Charcoal-stripped Fetal Calf Serum Research Articles

Cell Line Characteristics Predict Subsequent Resistance to Androgen Receptor-Targeted Agents (ARTA) in Preclinical Models of Prostate Cancer.

Treatment of prostate cancer (PCa) has changed considerably in the last decade due to the introduction of novel androgen receptor (AR)-targeted agents (ARTAs) for patients progressing on androgen deprivation therapy (ADT). Preclinical research however still relies heavily on AR-negative cell line models. In order to investigate potential differences in castration-resistant PCa (CRPC) growth, we set out to create a comprehensive panel of ARTA-progressive models from 4 androgen-responsive AR wild-type PCa cell lines and analyzed its androgen response as opposed to its ADT-progressive counterparts. Parallel cultures of VCaP, DuCaP, PC346C, and LAPC4 were established in their respective culture media with steroid-stripped fetal calf serum (FCS) [dextran-coated charcoal-stripped FCS (DCC)] without androgen (ADT) or in DCC plus 1 μM of the ARTAs bicalutamide, OH-flutamide, or RD162 (an enzalutamide/apalutamide analog). Cell growth was monitored and compared to those of parental cell lines. Short-term androgen response was measured using cell proliferation 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. qRT-PCR was performed to assess the mRNA expression of markers for AR signaling, steroidogenesis, glucocorticoid receptor (GR) signaling, epithelial-mesenchymal transition (EMT), and WNT signaling. Out of 35 parallel cultures per cell line, a total of 24, 15, 34, and 16 CRPC sublines emerged for VCaP, DuCaP, PC346C, and LAPC4, respectively. The addition of bicalutamide or OH-flutamide significantly increased CRPC growth compared to ADT or RD162. VCaP, DuCaP, and PC346C CRPC clones retained an AR-responsive phenotype. The expression of AR and subsequent androgen response were completely lost in all LAPC4 CRPC lines. Markers for EMT and WNT signaling were found to be elevated in the resilient PC346C model and CRPC derivatives of VCaP, DuCaP, and LAPC4. Although the resistant phenotype is pluriform between models, it seems consistent within models, regardless of type of ARTA. These data suggest that the progression to and the phenotype of the CRPC state might already be determined early in carcinogenesis.

Prostate cancer cell proliferation is influenced by LDL-cholesterol availability and cholesteryl ester turnover

BackgroundProstate cancer growth is driven by androgen receptor signaling, and advanced disease is initially treatable by depleting circulating androgens. However, prostate cancer cells inevitably adapt, resulting in disease relapse with incurable castrate-resistant prostate cancer. Androgen deprivation therapy has many side effects, including hypercholesterolemia, and more aggressive and castrate-resistant prostate cancers typically feature cellular accumulation of cholesterol stored in the form of cholesteryl esters. As cholesterol is a key substrate for de novo steroidogenesis in prostate cells, this study hypothesized that castrate-resistant/advanced prostate cancer cell growth is influenced by the availability of extracellular, low-density lipoprotein (LDL)-derived, cholesterol, which is coupled to intracellular cholesteryl ester homeostasis.MethodsC4-2B and PC3 prostate cancer cells were cultured in media supplemented with fetal calf serum (FCS), charcoal-stripped FCS (CS-FCS), lipoprotein-deficient FCS (LPDS), or charcoal-stripped LPDS (CS-LPDS) and analyzed by a variety of biochemical techniques. Cell viability and proliferation were measured by MTT assay and Incucyte, respectively.ResultsReducing lipoprotein availability led to a reduction in cholesteryl ester levels and cell growth in C4-2B and PC3 cells, with concomitant reductions in PI3K/mTOR and p38MAPK signaling. This reduced growth in LPDS-containing media was fully recovered by supplementation of exogenous low-density lipoprotein (LDL), but LDL only partially rescued growth of cells cultured with CS-LPDS. This growth pattern was not associated with changes in androgen receptor signaling but rather increased p38MAPK and MEK1/ERK/MSK1 activation. The ability of LDL supplementation to rescue cell growth required cholesterol esterification as well as cholesteryl ester hydrolysis activity. Further, growth of cells cultured in low androgen levels (CS-FCS) was suppressed when cholesteryl ester hydrolysis was inhibited.ConclusionsOverall, these studies demonstrate that androgen-independent prostate cancer cell growth can be influenced by extracellular lipid levels and LDL-cholesterol availability and that uptake of extracellular cholesterol, through endocytosis of LDL-derived cholesterol and subsequent delivery and storage in the lipid droplet as cholesteryl esters, is required to support prostate cancer cell growth. This provides new insights into the relationship between extracellular cholesterol, intracellular cholesterol metabolism, and prostate cancer cell growth and the potential mechanisms linking hypercholesterolemia and more aggressive prostate cancer.

