afimoxifene and Adenocarcinoma

Tamoxifen (TAM), used to treat estrogen receptor (ER)-positive breast cancer, is a well known estrogen antagonist in the breast, but a partial estrogen agonist in the endometrium. In addition, TAM metabolites, such as 4'-hydroxy-tamoxifen (4-OH-TAM), have been shown to be more potent than the parent compound. The objective of this study was to determine the effects of 4-OH-TAM and estradiol (E2) on two human endometrial adenocarcinoma cell lines, HEC-1B and HEC-1A. When HEC-1B cells were treated with lower concentrations (10-1,000 nM) of 4-OH-TAM or E2 for 1-3 days, no significant difference in the percentage of cell survival was observed among the varying concentrations. At higher 4-OH-TAM or E2 concentrations (1-100 µM), HEC-1B and HEC-1A cells responded similarly with an obvious decrease in cell growth noted at 10 and 100 µM 4-OH-TAM and 100 µM E2. In order to address the observed cell death, DNA laddering was performed at various time intervals with 4-OH-TAM (10 µM) or E2 (10 or 50 µM). DNA gel electrophoresis failed to show the typical laddering pattern (180-200 bp) observed in apoptosis. Furthermore, western blot analysis of caspase-8 and -3 failed to demonstrate caspase activity. These results suggest that apoptosis was not the underlying cellular mechanism of cell death. Due to the lack of apoptotic markers, a cytotoxic (cell death) effect was differentiated from a cytostatic (growth inhibition) effect confirming that cell death had occurred. In summary, micromolar concentrations of 4-OH-TAM induced a non-apoptotic cytotoxic effect in the endometrium; however further studies are needed to elucidate the cytotoxic pathway being utilized.

Topics: Adenocarcinoma; Apoptosis; Caspase 3; Caspase 8; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Endometrial Neoplasms; Estradiol; Female; Humans; Selective Estrogen Receptor Modulators; Tamoxifen

Pancreatic cancer is one of the deadliest cancers with poor survival rates and limited therapeutic options. To improve the understanding of this disease's biology, a prerequisite for the generation of novel therapeutics, new platforms for rapid and efficient genetic and therapeutic screening are needed. Therefore, a combined in vitro/in vivo hybrid shRNA assay was developed using isolated murine primary pancreatic ductal cells (PDCs), in which oncogenic Kras(G12D) could be activated in vitro by genomic recombination through 4OH-tamoxifen-induced nuclear translocation of Cre-ERT2 expressed under control of the ROSA26 promoter. Further genetic manipulation was achieved through selective and stable RNAi against the tumor suppressors p16(Ink4a) (CDKN2A) or Trp53 (TP53) using lentiviral gene delivery. Treatment of PDCs with 4OH-tamoxifen increased phosphorylation of ERK downstream of KRAS, and subsequent lentiviral transduction resulted in sustained target gene repression. Double-mutant PDCs were then reintroduced into the pancreata of NOD-SCID-gamma (NSG) mice and monitored for tumor growth. Orthotopic implantation of PDCs carrying the activated Kras(G12D)-allele and shRNA against p16(Ink4a) or Trp53 resulted in tumor growth, metastasis, and reduced survival of NSG mice. In contrast, Kras(G12D) alone was not sufficient to induce tumor growth.. The combinatory in vitro/in vivo approach described in this study allows for rapid and efficient identification of genes involved in carcinogenesis and opens new avenues for the development of therapeutic strategies to improve cancer treatment.

Topics: Adenocarcinoma; Animals; Carcinoma, Pancreatic Ductal; Genes, Tumor Suppressor; Heterografts; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Proto-Oncogene Proteins p21(ras); RNA, Small Interfering; Tamoxifen

Tamoxifen is a prodrug that is metabolically activated by 4-hydroxylation to the potent primary metabolite 4-hydroxytamoxifen (4OHT) or via another primary metabolite N-desmethyltamoxifen (NDMTAM) to a biologically active secondary metabolite endoxifen through a cytochrome P450 2D6 variant system (CYP2D6). To elucidate the mechanism of action of tamoxifen and the importance of endoxifen for its effect, we determined the anti-oestrogenic efficacy of tamoxifen and its metabolites, including endoxifen, at concentrations corresponding to serum levels measured in breast cancer patients with various CYP2D6 genotypes (simulating tamoxifen treatment).. The biological effects of tamoxifen and its metabolites on cell growth and oestrogen-responsive gene modulation were evaluated in a panel of oestrogen receptor-positive breast cancer cell lines. Actual clinical levels of tamoxifen metabolites in breast cancer patients were used in vitro along with actual levels of oestrogens observed in premenopausal patients taking tamoxifen.. Tamoxifen and its primary metabolites (4OHT and NDMTAM) only partially inhibited the stimulant effects of oestrogen on cells. The addition of endoxifen at concentrations corresponding to different CYP2D6 genotypes was found to enhance the anti-oestrogenic effect of tamoxifen and its metabolites with an efficacy that correlated with the concentration of endoxifen; at concentrations corresponding to the extensive metabolizer genotype it further inhibited the actions of oestrogen. In contrast, lower concentrations of endoxifen (intermediate and poor metabolizers) had little or no anti-oestrogenic effects.. Endoxifen may be a clinically relevant metabolite in premenopausal patients as it provides additional anti-oestrogenic actions during tamoxifen treatment.

Topics: Adenocarcinoma; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cytochrome P-450 CYP2D6; Female; Genetic Variation; Genotype; Humans; In Vitro Techniques; MCF-7 Cells; Premenopause; Tamoxifen

c-Src is an important adapter protein with oestrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2), which validates it as an attractive target for the treatment of breast cancer. A specific c-Src inhibitor, 4-amino-5-(4-chlorophenyl)-7(t-butyl)pyrazolo[3,4-d]pyrinidine (PP2), was utilised to block c-Src activity to identify targeted vulnerabilities affected by ER and HER2 in a panel of breast cancer cell lines.. ER, growth factor receptors and signalling pathways were detected by Western-blot. The DNA content of the cells was determined by using a DNA fluorescence quantitation kit. Cell cycles were analysed by flow cytometry.. The antiproliferative effect of PP2 closely correlated with the inhibition of c-Src mediated extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) and/or phosphoinositide 3-kinase (PI3K)/Akt growth pathways. Inhibition of c-Src tyrosine kinase predominantly blocked ER negative breast cancer cell growth, particularly the triple (i.e. ER, progesterone receptor (PR), and HER2) negative cells. In contrast, ER negative Sk-Br-3 cells with highest HER2 phosphorylation were resistant to PP2, in which hyper-activated HER2 directly regulated growth pathways. However, blocking c-Src recovered ER expression and down-regulated HER2 which made Sk-Br-3 cells regain responsiveness to 4-hydroxytamoxifen. The majority of ER positive cells were not sensitive to PP2 regardless of wild-type or endocrine resistant cell lines.. c-Src mediates the essential role of growth pathways in ER negative breast cancer cells. The ER positive and HER2 over-activation are two important predictive biomarkers for the resistance to a c-Src inhibitor. These data provided an important therapeutic rationale for patient selection in clinical trials with c-Src inhibitors in breast cancer.

