Cytosol and nuclear estrogen receptors (occupied and unoccupied sites) and progesterone receptors in human breast cancer

Summary Estrogen and progesterone receptor concentrations in cytosol and nucleus were measured in 21 primary breast cancer tumors. Twelve out of the 21 tumor samples were cytosol estrogen receptor positive, 8 of which contained only unoccupied estrogen binding sites in the cytosol, but 2 of the 9 estrogen receptor negative samples did contain cytosol binding sites already occupied by endogenous homone. Four other estrogen receptor negative tumors only showed nuclear binding sites. Only 3 of the 12 estrogen receptor positive tumors also contained progesterone receptors. All of these tumors also had estrogen receptor in the nucleus. However, three of the 17 progesterone receptor negative samples had progesterone receptor only in the nucleus. The present data indicate that 3 possible classes of false negative tumors can be encountered: 1) estrogen receptors occupied by endogenous hormone, 2) tumors containing only nuclear estrogen receptors, and 3) tumors having only nuclear progesterone receptors. Measurement of nuclear estrogen receptor together with the progesterone receptor provides further information on whether the estrogen receptor system is not only present but also functional, and should be of value in the prediction of hormone dependent breast cancer.     

Techniques de recherche des polymorphismes génétiques

Résumé: Le séquençage du génome humain est à lorigine de la découverte de millions de variations de séquence dans le génome humain. Ces variations de séquence dADN sont dans leur immense majorité limitées à des polymorphismes de nucléotide unique (Single Nucléotide Polymorphisms ou SNP); cette forme courante de polymorphisme se rencontre environ toutes les 1 000 bases dans le génome humain et 1,8 million de SNP sont actuellement répertoriés. Laccès à ces SNP dans les banques de données publiques a ouvert la possibilité détudier linfluence de ces polymorphismes répertoriés sur la prédisposition à certaines maladies génétiques ainsi que sur la réponse aux médicaments. Lidentification de ces nombreux SNP tient beaucoup aux progrès des techniques danalyse de lADN et nous décrivons, dans cette revue, différentes méthodes pouvant être utilisées pour létude de polymorphismes. Les techniques détude des SNP reposent sur deux principes fondamentaux: dune part la création ou lélimination par le SNP dun site de coupure pour une enzyme de restriction donnée, et dautre part la formation dun mésappariement due à la présence dune séquence variante lors dune hybridation. La technique originelle détude des SNP est le Southern blot couplé à une digestion enzymatique permettant détudier les polymorphismes de taille de fragments de restriction (RFLP). Ce procédé sest largement simplifié par larrivée de la technique de réaction en chaîne par polymerase (PCR). Dautres approches telles que la chromatographie à haute performance en conditions dénaturantes (dHPLC) ou la PCR en temps réel permettent une discrimination des allèles sauvages et variants. En revanche, le séquençage reste la seule technique permettant de déterminer la nature et la position des SNP connus et inconnus. Enfin, lapparition des techniques à haut débit permet de prévoir des changements déchelle considérables dans la recherche des SNP.     

Protein Kinase C ζ Isoform is Critical for Proliferation in Human Glioblastoma Cell Lines

Previous studies have confirmed that proliferation in glioblastoma cell lines can be blocked by non-isoform specific protein kinase C (PKC) inhibitors, e.g calphostin C, staurosporine. However, the exact mechanism of PKC involvement is poorly understood. The aim of this study was to explore the role of specific PKC isoforms in the aberrant growth of glioblastoma. Identification of the isoform(s) critical for proliferation in glioblastoma would present a better target for the design of chemotherapeutic strategies. To this end, we screened expression on PKC isoforms in four human glioblastoma cell lines both when proliferating and in a quiescent state using western assays. PKC isoforms , I, II and were found to be expressed in all cell lines. PKC was detected in three out of four cell lines and PKC was detected in one out of four cell lines. Quiescence of growth resulted in down-regulation of PKC. We examined the role of these isoforms by studying the effect of PKC isoform-specific inhibitors bisindolylmaleimide-I and Gö6976 on proliferation in a panel of four human glioblastoma cell lines. Inhibition of PKC and had no effect on proliferation, suggesting that previous studies targeting PKC may not be of therapeutic benefit. More significantly, it was shown that inhibition of PKC blocked proliferation. This suggests that the inhibition of PKC may be an important chemotherapeutic target for arresting growth in glioblastoma.     