Palmitoleate Is a Mitogen, Formed upon Stimulation with Growth Factors, and Converted to Palmitoleoyl-phosphatidylinositol

Controversial correlations between biological activity and concentration of the novel lipokine palmitoleate (9Z-hexadecenoate, 16:1) might depend on the formation of an active 16:1 metabolite. For its identification, we analyzed the glycerophospholipid composition of mouse Swiss 3T3 fibroblasts in response to 16:1 using LC-MS/MS. 16:1 was either supplemented to the cell culture medium or endogenously formed when cells were stimulated with insulin or growth factors as suggested by the enhanced mRNA expression of 16:1-biosynthetic enzymes. The proportion of 1-acyl-2-16:1-sn-phosphatidylinositol (16:1-PI) was time-dependently and specifically increased relative to other glycerophospholipids under both conditions and correlated with the proliferation of fatty acid (16:1, palmitate, oleate, or arachidonate)-supplemented cells. Accordingly, cell proliferation was impaired by blocking 16:1 biosynthesis using the selective stearoyl-CoA desaturase-1 inhibitor CAY10566 and restored by supplementation of 16:1. The accumulation of 16:1-PI occurred throughout cellular compartments and within diverse mouse cell lines (Swiss 3T3, NIH-3T3, and 3T3-L1 cells). To elucidate further whether 16:1-PI is formed through the de novo or remodeling pathway of PI biosynthesis, phosphatidate levels and lyso-PI-acyltransferase activities were analyzed as respective markers. The proportion of 16:1-phosphatidate was significantly increased by insulin and growth factors, whereas lyso-PI-acyltransferases showed negligible activity for 16:1-coenzyme A. The relevance of the de novo pathway for 16:1-PI biosynthesis is supported further by the comparable incorporation rate of deuterium-labeled 16:1 and tritium-labeled inositol into PI for growth factor-stimulated cells. In conclusion, we identified 16:1 or 16:1-PI as mitogen whose biosynthesis is induced by growth factors.

TRIM29 Functions as a Tumor Suppressor in Nontumorigenic Breast Cells and Invasive ER+ Breast Cancer

Tripartite motif-containing 29 (TRIM29) is a member of the TRIM protein family that has been implicated in hematologic and solid tumor cancers. We found that TRIM29 functions as a tumor suppressor in both the nontumorigenic MCF10A [estrogen receptor (ER)-/TRIM29+] breast cell line and the invasive MCF7 (ER+/TRIM29-) breast cell line. Silencing TRIM29 in MCF10A cells resulted in preneoplastic changes that included loss of polarity in three-dimensional culture, increased proliferation, anchorage-independent growth, and increased migration and invasion. Conversely, the introduction of TRIM29 into MCF7 cells caused reversion to a less aggressive phenotype by antagonizing the growth effect of 17β-estradiol. The interaction between TRIM29 and ER signaling in MCF7 cells was supported by a reduction in ERE binding in the presence of TRIM29 and suppression of ER-dependent gene expression of TFF1, FOS, and GREB1. By microarray analyses, we showed that younger women (<55 years of age) with early-stage, ER+ breast cancer who were given no adjuvant systemic therapy had a significantly lower risk of relapse when their tumor had high TRIM29 expression (P = 0.02). This effect was not observed in older women (>55 years of age) and thus may be due to menopause and loss of circulating estrogens. Our results suggest that loss of TRIM29 expression in normal breast luminal cells can contribute to malignant transformation and lead to progression of ER+ breast cancer in premenopausal women.

