The Mediet Project.

Preliminary evidence from a case control study of healthy postmenopausal women living in Palermo, Sicily, is presented to investigate the potential impact of a traditional Mediterranean diet on the risk of developing breast cancer. Of the 230 women who fulfilled specific eligibility criteria, 115 were enrolled in the study based on serum testosterone values equal to or greater than the median population value (0.14 microg/ml). Women were then individually randomized into a diet intervention (n = 58) and a control (n = 55) group. Women in the intervention group attended a weekly "cooking course" for 1 year, being trained by professional chefs in the correct use of the natural ingredients of the traditional Mediterranean diet, including whole cereals, legumes, seeds, fish, cruciferous vegetables, and many others. The intervention group was subsequently instructed to follow the learned diet at home, while the control group was only advised to increase the consumption of fruits and vegetables, as recommended by WHO. The following measures were taken at the beginning, middle, and end of the study: (a) fasting blood and 12-hour urine samples to assay defined hormonal endpoints; (b) height, weight, and circumference of the waist and hip; and (c) a food frequency and computerized 24-hour dietary recall questionnaire. After 1 year, both the control and the intervention groups showed satisfactory compliance rates (81 and 85%, respectively). In addition, preliminary results so far obtained reveal an unequivocal trend towards weight loss, a strong reduction in cholesterol levels, and a psychophysical feeling of well-being by women adopting the Mediterranean diet. The study is currently ongoing to verify the association of changes in serum and urine hormone levels and breast cancer risk in the intervention group.

Endocrine end-points in rheumatoid arthritis.

Our previous studies are reviewed and at the same time preliminary experimental observation to the topic of endocrine end-points in autoimmune disease is introduced. To this end, we have used rheumatoid arthritis (RA), including synovial fluids and primary cultures of synovial macrophages, as a model system in order to investigate (a) expression and subcellular localization of high-affinity sites of steroid binding in immune effector cells; (b) steroid metabolic profiles in both male and female RA patients, as compared to healthy subjects; and (c) activities of key steroid enzymes that govern intratissue accumulation of sex hormones. In RA tissues and cells, the concurrent evidence for (1) androgen and/or estrogen receptors, (2) high concentrations of biologically active steroids, (3) key enzymes of steroid metabolism, and (4) significant changes of estrogen to androgen ratio, all strongly suggests that individual immune cells, including synovial macrophages, may behave as steroid-sensitive cells, namely, they may represent a target for sex steroids, supporting the hypothesis of a potential endocrine regulation of the immune response also in RA disease. In this respect, definition of several endocrine end-points may have important implications for the treatment of rheumatic disease and other immunological disorders.

Alteration of oestradiol metabolism in myc oncogene-transfected mouse mammary epithelial cells.

Targeted overexpression of the c-myc oncogene induces neoplastic transformation in immortalized, non-tumorigenic mouse mammary epithelial cells (MMEC). Experiments in the present study were conducted to examine whether cellular transformation induced by c-myc oncogene is associated with altered metabolism of 17beta-oestradiol (E2). The parental, MMEC and the stable c-myc transfectant (MMEC/myc3) cell lines were compared for major oestrogen metabolic pathways, namely E2 and E1 interconversion, and C2- and C16alpha-hydroxylation by both high-pressure liquid chromatography (HPLC) analysis and the 3H release assay using specifically labelled [C2-3H]E2 or [C16alpha-3H]E2. The reductive conversion of E1 to E2 was about 14-fold and 12-fold higher than the oxidative conversion of E2 to E1 in MMEC and MMEC/myc3 cells respectively. However, in MMEC/myc3 cells, both reductive and oxidative reactions were decreased by about 32% and 12% relative to those seen in the parental MMEC cells (P = 0.0028). The extent of C16alpha-hydroxylation was increased by 164.3% (P < 0.001), with a concomitant 48.4% decrease (P < 0.001) in C2-hydroxylation in MMEC/myc3 cells; this resulted in a fourfold increase in the C16alpha/C2 hydroxylation ratio in this cell line. Thus, a persistent c-myc expression, leading to aberrant hyperproliferation in vitro and tumorigenesis in vivo, is associated with an altered oestrogen metabolism. However, it remains unclear whether this represents a result of oncogene expression/activation or is rather a consequence of phenotypic transformation of the cells.

