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Objective: It has been proposed that physiologic levels of estrogens stimulate immune responses whereas androgens suppress inflammatory reactions. Thus, prevalence of synovial androgens relative to estrogens would be favorable in rheumatoid arthritis (RA). We investigated synovial fluid (SF) concentrations of several estrogens and androgens and conversion products of the sex steroid precursor dehydroepiandrosterone (DHEA) in supernatants of mixed synoviocytes., Methods: SF steroid concentrations were measured by high performance liquid chromotography and mass spectrometry in 12 patients with RA and 8 subjects with traumatic knee injury (noninflammatory controls). Conversion of DHEA to downstream hormones was measured by thin-layer chromatography and phosphorimaging detection in 3 patients with RA and 3 patients with osteoarthritis (OA)., Results: Overall, SF concentration of free estrogens tended to be higher in RA patients versus controls (p < 0.06). Molar ratio of free SF estrogens/free SF androgens was elevated in RA compared to controls (1.17 +/- 0.32 vs 0.29 +/- 0.08, without unit; p = 0.017). The free SF concentration of the precursor androstenedione was significantly higher in RA patients than in controls (104.6 +/- 32.6 vs 30.4 +/- 0.4 ng/ml; p = 0.011), and SF estrone the aromatase conversion product of androstenedione was also elevated in RA compared to controls (13.6 +/- 2.6 vs 6.6 +/- 0.8 ng/ml; p = 0.035). The biologically active estrogen derivatives, 16a-hydroxyestrone and 4-hydroxyestradiol, were both higher in RA compared to controls (p = 0.085 and p = 0.044, respectively). In mixed RA synoviocytes, DHEA conversion yielded high local levels of 17beta-estradiol (708 pmol/l = 0.193 ng/ml) compared to testosterone (88 pmol/l = 0.026 ng/ml)., Conclusion: SF levels of estrogens relative to androgens are significantly elevated, while those of androgens are markedly reduced, in patients with RA compared to controls. This imbalance is most probably due to increased aromatase activity. Thus, an available steroid precursor, such as DHEA, may be rapidly converted to proinflammatory estrogens in the synovial tissue, which may in turn stimulate the inflammatory process in patients with RA.

We have investigated the activity and expression of aromatase enzyme in nontumoral, cirrhotic, and malignant human liver tissues and cells using both chromatographic and reverse transcription (RT)-PCR analyses. After 24- and 72-h incubation of tissue minces or hepatic cell lines with either testosterone or androstenedione as androgen precursor, human hepatocellular carcinoma (HCC) tissues and HepG2 hepatoma cells showed elevated aromatase activity, with estrogen formation rates being 20 and >95%, respectively, as opposed to nontumoral hepatic tissues and nonmalignant Chang liver (CL) cells, where no aromatase activity could be detected. Cirrhotic samples exhibited intermediate enzyme activity. Notably, exposure of HepG2 cells to the aromatase inhibitor Letrozole resulted in a striking decrease of estrogen formation, which became virtually absent at a Letrozole dose of 0.4 nM. RT-PCR analysis revealed markedly lower aromatase mRNA in both CL cells and nontumoral liver tissues, as compared with HepG2 cells and HCC samples. Cirrhotic specimens displayed variable transcript levels, in turn comparable with those observed in nontumoral or HCC tissues. Exon-specific RT-PCR showed prominent expression of exon I.3A-containing message and exon I.4-containing message in CL and HepG2 cells, as in nontumoral and HCC tissues, respectively. The present evidence implies that locally elevated estrogen formation in malignant human liver tissues and cells may have a role in the development and/or maintenance of human HCC, eventually leading to develop alternative strategies for treatment of HCC patients using antiaromatase agents.