Radioimmunoscintigraphy of Intracranial Glioma Xenograft with a Technetium-99m-Labeled Mouse Monoclonal Antibody Specifically Recognizing Type III Mutant Epidermal Growth Factor Receptor

The type III mutant epidermal growth factor receptor (EGFR) is expressed on the cell surface of a subset of glioma, but not of normal tissues. In this study, we investigated the in vivo kinetics of 3C10 mouse monoclonal antibody (mAb), specifically recognizing the type III mutant EGFR (EGFRvIII), using athymic nude mice bearing the intracranial glioma xenograft overexpressing the EGFRvIII.Human glioma cell line, U87MG, expressing the wild type EGFR and the transfectant, named U87MGEGFR, expressing the EGFRvIII, were transplanted subcutaneously or intracranially to nude mice. 3C10 mAb labeled with a technetium-99m (^99mTc) was intravenously injected into these nude mice and then the mice were sacrificed at 24h later, and the ^99mTc-uptake by xenografts and major normal organs was measured to determine the biodistribution of mAb. Furthermore, at 3, 6 and 24h after injection of ^99mTc-labeled 3C10 mAb, whole-body scintigraphy was obtained with a gamma camera to localize the tumor site.3C10 mAb significantly accumulated to U87MGEGFR xenografts transplanted subcutaneously or intracranially in nude mice, showing high tumor-to-blood ratio of 10.30 and 4.01, respectively. In contrast, uptake of control antibody in the intracranial tumor was as low as 0.43. In scintigrams, intracranially transplanted U87MGEGFR xenografts were detectable at 3h after injection of ^99mTc-labeled 3C10 mAb.These results suggest that intravenously injected 3C10 mAb specifically accumulated to the subcutaneous or intracranial glioma xenograft expressing the EGFRvIII and 3C10 mAb is a potential diagnostic and therapeutic agent for patients with gliomas expressing the EGFRvIII.     

Expression of TGFα in Meningiomas

The objective of this study was to examine the expression of transforming growth factor (TGF), a mitogen for many cell types, and its receptor in basic subtypes of meningiomas as well as in meningiomas of varying grade. Formalin-fixed tissues from 26 meningiomas including 15 benign (5 meningothelial, 5 transitional, and 5 fibrous variants), 6 atypical, and 5 malignant examples were immunohistochemically examined for both TGF protein and EGF/TGF receptor protein. In addition, in situ hybridization (ISH) was used to detect TGF mRNA expression. Immunostaining for TGF was strongest in fibrous and atypical meningiomas, followed closely by transitional and malignant tumors. Only weak reactivity was observed in the meningothelial variant. In all but 4 tumors (2 fibrous, 2 atypical), ISH showed TGF mRNA to be present, the signal being stronger in malignant than in conventional or atypical tumors. Lastly, immunostaining for EGF/TGF receptor was positive in all tumors studied. Strong TGF protein expression in meningiomas is commonly associated with fibrous morphology. Although the frequent detection of both TGF protein and its mRNA, as well as of EGF/TGF receptor within tumors of all type and grades, suggests that TGF serves to promote tumor growth, its possible role in tumorigenesis or malignant progression is uncertain.In summary, demonstration of these substances is of no utility in the classification or grading of this common tumor because the differences in their expression among the various meningioma subtypes were not statistically significant.     

«Coût éthique» du cancer et dignité

Résumé: La notion de «coût éthique» du cancer peut surprendre: est-elle recevable et pertinente dans le cadre dune approche humaine et sensible des réalités vécues par la personne malade et ses proches? Au-delà de considérations relevant de la santé publique, des soins, du suivi au cours de la maladie, de laccès aux traitements, on constate que vivre avec le cancer confronte à un cumul de menaces affectant la totalité de lexistence. Le parcours du malade est marqué par lobsédante succession de peurs diffuses, dentraves, daltérations physiques, de pertes, de renoncements, de vulnérabilités que lon peut comprendre comme autant de «coûts» qui simposent à elle. Il semble toutefois nécessaire de privilégier une approche en termes de dignité et de droits ne serait-ce que pour mieux identifier la nature des «coûts» induits par la maladie et concevoir à cet égard les responsabilités qui simposent à tous en termes de sollicitude et de solidarité.     