Expression of different 17beta-hydroxysteroid dehydrogenase types and their activities in human prostate cancer cells.

The 17beta-hydroxysteroid dehydrogenase (17betaHSD) enzyme system governs important redox reactions at the C17 position of steroid hormones. Different 17betaHSD types (no. 1-4) have been identified to date in peripheral human tissues, such as placenta, testis, and breast. However, there is little information on their expression and activity in either normal or malignant prostate. In the present work, we have inspected pathways of 17beta-oxidation of either androgen or estrogen in human prostate cancer cells (LNCaP, DU145, and PC3) in relation to the expression of messenger RNAs (mRNAs) for 17betaHSD types 1-4. These cell systems feature distinct steroid receptor status and response to hormones. We report here that high expression levels of 17betaHSD4 were consistently observed in all three cell lines, whereas even greater amounts of 17betaHSD2 mRNA were detected solely in PC3 cells. Neither 17betaHSD1 nor 17betaHSD3 mRNAs could be detected in any cell line. From a metabolic standpoint, intact cell analysis showed a much lower extent of 17beta-oxidation of both androgen [testosterone (T)] and estrogen [estradiol (E2)] in LNCaP and DU145 cells compared to PC3 cells, where a greater precursor degradation and higher formation rates of oxidized derivatives (respectively, androstenedione and estrone) were observed. Using subcellular fractionation, we have been able to differentiate among 17betaHSD types 1-4 on the basis of their distinct substrate specificities and subcellular localization. This latter approach gave rise to equivalent results. PC3 cells, in fact, displayed a high level of microsomal activity with a low E2/T activity ratio and approximately equal apparent Km values for E2 and T, suggesting the presence of 17betaHSD2. Dehydrogenase specific activity with both E2 and T was also detected, although at lower levels, in LNCaP and DU145 cells. No evidence for reductase activity could be obtained in either the soluble or microsomal fraction of any cell line. As comparable expression levels of 17betaHSD4 were seen in the three cell lines, 17betaHSD2 is a likely candidate to account for the predominant oxidative activity in PC3 cells, whereas 17betaHSD4 may account for the lower extent of E2 oxidation seen in both LNCaP and DU145 cells. This is the first report on the expression of four different 17betaHSD types in human prostate cancer cells. It ought to be emphasized that for the first time, analysis of different 17betaHSD activities in either intact or fractionated cells harmonizes with the expression of relevant mRNAs species.

Product of aromatase activity in intact LNCaP and MCF-7 human cancer cells.

We investigated conversion rates of androgens to estrogens in cultured, hormone-responsive prostate (LNCaP) and breast (MCF-7) human cancer cells. For this purpose, we adopted an intact cell analysis, whereby cells were incubated for different incubation times in the presence of close-to-physiological (1 nM) or supraphysiological (1 microM) concentrations of labelled androgen precursors, i.e. testosterone (T) and androstenedione (delta4Ad). The aromatase activity, as measured by estrogen formation, was detected in LNCaP cells (0.5 pmol/ml), even though to a significantly lower extent than in MCF-7 cells (5.4 pmol/ml), using 1 microM T after 72 h incubation. Surprisingly, LNCaP cells displayed a much higher aromatase activity when T was used as a substrate with respect to delta4Ad. In either cell line, T transformation to delta4Ad was relatively low, attaining only 2.8% in LNCaP and 7.5% MCF-7 cells. However, T was mostly converted to conjugates (over 95%), glucuronides and some sulphates, in LNCaP cells, whereas it was only partly converted to sulphates (<10%) in MCF-7 cells. Aromatase activity seems to be inconsistent in LNCaP cells, being strongly affected by culture conditions, especially by fetal calf serum (FCS). Further studies should assess the regulation of aromatase expression by serum or growth factors in different human cancer cells, also using anti-aromatase and/or anti-estrogen compounds, in different culture conditions.