Factors affecting cytotrophoblast cell viability and differentiation: Evidence of a link between syncytialisation and apoptosis

A relationship between cytotrophoblast differentiation (syncytialisation) and apoptosis is hypothesised to exist, but has not been clearly determined. To address this, we explored the effects of cAMP, an inducer of syncytialisation, on human choriocarcinoma cell differentiation and viability under three different culture conditions related to diverse survival status: no serum, 10% fetal calf serum or 10% charcoal-stripped fetal calf serum. 8-Br-cAMP increased BeWo cell viability in culture media without serum, but viability was decreased in a dose- and time-dependent manner when serum was present. The appearance of apoptotic nuclei fragments were only observed when BeWo cells were cultured in media containing serum combined with 8-Br-cAMP treatment. In addition, the ratio of FasL to Fas expression following treatment with 8-Br-cAMP increased by 20-fold in 10% charcoal-stripped fetal calf serum media and 65-fold 10% fetal calf serum media, and activation of caspase-3 also required media with serum. The markers of syncytialisation (syncytin 1 expression and human chorionic gonadotropin secretion) were induced significantly by 8-Br-cAMP, and were higher in 10% fetal calf serum media than in 10% charcoal-stripped fetal calf serum media, than in the absence of serum. Syncytia formation was stimulated by 8-Br-cAMP and this required serum in the media. We now show that factors contained within serum are necessary for cAMP-stimulated cytotrophoblast differentiation, that syncytialisation involves apoptotic events, and that a lack of serum based factors could switch the cellular program away from differentiation.

Abstract 4599: The RING E3 ligase BCA2 is regulated by estrogen and co-expressed with estrogen receptor in breast cancer cells

Abstract The Breast Cancer Associated gene 2 (BCA2) protein contains a RING H2 finger and a zinc finger near the N-terminus. The presence of the RING domain allows BCA2 to function as an E3 ligase. RING finger proteins play critical roles in mediating the transfer of ubiquitin and ubiquitin-like modifiers to substrates; such modifications are pivotal in regulating protein homeostasis and basic cellular processes such as gene-transcriptional regulation. The RING domain in BCA2 is known to act as an E3 ligase in its auto-regulation, and is suspected to play a role in the modification of other proteins. In a cohort of 945 primary invasive breast cancers examined for estrogen receptor-alpha (ER) and BCA2 expression, 67% were found to be ER-positive, 74% of which also exhibited strong nuclear BCA2 protein expression (p = 0.004). In this study, we examined potential mechanism(s) underlying BCA2 and ER co-expression at transcriptional and protein levels. Estrogen-responsive and non-responsive breast cancer cell lines were used as model systems. To investigate whether BCA2 is an estrogen responsive gene, BCA2 and ER-positive T47D breast cancer cells were cultured in medium containing charcoal-stripped fetal calf serum supplemented with 10 nM and 100 nM 17β-estradiol respectively over a 72-hour time course. Northern and Western blots showed a 17β-estradiol-associated induction of BCA2 mRNA and protein within 72 hrs. Moreover, when ER is ectopically or stably expressed in ER and BCA2-negative MDA-MB-231 cells, prominent BCA2 expression is detected, suggesting that ER is responsible for induction of BCA2 transcription. To investigate a possible interaction of BCA2 and ER at a transcriptional level, bioinformatic analysis of the BCA2 promoter region revealed ER and PR binding sites as well as possible sites of general transcription factors. Together our data indicates that BCA2 is upregulated in response to estrogen and that BCA2 expression can be rescued by the introduction of ER, suggesting that ER and BCA2 may be participating in crosstalk at the transcriptional level. Further investigations into this phenomenon are necessary to determine the possibility of the development of drugs targeting BCA2 in order to manipulate ER. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4599.

Hormonal regulation of β2‐adrenergic receptor level in prostate cancer

Androgen deprivation is the only effective systemic therapy available for patients with prostatic carcinoma, but is associated with a gradual transition to a hormone-refractory prostate cancer (HRCAP) in which ligand-independent activation of the androgen receptor has been implicated. The beta(2)-adrenergic receptor (beta(2)-AR) is a well-known activator of the androgen receptor. Prostatic cell lines were analyzed using cDNA micro-array, real time RT-PCR, radioligand binding assay, cAMP measurements, transfection and thymidine incorporation assay. Clinical specimens were studied by immunohistochemistry and Affymetrix microarrays. Here, we show that beta(2)-AR was transiently down-regulated both at mRNA- and protein levels when hormone-sensitive prostate cancer cells, LNCaP, were cultured in steroid stripped medium (charcoal-stripped fetal calf serum) or when the cells were treated with the anti-androgen, bicalutamide (Casodex). The number of beta-adrenergic receptors was modestly up-regulated in androgen independent cell lines (LNCaP-C4, LNCaP-C4-2 and DU145) compared to LNCaP. Triiodothyronine (T3) increased the level of beta(2)-AR and the effect of T3 was inhibited by bicalutamide. Immunohistochemical staining of human prostate specimens showed high expression of beta(2)-AR in glandular, epithelial cells and increased expression in malignant cells compared to benign hyperplasia and normal tissue. Interestingly, beta(2)-AR mRNA was strongly down-regulated by androgen ablation therapy of prostate cancer patients. The level of beta(2)-AR was increased by T3 in prostatic adenocarcinoma cells and reduced in prostate cancer patients who had received androgen ablation therapy for 3 months.