Polyamine involvement in the secretion and action of TGF-α in hormone sensitive human breast cancer cells in culture

These experiments were designed to test polyamine (PA)* involvement in the secretion and action of transforming growth factor (TGF-) in hormone responsive MCF-7 breast cancer cells in liquid culture. At the same time, we evaluated the influence of culture conditions (with serum vs. serum depleted) and subclonality of MCF-7 cells on PA involvement in estrogen (E₂) and TGF- stimulated cell proliferation. Despite inducing a profound suppression of cellular PA levels and inhibiting basal and E₂-stimulated growth, administration of the PA synthesis inhibitor -difluoromethylornithine (DFMO) did not influence either basal or E₂-induced TGF- secretion. In the same experiments, on the other hand, addition of DFMO completely blocked the growth stimulatory effect of exogenous TGF-. However, when the culture conditions were changed to serum-free medium, TGF- and E₂-induced cell proliferation was affected modestly or not at all by DFMO administration, despite similar suppression of cellular ornithine decarboxylase (ODC) activity and PA levels. In addition, different clones of MCF-7 cells differed in their sensitivity to the antiproliferative effect of DFMO as well as in basal levels of ODC activity and PA. We conclude that PAs are not involved in basal or E₂-stimulated TGF- secretion in MCF-7 breast cancer cells. On the other hand, PAs do seem to be important mediators of TGF- and E₂-induced breast cancer cell proliferation, though the degree of such involvement appears to be influenced by serum factors and clonal variability of MCF-7 cells.     

Synergistic effect of 3′-deoxyadenosine N 1-oxide and adenosine deaminase inhibitors on growth of Ehrlich ascites tumor cells in vivo

Summary The simultaneous administration of 3-deoxyadenosine N ^l-oxide (3-dANO) and the adenosine deaminase inhibitors erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) or 2-deoxycoformycin (2-dCF) to mice bearing Ehrlich ascites tumor cells resistant to 3-dANO resulted in 80%–90% inhibition of tumor growth in vivo. 3-dANO and 2-dCF increased the survival time of tumor-bearing mice by a factor of 2. In vitro studies showed that the 3-dANO resistant Ehrlich cells initiate the metabolism of 3-dANO by a reduction to 3-deoxyadenosine, which is converted primarily to 3-deoxyinosine by adenosine deaminase and, to a small extent, phosphorylated to the cell toxic agent 3-dATP. By the addition of EHNA or 2-dCF it was possible to block the formation of 3-deoxyinosine, resulting in a profound stimulation in the accumulation of 3-dAtP. The development of resistance to 3-dANO was studied in cell cultures and found to be accompanied by changes in the enzyme activities of the reductase, the adenosine kinase, and the adenosine deaminase.Abbreviations 3-dANO 3-deoxyadenosine N ^l-oxide - 3-dA 3-deoxyadenosine - 3-dI 3-deoxyinosine - 3-dATP 3-deoxyadenosine triphosphate - EHNA erythro-9-(2-hydroxy-3-nonyl) adenine - 2-dCF 2-deoxycoformycin This work was supported by the Danish Medical Research Council, Gerda and Åge Haensch Foundation, Dirktør Åge Henriksens Foundation, P. Carl Petersens Foundation and the Danish Cancer Society     

Retinoid Receptors in Human Breast Carcinoma: Possible Modulators of in Situ Estrogen Metabolism

Retinoid receptors (retinoic acid (RARs) and retinoid X (RXRs) receptors) were immunolocalized in 32 human invasive ductal breast carcinomas. These findings were correlated with clinicopathological parameters to study their biological significance in breast carcinoma. Retinoid receptor immunoreactivity, except for RXR, was detected in the nuclei of carcinoma cells. Percentage of positive cases were RAR 81%, RAR; 6%, RAR; 28%, RXR; 81%, and RXR; 59%. A significant correlation was detected between RAR labeling index (LI), and RXR LI (r=0.667, p<0.001). Results from immunoblotting performed in three cases were consistent with those of immunohistochemistry. There was a significant correlation between RAR LI and 17-hydroxysteroid dehydrogenase (17-HSD) type 1 immunoreactivity (p<0.05). A significant correlation was also detected between RAR (r=0.413, p=0.019) or RXR (r=0.429, p=0.014) LI, and estrogen receptor (ER) LI. In T-47D breast cancer cells, which express RAR, RXR and ER, 17-HSD reductive activity increased 1.76-fold (p<0.001), five days following treatment with 10nM retinoic acid. These data suggest that retinoid receptors modulate various effects of retinoids, including estrogen metabolism in human breast carcinomas.     