17 beta-hydroxysteroid oxidoreductase activity in intact cells significantly differs from classical enzymology analysis.

This paper summarizes our most recent results of steroid enzyme studies on cultured breast and endometrial cancer cells. It deals mainly with estrogen 17 beta-hydroxysteroid oxidoreductase (17 beta HSOR) activity, which presides over estradiol (E2) and estrone (E1) interconversion, a major metabolic pathway of estrogens. Assessment of either the oxidative or reductive component of 17 beta HSOR was carried out on intact cells by means of an original approach based on reverse phase-high performance liquid chromatography and radioactive detection on line. This system allows the continuous monitoring of both precursor degradation and formation of several radiometabolites to assess rates and direction of steroid metabolism. Overall, hormone-responsive, estrogen receptor (ER)-positive cells, regardless of whether they were derived from breast (MCF7) or endometrial (Ishikawa) tumor tissues, showed a prevalence for reductive metabolism (E1-->E2), whilst oxidative pathways (E2-->E1) were largely dominant in non-responsive, ER-poor mammary (MDA-MB231) and endometrial (HEC-1A) cells. The above estimates of 17 beta HSOR activity were at variance with those obtained using the classical enzymology approach, not only in quantitative terms (being markedly lower using intact cell analysis), but also because the prevalent direction of estrogen metabolism was often reversed. Although striking methodological differences may well account for this discrepancy, intact cell analysis is undoubtedly more similar to the in vivo state than the artificial requirements of classical enzymology procedures.

Steroid-growth factor interaction in human prostate cancer. 2. Effects of transforming growth factors on androgen metabolism of prostate cancer cells.

The ability of human prostate cancer cells to metabolize androgens was assessed through administration of physiological concentration (0.5-10 nM) of tritiated testosterone (T) as precursor and one-step analysis of both T degradation and products' formation by reverse-phase HPLC and on-line radioactive detection after either 24 h or 72 h incubation. Overall, different prostate cancer cells degraded T quite differently, favoring alternatively reductive or oxidative metabolic pathways. In particular, both LNCaP and DU145 cells retained high levels of unconverted T, with a limited production of androstenedione and its 17-keto derivatives and relatively high amounts of dihydrotestosterone (DHT) and 3 alpha-androstanediol (3 alpha-diol). In contrast, PC3 cells quickly degraded T and exhibited high formation rates of androstenedione and 17-keto metabolites, while neither dihydrotestosterone nor 3 alpha-diol were detected after short or longer incubation times. The effects of both TGF alpha (50 ng/mL) and TGF beta 1 (5 ng/mL) on rates and direction of T metabolism were also explored. In LNCaP cells TGF alpha induced a significant (P < 0.04) decrease of the reductive metabolism of T with a corresponding enhancement of the oxidative pathway (P < 0.002), while TGF beta 1 did not significantly affect T metabolism. On the other hand, both reductive and oxidative pathways were only partially influenced by either growth factor in DU145 and PC3 cells, although TGF alpha significantly raised 5 alpha-androstanedione formation and reduced androsterone production in DU145 cells. All the above evidence was confirmed at both 24 h and 72 h or using increasing doses of TGF alpha and TGF beta 1, a peak activity of 50 ng/mL and 5 ng/mL, respectively, being generally encountered. Overall, our data suggest that TGFs may have a role in the growth regulation of hormone-responsive prostate tumor cells through changes of the intracellular contents of biologically active androgen metabolites.

17 beta-Hydroxysteroid dehydrogenase activity in endometrial cancer cells: different metabolic pathways of estradiol in hormone-responsive and non-responsive intact cells.

