than absolute, implying that the normal mechanism is functioning but impaired. Furthermore, there is at least a 20-fold difference in the responsiveness to glucocorticoid inhibition between pituitary and extra-pituitary tumour cells in vitro, which may signify a difference in the underlying mechanism in these two cell types.

We previously identified a novel pathway of testosterone action via the androgen receptor (AR) in bone marrow mesenchymal precursor cells (BM-PCs) to negatively regulate fat mass and improve metabolic function in male mice. This was achieved using our PC-AR Gene Replacement mouse model in which the AR is only expressed in BM-PCs and deleted in all other tissues. We hypothesise that the markedly reduced fat mass and increased insulin sensitivity of PC-AR Gene Replacements will confer protection from diet-induced overweight and obesity. To test this, 6-week-old male PC-AR Gene Replacements and controls (WT, global-AR knockouts (KOs)) were fed a chow or high-caloric diet (HCD) for 8 or 18 weeks. Following 8 weeks (short-term) of HCD, WT and Global-ARKOs had markedly increased subcutaneous white adipose tissue (WAT) and retroperitoneal visceral adipose tissue (VAT) mass compared to chow-fed controls. In contrast, PC-AR Gene Replacements were resistant to WAT and VAT accumulation following short-term HCD feeding accompanied by fewer large adipocytes and upregulation of expression of the metabolic genes Acaca and Pnlpa2. Following long-term HCD feeding for 18 weeks, the PC-AR Gene Replacements were no longer resistant to increased WAT and VAT adiposity, however, maintained their improved whole-body insulin sensitivity with an increased rate of glucose disappearance and increased glucose uptake into subcutaneous WAT. In conclusion, the action of testosterone via the AR in BM-PCs to negatively regulate fat mass and improve metabolism confers resistance from short-term diet-induced weight gain and partial protection from long-term diet-induced obesity in male mice.