Tissue inhibitor of metalloprotease (TIMP)‐1 and proliferative behaviour of clonal breast cancer cells

In the present paper, we have examined whether human tissue inhibitor of metalloprotease1 (hTIMP1) is able to exert a growth factorlike effect on two clonal cell lines (BC3A and BC61), isolated from a parental line of human breast carcinoma cells (8701BC), and endowed with different growth and invasive behaviour in vitro and in nude mouse. The data obtained indicate that only the more tumorigenic clonal cell line (BC61) is responsive to hTIMP1 treatment by increasing its proliferative rate in a dosedependent manner. It was also found that BC61 cells selectively express a transmembrane protein of about 80kDa able to bind hTIMP1 in vitro and in vivo with high affinity (K_d of 0.07 ± 0.004 nM), and that treatment of BC61 cells with a proliferationpromoting concentration of hTIMP1 is able to stimulate tyrosinetargeted phosphorylation. The cumulative results obtained strongly support the hypothesis that hTIMP1, classically regarded as a collagenase inhibitor, may be a crucial element of the extracellular signalling network during breast cancer development by controlling cell growth phenotype in autocrine and paracrine manner, and that intratumoural heterogeneity for the biological response to TIMP1 may exist within the composite cell population of the primary tumour site.     

The α6β1 and α6β4 integrins in human prostate cancer progression

Summary Prostatic secretions are formed by glands composed of basal and luminal cells and surrounded by a basal lamina. The normal basal cells express several integrins (extracellular matrix receptors) including alpha 2, 3, 4, 5, 6, v, beta 1 and beta 4. These integrin units are polarized at the base of the cells adjacent to the basal lamina. The integrin alpha 6 beta 4 is associated with hemidesmosomal-like structures.The natural history of prostate cancer involves the presence of prostatic intraepithelial neoplasia (PIN) lesions (considered precursor lesions), carcinomain situ and invasive carcinoma. Hemidesmosomal proteins and the 31 and 61 integrins (laminin receptors) are retained in the early PIN lesions. Expression of the integrins 2, 4, 5, v and 4 is lost in carcinoma. The 31 and 61 integrins remain associated with invasive carcinoma, the latter being predominant. Integrin expression in carcinoma is diffuse in the plasma membrane and not restricted to the basal aspects of the cell. The 61 integrin is fully functional as judged by an ability to adhere to laminin and contains the wild type 6A cytoplasmic signaling domain. The 61 integrin is a leading candidate for conferring the invasive phenotype in prostatic carcinoma.Tumor cells with high expression of 6 integrin are more invasive when tested in a SCID mouse model system. Following intraperitoneal injection, the human tumor cells invade the mouse diaphragm and move through the muscle on the surface of the laminin coated muscle cells. Our current working hypothesis is that the production of 61 and laminin in human tumor cells contributes to the invasive phenotype. Invasion could occur on the surfaces of laminin coated structures such as the nerves, blood vessels or muscle and account for the known patterns of human prostate tumor progression. Blockage of the expression or function of 61 or laminin or preventing the loss of 4 would be essential steps in confining the carcinoma to the prostate gland where conventional treatment has already proven effective.     

Oestrogen receptor concentration in primary breast cancer and axillary node metastases

Summary The primary tumour and 1–3 invaded, axillary nodes from each of 24 patients were examined both histologically for the proportion of the specimen constituted by malignant epithelial cells (cellularity index) and biochemically for oestrogen receptor concentration. Both malignant epithelial cell content and oestrogen receptor concentration were significantly higher in the nodal metastases than in the primary tumours, malignant cells constituting approximately half of the former tissue and three quarters of the latter. On average, receptor concentrations were 1.6 × (protein basis) to 2.3 × (wet weight basis) higher in nodes than in the primary tumours, probably due at least in part to the difference in cellularity. When, to eliminate the effect of the latter, receptor concentration in each tumour deposit was corrected using the appropriate cellularity index, the difference in receptor concentration between primary and node was significantly diminished, but not quite eliminated. In one patient, progestogen receptor concentrations were also studied and found to be higher in the nodes than in the primary tumour. If the actual quantity of receptor is to be used for predictive/prognostic purposes, then either a different cut-off point should be used for invaded nodes from that used for assessment on the primary tumour, or receptor concentrations should be corrected for differences in cellularity. Reprints: No reprints are supplied by this Department.     