Topics: Adenocarcinoma; Antineoplastic Agents; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Division; Cell Line, Tumor; DNA, Neoplasm; Drug Resistance, Neoplasm; Estrogen Receptor Modulators; Estrogens; Female; Humans; MAP Kinase Signaling System; Molecular Targeted Therapy; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Phosphatidylinositol 3-Kinases; Progesterone; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins pp60(c-src); Pyrimidines; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Signal Transduction; Tamoxifen

We have isolated from human breast cancers several mutations in the Helix 12 component of activation function 2 (AF-2) in the estrogen receptor alpha (ERalpha). We used a novel approach to detect changes in the hormone-binding domain of ERalpha, based on the evidence that antiestrogens, such as 4-hydroxytamoxifen (ZOHT) and ICI 182,780, block the function of ERalpha by binding and folding the AF-2 transcriptional domain in a way that inhibits its association with coactivator proteins. We have identified a Helix 12 mutation, M543V, which leads to greater ERalpha transcription with ZOHT and other antiestrogens (including 1,1-dichloro-2,2,3-triarylcyclopropanes, DTACs) than with 17-beta estradiol (E2). We also found an independent mutation at the same position, M543I, which did not show this inverted ligand phenotype. In comparison to further Helix 12 mutations made in vitro, it appears that relative hydrophobicity of the amino acid side chains on the inner face of Helix 12 is key to maintaining the transcriptionally active, agonist conformation with bound E2. This active conformation can be induced, resulting in increased transcription, by adding excess p160 coactivator AIB1 in transcriptional assays with E2-bound receptors, while the ZOHT-bound receptors were not further activated by AIB1. Other experiments show that the cross talk between ERalpha and AP-1 protein from AP-1-binding sites is not dependent on Helix 12 integrity. We show that two alleles containing a proline substitution in Helix 12 that inactivate AF-2 function of ERalpha at EREs have little negative effect on function through AP-1 elements, supporting a prominent role for the N-terminal AF-1 of ERalpha in AP-1/ERalpha transcriptional cross talk.

Topics: Adenocarcinoma; Amino Acid Motifs; Amino Acid Substitution; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Estradiol; Estrogen Receptor alpha; Estrogens; Female; Fulvestrant; Humans; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Point Mutation; Protein Binding; Protein Conformation; Protein Interaction Mapping; Protein Structure, Tertiary; Recombinant Fusion Proteins; Selective Estrogen Receptor Modulators; Tamoxifen; Transcription Factor AP-1; Transfection

The role of estrogens in the increased risk of lung adenocarcinoma in women remains uncertain. We reported that lung adenocarcinoma cell lines from female, but not male, patients with non-small cell lung cancer respond proliferatively and transcriptionally to estradiol (E(2)), despite equal protein expression of estrogen receptors (ER) alpha and beta. To test the hypothesis that nuclear localization of ER alpha corresponds to genomic E(2) activity in lung adenocarcinoma cells from females, cell fractionation, immunoblot, and confocal immunohistochemical microscopy were performed. We report for the first time that E(2) increases phospho-serine-118-ER alpha (P-ser118-ER alpha) and cyclin D1 (CCND1) nuclear colocalization in H1793, but not A549 lung adenocarcinoma cells, derived from a female and male patient, respectively. ER beta was primarily in the cytoplasm and mitochondria, independent of E(2) treatment, and showed no difference between H1793 and A549 cells. E(2) induced higher transcription of endogenous ER alpha-regulated CCND1 in H1793 than in A549 cells. Likewise, higher rapid, non-genomic E(2)-induced extracellular signal-regulated kinase 1/2 activation was detected in H1793 compared with A549 cells, linking extracellular signal-regulated kinase activation to increased P-ser118-ER alpha. Furthermore, E(2) increased cyclin D1 and P-ser118-ER alpha nuclear localization in H1793, but not A549 cells. Together, our results indicate that nuclear localization of P-ser118-ER alpha provides one explanation for sex-dependent differences in E(2)-genomic responses in lung adenocarcinoma cell lines.

Topics: Adenocarcinoma; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Enzyme Activation; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Fluorescence; Fulvestrant; Genome, Human; Humans; Lung Neoplasms; Male; Microscopy, Confocal; Mitogen-Activated Protein Kinases; Mutant Proteins; Phosphoserine; Protein Transport; Sex Characteristics; Signal Transduction; Subcellular Fractions; Tamoxifen

Although the net benefits of tamoxifen in adjuvant breast cancer therapy have been proven, the recurrence of the cancer in an aggressive and hormone independent form has been highly problematic. We previously demonstrated the important role mitochondrial DNA (mtDNA) plays in hormone-independence in prostate cancer. Here, the role of mtDNA in breast cancer progression was investigated. We established hydroxytamoxifen (4-OHT) resistant HTRMCF by growing MCF-7, human breast adenocarcinoma cells, in the presence of 4-OHT. HTRMCF was cross-resistant to 4-OHT and ICI182,780 concurrent with the depletion of mtDNA. To further investigate the role of mtDNA depletion, MCF-7 was depleted of mtDNA by treatment with ethidium bromide. MCF Rho 0 was resistant to both 4-OHT and ICI182,780. Furthermore, cybrid (MCFcyb) prepared by fusion MCF Rho 0 with platelet to transfer mtDNA showed susceptibility to antiestrogen. Surprisingly, after withdrawal of 4-OHT for 8 weeks, HTRMCF and their clones became susceptible to both drugs concurrent with a recovery of mtDNA. Herein, our results substantiated the first evidence that the depletion of mtDNA induced by hormone therapy triggers a shift to acquired resistance to hormone therapy in breast cancer. In addition, we showed that mtDNA depletion can be reversed, rendering the cancer cells susceptible to antiestrogen. The fact that the hormone independent phenotype can be reversed should be a step toward more effective treatments for estrogen-responsive breast cancer.

Topics: Adenocarcinoma; Antineoplastic Agents, Hormonal; Blotting, Southern; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Disease Progression; DNA, Mitochondrial; DNA, Neoplasm; Drug Resistance, Neoplasm; Estradiol; Estrogen Receptor Modulators; Ethidium; Female; Flow Cytometry; Fulvestrant; Humans; Neoplasms, Hormone-Dependent; Reverse Transcriptase Polymerase Chain Reaction; Tamoxifen

Pharmacological control is a desirable safety feature of oncolytic adenoviruses (oAdV). It has recently been shown that oAdV replication may be controlled by drug-dependent transcriptional regulation of E1A expression. Here, we present a novel concept that relies on tamoxifen-dependent regulation of E1A activity through functional linkage to the mutated hormone-binding domain of the murine estrogen receptor (Mer). Four different E1A-Mer chimeras (ME, EM, E(DeltaNLS)M, MEM) were constructed and inserted into the adenoviral genome under control of a lung-specific surfactant protein B promoter. The highest degree of regulation in vitro was seen for the corresponding oAdVs Ad.E(DeltaNLS)M and Ad.MEM, which exhibited an up to 100-fold higher oAdV replication in the presence as compared with the absence of 4-OH-tamoxifen. Moreover, destruction of nontarget cells was six- and 13-fold reduced for Ad.E(DeltaNLS)M and Ad.MEM, respectively, as compared with Ad.E. Further investigations supported tamoxifen-dependent regulation of Ad.E(DeltaNLS)M and Ad.MEM in vivo. Induction of Ad.E(DeltaNLS)M inhibited growth of H441 lung tumors as efficient as a control oAdV expressing E1A. E(DeltaNLS)M and the MEM chimeras can be easily inserted into a single vector genome, which extends their application to existing oAdVs and strongly facilitates in vivo application.