Downregulation of Thymosin β4 Expression by Androgen in Prostate Cancer LNCaP Cells

Androgen ablation therapy is an effective treatment for advanced prostate cancer, but the tumor often progresses toward a more aggressive phenotype. We determined the changes in genes associated with the malignant progression and found increased thymosin beta4, involved in tumor metastasis, in androgen-sensitive LNCaP cells grown in the medium with androgen-deficient, charcoal-stripped fetal calf serum. The mRNA expression of thymosin beta4 was determined by real-time polymerase chain reaction analysis. The transcriptional activity of thymosin beta4 was measured by luciferase assay using reporter plasmid containing 5'-flanking region of thymosin beta4. Thymosin beta4 mRNA expression was increased in LNCaP cells in the androgen-deficient condition and decreased by dihydrotestosterone treatment. Androgen receptor antagonist bicalutamide inhibited thymosin beta4 expression in a dose-dependent manner. In androgen receptor-negative PC-3 cells, no significant effects on thymosin beta4 gene expression were observed. The regulation of thymosin beta4 mRNA expression by androgen is due to the transcriptional activation. Deletion analysis revealed that the region between -83 bp and -46 bp of the thymosin beta4 gene is responsible for the regulation of the transcriptional activity by androgen. Thymosin beta4 expression is negatively controlled at the transcriptional level by androgen.

Extraction-Dependent Effects of American Ginseng (Panax quinquefolium) on Human Breast Cancer Cell Proliferation and Estrogen Receptor Activation

Ginseng root extracts and the biologically active ginsenosides have been shown to inhibit proliferation of human cancer cell lines, including breast cancer. However, there are conflicting data that suggest that ginseng extracts (GEs) may or may not have estrogenic action, which might be contraindicated in individuals with estrogen-dependent cancers. The current study was designed to address the hypothesis that the extraction method of American ginseng (Panax quinquefolium) root will dictate its ability to produce an estrogenic response using the estrogen receptor (ER)-positive MCF-7 human breast cancer cell model. MCF-7 cells were treated with a wide concentration range of either methanol-(alc-GE) or water-extracted (w-GE) ginseng root for 6 days. Cells were grown in media containing either normal or charcoal-stripped fetal calf serum to limit exposure to exogenous estrogen. Thus, an increase in MCF-7 cell proliferation by GE indicated potential estrogenicity. This was confirmed by blocking GE-induced MCF-7 cell proliferation with ER antagonists ICI 182,780 (1 nM) and 4-hydroxytamoxifen (0.1 microM). Furthermore, the ability of GE to bind ERalpha or ERbeta and stimulate estrogen-responsive genes was examined. Alc-GE, but not w-GE, was able to increase MCF-7 cell proliferation at low concentrations (5-100 microg/mL) when cells were maintained under low-estrogen conditions. The stimulatory effect of alc-GE on MCF-7 cell proliferation was blocked by the ER antagonists ICI 182,780 or 4-hydroxyta-moxifen. At higher concentrations of GE, both extracts inhibited MCF-7 and ER-negative MDA-MB-231 cell proliferation regardless of media conditions. Binding assays demonstrated that alc-GE, but not w-GE, was able to bind ERalpha and ERbeta. Alc-GE (50 microg/mL) also induced an approximate 2.5-fold increase in expression of the estrogen-responsive pS2 gene, as well as progesterone receptor (PgR) gene expression, whereas w-GE was without effect. These data indicate that low concentrations of alc-GE, but not w-GE, elicit estrogenic effects, as evidenced by increased MCF-7 cell proliferation, in a manner antagonized by ER antagonists, interactions of alc-GE with estrogen receptors, and increased expression of estrogen-responsive genes by alc-GE. Thus, discrepant results between different laboratories may be due to the type of GE being analyzed for estrogenic activity.