Purpose: The main goal of our study was to assess estrogen contents of breast tumor tissues, having different estrogen receptor status, in relation to long-term follow-up of patients., Experimental Design: Twenty-one breast cancer cases, all collected from January 1986 to January 1988 at the M. Ascoli Cancer Hospital Centre in Palermo, were included in the study and compared with 6 healthy women as a control group. Average follow-up time of patients was 144 +/- 10 months. The estrogen receptor status of tissues was determined by both ligand binding and immunohistochemical assays. A high performance liquid chromatography-based approach, jointly with gas chromatography/mass spectrometry, was used to identify and measure main estrogens, various hydroxyestrogens, and their methoxy derivatives in both normal and tumor tissues., Results: Although variable concentrations of hydroxylated estrogens were detected, they consistently accounted for >80% of all of the estrogens. Significantly greater amounts of both 2- and 4-hydroxyestradiol, along with a marked increase of 16 alpha-hydroxyestrone (OHE(1)), were observed in cancer with respect to normal breast tissues. A significant positive association was observed with elevated 16 alpha OHE(1) (P = 0.015) in patients alive, leading to significantly lower (P = 0.043) 2OHE(1):16 alpha OHE(1) ratio values. Conversely, ratio values of 4:2 hydroxy+methoxy estrogens was significantly lower (P = 0.006) in deceased patients. Using cutoff values of 1.2 for 4:2 hydroxy+methoxy ratio and 150 fmol/mg tissue for 16 alpha OHE(1) we achieved a clear-cut separation of patients, with over-cutoff patients having 147 months and under cutoff patients showing only 47 months median survival time (P = 0.00008)., Conclusions: Our data imply that individual hydroxyestrogens may have a distinct role in the onset and the clinical progression of breast cancer, with greater 16 alpha OHE(1) levels being in turn associated to cancer with respect to normal tissues and to a prolonged survival of breast cancer patients.

Sex steroid hormones are thought, among several other risk factors, to play a role in liver malignancies. For example, from epidemiological studies in hepatocellular carcinoma (HCC), a clear disadvantage for male sex is evident. In addition, elevated levels of serum testosterone (T) and increased T to Estradiol (E(2)) ratio have been reported to predict an increased risk of HCC for male cirrhotic patients. On the other hand, palliative treatment of liver cancer patients with anti-hormones has been widely used in the past. However, the molecular mechanism(s) underlying sex steroid action on either normal or transformed liver cells, have not yet been fully clarified, nor endocrine discriminants have been satisfactorily assessed for an adequate characterization of liver cancer tissues. In this paper, we report studies on hormonal status of human liver tissues and cells, especially focusing on androgens, to better define endocrine end-points of interest for HCC. A consistent evidence from ex vivo or in vitro systems strongly suggests that high affinity binding sites of androgens are expressed at sufficient concentrations to induce a biological response in either normal or phenotipically transformed hepatocytes; in the latter, however, high heterogeneity and/or more scattering concentrations were encountered. Further, experimental data seem to suggest that lack of response to androgens may be due to a rapid metabolic conversion of steroids by neoplastic tissues and cells. Cancer hepatocytes privilege in fact 5beta more than 5alpha metabolic pathway of androgens. This may eventually lead biologically active androgens to be transformed into less active derivatives, as it occurs for T which is massively converted (>90% at 6 h) thus hindering the whole mechanism of action of androgens., (Copyright 2002 Elsevier Science Ireland Ltd.)

Expression of gap-junction proteins connexins (Cx), specifically Cx43, Cx32, and Cx26, in both nontumorigenic (RWPE-1) and tumorigenic (RWPE-2) human prostate epithelial cells as well as in two cell clones (WPEI-7 and WPEI-10) originating from the RWPE-1 cell line was investigated. The aim was to determine whether individual connexins are differentially expressed in cultured cells. Western blot analysis revealed striking differences in the expression of individual connexins in the cell lines studied. In particular, Cx43 is largely expressed in RWPE-1 and WPEI-10 cells, whereas Cx32 is expressed predominantly in RWPE-2 and WPEI-7 cells. In addition, both forskolin and estrone increase Cx43 expression levels in WPEI-10 cells, with no apparent effect on WPEI-7 cells. Conversely, forskolin and especially estrone induce a marked increase of Cx32 in WPEI-7 cells, whereas Cx32 expression is limitedly affected by both agents in WPEI-10 cells. Overall, expression levels of Cx43 and Cx32 appear to be inversely related, with RWPE-1 and WPEI-10 cells having a significantly higher Cx43 to Cx32 ratio than that observed in RWPE-2 and WPEI-7 cells. We recently reported that junctional communication could be rescued in RWPE-1 cells by either forskolin or estrone and that restoration of GJIC is associated with an increase of Cx43 or a decrease of Cx32, or both, eventually leading to a marked rise of the Cx43 to Cx32 ratio. Studies are currently ongoing in our laboratories to assess the potential effect of agents increasing the Cx43 to Cx32 ratio on GJIC activity in these systems.