In this paper we report that two human long-term endometrial cancer cell lines, Ishikawa and HEC-1A, exhibit quite different abilities in metabolizing estrogens. As a matter of fact, incubation of Ishikawa cells with close-to-physiological concentrations of estradiol (E2) as precursor resulted in: (1) elevated formation (up to 90%) of E2-sulphate (E2-S), using lower precursor concentrations; (2) very limited conversion to estrone (E1) (< 10% at 24 h incubation), as either free or sulphate; and (3) low but consistent production of other estrogen derivatives, such as 2-hydroxy-estrogens and estriol. Conversely, scant amounts (if any) of E2-S were found in HEC-1A cells, while no detectable formation of other estrogen metabolites could be observed after 24 h. On the other hand, E1 production was significantly greater (nearly 60% at 24 h) than in Ishikawa cells, a large proportion of E1 (over 50% of the total) being formed after only 6 h incubation using time-course experiments. The hypothesis that E2 metabolism could be minor in Ishikawa cells as a consequence of the high rate of E2-S formation encountered is contradicted by the evidence that conversion to E1 also remains limited in the presence of much lower E2-S amounts, seen using higher molar concentrations of precursor. Overall, we observe that 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity diverges significantly in intact Ishikawa and HEC-1A endometrial cancer cells. This difference could not merely be accounted for by the diverse amounts of substrate (E2) available to the cells, nor may it be imputed to different levels of endogenous estrogens. It should rather be sought in different mechanisms controlling 17 beta-HSD activity or, alternatively, in the presence of distinct isoenzymes in the two different cell types.

Oxidative and reductive pathways of estrogens in hormone responsive and non-responsive human breast cancer cells in vitro.

In order to measure the formation and degradation rates of estradiol by human breast cancer cells, after assessing the biochemical basis of hormone responsiveness and growth response to estrogens, we considered both responsive, estrogen receptor (ER) positive, and non-responsive, ER-negative, breast cancer cell lines, i.e. MCF7, ZR75-1 and MDA-MB231. To this end, we employed a novel "intact cell" approach which allows us, after 24 h incubation, to analyze several enzyme activities in sequence, concurrently with the monitoring of labeled precursor degradation. Our investigations led to the following evidence: (a) the reductive activity of the 17 beta-hydroxysteroid oxoreductase (17 beta-HSOR) appears to be higher than the oxidative only in responsive, ER-rich MCF7 and ZR75-1 cells, as also previously observed by others; (b) this activity is, on the contrary, much lower in MDA-MB231 cells and other unresponsive, ER-poor breast cancer cell lines; (c) conversely, the oxidative activity shows an opposite pattern, being limited in MCF7 and ZR75-1 cells and much higher in MDA-MB231 cells. Overall, a 17 beta-HSOR reductive pathway prevails in both MCF7 and ZR75-1 cells, whilst the oxidative pathway is prevalent in MDA-MB231 cells, leading to a large formation of estrone that is no further metabolized, at least in the experimental conditions used. Our results may provide a likely explanation of previous data on the different estrogen content of breast tumor tissues.

Androgen metabolism and inhibition of interleukin-1 synthesis in primary cultured human synovial macrophages.

The presence of androgen receptors on synovial macrophages in human normal and rheumatoid synovial tissues has been described previously. It is now reported that primary cultured human macrophages obtained from normal and rheumatoid synovia express functional androgen receptors. We have investigated the capacity of cultured macrophages to metabolize androgens and have found that these cells were capable of metabolizing testosterone to the bioactive metabolite dihydrotestosterone. Therefore, macrophages contain the key enzymes of steroidogenesis, in particular the 5alpha-treductase. Furthermore, interleukin-1beta production by primary cultured rheumatoid macrophages was analysed, following exposure to physiological concentrations of testosterone (10(-8) M). A significant decrease of IL-1beta levels in conditioned media after 24 h (p < 0.05) was observed. It is concluded that androgens may act directly on human macrophages and may interfere with some of their functions via receptor-dependent mechanisms.

Different conversion metabolic rates of testosterone are associated to hormone-sensitive status and -response of human prostate cancer cells.