Human Constitutive Androstane Receptor Mediates Induction of CYP2B6 Gene Expression by Phenytoin

Compared with its rodent orthologs, little is known about the chemical specificity of human constitutive androstane receptor (hCAR) and its regulation of hepatic enzyme expression. Phenytoin (PHY), a widely used antiepileptic drug, is a potent inducer of CYP2B6 in primary human hepatocytes, but does not activate human pregnane X receptor (PXR) significantly in cell-based transfection assays at the same concentrations associated with potent induction of CYP2B6. Based on this observation, we hypothesized that PHY may be a selective activator of hCAR. In primary human hepatocytes, expression of CYP2B6 reporter genes containing phenobarbital-responsive enhancer module (PBREM) or PBREM/xenobiotic-responsive enhancer module (XREM) response elements were activated up to 14- and 28-fold, respectively, by 50 microm PHY. By contrast, parallel experiments in HepG2 cell lines co-transfected with an hPXR expression vector did not show increased reporter activity. These results indicated that a PXR-independent pathway, which is retained in primary hepatocytes, is responsible for PHY induction of CYP2B6. Further experiments revealed that PHY effectively translocates hCAR from the cytoplasm into the nucleus in both primary human hepatocytes and CAR(-/-) mice. Compared with vehicle controls, PHY administration significantly increased CYP2B6 reporter gene expression, when this reporter construct was delivered together with hCAR expression vector into CAR(-/-) mice. However, PHY did not increase reporter gene expression in CAR(-/-) mice in the absence of hCAR vector, implying that CAR is essential for mediating PHY induction of CYP2B6 gene expression. Taken together, these observations demonstrate that, in contrast to most of the known CYP2B6 inducers, PHY is a selective activator of CAR in humans.

Suppression Versus Induction of Androgen Receptor Functions by the Phosphatidylinositol 3-Kinase/Akt Pathway in Prostate Cancer LNCaP Cells with Different Passage Numbers

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway controls several important biological functions, such as cell growth regulation, apoptosis, and migration. However, the way in which PI3K/Akt controls androgen receptor (AR)-mediated prostate cancer cell growth remains unclear and controversial. Here, we demonstrate that the PI3K/Akt pathway regulates AR activity in a cell passage number-dependent manner. Specifically, PI3K/Akt pathway can suppress AR activity in androgen-dependent LNCaP cells with low passage numbers. In contrast, it can also enhance AR activity in LNCaP cells with high passage numbers. Furthermore, we also demonstrate that insulin-like growth factor-1 can activate the PI3K/Akt pathway that results in the phosphorylation of AR at Ser210 and Ser790. The consequence of these events may then change the stability of AR protein. Together, our results demonstrate that the PI3K/Akt pathway may have distinct mechanisms to modulate AR functions in various stages of prostate cancer cells and that a combined therapy of antiandrogens and anti-PI3K/Akt inhibitors may be worth considering as a future therapeutic approach to battle prostate cancer.

An in vitro long-term culture model for normal human mammary gland: expression and regulation of steroid receptors.

Steroids and their nuclear receptors play crucial roles in the development and maintenance of normal functions of the human mammary gland (HMG). They have also been implicated in breast carcinogenesis. However, the study of steroid action in normal HMG has been hampered by experimental difficulties. By using a newly established in vitro long-term culture method, we successfully cultured normal HMG tissue for more than 2 months without detriment to its morphology or steroid receptor expression. Expression of the cellular structural and extracellular matrix proteins was similar to that prior to culture, and HMG tissue retained its properties of steroid receptor expression and regulation. Addition of 17-beta estrogen to mammary tissues markedly increased the expression of progesterone receptor (PR) but only slightly affected that of the estrogen receptor (ER). Medroxyprogesterone acetate down-regulated the expression of PR within 24-48 h and also increased the expression of androgen receptor. When HMG tissue was cultured in medium containing normal or dextran-coated charcoal-stripped fetal calf serum or normal human serum, the expression and regulation of steroid hormone receptors were similar, although different in extent. When serum was omitted, the morphology of HMG was normal after 1 week, but the expression and regulation of ER and PR were altered. Thus, as HMGs retain the capacity to express steroid receptors in culture, this long-term culture system is probably a good model for studying the regulation of the mammary gland by steroids.