Topics: Adenocarcinoma; Adenovirus E1A Proteins; Animals; Cytopathogenic Effect, Viral; Female; Gene Expression Regulation; Genetic Engineering; Genetic Therapy; HeLa Cells; Humans; Lung Neoplasms; Mice; Mice, Nude; Receptors, Estrogen; Recombinant Proteins; Tamoxifen; Transcription, Genetic; Tumor Cells, Cultured; Virus Replication

Cytochrome P450 monooxygenase 3A4 (CYP3A4) and P-glycoprotein, encoded by multidrug resistance 1 (MDR1) gene, are responsible for the metabolism of endogenous steroids, prescribed drugs, and xenobiotics. Both genes are regulated by steroid and xenobiotic receptor (SXR), a member of nuclear hormone receptors. Various endogenous steroids and drugs function as ligands of SXR. Although CYP3A4, MDR1, and SXR are expressed mainly in the liver and the small intestine, these gene products are also expressed in breast cancer cells. Because tamoxifen (TAM) is known to be metabolized by CYP3A4 and P-glycoprotein, we investigated the effect of TAM on these SXR-targeted genes in breast cancer cells. Transient transfection-based reporter gene assays showed 4-hydroxy TAM activated the SXR-mediated transcription through CYP3A4 and MDR1 promoters in a ligand- and receptor concentration-dependent manner. We confirmed the binding of 4-hydroxy TAM to SXR by ligand binding assay. Moreover, semiquantitative RT-PCR studies revealed that 4-hydroxy TAM activated the expression of CYP3A4 and MDR1 mRNA in MCF-7 cells. These results suggest that TAM induces CYP3A4 and MDR1 gene expression through SXR, which may affect TAM metabolic pathway in breast cancer cells.

Topics: Adenocarcinoma; Antineoplastic Agents, Hormonal; ATP Binding Cassette Transporter, Subfamily B, Member 1; Breast Neoplasms; Cell Line, Tumor; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Estrogen Antagonists; Gene Expression Regulation, Neoplastic; Genes, MDR; Humans; Pregnane X Receptor; Receptors, Steroid; Retinoid X Receptors; RNA, Messenger; Tamoxifen; Transcription, Genetic

Estrogen 17beta-estradiol (E2) rapidly modulates several signaling pathways related to cell growth, preservation, and differentiation. The physiological role of these nongenomic effects with regard to downstream outcomes, and the relationship with transcriptional estrogen activity are unclear. Furthermore, the ability of selective estrogen receptor modulators (SERMs) to trigger nongenomic actions is largely unknown. To determine whether estrogen receptor (ER) ligands exert nongenomic activity in endometrial adenocarcinoma cells, and whether this activity affects transcription and DNA synthesis, we challenged human Ishikawa cells with E2 or partial ER agonists 4-hydroxytamoxifen (OHT) and raloxifene (ral). Serum-starved Ishikawa cells exposed for 5 min to 0.1 nM E2 showed induced phosphorylation of MAPK (ERK1/2). Ral and 4-OHT each at 1 nM also stimulated ERK in a rapid transient manner. E2 and 4-OHT induced proto-oncogene c-fos mRNA expression in Ishikawa cells within 30 min, but ral had no effect. In contrast to nongenomic action, only E2 stimulated expression of an estrogen response element (ERE)-driven luciferase (LUC) reporter gene. To examine DNA synthesis, [(3)H]-thymidine incorporation was measured in serum-starved cultures exposed to E2 or partial agonists for 2 d. E2 at 1 nM stimulated thymidine uptake in an ERK-dependent manner, but 1 nM 4-OHT, 1 nM ral, and 0.1-nM concentrations of E2 had no significant effects. Taken together, these data indicate that both nongenomic and direct transcriptional ER effects are likely required to promote DNA synthesis.

Topics: Adenocarcinoma; DNA; Endometrial Neoplasms; Enzyme Activation; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Gene Expression; Humans; Luciferases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Mutation; Proto-Oncogene Mas; Proto-Oncogene Proteins c-fos; Raloxifene Hydrochloride; Receptors, Estrogen; Recombinant Fusion Proteins; Response Elements; Tamoxifen; Transfection; Tumor Cells, Cultured

Estrogens promote cell proliferation in normal and transformed mammary epithelial cells by inducing expression of hormone-responsive genes involved in the cell cycle. The action of antiestrogens is therefore central in regard to their potent inhibitory effects on estrogen-induced cell growth. We used normal human epithelial breast cells from primary cultures (HBE cells) to study hormonal (estrogen and antiestrogen) regulation on 3 key proteins involved in the apoptotic process: Bcl-2, p53 and caspase-3. The mammary adenocarcinoma cell line, MCF-7, was also used to study the molecular regulation of Bcl-2. In both HBE and MCF-7 cells, we found that estradiol (E2) induced an increase in Bcl-2 mRNA levels. This effect was counteracted in the presence of a pure antiestrogen, ICI 182780 (ICI). Alone, ICI did not modify either the Bcl-2 protein or mRNA levels in HBE cells, whereas in MCF-7, a strong downregulation of Bcl-2 mRNA was observed. In parallel, in HBE cells, we observed that E2 caused a decrease in p53 and caspase-3 protein levels, whereas ICI alone increased p53 and caspase-3 protein levels. The ICI effects on p53 and caspase-3 were partially counteracted by E2. Under the same experimental conditions, ICI exerts a potent pro-apoptotic effect, which was not counteracted by E2. In contrast, 4-hydroxytamoxifen was slightly weaker as a pro-apoptotic agent in HBE cells and its effects were reversed by E2. We demonstrate that in HBE cells, ICI reverses the anti-apoptotic action of E2 and alone acts as a highly potent pro-apoptotic molecule. These results provide new insight into treatment for breast cancer prevention.