New approach to metabolism of 5′-deoxy-5-fluorouridine in humans with fluorine-19 NMR

Summary The metabolism of 5-deoxy-5-fluorouridine (5dFUrd), an antitumor fluoropyrimidine, has been investigated in human biofluids (blood, plasma, urine) using a new method: fluorine-19 NMR spectrometry. This method allows direct study of the biological sample and simultaneous identification of all the fluorinated metabolites. In the blood of a patient treated with 5dFUrd during a 6-h continuous perfusion, we observed unmetabolized 5dFUrd, 5-fluorouracil, 5,6-dihydrofluorouracil, and another metabolite which has not previously been reported -fluoro--alanine. The two major metabolites in urine are unmetabolized 5dFUrd and -fluoro-alanine.     

Studies on the mechanism of cytotoxicity of 3′-deoxyadenosine N1-oxide in different strains of Ehrlich ascites tumor cells

Summary The correlation between the metabolic processing of 3-deoxyadenosine N ¹-oxide (3-dANO) in vitro and its effect on tumor growth in vivo has been investigated in seven different strains of Ehrlich ascites tumor cells.The metabolism of 3-dANO is initiated by reduction to 3-deoxyadenosine (3-dA). This process is the rate-limiting process. The 3-dA does not accumulate, but is converted to 3-deoxyadenosine triphosphate (3-dATP) or 3-deoxyinosine (3-dI). The ratio between 3-dATP and 3-dI inosine corresponds to the ratio between the activities of adenosine kinase and adenosine deaminase in the cell.Two of the cell lines were markedly inhibited by 3-dANO in vivo. In these cells the accumulation of 3-dATP was 1.4–2.2 nmol/h per mg cells, which accounts for the major part of the metabolized 3-dANO. Five of the cell lines were not inhibited by 3-dANO and the formation of 3-dATP was 5–10 times less in these than in the sensitive strains. The low level of 3-dATP is caused primarily by a low ratio between the activities of adenosine kinase and adenosine deaminase, which is 15 time less than in the sensitive cell lines. The rate of reduction of 3-dANO seems to be of minor importance.These results indicate a correlation between the inhibition of tumor growth by 3-dATP and the ability of the cell to accumulate 3-dATP from 3-dANO and show that this conversion is determined solely by the rate of reduction of 3-dANO (3-dANO reductase activity) and the ratio between the activities of adenosine kinase and adenosine deaminase in the cell. Consequently, the estimation of these enzyme activities in cell lysate of a given tumor can be used to predict whether the tumor is susceptible to inhibition by 3-dANO.Abbreviation 3-dANO 3-deoxyadenosine N ¹-oxide - 3-dA 3-deoxyadenosine - 3-dI 3-deoxyinosine - 3-dATP 3-deoxyadenosine triphosphate This work was supported by The Danish Medical Research Council, Gerda and Åge Haensch Fond, Direktor Åge Henriksens Fond, P. Carl Petersens Fond and the Danish Cancer Society     

Estradiol stimulates synthesis of a major intracellular protein in a human breast cancer cell line (MCF-7)