Development and Characterization of a Progressive Series of Mammary Adenocarcinoma Cell Lines Derived from the C3(1)/SV40 Large T-antigen Transgenic Mouse Model

We have developed four new mammary adenocarcinoma cell lines from the C3(1)/SV40 Large T-antigen (Tag) transgenic mouse model: M28N2 and M27H4 (weakly tumorigenic), M6 (carcinoma), and M6C (metastatic). The C3(1) promoter directs Tag expression to the mammary epithelium and 100% of female C3(1)/Tag transgenic mice develop mammary adenocarcinoma in a predictable and progressive manner. The cell lines we developed from this model are demonstrated to be of epithelial origin and display growth rates, both in vitro and following subcutaneous inoculation into nude mice, that are consistent with their representative stage of tumor progression. The more tumorigenic cell lines, M6 and M6C, both express the sodium/iodide symporter, a mammary carcinoma cell marker with potential therapeutic and diagnostic applications. All of the cell lines express estrogen receptor (ER) alpha and ER beta mRNA, and Western blot analysis demonstrates that the ER alpha protein is down-regulated in the M6 and M6C cell lines. M28N2 cells also express progesterone receptor (PgR), which is very unusual in a mouse mammary carcinoma cell line. In addition, all of the cell lines display growth inhibition when plated in media supplemented with charcoal-stripped fetal calf serum (CS FBS). When CS FBS is supplemented with beta estradiol or the progestin MPA, no significant difference in growth rates is observed relative to growth in CS FBS. The development and characterization of a progressive series of new mammary carcinoma cell lines will aid in the study of mammary carcinoma progression both in vitro and in vivo.

Alterations of prostate biomarker expression and testosterone utilization in human LNCaP prostatic carcinoma cells by garlic-derived S-allylmercaptocysteine.

This study determined the effects of S-allylmercaptocysteine (SAMC), a phytoconstituent from garlic, on the expression of androgen-responsive biomarkers, prostate specific antigen (PSA), and prostate specific membrane antigen (PSMA), in human prostatic carcinoma cells (LNCaP). Secretion of PSA was determined as well as the activity of PSMA measured as a function of its ability to hydrolyze poly-gamma-glutamated folate and N-acetylaspartylglutamate (NAAG). Folate hydrolase capacity was also determined in SAMC-treated cells grown in charcoal stripped fetal calf serum (CS-FCS). In addition, testosterone disappearance was measured from culture media of SAMC-treated LNCaP and PC-3 cells as well as from cell free lysates. PSA secretions were significantly decreased compared to control values at 1 day (8.4 +/- 2.6 vs. 18.9 +/- 1.7, P < 0.01), 4 days (18.9 +/- 5.3 vs. 73.8 +/- 4. 4, P < 0.001), and 6 days (35.6 +/- 2.1 vs. 96.5 +/- 17.9 ng/10(5) cells, P < 0.01; mean +/- SD). By contrast, PSMA activity measured as either folate hydrolase or NAAG dipeptidase (NAALADase) activity increased in cells treated with SAMC. PSMA-folate hydrolase activity in SAMC-treated cells grown in CS-FCS increased beyond that observed in cells grown in CS-FCS alone. Pre-exposure of LNCaP cells to SAMC resulted in enhanced rate of testosterone disappearance from culture media at 6 hr (P < 0.01) and at 48 hr (P < 0.001) compared to media from cells not previously exposed to SAMC. Results similar to these were also observed in androgen-independent PC-3 cells treated with SAMC. In lysates of SAMC-treated LNCaP cells, the rate of testosterone catabolism was twice that from phosphate buffered saline (PBS)-treated cells. SAMC-treated LNCaP cells grown in media supplemented with testosterone temporarily exhibited enhanced growth over a 2 day period but cell numbers declined later to levels similar to those of SAMC treatment. These results show that SAMC exhibits differential effects on recognized biomarkers for LNCaP cells similar to those produced by androgen deprivation and strongly suggests that this effect may be mediated, in part, by diminishing the trophic effects of testosterone, likely by converting it to metabolites less reactive toward androgen receptors.

Effects of epidermal growth factor (EGF) on the development of EGF‐receptor (EGF‐R) binding in fetal rabbit lung organ culture