Topics: Adenocarcinoma; Antineoplastic Agents, Hormonal; Apoptosis; Breast; Breast Neoplasms; Caspase 3; Caspases; Cells, Cultured; Enzyme Induction; Epithelial Cells; Estradiol; Estrogen Receptor Modulators; Estrogens; Female; Fulvestrant; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Genes, p53; Humans; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; RNA, Neoplasm; Stimulation, Chemical; Tamoxifen; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Estrogen receptors are phosphoproteins which can be activated by ligands, kinase activators, or phosphatase inhibitors. Our previous study showed that p38 mitogen-activated protein kinase was involved in estrogen receptor activation by estrogens and MEKK1. Here, we report estrogen receptor-dependent p38 activation by estrogens in endometrial adenocarcinoma cells and in vitro and in vivo phosphorylation of the estrogen receptor alpha mediated through p38. The phosphorylation site was identified as threonine-311 (Thr(311)), located in helix 1 of the hormone-binding domain. The mutation of threonine-311 to alanine did not affect estrogen binding of the receptor but compromised its interaction with coactivators. Suppression of p38 activity or mutation of the site inhibited the estrogen-induced receptor nuclear localization as well as its transcriptional activation by estrogens and MEKK1. The inhibition of the p38 signal pathway by a specific chemical inhibitor blocked the biological activities of estrogens in regulating endogenous gene expression as well as endometrial cancer cell growth. Our studies demonstrate the role of estrogen receptor phosphorylation induced by the natural ligand in estrogen receptor's cellular distribution and its significant contribution to the growth-stimulating activity of estrogens in endometrial cancer cells.

Topics: Active Transport, Cell Nucleus; Adenocarcinoma; Amino Acid Sequence; Animals; Endometrial Neoplasms; Enzyme Activation; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Fatty Acids, Unsaturated; Female; Humans; Ligands; MAP Kinase Kinase Kinase 1; Mitogen-Activated Protein Kinases; Molecular Sequence Data; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Protein Sorting Signals; Receptors, Estrogen; Signal Transduction; Tamoxifen; Threonine; Transcription, Genetic

Oncolytic adenoviruses with restricted replication can be produced if the expression of crucial transcription units of the virus is controlled by tissue- or tumor-specific promoters. Here we describe a method for the rapid incorporation of exogenous promoters into the E1A and E4 regions of the human adenovirus type 5 genome. Using this system, we have generated AdEHT2 and AdEHE2F, two conditionally replicative adenoviruses for the treatment of breast cancer. The expression of the E1A gene in both viruses is controlled by a minimal dual-specificity promoter that responds to estrogens and hypoxia. The tight regulation of E1A expression correlated with the ability of these viruses to replicate and kill human cancer cells that express estrogen receptors, or are maintained under hypoxic conditions. The telomerase reverse transcriptase (TERT) promoter and the E2F-1 promoter are preferentially activated in cancer cells. They were introduced into the E4 region of AdEHT2 and AdEHE2F, respectively. The telomerase core promoter failed to block the replication of the virus in telomerase-negative cells. In contrast, AdEHE2F was attenuated in nontransformed quiescent cells growing under normoxic conditions, suggesting that an intact pRB pathway with low levels of E2F transcription factors acts as a negative modulator for the virus. These data indicate that the simultaneous regulation of E1A and E4 viral transcription units by the appropriate combination of promoters can increase the tumor selectivity of oncolytic adenoviruses.

Topics: Adenocarcinoma; Adenovirus E1A Proteins; Adenovirus E4 Proteins; Adenoviruses, Human; Animals; Breast Neoplasms; Cell Hypoxia; Cytopathogenic Effect, Viral; DNA-Binding Proteins; Estradiol; Estrogens; Female; Fibroblasts; Gene Expression Regulation, Viral; Genes, Synthetic; Genetic Therapy; Genetic Vectors; HeLa Cells; Humans; Lung Neoplasms; Mice; Mice, Nude; Neoplasms, Hormone-Dependent; Promoter Regions, Genetic; Receptors, Estrogen; Tamoxifen; Telomerase; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Virus Replication; Xenograft Model Antitumor Assays

Tamoxifen is an estrogen receptor (ER)-antagonist that is widely used for the treatment of breast cancer, although it increases the risk of endometrial cancer. The mechanism mediating the stimulatory effect of tamoxifen on endometrial cancer is presently unknown. In this study we examined the effects of tamoxifen on Ishikawa 3H-12 endometrial cancer cells and MCF-7 breast cancer cells. Ishikawa cell growth was stimulated by 4-hydroxytamoxifen and accompanied by increased transcriptional activity of the endogenous ER. These stimulatory effects did not occur in MCF-7 cells. The relative transcriptional activity of the activation function (AF) 1 domain of ERalpha compared with that of the AF2 domain was 4-fold higher in Ishikawa cells than in MCF-7 cells. Mitogen-activated protein (MAP) kinase, which stimulates the transcriptional activity of AF1, was constitutively activated in Ishikawa cells, but not in MCF-7 cells. These observations suggest that the constitutively activated MAP kinase-signaling pathway in Ishikawa cells enhances the transcriptional activity of ERalpha via the AF1 domain. This ERalpha activation pathway may be involved in the stimulatory effect of tamoxifen on the development and/or progression of endometrial cancer.

Topics: Adenocarcinoma; Breast Neoplasms; Carcinoma; Endometrial Neoplasms; Endometrium; Estradiol; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Models, Biological; Neoplasms, Hormone-Dependent; Nuclear Receptor Coactivator 3; Protein Structure, Tertiary; Receptors, Estrogen; Selective Estrogen Receptor Modulators; Signal Transduction; Tamoxifen; Transcription Factors; Transcriptional Activation; Tumor Stem Cell Assay

Proliferation of MCF-7 cells is estrogen dependent and antiestrogen sensitive. In the absence of estrogens or presence of antiestrogens MCF-7 cells arrest in the G1 phase of the cell cycle, and this arrest is associated with an accumulation of the active, hypophosphorylated form of the retinoblastoma protein (pRb). Because active pRb negatively regulates passage from G1 to S phase, this suggests that pRb is a crucial target of estrogen action, and that its inactivation might lead to antiestrogen resistance. We tested this hypothesis by expressing viral tumor antigens (T antigens), which bind and inactivate pRb, in MCF-7 cells, and determining the effects on cell proliferation in the presence of antiestrogens. The results of these experiments demonstrate that T antigen expression confers antiestrogen resistance to MCF-7 cells. Using a panel of mutant T antigens, we further demonstrate that the pRb-binding, but not the p53 binding domain is required to confer antiestrogen resistance. Thus, pRb is an important target of estrogen action, and its inactivation can contribute to the development of antiestrogen resistance.

Topics: Adenocarcinoma; Antigens, Polyomavirus Transforming; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cyclin D1; Drug Resistance, Neoplasm; Estradiol; Estrogen Receptor Modulators; Estrogens; Female; Fulvestrant; G1 Phase; Humans; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Protein Structure, Tertiary; Recombinant Fusion Proteins; Retinoblastoma Protein; Tamoxifen; Transfection; Tumor Cells, Cultured

Antiestrogen tamoxifen (Tam) is the most prescribed drug for the treatment of estrogen receptor (ER)-positive breast cancers. It is also used in long-term clinical trials with encouraging preliminary results as a chemopreventive agent for breast cancer. The effect of Tam on ER-negative cancers, however, is unclear. Here we reported that paclitaxel and 4-hydroxytamoxifen (4-HT) have a synergistic cytotoxic effect on the ER-negative colon cancer cell line HCT15, which is refractory to paclitaxel alone. Our results showed that 4-HT at submicromolar concentrations effectively enhanced the antiproliferative effect of paclitaxel. In addition, at 1/10 of the paclitaxel concentrations used for HCT15, 4-HT and paclitaxel also showed synergistic effect on NCI H460, an ER-negative lung cancer cell line. For both cell lines, the effective concentration for paclitaxel to inhibit cell growth was 1 log lower in the combination treatment than the concentration used in the single treatment. Cell cycle analysis showed that the combination of paclitaxel and 4-HT increased the G2/M population and resulted in the increase of apoptosis in both cell lines. Enhanced early release of cytochrome c from mitochondria may be the apoptotic pathway activated in the combination treatment in HCT15 cells.