The incidence of hepatocellular carcinoma is increasing in many countries. The estimated number of new cases annually is over 500,000, and the yearly incidence comprises between 2.5 and 7% of patients with liver cirrhosis. The incidence varies between different geographic areas, being higher in developing areas; males are predominantly affected, with a 2:3 male/female ratio. The heterogeneous geographic distribution reflects the epidemiologic impact of the main etiologic factors and environmental risk, which are the hepatitis B (HBV) and hepatitis C (HCV) viruses. The percentage of cases of hepatocellular carcinoma attributable to HBV worldwide is 52.3% and is higher in Asia where the seroprevalence of HBsAg in the population is high. However, the vaccination campaign against this virus in some eastern countries has tended to lower the incidence of new cases of hepatocellular carcinoma. The percentage of cases of hepatocellular carcinoma attributable to HCV is 25%, and it is more prevalent in Japan, Spain, and Italy where the association between hepatocellular carcinoma and antibodies to HCV ranges between 50 and 70%. In most cases hepatocellular carcinoma develops in cirrhotic livers, where the persistent proliferation of liver cells represents the key factor of progression to hepatocellular carcinoma independent of the etiology. Another minor risk factor is aflatoxin B1 consumption, which is responsible for most cases of hepatocellular carcinoma in Africa, where the consumption of contaminated foods is common. Other known risk factors are some hereditary diseases, such as hemochromatosis, porphyria cutanea tarda, hereditary tyrosinemia, and alpha1 anti-trypsin deficiency. The natural history of hepatocellular carcinoma is heterogeneous and is influenced by nodule dimension, the mono- or plurifocality of lesions at diagnosis, the growth rate of the tumor, and the stage of the underlying cirrhosis. Available data to date suggest that tumor growth in a cirrhotic liver is variable and that the time in which a lesion in undetectable until it becomes 2 cm is between 4 and 12 months. Therefore, the suggested interval for surveillance screening with ultrasound in patients with liver cirrhosis has been set at 6 months. Patients who should benefit from screening programs are those who would be treated with curative therapy if diagnosed with hepatocellular carcinoma. Thus, the ideal target population should be limited to Child-Pugh's class A cirrhotic patients without significant comorbidity.

Background: Gap-junction-mediated intercellular communication (GJIC) is required for normal development and tissue homeostasis. However, the role of GJIC in human prostate carcinogenesis and progression remains ill-defined., Methods: The ability of hormones, anti-hormones, and the anti-hypertensive drug, forskolin, to restore GJIC in non-tumorigenic (RWPE-1 and PWR-1E) and malignant (RWPE-2, LNCaP, DU-145) human prostate epithelial cell lines, was examined by Scrape-Loading/Dye Transfer (SL/DT) and Fluorescence Recovery After Photobleaching (FRAP) methods using an Ultima laser cytometer., Results: Results from both assays show that PWR-1E, RWPE-2, LNCaP, and DU-145 cells have weak or absent GJIC activity. However, the non-tumorigenic RWPE-1 cells showed restoration of some GJIC (nearly 10%) after 1 hr in the FRAP assay. Forskolin and estrone, which increase intracellular cAMP levels, induced a significant and consistent increase (2.8- and 4.4-fold, respectively) in cell-to-cell communication only in the non-tumorigenic RWPE-1 cells. Furthermore, estrone induced a two-fold increase in connexin 43 (Cx43) and a 30% decrease in Cx32 expression, while forskolin caused a 50% reduction in Cx32 with no effect on Cx43 expression in RWPE-1 cells., Conclusions: These data suggest that agents that increase Cx43:Cx32 ratio may be used to restore GJIC in junctionally-deficient, non-tumorigenic immortalized cells, thus providing insights into potential mechanisms responsible for the multistep carcinogenesis in the human prostate., (Copyright 2002 Wiley-Liss, Inc.)

In the present work, we have inspected expression of estrogen receptors (ER) and their regulation by the phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) in a leukemic cell line, the THP-1 cells, using multiple experimental approaches. Firstly, ligand binding assay (LBA) revealed that control (unstimulated) THP-1 cells express type I (high affinity, limited capacity) ER in the nuclear fraction only, whilst treatment of cells with TPA resulted in the appearance of type I ER in the soluble fraction as well, with the 50 ng/ml dose and the 48 h incubation time being the most effective experimental condition. A concomitant increase of type II ER was also seen in both soluble and nuclear cell fractions. Unstimulated THP-1 cells were found to be ER negative by immunocytochemistry; conversely, cells exposed to 50 ng/ml TPA for 48 h stained positively for ER, with the majority of cells having a strong nuclear staining. Scrutiny of ER mRNA expression using reverse transcriptase-polymerase chain reaction showed the presence of a wild type ER transcript in both control and TPA-treated THP-1 cells, though levels of ER mRNA were found to be comparatively higher in the latter. This combined evidence would imply that the TPA-induced differentiation of THP-1 cells is accompanied by the rise of high affinity (type I) ER, suggesting that estrogens may play a role in the regulation of macrophage activity during the inflammatory and/or the immune response., (Copyright 2001 Wiley-Liss, Inc.)

