The 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase (3beta-HSD) isoenzymes are responsible for the oxidation and isomerization of Delta(5)-3beta-hydroxysteroid precursors into Delta(4)-ketosteroids, thus catalyzing an essential step in the formation of all classes of active steroid hormones. In humans, expression of the type I isoenzyme accounts for the 3beta-HSD activity found in placenta and peripheral tissues, whereas the type II 3beta-HSD isoenzyme is predominantly expressed in the adrenal gland, ovary, and testis, and its deficiency is responsible for a rare form of congenital adrenal hyperplasia. Phylogeny analyses of the 3beta-HSD gene family strongly suggest that the need for different 3beta-HSD genes occurred very late in mammals, with subsequent evolution in a similar manner in other lineages. Therefore, to a large extent, the 3beta-HSD gene family should have evolved to facilitate differential patterns of tissue- and cell-specific expression and regulation involving multiple signal transduction pathways, which are activated by several growth factors, steroids, and cytokines. Recent studies indicate that HSD3B2 gene regulation involves the orphan nuclear receptors steroidogenic factor-1 and dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1). Other findings suggest a potential regulatory role for STAT5 and STAT6 in transcriptional activation of HSD3B2 promoter. It was shown that epidermal growth factor (EGF) requires intact STAT5; on the other hand IL-4 induces HSD3B1 gene expression, along with IL-13, through STAT 6 activation. However, evidence suggests that multiple signal transduction pathways are involved in IL-4 mediated HSD3B1 gene expression. Indeed, a better understanding of the transcriptional factors responsible for the fine control of 3beta-HSD gene expression may provide insight into mechanisms involved in the functional cooperation between STATs and nuclear receptors as well as their potential interaction with other signaling transduction pathways such as GATA proteins. Finally, the elucidation of the molecular basis of 3beta-HSD deficiency has highlighted the fact that mutations in the HSD3B2 gene can result in a wide spectrum of molecular repercussions, which are associated with the different phenotypic manifestations of classical 3beta-HSD deficiency and also provide valuable information concerning the structure-function relationships of the 3beta-HSD superfamily. Furthermore, several recent studies using type I and type II purified enzymes have elegantly further characterized structure-function relationships responsible for kinetic differences and coenzyme specificity.

Molecular Biology of the 3β-Hydroxysteroid Dehydrogenase/Δ5-Δ4 Isomerase Gene Family

The presence of mRNA transcripts for cytokines in normal and neoplastic human breast tissue has been investigated. Using reverse transcriptase-linked polymerase chain reaction (RT-PCR), we have specifically screened for the following cytokines: interleukin (IL)-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, tumour necrosis factor (TNF)-α, TNF-β and interferon (IFN)-γ. No significant differences in expression of IL-1α, IL-1β, IL-4, IL-6, TNF-α or TNF-β were observed between the 2 groups of tissues. However, there was a significant difference in expression of IL-8 transcripts (p = 0.0017) which was higher in the neoplastic population. Transcripts for IL-2, IL-3, IL-5, IL-7 and IFN-γ were not detected in either group. There was no evidence of associations between cytokine expression and tumour histological grade, patient age or lymph node metastases. Correlating tumour types with specific cytokine transcripts revealed high expression of IL-8, and to a lesser extent, IL-8 and TNF-β irrespective of tumour origin. Analysis of primary epithelial and stromal cultures derived from both types of tissue showed that increased levels of IL-8, but not IL-6, were secreted by cells obtained from tumours. Thus, breast tissue of both normal and neoplastic origin expresses a wide range of cytokines. Increased or aberrant expression of cytokines, in particular IL-8, may be involved in the development/progression of breast cancer. Int. J. Cancer 72:937–941, 1997. © 1997 Wiley-Liss, Inc.

Expression of cytokine messenger RNA in normal and neoplastic human breast tissue: Identification of interleukin-8 as a potential regulatory factor in breast tumours

I. Introduction II. Peripheral Estrogen Synthesis and Blood and Tissue Estrogens in Breast Cancer A. Peripheral estrogen synthesis B. Aromatase C. Estrone sulfatase D. E2DH E. Origins of breast tumor estrogens III. Influence of Breast Tumor Location on Enzyme Activities in Adjacent Tissues IV. Proposed Model for the Regulation of Estrogen Synthesis in Breast Tumors V. Evidence in Support of Proposed Model of Cytokine Regulation of Estrogen Synthesis A. Identification of factors that stimulate estrogen synthesis B. Potentiation of cytokine stimulation of estrogen synthesis VI. The Role of Soluble Cytokine Receptors in Cytokine Action A. Mechanism of cytokine action B. Regulation of gp80 and gp130 C. Regulation of IL-6 sR shedding VII. Origin of Estrogen-Stimulating Factors in Breast Tumors A. The role of cells of the immune system B. Macrophages and lymphocytes in breast tumors VIII. Regulation of Lymphocyte Cytokine Production A. Th1 and Th2-helper cells B. Dehydroepiandrosterone (DHA) and glucocorticoids...