Topics: Adenocarcinoma; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Large Cell; Cell Division; Colorectal Neoplasms; Cytochrome c Group; Drug Synergism; Drug Therapy, Combination; Estrogen Receptor Modulators; G2 Phase; Humans; Lung Neoplasms; Mitochondria; Mitosis; Paclitaxel; Receptors, Estrogen; Tamoxifen; Tumor Cells, Cultured

Transforming growth factor-alpha (TGF-alpha) contributes to the progression of mammary carcinogenesis in part through synergistic augmentation of estradiol (E2) action. To investigate this further, we sought to determine (1) whether the duration of TGF-alpha treatment might influence the nature of the TGF-alpha/E2 interaction, and (2) whether TGF-alpha would behave in a similar manner when combined with phytoestrogens. To this end, we transfected T47-D breast cancer cells with an estrogen-responsive reporter and then treated the cells (for 4-48 h) with varying concentrations of TGF-alpha, E2, the antiestrogen 4-hydroxy-tamoxifen (HOT), and/or one of three phytoestrogens. Our findings revealed that TGF-alpha has short-term synergistic and long-term inhibitory effects on E2- and phytoestrogen-regulated gene expression. Furthermore, this secondary inhibition of E2 action by TGF-alpha was similar in magnitude to that imposed by HOT. These findings demonstrate a novel role for TGF-alpha and invite reevaluation of current models regarding TGF-alphas interactions with E2 in breast cancer cells. Our results also raise the possibility that phytoestrogens, which interact with TGF-alpha in a manner conceptually identical to that of E2, may subserve a regulatory function in breast cancer cells.

Topics: Adenocarcinoma; Breast Neoplasms; Drug Synergism; Estrogen Antagonists; Estrogens; Estrogens, Non-Steroidal; Gene Expression Regulation, Neoplastic; Humans; Isoflavones; Luciferases; Phytoestrogens; Plant Preparations; Receptors, Estrogen; Tamoxifen; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Estrogenic activities of testosterone (T) and 5a-dihydrotestosterone (DHT) were detected and measured by using their specific stimulatory effects on alkaline phosphatase (AP) activity in human endometrial adenocarcinoma cells of the Ishikawa Var-1 line. These two physiologic androgens were able to induce, at microM concentrations, estrogenic effect believed to be mediated by the estrogen receptor (ER) since the antiestrogens ICI-164384 and 4-hydroxytamoxifen (OHTam), but not the antiandrogens hydroxyflutamide (OHFl) or cyproterone acetate (CPA), reversed that effect. By using another in vitro bioassay, based on the progestin-specific stimulation of AP activity in cells of the T47D human breast cancer line, progestagenic activity was detected and measured in T, DHT and three synthetic androgens: nandrolone (19-nortestosterone). 7 alpha-methyl 19-nortestosterone (MENT) and mibolerone (7 alpha, 17 alpha-dimethyl 19-nortestosterone) (DMNT). While progestagenic effects of T and DHT required relatively high concentrations (microM levels), the synthetic androgens stimulated AP activity at nM or pM levels. These effects seem to be mediated by the progesterone receptor (PR), since they are completely abolished by the antiprogestins RU-486, ZK-98299 and ZK-112993, but not by the antiandrogen OHFl. These simple in vitro bioassays, expressing biological effects of the test compounds in human cells in culture, revealed dual or multiple hormonal activities coexisting in a single compound and provide quantitative information of considerable pharmacological importance concerning the complex actions of drugs.

Topics: Adenocarcinoma; Alkaline Phosphatase; Androgen Antagonists; Androgens; Breast Neoplasms; Cyproterone Acetate; Endometrial Neoplasms; Estradiol; Estrogen Antagonists; Female; Flutamide; Gonanes; Humans; Mifepristone; Polyunsaturated Alkamides; Progestins; Tamoxifen; Testosterone; Testosterone Congeners; Tumor Cells, Cultured

We investigated the effects of estrogens and other steroid hormones on c-erbB2 gene expression in Ishikawa human endometrial adenocarcinoma cells. We have found that the c-erbB2/NEU transcripts are present in the Ishikawa endometrial cell line as well as in human endometrial adenocarcinoma cells. Both cell types express the 4.6 and 2.3 kb c-erbB2 mRNAs. Estradiol significantly increased in a time- and dose-dependent manner the content of c-erbB2 mRNA and the concentration of NEU protein in Ishikawa cell extracts, while progesterone was devoid of any activity. The effect of estradiol was partially reversed by the antiestrogen 4-hydroxytamoxifen, which, however, given alone exhibited agonist effects. Glucocorticoid dexamethasone augmented in a time- and dose-dependent fashion the content of c-erbB2 mRNA and the concentration of NEU protein in Ishikawa cell extracts. The antiglucocorticoid RU 486 acted as a glucocorticoid agonist increasing c-erbB2 gene activation. To our knowledge, this is the first report documenting the induction by steroid hormones of c-erbB2 gene expression in neoplastic human endometrial cells. Our data support the hypothesis that the oncogenic effect of estrogens on human endometrial cells may be partially mediated by its effect on the expression of the c-erbB2 proto-oncogene. The finding that glucocorticoids may induce endometrial c-erbB2 gene expression suggests that they may participate in the emergence of uterine neoplasias.

Topics: Adenocarcinoma; Dexamethasone; Endometrial Neoplasms; Endometrium; Estrogens; Female; Gene Expression Regulation, Neoplastic; Glucocorticoids; Humans; Proto-Oncogene Mas; Receptor, ErbB-2; RNA, Messenger; RNA, Neoplasm; Tamoxifen; Transcriptional Activation; Tumor Cells, Cultured

We have previously shown that estrogen and progestins regulate both cellular proliferation and transforming growth factor (TGF) expression in human endometrial adenocarcinoma cells in vitro. In the current study we examined the regulation of TGF-alpha and -beta 1 expression in endometrial adenocarcinoma xenografts. Four human endometrial adenocarcinoma cell lines were inoculated into female BALB/c nude mice. Administration of 17 beta-estradiol (E2) increased tumor size in intact mice inoculated with Ishikawa, HEC-50 and HEC-1B cells but inhibited growth of HEC-1A xenografts. 4-Hydroxy tamoxifen (OH-Tam) had similar effects to E2 in animals carrying Ishikawa and HEC-1A cell xenografts but had no significant effect on growth of HEC-50 or HEC-1B xenografts. In intact mice inoculated with OH-Tam pellets and Ishikawa cells, the tumors were larger and had lower levels of TGF-alpha mRNA than in untreated or E2 treated mice. In mice carrying Ishikawa, HEC-50 and HEC-1B cell xenografts none of the hormones or agents tested altered TGF-beta 1 mRNA levels. In contrast, both E2 and OH-Tam significantly increased xenografts TGF-beta 1 mRNA levels in HEC-1A xenografts as well as significantly reduced tumor size. Medroxyprogesterone acetate (MPA) had no effect on tumor size of Ishikawa, HEC-1A and HEC-1B cell cell xenografts but significantly increased the size of HEC-50 xenografts. MPA significantly reduced TGF-alpha expression in Ishikawa cell xenografts but had no effect in the other cell xenografts. MPA had no effect on TGF-beta 1 expression in any of the xenografts. These observations demonstrate a discordance between the hormonal effects on TGF expression and cellular proliferation and argue against a major role for the TGFs in regulation of human endometrial adenocarcinoma cell proliferation in vivo.