Epidermal growth factor (EGF) causes gender- and development-specific changes in fetal lung surfactant synthesis. We hypothesized that the effects of EGF on development of surfactant synthesis are related to effects on EGF receptor (EGF-R) expression. We prepared sex-specific fetal rabbit lung organ cultures on gestational days 21 and 24 (term = 31 days) in Waymouth's medium + 10% charcoal-stripped fetal calf serum as control or with added EGF (10 ng/mL). After 3, 5, and 7 days of culture, we measured specific EGF-R binding in fetal lung plasma membrane preparations. Analysis of variance (ANOVA) revealed significant effects of fetal gender (P = 0.0003), time in culture (P = 0.01), and EGF treatment (P = 0.0003) on EGF specific binding. In control cultures from days 21 and 24 (both male and female), EGF specific binding tended to decrease with time in culture. Specific binding in EGF-treated female 21-day cultures was significantly higher than in controls, both after 5 days (184% of control, P = 0.007) and after 7 days (151% of control, P = 0.01; Bonferroni multiple comparisons) of treatment, whereas males exhibited no response to EGF treatment. As opposed to these effects in 21-day cultures, EGF had little effect on 24-day cultures. We conclude that EGF affects the expression of the EGF-R on EGF specific binding in the fetal lung. The development of surfactant synthesis in the fetal lung may be controlled by upregulation of the EGF-R. Pediatr Pulmonol. 2000; 29:27–33. © 2000 Wiley-Liss, Inc.

Estrogen synthesis in human colon cancer epithelial cells

Epidemiological and experimental data suggest an involvement of estrogen in the development and progression of colorectal cancer. In order to determine whether local synthesis of estrogen occurred in human colonic cancer cells, two colorectal cancer cell lines, HCT8 and HCT116, were evaluated for gene expression and enzyme activity of cytochrome P450 aromatase. In addition, the effect on aromatase expression of charcoal-stripped fetal calf serum, of quercetin and genistein and of tamoxifen and raloxifene was investigated in both cell lines. RT-PCR analysis revealed that colorectal adenocarcinoma cell lines contain aromatase as a major component. The conversion of [ 3H]-androstenedione to estrone and labeled water was dose-dependently inhibited by 4-hydroxyandrostenedione and obeyed Michaelis–Menten kinetic with apparent Km values of ∼20 nM and V max values of approx. 200 and 500 fmol/mg protein/h for HCT8 and HCT116 cells, respectively. After 24 h incubation, genistein (1 μM) significantly increased aromatase activity in HCT8 cells, with no effect on HCT116 cells. In accord with previous observation in reproductive tissues, quercetin (1 μM) significantly inhibited the enzyme activity in both cell lines. Also tamoxifen (100 nM) acted as inhibitor, while raloxifene (10 nM) decreased the enzyme activity only in HCT116 cells. The aromatase gene expression modulation by these effective agents was consistent with their effects on enzyme activity. These findings demonstrate for the first time that colorectal adenocarcinoma cell lines express aromatase. Interestingly, the enzyme activity was inhibited by quercetin, one major dietary flavonoid, by tamoxifen, a hormonal therapeutic agent for breast cancer, and by raloxifene, used in the prevention of postmenopausal osteoporosis.

An Upstream Regulator of the Glycoprotein Hormone α-Subunit Gene Mediates Pituitary Cell Type Activation and Repression by Different Mechanisms

Targeting of alpha-subunit gene expression within the pituitary is influenced by an upstream regulatory region that directs high level expression to thyrotropes and gonadotropes of transgenic mice. The same region also enhanced the activity of the proximal promoter in transfections of pituitary-derived alpha-TSH and alpha-T3 cells. We have localized the activating sequences to a 125-bp region that contains consensus sites for factors that also play a role in proximal promoter activity. Proteins present in alpha-TSH and alpha-T3 cells as well as those from GH3 somatotrope-derived cells interact with this region. The upstream area inhibited proximal alpha-promoter activity by 80% when transfected into GH3 cells. Repression in GH3 cells was mediated through a different mechanism than enhancement, as supported by the following evidence. Reversing the orientation of the area resulted in a loss of proximal promoter activation in alpha-TSH and alpha-T3 cells but did not relieve repression in GH3 cells. Mutation of proximal sites shown to be important for activation had no effect on repression. Finally, bidirectional deletional analysis revealed that multiple elements are involved in activation and repression and, together with the DNA binding studies, suggests that these processes may be mediated through closely juxtaposed or even overlapping elements, thus perhaps defining a new class of bifunctional gene regulatory sequence.