The main goal of the present work was to compare the ability of human prostate cancer (PCa) cells to metabolize testosterone (T) in living conditions. To this end we studied three different human PCa cell lines (LNCaP, DU145 and PC3) having different hormone-sensitive status and capability of response to androgens. We used an original approach which allows the evaluation of conversion metabolic rates in growing cells after administration of labeled steroid precursor (presently T), at physiological concentrations (1-10 nM). Analysis of both precursor degradation and formation of several products was carried out using reverse phase-high performance liquid chromatography (RP-HPLC) and "on line" radioactive detection. Comparison of the three human PCa cells revealed that their metabolic aptitude differed in many respects: (i) rates of precursor degradation, (ii) different products' formation, and (iii) extent of conjugate production. In detail, PC3 cells quickly degraded T and exhibited high formation rates of androstenedione (A-4-ene-Ad); both DU145 and LNCaP cells mostly retained high levels of unconverted T, with a limited production of A-4-ene-Ad and its 17-keto derivatives (if any). Either LNCaP or DU145 cells generated a relatively high amount of dihydrotestosterone (DHT). In contrast, neither DHT nor its main metabolites were detected in PC3 cells at both short and longer incubation times. As expected, T degradation and A-4-ene-Ad production were highly correlated (r = 0.97; P < 0.03); similarly, A-4-ene-Ad and DHT formation showed a negative, significant correlation. Negligible production of conjugates was noted in both PC3 and DU145 cells, whilst it was remarkable in LNCaP cells (ranging from 43 to 57%). Overall, our data indicate that human PCa cells degrade T quite differently, favoring alternatively reductive or oxidative patterns of androgen metabolism.

Evidence for soluble and nuclear site I binding of estrogens in human aorta.

The purpose of this study was to establish the estrogen receptor (ER) expression and content in human aorta fragments removed at the time of by-pass surgery. To this end, we adopted a radioligand binding assay to evaluate either soluble (S) or nuclear (N) ER using dextran-coated charcoal (DCC) and filtration methods, respectively. To better define the intratissular distribution and content of ER, we also measured the presence of a 27 kDa heat shock protein (HSP27), a well established ER-associated protein, using D5 monoclonal antibody. Finally, we analysed the different molecular isoforms of both S and N ER using size exclusion-high performance liquid chromatography (SE-HPLC). High affinity (type I) sites of estrogen binding were detected in 17 out of 19 samples in either S or N fraction, although only 9 out of 19 cases displayed site 1 ER in both cell compartments. ER levels in aortic tissues, detected by radioligand method, compare well with those we have found in other hormone-sensitive human cancer tissues and cells. SE-HPLC analysis revealed two main receptor isoforms in the soluble fraction, having 65 kDa and 18 kDa molecular mass, while a minor component of 29 kDa was also found; the nuclear fraction displayed again two major components of 38 and 23 kDa. Using the HSP27 immunohistochemistry we observed a major staining occurring in smooth muscle cells (SMC), with an increasing intensity towards the lumen. All samples, including the ER negative ones, exhibited some degree of histochemical staining. Using an arbitrary cut-off value, 7 out of 12 samples displayed a highly positive staining, 6 of which showed nuclear ER. Furthermore, SE-HPLC separation indicated the presence of a 64.9 kDa component in the soluble fraction, according to the well known relative molecular mass of ER. Following HSP27 immunohistochemistry, the overall staining intensity in aortic SMC approaches that seen in endometrial and breast epithelia, whilst the muscle ER content is generally lower. Although our data are compatible with a direct role of estrogens in arterial function, the extent of the link with arterial disease remains to be established.

Relationship between the concentrations of estriol sulfate and estrone sulfate in human breast cyst fluid.

Estriol-3-sulfate (E3S) is present in human breast cyst fluid (BCF) in median levels of 8.7-10.4 nmol/L, yet is barely detectable in the serum (less than 0.034 nmol/L). The source of this huge concentration of E3S is unknown. It may accumulate from blood by active transport or be synthesized and concentrated within the cyst. Since estrone sulfate (E1S) and its possible precursor, dehydroepiandrosterone sulfate (DHEAS) are elevated in BCF, E3S may originate via 16 alpha-hydroxylation of E1S. The present study examined the correlations between the levels of DHEAS and E1S with those of E3S in BCF. The sodium and potassium ions were also quantified and related to the steroid concentrations. By linear regression analysis of log-normalized data there was a highly significant correlation between the concentrations of E1S and E3S (n = 355, r = 0.690, P less than 0.001) and between DHEAS and E3S (n = 361, r = 0.577, P less than 0.001). The BCF were classified according to their K/Na ion ratios: type 1, greater than 1.0, type II, less than 0.25, and type III, 0.25-1.0. By Student's t test, the concentrations of E3S differed between each BCF Type (P less than 0.002). This was also true for E1S and DHEAS. Type 1 cysts were associated with the highest estrogen sulfate levels and type II with the lowest levels. The possible physiological importance of this observation resides in reports that the BCF type expressing the highest steroid concentrations has been related to an aporcine-like epithelial lining of the cyst wall and a somewhat higher risk for developing breast cancer. The results suggest that E3S in BCF may originate from E1S, but alternate mechanisms are not precluded.