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Breast Cancer and the Role of Cytokines in Regulating Estrogen Synthesis: An Emerging Hypothesis

There is now substantial evidence that many cytokines can influence the function of endocrine tissues (see Kennedy & Jones, 1991). These include interleukin1 a (IL-1 a ) and interleukin-l@ (IL-l@), interleukin-2 (IL-2), interleukin-6 (IL-6), tumour necrosis factor-a (TNF-a), y-interferon (INF-7) and transforming growth factor-@ (TFG-@). IL-6 is a pleiotrophic cytokine which has a systemic role in infections and inflammatory disorders as part of the acute phase response but is also released locally in normal, inflamed and neoplastic tissues. IL-6 was initially found to be released by a human T-cell hybridoma, T-lymphocytes and by activated B-lymphocytes but has since been found to be produced by a variety of other cell types such as fibroblasts, endothelial cells, macrophages and by many different tumour cells (Kishimoto, 1989; Akira & Kishimoto, 1992). IL-6 is one of the major substances released during the acute phase response and promotes the release of other acute phase proteins from the liver. IL-6 is a glycoprotein with a molecular weight heterogeneity of 20-30 kDa, the human form consisting of 212 amino acids. Granulocyte-colony stimulating factor (G-CSF) is the only protein which shows a significant homology with IL-6. IL-6 acts at a specific receptor which is also a glycoprotein, having extracellular and intracellular components. The IL-6 receptor possesses a similar binding domain to that of the growth hormone, prolactin and erythropoietin receptors (Bazan, 1989). The major part of the receptor is extracellular and on activation by IL-6 binds to another membrane glycoprotein, gp130 (Kishimoto et al., 1992). It is the gp130 molecule which transduces the IL-6 signal although the mechanism by which this occurs has not been elucidated. There is, however, some recent evidence that in human trophoblasts, IL-6 activates a tyrosine kinasedependent signalling mechanism in the release of hCG (Neki et ul., 1993). In various cell types, the production of IL-6 is affected by several common factors which include IL-la, IL-I@, bacterial endotoxin lipopolysaccharide, y-interferon, platelet derived growth factor and prostaglandins, each of which has a stimulatory effect, and glucocorticoids which are inhibitory. The 5’ promoter region of the IL-6 gene has three functional cis-regulatory elements (Kishimoto, 1989) which comprise cyclic AMP-responsive, glucocorticoid-responsive and multi-response elements. The muti-response element can be activated by IL1, TNF-a, serum, phorbol esters and IL-6. IL-6 gene expression is activated in a number of human diseases which include multiple myeloma, melanomas, meningiomas, cardiac myxomas, Kaposi’s sarcoma, Castleman’s disease, mesangial proliferative glomerulonephritis, rheumatoid arthritis and psoriasis (Akira & Kishimoto, 1992). In multiple myeloma, mesangial proliferative glomerulonephritis, Kaposi’s sarcoma and some lymphoma and leukaemia cell lines, IL-6 acts in vitro as an autocrine growth factor (Akira & Kishimoto, 1992), whereas in human breast carcinoma cell lines IL-6 inhibits growth (Chen et al., 1991). There are close interactions between the immune and endocrine systems with cytokines acting as hormonal messengers. The raised plasma level of IL-6 can directly affect the hypothalamic-pituitary axis and other endocrine glands to induce changes in hormone secretion. More recently, there is convincing evidence that IL-6 is produced within endocrine glands and specifically in some instances by endocrine cells both normal and neoplastic (Tzble 1 ) . The functions of IL-6 in the endocrine system are not at present clear but it can affect hormone production and there is early evidence that it may control cell growth and apoptosis (cell death). The synthesis and release of IL-6 is partly regulated by hormones, particularly by glucocorticoids. The purpose of this article is to review the current knowledge of IL-6 production and actions within the endocrine system and try to explore its potential roles in normal and disease processes.

Interleukin-6 an endocrine cytokine

As experimental models for breast cancer, most studies rely on established human breast cancer cell lines. However, many of these lines were established over 20 years ago, many from pleural effusions rather than the primary tumour, so the validity of using them as representative models is questionable. This paper describes our experiences, over a 3-year period, in establishing short-term epithelial-cell-enriched preparations from primary breast tumours based on differential centrifugation followed by culture in selective media. Epithelial cells were successfully cultured from 55% of samples, but culture success did not appear to be correlated with tumour histology, stage, grade or node status. Epithelial cell-enriched cultures were immunopositive for broad-spectrum cytokeratin and epithelial membrane antigen (EMA). Positivity for keratin 19 confirmed that the cultures contained tumour-derived cells, which additionally showed significantly higher activity of the reductive pathway of the steroid-converting enzyme 17beta-hydroxysteroid dehydrogenase type I. That the cultures contained tumour and not normal epithelial cells was further substantiated by the complete absence of the calmodulin-like gene NB-1 in tumour-derived cultures; this is only associated with normal breast epithelia. Eighty-five per cent of cultures established from oestrogen receptor (ER)-positive tumours expressed ER in vitro; this was functional in 66% of cultures, although ER-positive phenotype was gradually lost over time. In conclusion, epithelial cells can be isolated and maintained as short-term cultures from primary breast tumours irrespective of histopathological or clinical details, providing a model system with a greater biological and clinical relevance than breast cancer cell lines.

Short-term primary culture of epithelial cells derived from human breast tumours.

Interactive effects of interleukin-6, 17β-estradiol and progesterone on growth and 17β-hydroxysteroid dehydrogenase activity in human breast carcinoma cells

B. Rafferty

Papers

Interactive effects of interleukin-6, 17β-estradiol and progesterone on growth and 17β-hydroxysteroid dehydrogenase activity in human breast carcinoma cells