Objective: To investigate gap-junctional intercellular communication (GJIC) in LNCaP and DU145 human prostate cancer cells., Study Design: Normal rat liver F344 (WB1) cells were used as positive controls. Functional GJIC was inspected using either the scrape-loading/dye transfer (SL/DT) method or fluorescence recovery after photobleaching (FRAP) analysis. In the former, GJIC activity was expressed as a measure of the extent of diffusion of Lucifer Yellow after cell monolayers were scraped using a surgical blade and exposed to dye for a few minutes at room temperature. In the latter, cells were incubated for 15 minutes at 37 degrees C with 5,6-carboxyfluorescein diacetate dye and the dye transfer visualized by photobleaching individual cells with a 488-nm laser and monitoring the recovery of fluorescence using a laser cytometer., Results: The preliminary results obtained indicate that neither LNCaP nor DU145 cells have functional GJIC, while, as expected, WB1 cells show unimpaired GJIC activity. Equivalent results were consistently obtained using either SL/DT or the FRAP approach. However, using FRAP analysis, DU145 cells only showed weak recovery of fluorescence after a total observation interval of 15 minutes., Conclusion: The present data, though preliminary, suggest that disruption of GJIC may play a role in development of malignancy in the human prostate.

Objective: To assess estrogen and progesterone receptor presence in human breast tumors using immunocytochemical analysis., Study Design: For both estrogen (ER) and progesterone (PR) receptor assay, percent of stained cells and intensity of staining were estimated on a series of 251 consecutive breast cancer cases from the M. Ascoli Cancer Hospital Center in Palermo using the CAS 200 image analysis system., Results: Cytochemical assay revealed a differential distribution of both ER and PR, by menopausal status of the patients; premenopause (PreM) was mostly ER negative (63%), and postmenopause (PostM) > 10 years was mostly ER and PR positive (64%). The percent of cells stained for ER was significantly different between PreM and PostM patients when they were considered as a whole. By contrast, no difference emerged for PR staining among menopausal groups. Overall, patients whose tumors were PR positive showed a significantly (P < .03) longer interval free of relapse., Conclusion: The present results suggest that PRs behave as better indicators than ERs of early relapse in breast cancer patients. Further studies, with longer follow-up, are needed, however, to validate this concept.

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.

In the present study we have inspected estrogen metabolism in cultured human prostate cancer cells (LNCaP, DU145, PC3), in relation to the expression of mRNAs for different 17 beta hydroxysteroid dehydrogenase (17 beta HSD) enzymes (from 1 to 4). Using an intact cell analysis, we have compared precursor degradation and product formation after incubation of cells with physiological amounts of radioactive E2 or estrone (E1) for 24-72 h and subsequent reverse-phase high performance liquid chromatography analysis. The LNCaP and DU145 cells only partly converted E2 to E1 (26 and 13% at 72 h, respectively), giving rise to an appreciable production of E2 from E1 (nearly 20% in all cases). Conversely, PC3 cells revealed a massive E2 oxidation to E1 (up to 90% by 72 h) and a scant formation of E2 (<2%) from E1. In addition, an appreciable formation of 16 alpha OHE1 was seen in either PC3 (11%) or DU145 (5%) cells. respectively using E2 or E1 as precursor. All three cell lines exhibited marked amounts of 17 beta HSD4 mRNA species, whilst even greater amounts of 17 beta HSD2 transcript were found in PC3 cells only. No mRNA for either 17 beta HSD1 or 17 beta HSD3 could be detected in any cell line. The present evidence indicates that pathways of estrogen metabolism are distinctly governed in prostate cancer cells depending on their endocrine status, being associated with a differential expression of mRNA for different 17 beta HSD enzymes.

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.

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.

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.

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.

There is convincing evidence that a reduced expression of the E-cadherin cell-cell adhesion molecule associates with low tumor grade and poor prognosis in prostate cancer patients. However, little is known on how E-cadherin levels are regulated in human prostate cancer cells. We have inspected the effect of both androgens and estrogen on the expression of E-cadherin in the hormone-responsive LNCaP prostate tumor cell line, which is endowed with both androgen and estrogen receptors. Using both Dot Blot analysis and immunocytochemistry we have observed that either steroid significantly increased E-cadherin levels in these cells; this effect was not reversed by the simultaneous addition of the relevant antagonist, hydroxyflutamide or ICI-182,780.