Effects of Tamoxifen, Toremifene and Chloroquine on the Lysosomal Enzymes in Cultured Retinal Pigment Epithelial Cells

Retinal pigment epithelial cells carry out phagocytosis and digestion of material shed from the photoreceptor outer segments. In this process, the integrity of lysosomal enzymes is of major importance. In the present study the effects of tamoxifen, toremifene and chloroquine on the activity of two lysosomal enzymes (cathepsin D and N-acetyl-beta-D-glucosaminidase) in the retinal pigment epithelial cells were studied. Retinal pigment epithelial cells from pig eyes were cultured for two weeks in Dulbecco's Modified Eagle Medium, after which the cells were exposed to 1-40 microM concentrations of tamoxifen citrate, toremifene citrate and chloroquine diphosphate. To eliminate possible medium-borne oestrogenic mechanisms, the test was repeated using phenol red-free medium with charcoal-stripped fetal calf serum. The exposure time was one week, after which the lysosomal enzymes cathepsin D and N-acetyl-beta-glucosaminidase were determined. Cellular injuries were assessed by quantifying the leakage of lactate dehydrogenase into the culture medium. Cathepsin D and N-acetyl-beta-D-glucosaminidase showed different sensitivities to tamoxifen, toremifene and chloroquine. The main lysosomal protease cathepsin D was more sensitive than N-acetyl-beta-D-glucosaminidase to the effects of tamoxifen and toremifene, possibly due to their antioestrogenic properties. The phenol red-free medium with charcoal-stripped serum seemed to make the drugs more effective than the reference medium. Chloroquine had only a minor effect on the lysosomal protease cathepsin D, but a clearer effect could be seen on N-acetyl-beta-glucosaminidase.

Regulation of epidermal growth factor receptor by androgens in human endometrial cells in culture.

Women with polycystic ovaries (PCO) have a thicker endometrium than women with normal ovaries. This cannot be due to unopposed oestrogen, as it occurs in ovulatory cycles. Androgens may be involved, as these are raised in women with PCO. The effects of steroids are partly mediated by growth factors and their receptors. The aim of this study was to investigate the effect of androgens on epidermal growth factor (EGF) receptor in human endometrium. Endometrium was enzymatically dispersed and glands and stromal cells separated. Cells were incubated in Ham's F10 medium supplemented with 5% charcoal-stripped fetal calf serum and either androgens or vehicle. Specific binding of [125I]-labelled EGF was measured. Testosterone and dihydrotestosterone (DHT) (10 micromol/l) increased EGF receptor concentration (control 100 +/- 9%, testosterone 196 +/- 23% control; control 100 +/- 1%, DHT 244 +/- 6% control) but did not alter receptor affinity. The effect of testosterone was inhibited by the anti-androgen hydroxyflutamide, but not by the antioestrogen ICI182780 nor the aromatase inhibitor 4-hydroxyandrostenedione. EGF receptor levels were increased by androstenediol (control 100 +/- 2%, androstenediol 120 +/- 10% control) but not by androstanediol, dehydroepiandrosterone (DHA), DHA sulphate or androstenedione. Testosterone and DHT increased EGF receptor concentrations in glandular epithelium (control 100 +/- 24%, testosterone 147 +/- 5%, DHT 185 +/- 30% control). These data suggest that androgens may have an effect on the endometrium via an increase in EGF receptor concentrations.

Involvement of EGF in medroxyprogesterone acetate (MPA)-induced mammary gland hyperplasia and its role in MPA-induced mammary tumors in BALB/c mice

In previous papers we have demonstrated that sialoadenectomy inhibited MPA-induced mammary tumorigenesis in BALB/c mice. To further explore the role of EGF in this experimental model, we evaluated its effects on mammary glands of sialoadenectomized (sialox) MPA-treated female mice and on tumor growth. MPA-treated sialox mice were injected s.c. ( n=3) or not ( n=6) with 5 μg EGF every 36 h for 45 days; MPA-treated sham-operated mice were used as controls ( n=6). Mammary glands from sialox MPA-treated mice are considerably less developed as compared with sham-operated animals. The exogenous administration of EGF restores the usual MPA-induced growth pattern of the glands, thus confirming a role for EGF either in mediating or cooperating with MPA in inducing the mammary architectural changes observed in MPA-treated mice. On the other hand, primary cultures of progestin-dependent (PD) ductal mammary adenocarcinoma in vivo tumor lines and of lobular progestin-independent (PI) tumor lines were used to evaluate the effect of EGF on tumor growth. In vitro EGF was found to stimulate cell proliferation of lobular PI tumor cells and of fibroblastic cells from both types of tumors at concentrations higher than 0.1–0.5 ng/ml and in the presence of 1–5% of charcoal-stripped fetal calf serum. Conversely, no proliferative effects were observed in ductal PD cells under the same experimental conditions, regardless of the presence of 10 nM MPA. It can be concluded that although EGF plays an important role in MPA-induced mammary carcinogenesis, it is not necessary in PD tumor growth.