Topics: Adenocarcinoma; Animals; Blotting, Northern; Cell Division; Dexamethasone; Dihydrotestosterone; Endometrial Neoplasms; Estradiol; Female; Gene Expression Regulation, Neoplastic; Hormones; Humans; Medroxyprogesterone Acetate; Mice; Mice, Inbred BALB C; Tamoxifen; Transforming Growth Factor alpha; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

Some of the isoflavonoids present in human diet as well as in urine are expected to exert biologic effects as they have been reported to bind to estrogen receptors and to be estrogenic in other species. This report describes the in vitro assessment of estrogenic effects of isoflavonoids using human endometrial cells and tissue. The relative estrogenic potencies (EC50 values) of estradiol, 3 dietary isoflavonoids (coumestrol, genistein and daidzein) and one of their metabolites (equol), were estimated by using a recently developed multiwell plate in vitro bioassay based on the estrogen-specific enhancement of alkaline phosphatase (AlkP) activity in human endometrial adenocarcinoma cells of the Ishikawa-Var I line. The maximal AlkP activity elicited by the isoflavonoids tested was as high as that achieved with estradiol and their effects were suppressed by the antiestrogens 4-hydroxytamoxifen and ICI 164,384. These results indicate that estradiol and the isoflavonoids exert their effects on AlkP by similar interactions with the estrogen receptor, with potencies depending on binding affinities. The estrogenic effect of equol was confirmed by another in vitro bioassay, based on the estrogen-stimulated enhancement of prostaglandin F2 alpha output by fragments of human secretory endometrium.

Topics: Adenocarcinoma; Alkaline Phosphatase; Biological Assay; Chromans; Coumestrol; Dinoprost; Endometrial Neoplasms; Endometrium; Equol; Estradiol; Female; Genistein; Humans; Isoflavones; Polyunsaturated Alkamides; Tamoxifen; Tumor Cells, Cultured

We have examined the effects of medroxyprogesterone acetate (MPA) and 4-hydroxytamoxifen (OH-TAM) on the cell proliferation and the expression of TGF-alpha and TGF-beta genes in Ishikawa cells and HEC-50 human endometrial adenocarcinoma cells. The effects of exogenous TGF-alpha, TGF-beta and anti-EGF receptor monoclonal antibody on cell proliferation were also determined. Antisense oligonucleotides were used to determine the effects of endogenous expression of TGF-alpha and TGF-beta. In both cell lines, MPA resulted in a time and dose-dependent inhibition of cell proliferation whereas OH-TAM had no effect on HEC-50 cell proliferation. The relative abundance of TGF-alpha mRNA was significantly reduced by MPA in Ishikawa cells but not in HEC-50 cells. In Ishikawa cells, a reduction in TGF-alpha mRNA abundance was observed with OH-TAM under conditions where both inhibition and stimulation of cell proliferation were demonstrated. Anti-EGF receptor monoclonal antibody inhibited Ishikawa cell growth but had little effect on HEC-50 cell proliferation. Exogenous TGF-alpha stimulated proliferation of both cell lines whereas exogenous TGF-beta inhibited proliferation of Ishikawa cells but stimulated proliferation of HEC-50 cells. Antisense oligonucleotides to TGF-beta inhibited proliferation of HEC-50 cells. From these data we conclude that the antiproliferative effects of progestins and OH-TAM on endometrial cancer cells appear to be mediated by different mechanisms.

Topics: Adenocarcinoma; Antineoplastic Agents; Cell Division; Cell Line; Endometrial Neoplasms; Estrogen Antagonists; Female; Gene Expression Regulation, Neoplastic; Humans; Medroxyprogesterone; Medroxyprogesterone Acetate; Oligonucleotides, Antisense; Steroids; Tamoxifen; Transforming Growth Factor alpha; Transforming Growth Factor beta

While antiestrogens are useful agents in the treatment of breast cancer, the usefulness of these agents in the treatment of endometrial cancer remains controversial. There is some concern that the currently available antiestrogens may have partial agonist activity in uterine tissue. To better understand the mechanisms by which estrogens and antiestrogens modulate growth of endometrial adenocarcinoma cells, we have compared the effects of 17-beta estradiol and three antiestrogens, 4-hydroxytamoxifen (OH-TAM), ICI 164384, and LY 117018 on proliferation and transforming growth factor (TGF) mRNA accumulation in two human endometrial adenocarcinoma cell lines. In HEC-50 cells, neither estradiol nor anti-estrogens had any effect on cell proliferation or TGF mRNA abundance under estrogen-depleted culture conditions [basal medium containing 1% twice charcoal-treated fetal bovine serum (ctFBS)] or in the presence of estrogen (basal medium containing 5% fetal bovine serum). At very high concentrations, both estradiol and OH-TAM caused a small decrease in HEC-50 cell proliferation in medium containing 5% serum. In contrast, the antiestrogens had different effects on Ishikawa cells, depending upon the culture conditions. In medium containing 5% fetal bovine serum, the antiestrogens inhibited cell proliferation and significantly decreased TGF-alpha mRNA abundance and TGF-alpha secretion. OH-TAM was more potent than the other antiestrogens. Under these culture conditions, estradiol had no effect on cell proliferation or TGF-alpha mRNA levels but increased TGF-alpha secretion. In medium supplemented with 1% ctFBS, estradiol increased cell proliferation and TGF-alpha mRNA (2.72-fold, P less than 0.005) and TGF-alpha secretion (700 +/- 156 versus 250 +/- 23 pg/10(6) cells/24 h, P less than 0.05), whereas OH-TAM, which also stimulated cell proliferation, reduced TGF-alpha mRNA abundance (P less than 0.05) but had no significant effect on TGF-alpha secretion. Under these conditions, ICI 164384 and LY 117018 had no effect on either cell proliferation or TGF-alpha expression. Estradiol treatment decreased, whereas OH-TAM increased, epidermal growth factor receptors in Ishikawa cells. Both estradiol and the antiestrogens decreased TGF-beta 1 mRNA abundance when cells were grown in media containing 1% ctFBS. In summary, the response of human endometrial adenocarcinoma cells to estrogen and antiestrogens varied between cell lines and was dependent upon the culture conditions use