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.

We report that growth of LNCaP human prostate cancer cells is significantly stimulated (up to 120% above control) by physiological estradiol (E2) concentrations. This growth increase appears to be comparable to that induced by either testosterone or dihydrotestosterone, as also reported by others. This paper presents novel illustrative evidence for estrogen-binding proteins and messenger RNA transcripts in LNCaP cells. In fact, 1) the reverse transcriptase-polymerase chain reaction system documented normal messenger RNA for estrogen receptors (ER); 2) the radioligand binding assay allowed the detection of high affinity, reduced capacity binding sites in both soluble and nuclear cell fractions; and 3) the immunocytochemical analysis showed a consistently intensive staining for both ER and progesterone receptors. Compared to other human estrogen-responsive mammary cancer cells, MCF7 and ZR75-1, ER expression in LNCaP cells was not significantly lower, as shown by levels of the ER transcripts, number of sites per cell, or femtomoles per mg DNA as well as the percentage and intensity of immunocytochemical staining. A relative estimate of ER expression obtained by matching LNCaP with another human prostate cancer cell line, PC3, always displayed significantly and consistently higher levels in LNCaP cells. The detection of relatively high type I ER content in either cell compartment of LNCaP cells was paralleled by a highly intensive staining for progesterone receptors. In addition, evidence that the synthetic androgen R1881 did not compete for type I binding of E2 and that any E2-induced growth was completely reversed by the pure antiestrogen ICI-182,780, but unaffected by the antiandrogen Casodex, clearly suggests that the biological response of LNCaP cells to E2 is mediated via its own receptor.

We have studied the response to oestrogen and expression of oestrogen receptors in responsive LNCaP and androgen non-responsive PC3 human prostate cancer cell lines. Growth of LNCaP cells is significantly stimulated by physiological concentrations of oestradiol; this growth increase appears to be comparable to that induced by either testosterone or dihydrotestosterone. In contrast, oestradiol significantly inhibits the proliferation of PC3 cells. We also present novel evidence for functional oestrogen binding in LNCaP cells. This evidence was first obtained by means of radioligand binding assays and was further corroborated using: (a) immunocytochemical analysis of oestrogen and progesterone receptors; (b) reverse transcriptase polymerase chain reaction of oestrogen receptor mRNAs; and (c) immunofluorescence of the 27 kDa heat shock protein (Hsp27), which has been reported to be a marker of functional oestrogen receptors. There appeared to be significantly and consistently lower levels of oestrogen receptor expressed in PC3 cells than in LNCaP cells. The observation that oestradiol-induced growth of LNCaP cells is completely reversed by the pure anti-oestrogen ICI 182,780 clearly implies that the biological response of these cells to oestradiol is mediated mainly via its own receptor. On the other hand, use of a neutralizing antibody against transforming growth factor (TGF)-beta 1 results in a remarkable increase in the growth of PC3 cells; this effect is almost completely abolished after the addition of oestradiol. This suggests that the oestradiol-induced growth inhibition may be mediated by TGF-beta 1. These results suggest that the current model for hormone-dependence of human prostatic carcinoma should be revised. This is of special concern, because recent data indicate that prostate cancer has become the most prevalent cancer and the second principal cause of cancer death in western countries.

In order to better define potential mechanisms of growth regulation in human prostate cancer cells, we have compared biological responses (such as short-term response to both transforming growth factor alpha and beta; TFG alpha and TFG beta) in relation to hormone sensitivity of LNCaP, DU145, and PC3 cells. Androgen receptor (AR) and epidermal growth factor receptor (EGF-R) content of each cell line was also investigated. In addition, expression of EGF, TGF alpha, and TGF beta was evaluated through immunofluorescent staining. Growth of androgen non-responsive PC3 cells was stimulated by TGF alpha (about 35%) and inhibited by TGF beta (more than 50%), with respect to controls, after 48 h exposure. Conversely, AR-positive, hormone-responsive LNCaP cells proved to be poorly sensitive, at least short-term, to either growth factor. Furthermore, high levels of both EGF-R and TGF alpha, and a fairly high amount of EGF, were found in DU145 cells and, to a lesser extent, in LNCaP cells; in contrast, PC3 cells exhibited low expression levels of both receptors (EGF-R) and ligands (EGF, TGF alpha), but displayed remarkable TGF beta binding and relatively high levels of endogenous TGF beta. Overall, these results suggest a differential sensitivity to TGF alpha and TGF beta by prostate cancer cells; TGF alpha response seems not to be proportional to the EGF-R content of individual cell lines.

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)

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.