Summary Previous studies have shown that synthesis of a 24,000 molecular weight (24k) protein (7) and the mRNA activity coding for a 24k protein (8) are selectively stimulated by estradiol in MCF-7 human breast cancer cells. Utilizing a double-label electrophoresis technique to measure the relative rates of specific protein synthesis, the present study relates further characterization of this estrogen response. The 24k protein was found to be stimulated by estradiol provided cells were preincubated with antiestrogen. Under these conditions, antiestrogens inhibit cell growth and addition of estradiol reverses growth inhibition (estrogen growth rescue). As a control experiment, specific protein synthesis was measured in another growth rescue model produced by withdrawing serum from the medium to inhibit growth and then stimulating cells by readdition of serum (serum growth rescue). This failed to stimulate synthesis of a 24k protein, implying that the effect observed following estrogen rescue was not a non-specific result of growth stimulation. Increased 24k synthesis was found to be both time and estrogen-dose dependent and required specifically those steroid hormones which interact with the estrogen receptor. Moreover, the dose response curve for 24k stimulation paralleled closely the dose response for estrogen stimulation of nuclear estrogen receptor processing, an event correlated with another estrogenic response in MCF-7 cells, induction of progesterone receptor. These findings suggest that estradiol itself directly stimulates synthesis of the 24k protein through interaction with the estrogen receptor system. Further separation of double-label cytosols by two-dimensional electrophoresis resolved the 24k protein into two isoelectric species with pI's in the range of 6.7–6.9 and 6.4–6.7, the latter being the most abundant or rapidly labeling protein. Based on the percentage of total cytosol incorporation contained in individual two-dimensional gel spots, the major species of 24k represented in the fully stimulated cell (³H-counts) about 1.6% of newly synthesized protein. It also represented about 0.7% of newly synthesized protein in the unstimulated (¹⁴C-counts) cell, which indicates that 24k is synthesized constitutively. Because of its relative abundance, the 24k protein may be a suitable end product for investigating the mechanisms of estrogen action in human breast cancer.Abbreviations used are PgR, progesterone receptor ER estrogen receptor - DHT dihydrotestosterone - Hepes N¹-2-hydroxyethylpiperazine-N¹-ethane sulfonic acid - MEM Eagle's minimum essential medium - TCA trichloroacetic acid - SDS sodium dodecylsulfate; nafoxidine, (1-(2-[p-(3,4-dihydro-6-methoxy-2phenyl-1-naphthyl) Phenoxy]ethyl) pyrrolidine hydrochloride; Upjohn); tamoxifen, (1-p--dimethylaminoethyoxyphenyl-trans-1,2-diphenylbut-1-ene; ICI-46474); CI-628,1-[2-[p[-(p-methoxyphenyl)--nitrostyryl]-phenoxy]ethyl]pyrrolidine monohydrate monocitrate Address for reprints: William L. McGuir, M.D., Department of Medicine, University of Texas Health Science Center 7703 Floyd Curl Drive. San Antonio, TX 78284.     

Immunological detection and quantitation of alpha transforming growth factors in human breast carcinoma cells

Summary Alpha transforming growth factors (TGFs) were immunologically detected in the concentrated conditioned medium (CM) prepared from four human breast cancer cell lines and from primary cultures of human mammary epithelial cells, and in the tissue extracts prepared from normal, benign, and malignant breast biopsies. Immunoreactive TGFs were quantitated by a competitive radioimmunoassay (RIA) using affinity-purified polyclonal sheep anti-rat TGF antibodies which react with human TGF but not with human epidermal growth factor (EGF). The relative level of RIA-detectable TGFs in the CM from the breast cancer cell lines MCF-7, ZR-75-1, T47-D, and MDA-MB-231, and from the CM of primary cultures of human mammary epithelial cells, ranged from 0.02 to 0.85 ng/ml. MCF-7 or ZR-75-1 cells grown in the presence of 17-estradiol (10^–8 M) for 48 h were found to release two- to three-fold more TGFs into their CM than the same cells grown in the absence of estrogen. In detergent extracts prepared from normal breast tissue, a benign fibrocystic lesion, fibroadenomas and primary breast carcinomas, the relative TGF concentrations were found to range from 1.5 to 6 ng/mg cell protein. No significant correlations were found between the TGF levels and the pathological state of the tissues, the estrogen receptor status of the tumors, or the relative amounts of theras gene protein p21ras in the tissues as determined by Western immunoblot analysis. The question of biological relevancy of TGF for human mammary tumors will require further studies on (a) synthesis and turnover of TGF, (b) the relationship between immunoreactivity and biological activity of TGF, and (c) differences in biological responsiveness of mammary tumor cells.     

Multiple actions of synthetic ‘progestins’ on the growth of ZR-75-1 human breast cancer cells: Anin vitro model for the simultaneous assay of androgen,progestin, estrogen,and glucocorticoid agonistic and antagonistic activities of steroids