Gas chromatography/mass spectrometry of catechol estrogens.

Catecholestrogens (CCEs), namely 2- or 4-hydroxyestradiol and hydroxyestrone, are highly polar, reactive, and extremely labile estrogen metabolites in many experimental conditions. For these reasons, indirect assay methods mainly have been used. Some experimental evidence suggests that CCEs are synthesized and biologically active mostly in target cells. At this level, unfortunately, the indirect assays cannot be used. We present a method of gas chromatographic/mass spectral (GC/MS) analysis for the identification of individual CCEs; the major fragmentation ions of authentic estrogen standards as trimethylsilylether derivatives, and the MS patterns of the major CCEs, namely, 2-hydroxyestradiol and hydroxyestrone, are included. Few examples of CCEs detected in human breast cancer tissues and in breast cyst fluids are reported. Sample extracts were submitted to reversed-phase, high-performance liquid chromatography (RP-HPLC) and were quantified by "on line" electrochemical (EC) detection; thereafter, either crude extracts or single eluted peaks were submitted to GC/MS, by which detection limits of less than 5 pmol were attained. As expected, the molecular ion was the most relevant molecule in all but one case. On the contrary, the other relative intensities of major fragmentation ions M -15, M -30, M -90, and M -15 + (-90) were unevenly distributed, although represented in the majority of cases. In all cases, the GC/MS of peak fractions, purified by RP-HPLC and UV detection, confirmed the results of liquid chromatographic analysis combined with EC detection. In contrast, GC/MS of crude extracts was not equally satisfactory. Comparison of a liquid chromatography system with EC detection and the GC/MS approach revealed some inconsistency in quantitation of individual CCEs.(ABSTRACT TRUNCATED AT 250 WORDS)

Simple approach to measure metabolic pathways of steroids in living cells.

A simple, rapid approach to the study of conversion rates and metabolic patterns of the steroids testosterone and estradiol is presented. It includes an optimized isocratic high-performance liquid chromatographic procedure in the reversed-phase mode and radioactive on-line detection. The purpose was to estimate the activity of key enzymes of steroid pathways, such as 17 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase, in in vivo conditions. Using this system, we obtained good efficiency and linearity of radio detection, under continuous flow conditions. Sensitivity limits were of the order of 50 and 70 cpm for [3H]estradiol and [14C]estrone, respectively, even though the efficiency was quite dissimilar (17.3% versus 56.2%). The applicability of this approach to studies of steroid metabolic pathways in growing cancer cells in culture is illustrated with examples of the conversion rates of both testosterone and estradiol. The high reproducibility (coefficients of variation of 2.7 and 5.1% for 3H and 14C, respectively) and good extraction efficiency (ranging from 86 to 94%) indicate the feasibility and reliability of this approach.

Prostate long-term epithelial cell lines. Biological and biochemical features.