Topics: Adenocarcinoma; Analysis of Variance; Cell Division; Cell Line; Dose-Response Relationship, Drug; Endometrial Neoplasms; ErbB Receptors; Estradiol; Estrogen Antagonists; Female; Gene Expression; Humans; Kinetics; Polyunsaturated Alkamides; Pyrrolidines; RNA, Messenger; Tamoxifen; Thiophenes; Transforming Growth Factor alpha; Tumor Cells, Cultured

The effects of the transforming growth factor-beta 1 (TGF-beta 1) and epidermal growth factor (EGF) on the growth of cells from 2 endometrial cancer lines, Ishikawa and HEC-50 were evaluated by measuring rates of DNA synthesis and changes in cell numbers during culture. EGF at 17 and 1.7 nM concentrations consistently enhanced HEC-50 cell proliferation. TGF-beta 1 inhibited Ishikawa cell proliferation but, unexpectedly for epithelium-derived cells, stimulated HEC-50 cell growth. This effect is of interest as it indicates that endometrial cells can acquire an altered responsiveness to a growth inhibitor during the process of malignant transformation. Northern blot analyses showed expression of TGF-alpha, TGF-beta 1 and EGF receptors mRNA in both cell lines. Neither estradiol (E2) nor 4-hydroxytamoxifen (OHTam) affected mRNA levels for either TGF-alpha or TGF-beta in HEC-50 cells, a line unresponsive to E2 for proliferation. In Ishikawa cells, previously shown to respond to both E2 and OHTam by increasing proliferation rates, E2 increased TGF-alpha mRNA and reduced TGF-beta mRNA levels. OHTam lowered the levels of both mRNA species, although the effect was greater on TGF-beta than TGF-alpha mRNA. These data are consistent with, but do not prove, the existence of a possible autocrine regulation by TGF-alpha and TGF-beta of human cancer cell proliferation, which might be under E2 influence in Ishikawa cells.

Topics: Adenocarcinoma; Blotting, Northern; Cell Division; DNA; Endometrial Neoplasms; Epidermal Growth Factor; Estrogen Antagonists; Female; Humans; RNA, Messenger; Tamoxifen; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

The steroid hormone receptor content of 32 malignant ovarian tumors was compared with the in vitro effectiveness of 4 hydroxytamoxifen (OH-TAM) and medroxy-progesterone acetate (MPA) tested in the Human Tumor Colony Forming Assay (HTCFA). The sensitivity for the receptor determination was 5 fmol/mg cytosol protein. Estrogen receptors (ER) and progesterone receptors (PR) were found in 15 (47%) and 13 (41%) of the tumors respectively. As standard criteria for the HTCFA, a minimum of 30 colonies with a diameter of more than 60 microns and 100 microns was used in the control group. The in-vitro sensitivity of ovarian tumors to OH-TAM and MPA was independent on the ER or PR content, and amounted to 9% for OH-TAM and 6% for MPA. However, all 12 ER-PR-tumors proved resistant to OH-TAM and MPA. 18 ovarian tumors showed a sufficient colony growth, even in the size class exceeding 100 microns. With a minimum colony size of 60 microns and 100 microns, 17% and 33% respectively were sensitive to OH-TAM. A similar effect on the proliferative capacity of the Tumor Colony Forming Units (TCFUs), unrelated to PR, was observed with MPA. Dependent on colony size, we found an increasing sensitivity against MPA from 11% to 22%. The in-vitro effectiveness of both OH-TAM and MPA in the clonogenic assay of malignant ovarian tumors was certainly not as potent as suggested by the results obtained in biochemical steroid hormone receptor analysis. To prove the hormonal response in the HTCFA, it is necessary to determine number and size of the colonies as an expression of their proliferative potential.

Topics: Adenocarcinoma; Colony-Forming Units Assay; Cystadenocarcinoma; Female; Granulosa Cell Tumor; Humans; Krukenberg Tumor; Medroxyprogesterone; Medroxyprogesterone Acetate; Neoplasms, Hormone-Dependent; Ovarian Neoplasms; Receptors, Estrogen; Receptors, Progesterone; Tamoxifen; Tumor Cells, Cultured

The effects of trans-4-hydroxytamoxifen (OHTam) on proliferation of cells of the Ishikawa human endometrial adenocarcinoma line were studied under serum-free, phenol red-free conditions and compared to those of estradiol. The addition of OHTam (1 microM) to basal medium (BM), consisting of equal parts of Dulbecco's modified Eagle's medium and Ham's F-12 with additional glutamine and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, resulted in significant increases in cell numbers relative to controls. These effects were even greater than those obtained with estradiol (10 nM-1 microM) or 1% charcoal-treated fetal bovine serum (ctFBS). Addition of 1% ctFBS to BM containing 1 microM OHTam further increased cell numbers whereas addition of estradiol (10 nM) did not do so. The stimulation of growth was positively correlated with OHTam concentrations in the range of 10 nM to 1 microM. Dissociation of estradiol and OHTam proliferative effects was observed in a variant of Ishikawa cells in which estradiol did not increase proliferation while OHTam had a strong stimulatory effect. The growth-promoting effects of OHTam were also observed in BM containing 5% or 15% ctFBS. In contrast, in parallel experiments in which BM was replaced by minimal essential medium (Eagle's) with Earle's salts, OHTam (1 microM) did not stimulate proliferation under these conditions and acted as an antiestrogen, inhibiting the proliferative effects of estradiol. These results illustrate marked effects of medium composition on proliferation and antiestrogenic actions of OHTam. Alkaline phosphatase activity was strongly stimulated by estradiol (10 nM) but only very weakly affected by OHTam (1 microM); at these concentrations, OHTam inhibited the effect of estradiol, both in serum-free BM and in minimal essential medium plus 15% ctFBS, demonstrating dissociation in its actions on proliferation and on enzymatic activity. These findings suggest that OHTam may stimulate the proliferation of particular clones of endometrial cancer cells in human tumors. They also suggest that OHTam can exert effects not mediated by the estrogen receptor system, or form OHTam-estrogen receptor agonistic complexes unlike those resulting from estradiol-estrogen receptor interactions. Clearly, Ishikawa cells provide a useful model to investigate mechanisms of action of antiestrogens.

Topics: Adenocarcinoma; Alkaline Phosphatase; Cell Division; Estradiol; Female; Fetal Blood; Humans; Tamoxifen; Tumor Cells, Cultured; Uterine Neoplasms

Effects of 4-hydroxytamoxifen, a major metabolite of tamoxifen, on the proliferation of cancer cells from human endometrial adenocarcinomas obtained by hysterectomy were investigated in primary culture. Competitive binding studies showed that 4-hydroxytamoxifen effectively binds to cytoplasmic estrogen receptors (ER) in uterine adenocarcinomas. Of 20 endometrial adenocarcinomas examined, five tumors were successfully grown in primary cell culture. The addition of 4-hydroxytamoxifen (1 nmol/l to 1 mumol/l) in a medium supplemented with estrogen-free serum resulted in a dose-dependent inhibition of the growth of cancer cells in two tumors having ER. However, 4-hydroxytamoxifen did not affect the growth in the culture system of the remaining three tumors, in which ER were absent in two tumors but were present in one. These results strongly suggest that tamoxifen has a direct growth-inhibitory effect on human endometrial adenocarcinoma possibly through ER in the tumor.