This study was designed to assess the multiple steroid receptor mediated activities of a series of synthetic progestins on breast cancer cell growth, using the human ZR-75-1 cell line which possesses functional estrogen (ER), androgen (AR), and glucocorticoid (GR) receptors as well as progesterone (PgR) receptors. Four 17-hydroxyprogesterone derivatives (chlormadinone acetate, CMA; cyproterone acetate, CPA; medroxyprogesterone acetate, MPA; and megestrol acetate, MGA) and two 19-nortestosterone derivatives (norethindrone, NRE, and norgestrel, NRG) were thus investigated.Based on the requirement of estrogens for PgR-mediated antiproliferative effects and the reversal of PgR-mediated action by insulin, it was found that although all progestins could inhibit ZR-75-1 cell growth through the PgR at low concentrations, the relative contribution of this receptor in cell growth control is highly variable between compounds. The quantitative importance of PgR-mediated inhibition of cell proliferation was inversely related to the amplitude of the androgenic effects induced by the compounds, the AR-mediated effects increasing in the order CPA < MGA < CMA < NRE < NRG < MPA. The specificity of these androgenic effects is further supported by their reversal upon addition of the antiandrogen hydroxyflutamide. In addition, the 17-hydroxyprogesterone derivatives, but not the 19-nortestosterone derivatives, had glucocorticoid activities at high (micromolar) concentrations, as shown by reversal of growth inhibition by the antagonist RU486 in the presence of saturating concentrations of 5-dihydrotestosterone. All progestins tested, except MPA and NRE, also had some antiglucocorticoid activity, NRG being the most potent in this respect. Finally, NRE and NRG exerted a marked mitogenic effect in estrogen-free medium which was clearly mediated through the ER as shown by the competitive reversal of their action by the steroidal antiestrogen EM-139.The present results show that growth measurements of the human breast cancer cells ZR-75-1 permit, with the appropriate steroid additions, the assay of progestin, androgen, estrogen, and glucocorticoid agonistic as vell as antagonistic activities of test compounds. The present study shows, somewhat surprisingly, that while the AR is almost completely responsible for the action of MPA at low concentrations, the majority of the action of NRE, NRG, and MGA is also exerted through AR, while the androgenic action of CPA plays a lower role in the growth inhibition induced by this compound. Such a model should be of great help in designing more specific steroid drugs and in better understanding the role of the different steroid classes which can be used to control the growth of hormone-sensitive cancer. The present data also indicate that progestin is an inappropriate name for MPA, NRE, NRG, MGA, CMA, and CPA, which all possess other and sometimes more potent steroidal activites than those related to interaction with the progesterone receptor.Abbreviations CMA chlormadinone acetate [17-acetoxy-6-chloropregna-4, 6-dien-3, 20-dione] - CPA cyproterone acetate [17-acetoxy-6-chloro-1,2-methylene-pregna-4, 6-dien-3, 20-dione] - DEX dexamethasone [9-fluoro-11, 17, 21-trihydroxy-16-methyl-pregna-1, 4-dien-3, 20-dione] - DHT 5-dihydrotestosterone [17-hydroxy-5-androstan-3-one] - E₂ estradiol [estra-1, 3, 5 (10)-trien-3, 17-diol] - EM 139 [N-n-butyl-N-methyl-11-(16-chloro-3, 17-dihydroxyestra-1, 3, 5 (10)-triene-7-yl) undecanamide] - MGA megestrol acetate [17-acetoxy-6-methylpregna-4, 6-dien-3, 20-dionel] - MPA medroxyprogesterone acetate [17-acetoxy-6-methylpregn-4-en-3, 20-dione] - NRE norethindrone [17-hydroxy-19-nor-17-pregn-4-en-20-yn-3-one] - NRG norgestrel [13-ethyl-17-hydroxy-18, 19-dinor-17-pregn-4-en-20-yn-3-one] - OHF hydroxyflutamide (SCH 16423) [, , -trifluoro-2-methyl-4-nitro-m-lactotoluidide] - R1881 methyltrienolone [17-hydroxy-17-methyl estra-4, 9, 11-trien-3-one] - R5020 promegestone [17, 21-dimethyl-19-norpregna-4, 9-dien-3, 20-dione] - RU486 [17-hydroxy-11-(4-dimethylaminophenyl)-17-(1-propynyl)-estra-4, 9-dien-3-one] - triamcinolone acetonide [9-fluoro-11, 21-dihydroxy-16, 17(1-methylethylidenebis oxy) pregna-1, 4-dien-3, 20-dione]     

Involvement of nuclear steroid/thyroid/retinoid receptors and of protein kinases in the regulation of growth and of c‐erbB and retinoic acid receptor expression in MCF‐7 breast cancer cells