This review reports studies on long-term prostate cell lines using multiple experimental approaches. The main goal was to investigate the metabolism of testosterone (T) through in vitro conversion rates. Extensive studies were also carried out on growth curves, tritiated thymidine incorporation, and morphometry by either hormone-responsive or hormone-unresponsive, normal and neoplastic human (PC3 and DU-145) and canine (CAPE and CPA) cell lines. All of them were characterized for their content of both soluble and nuclear androgen receptors. Receptor studies at site I binding in both soluble and nuclear fractions were carried out to establish the hormone sensitivity status of cells. In two prostate epithelial cells, steroid metabolic conversions in vitro show predominantly an oxidative metabolism of T, forming mainly androstenedione. Conversion rates were greater than 50% in the first 24 hours and still higher after 72 hours. At the same time and under exactly the same experimental conditions, the other cells showed metabolic pathways in which reductive metabolism prevails, dihydrotestosterone (DHT) being the prevalent metabolite. Different metabolic patterns of steroids of several cell lines relate to the hormone sensitivity status of the cells; steroid receptor-endowed cells are maintaining higher levels of unconverted precursor than are receptor-empty cells. In fact, hormone-sensitive cells, such as cancer canine CPA and human DU-145, produced DHT early through slowly converting T. On the contrary, unresponsive cells such as human cancer cells PC3 and normal canine CAPE quickly metabolize T, but DHT formation was not observed. These significant differences between cells are highly reproducible provided the proportion between cell number and molar concentration of precursors is constant. Differences we observe cannot be attributed to different experimental conditions. Cell viability, extraction efficiency, and all other parameters used for monitoring cell growth kinetics do not substantiate these reported significant differences in metabolic abilities of cells. The divergent steroid metabolic pathway we observe in different prostate long-term cells appears to be an intrinsic, consistent, highly reproducible property of each cell line.

Steroid patterns of benign breast disease.

We briefly review some biochemical aspects of benign breast disease (BBD), mainly focusing on free and conjugate estrogen content of breast cyst fluid (BCF), also in relation to cyst type. Evidence is reported that high K(+)-type I-cysts clearly associate with low Cl- levels and accumulate significantly higher quantities of dehydroepiandrosterone sulfate (DHAS) and estrone-3-sulfate (E1S). In spite of the limited number of cases, both increasing DHAS and E1S levels correlate with the increment of K+ to Na+ ratio. A positive correlation was also found between DHAS and E1S. Using electrochemical detection (ECD) on-line to high performance liquid chromatography (HPLC) in the reverse phase mode, we also studied the free estrogen profile. We observed that in type I BCF there are significantly increased amounts of free estrone (E1). The E1S to E1 ratio was significantly different in the two cyst subpopulations; again, a positive correlation was found between free and sulfated E1 (r = 0.820, p less than 10(-6). This last, together with other experimental observations, allows us to hypothesize that in BCF a main pathway of steroids should be E1S----E1. Besides, high specific activity of sulfatase, as well as beta-glucuronidase enzymes, has been demonstrated for BBD. Preliminary information is also reported concerning the BCF pattern of free estrogens, including the highly polar ones, i.e., catecholestrogens (CCE) and the parent methoxy (MeO) conjugates, which represent, in BCF, a predominant portion of all free estrogens. Both CCE levels and ratios appear unevenly distributed in the two different cyst types. In addition, some BCFs show very high concentrations of 16 alpha-OH-E1. Further studies are needed to answer the main question: whether estrogen patterns could represent additive parameters to further categorize breast cystic disease (BCD) or whether they are of minor interest to determine patients' risk of developing breast cancer.

Soluble and nuclear oestrogen receptor status of advanced endometrial cancer in relation to subsequent clinical prognosis.

Both soluble and nuclear oestrogen receptors have been measured in at least two separate sections from 72 endometrial cancers and 12 normal endometria. Concentration of oestrogen receptor is shown to be, in our hands, more meaningful when expressed per unit DNA than per unit protein, whether for soluble or nuclear receptor. Endometrial cancer cells from the central part of the tumour are shown to be receptor negative more frequently than those from peripheral tumour. Thus, in large cancers, biopsies from different areas are required before a tumour can be correctly designated as receptor positive, heterogeneous or receptor negative. The intratumoral variation of receptor status may relate to poor prognosis, since patients with homogeneous receptor-positive disease survive significantly longer than those with tumours showing either heterogeneous distribution of receptor or homogeneous absence of receptor. Intratumoral variation in receptor status is found to be more common in the group of patients who are within 7 years of their menopause, than in older patients.