Topics: Adenocarcinoma; Binding, Competitive; Cytosol; Drug Evaluation, Preclinical; Female; Humans; Receptors, Estrogen; Tamoxifen; Tumor Cells, Cultured; Uterine Neoplasms

As previously reported, ovarian epithelial carcinomas may respond to endocrine therapy. We examined the direct effect of progesterone, medroxyprogesteroneacetate, gestoneron, 17-beta-estradiol, tamoxifen, 4-OH-tamoxifen, or N-desmethyltamoxifen on the proliferative capacity of ovarian carcinoma cells by means of the colony assay described by Hamburger and Salmon. The growth rate of 25 tested tumors (ascitic fluid, primary tumor, metastases) was 68%. The plating efficiency was 0.078%. Beside the drug testing estrogen and progesterone receptor levels were determined. The inhibition of colony survival was slightest with 17-beta-estradiol, more pronounced with medroxyprogesteroneacetate, gestoneron, N-desmethyltamoxifen, and progesterone, and greatest with 4-OH-tamoxifen and tamoxifen. Significant and dose-dependent inhibition of greater than 70% was observed with tamoxifen and 4-OH-tamoxifen in 80% of the tested tumors. There was no significant correlation between the in vitro responsiveness and the level of hormonal act not only via an estrogen receptor but also via an antiestrogen-binding site.

Topics: Adenocarcinoma; Aged; Cell Division; Cells, Cultured; Estradiol; Estrogen Antagonists; Ethanol; Female; Gestonorone Caproate; Humans; Medroxyprogesterone; Medroxyprogesterone Acetate; Middle Aged; Neoplasm Metastasis; Neoplastic Stem Cells; Ovarian Neoplasms; Progesterone; Receptors, Estrogen; Receptors, Progesterone; Tamoxifen

The effects of estradiol on DNA polymerase alpha activity were investigated in an estrogen-responsive human endometrial cancer cell line (Ishikawa) derived from a well differentiated endometrial adenocarcinoma. These cells are known to respond to estradiol by increasing progesterone receptor levels, alkaline phosphatase activity, and cell density. Four- to 5-fold increases in DNA polymerase alpha activity occurred when estradiol was added to cultures of Ishikawa cells in medium containing charcoal-treated fetal bovine serum. Maximal stimulation was achieved at 18 h during incubations with 10(-8) M estradiol, but significant effects also were found with 10(-9) and 10(-6) M. These effects were almost completely counteracted by a 100-fold excess of 4-hydroxytamoxifen. At 10(-6) M, the antiestrogen had no influence on the basal levels of DNA polymerase alpha. Medroxyprogesterone acetate (10(-6) M) was ineffective as either an enhancer of enzymatic activity or an antiestrogen when tested in combination with 10(-8) M estradiol, even in the presence of appreciable levels of specific progesterone binders. The responsiveness of the Ishikawa cells to estrogen contrasts with the lack of effects of estradiol on DNA polymerase alpha activity in another human endometrial adenocarcinoma cell line (HEC-50).

Topics: Adenocarcinoma; Cell Line; DNA Polymerase II; Enzyme Activation; Estradiol; Female; Humans; Medroxyprogesterone; Medroxyprogesterone Acetate; Tamoxifen; Uterine Neoplasms

The influence of 17 beta-estradiol on insulin receptor distribution was studied in primary cultures of R3230AC mammary tumors prepared from intact or ovariectomized Fischer rats. [125I] Insulin binding to plasma membrane (IRS) and to solubilized cells (IRt) was measured, and intracellular insulin binding (IRi) was calculated by the difference between the two; calculated IRi agreed well with measured IRi of solubilized cells that were pretreated with trypsin to remove IRs. Scatchard analysis of saturation studies on IRs and IRt displayed typical curvilinearity and were roughly parallel with comparable Ke and Kd values for high affinity sites, but tumors from ovariectomized rats displayed more sites per cell. Insulin receptors relative to time in culture showed an early decline in IRt and IRs for both types of cells, followed by a gradual return to similar IRt values; in both types of cells, however, the proportion of IRs was increased compared to their initial distribution. Cells from both types of host animals displayed an insulin dose-related down-regulation with both IRt and IRs decreased. Short exposure (24 h) to 17 beta-estradiol in vitro also reduced IRs and IRt, but to a lesser extent than insulin, whereas longer exposure to estradiol (48 h) caused a continued reduction in IRs but not IRt. Compared to cells exposed to estradiol for 24 h in vitro, cultured cells treated with progesterone demonstrated an increased IRs, a marginal effect upon exposure to higher doses of testosterone, and reduced IRt, IRs, and IRi in response to dexamethasone. Monohydroxytamoxifen, an antiestrogen, displayed an unusual pattern, inducing a reduction in IRt and IRs at the lower doses but not at the higher doses studied. Degradation of [125I]insulin was examined in cells that were insulin down-regulated in the absence or presence of various levels of estradiol; the steroid hormone appeared to reduce degradation of [125I]insulin when lower levels of insulin were employed. The presence of estradiol in the medium enhanced the reappearance of IRt during the first 10 h after removal of the insulin that caused down-regulation. In conjunction with our previous observations in vivo, we conclude that 1) estradiol in vitro can decrease (down-regulate) insulin receptors on the plasma membrane of R3230AC mammary tumors; 2) the steroid may reduce degradation of internalized [125I]insulin; and 3) the steroid may enhance insulin receptor reappearance after insulin down-regulation. These

Topics: Adenocarcinoma; Animals; Castration; Cell Membrane; Cells, Cultured; Dexamethasone; Estradiol; Estrogen Antagonists; Female; Insulin; Kinetics; Mammary Neoplasms, Experimental; Progesterone; Rats; Receptor, Insulin; Tamoxifen; Testosterone; Trypsin

This study demonstrates in vivo and in vitro properties of the non-steroidal antiestrogens tamoxifen (TAM), 4-OH-tamoxifen (4-OH-TAM) and 3-OH-tamoxifen (K 060 E). In immature rabbit uteri 4-OH-TAM and K 060 E bound to the respective estrogen receptors with a ten-fold higher affinity than TAM. Furthermore, K 060 E exhibited less agonistic (estrogenic) but higher antagonistic (antiestrogenic) activity in the immature rat uterus than TAM and 4-OH-TAM (change of uterine weight). The ratio of agonistic vs antagonistic effect of K 060 E was distinctly lower than in TAM and 4-OH-TAM. In addition, K 060 E reduced by approximately 45% the growth of the transplantable Fisher rat mammary tumor (R 3230 AC) as compared with TAM (33%). We assume that, due to the higher antitumor activity, K 060 E (3-OH-TAM) is a better antiestrogen than TAM.

Topics: Adenocarcinoma; Animals; Binding, Competitive; Dose-Response Relationship, Drug; Estradiol; Estrogen Antagonists; Female; Mammary Neoplasms, Experimental; Organ Size; Rabbits; Rats; Rats, Inbred Strains; Receptors, Estrogen; Tamoxifen; Uterus