Nuclear steroid/thyroid/retinoid receptors and cerbB membrane receptor tyrosine kinases control epithelial growth and differentiation. Retinoid receptors can dimerize with the vitamin D receptor, the glucocorticoid receptor or the thyroid receptor. Furthermore, multiple cerbB receptor dimers have been identified. It has been shown that some of these receptor pathways communicate with each other via crossconnected regulatory networks. Molecular interactions between retinoid receptors or estrogen receptors (ER) and cerbB2, and between ER and retinoic acid receptor(RAR) have been reported. Here, we demonstrate the effects of steroids/thyroids/retinoids and of activators of protein kinase A (forskolin, Forsk) and C (12Otetradecanoylphorbol13acetate, TPA), on growth and expression of cerbB and RARs in MCF7 breast cancer cells, which contain high levels of RAR and , and which express significant amounts of cerbB2 and 3. All transretinoic acid (tRA), the antiestrogen ICI 182 780 (ICI), Forsk and TPA reduced, whereas triiodothyronine and 17estradiol (E₂) stimulated cell growth. Flow cytometry revealed that tRA and E₂ reduced cerbB2 and 3, whereas tamoxifen, Forsk and TPA upregulatedcerbB2. cerbB3 was coregulated with cerbB2. Northern analysis demonstrated that RAR was downregulated by dexamethasone, ICI, and TPA, whereas vitamin D₃ and E₂ upregulated RAR. RAR expression was less responsive to such treatment, being reduced only by ICI and Forsk. These data indicate that nuclear receptor and protein kinase signaling communicate with each other and control the expression of RARs and cerbB receptors. Efficient growth control requires the coordinated interplay of both receptor systems.     

Reduced cardiotoxicity and increased cytotoxicity in a novel anthracycline analogue, 4′-amino-3′-hydroxy-doxorubicin

Summary The acute and chronic cardiotoxicity and cytotoxicity of the novel doxorubicin (DXR) derivative 4-amino-3-hydroxy-DXR were compared with those of 4-deoxy-DXR and DXR. In the acute cardiotoxicity study, the ECG and hemodynamic changes recorded in anesthetized rats that had been treated i.v. with 10 mg/kg 4-amino-3-hydroxy-DXR or 8.6 mg/kg 4-deoxy-DXR were significantly less severe than those caused by 13 mg/kg DXR. In the chronic cardiotoxicity study, rats received 3 weekly i.v. injections of 3 mg/kg DXR, 3 mg/kg 4-amino-3-hydroxy-DXR, or 2 mg/kg 4-deoxy-DXR during the first 14 days of the study and were observed for an additional 35-day period. DXR induced severe cardiomyopathy that was characterized by ECG changes in vivo (ST-segment widening and T-wave flattening) and by impairment of the contractile responses (F _max,±dF/dt _max) to adrenaline of hearts isolated from treated animals. 4-Deoxy-DXR caused a progressive enlargement of the ST segment in vivo and a significant impairment of the-dF/dt _max value in vitro, which were less severe than those produced by DXR. The least cardiotoxic drug was 4-amino-3-hydroxy-DXR, which induced minor ECG changes without causing significant alterations in the contractile responses of isolated hearts to adrenaline. On the basis of the drug concentration required to inhibit 50% of the colony formation (IC₅₀) of cell lines in vitro, 4-amino-3-hydroxy-DXR was less active than 4-deoxy-DXR but at least twice as active as DXR against human cancer and murine transformed cell lines. These data indicate that 4-amino-3-hydroxy-DXR is significantly less cardiotoxic and more cytotoxic than DXR.     

Ligand-Induced Regulation of ERα and ERβ is Indicative of Human Breast Cancer Cell Proliferation

Two estrogen receptors (ER), ER and ER, are expressed in breast cancer but their role in treatment response is unclear. The overall objective of this study was to determine if the presence of ER protein in breast cancer cell lines is an indicator of a poor prognosis based on cell proliferation. In addition, we determined the effect of estradiol (E2) and selective estrogen receptor modulators (SERMs), such as tamoxifen and genistein, on ER and ER protein regulation, to help in the understanding of the mechanism behind their role in modulating cell proliferation. Using western blot and immunofluorescence analysis, the ER positive cell lines, MCF-7 and T47D, were found to contain both ER and ER, and thus were used as model systems. E2 and genistein, which increased cell proliferation in both cell lines, induced an up regulation of ER in both cell lines. This suggests that an estrogenic response in breast cancer cells is indicated by an increase in ER expression. Tamoxifen decreased cell proliferation in both cell lines, while up regulating ER in both cell lines, suggesting that antiestrogenic response is indicated by an increase in ER expression. Although a change in the ER/ER ratio may play a role in the effect seen in cell proliferation, this study indicates that ER is a poor prognosticator of cell proliferation in breast cancer and that ER is a positive prognosticator of responsiveness to antiestrogen treatment.