Estrogen-induced cell transformation and DNA adduct formation in cultured Syrian hamster embryo cells

To study the possible involvement of DNA damage in cell transformation induced by estrogens, we examined whether DNA adduct formation is elicited in cultured Syrian hamster embryo (SHE) cells treated with estrogens and their derivatives by means of the ³²P-postlabeling assay. Morphological transformation of the cells was induced by treatment with diethylstilbestrol (DES) at 1–10 μg/mL for 24 h but not by treatment with its derivatives trans, trans-dienestrol (α-DIES) or cis, cis-dienestrol (β-DIES) at 1–10 μg/mL for 24 h. Similarly, DNA adduct formation was elicited by exposure of SHE cells to DES at 1–10 μg/mL for 24 h but not by either α-DIES or β-DIES. Treatment of SHE cells with DES at 1–10 μg/mL for 2 h in the presence of exogenous metabolic activation with rat liver post-mitochondrial supernatant enhanced morphological transformation in a dose-dependent manner. Our previous studies have demonstrated that exposure of SHE cells to DES under the same conditions with exogenous metabolic activation induces somatic mutations at the Na⁺/K⁺ ATPase locus. Therefore, we examined whether with exogenous metabolic activation DES induced DNA adduct formation in SHE cells. DNA adducts were not detected when SHE cells were treated with DES at 1–10 μg/mL for 2 h in the presence of exogenous metabolic activation. Treatment with 17β-estradiol (E₂), 2-hydroxyestradiol (2-OH E₂), or 4-hydroxyestradiol (4-OH E₂) at 1 μg/mL for 24 h induced DNA adduct formation in the cells, in parallel with the induction of cell transformation. The rank order of DNA adduct formation was 4-OH E₂ > 2-OH E₂ > E₂. The results indicate that estrogens induce DNA adduct formation in cultured SHE cells, but the induction may not be the only mechanism relevant to the initiation of cell transformation. © 1996 Wiley-Liss, Inc.     

Modulatory effect of tamoxifen and ICI 182,780 on adriamycin resistance in MCF-7 human breast-cancer cells

In this study the ability of the new pure anti-estrogen ICI 182,780 to modulate the cytotoxic action of adriamycin (ADR) on parental and ADR-resistant MCF-7 (MCF-7 ADRr) human breast-cancer cells was investigated and compared with that of tamoxifen (TAM). TAM enhanced ADR cytotoxicity in MCF-7 ADRr cells in a dose-related manner, but this effect was slight or absent in MCF-7 WT. In contrast, ICI 182,780 was able to enhance ADR toxicity both in MCF-7 ADRr and in the parental cell line. ICI 182,780 was up to 2.5-fold more effective than TAM in reducing the IC₅₀ of ADR in MCF-7 ADRr cells. Analysis of the data by the isobole method showed that the combination ADR/TAM and ADR/ICI 182,780 produced synergistic anti-proliferative activity in MCF-7 ADRr cells. Because ADR resistance in these cells is associated with the expression of high levels of P-glycoprotein (Pgp), we evaluated the effect of anti-estrogens on Pgp expression and activity. Both ICI 182,780 and TAM failed to modulate Pgp expression as assessed by flow cytometry and Western-blot analysis, performed using the monoclonal antibodies MM4.17 and C219, which are specific for an external or an internal determinant respectively. Pgp activity was investigated by flow cytometry measuring the extrusion of ADR and the cationic dye Rhodamine 123 (Rh 123). ICI 182,780, but not TAM, reduced the activity of Pgp in MCF-7 ADRr cells. Flow cytometry was also used to investigate cell-cycle modifications induced by ADR in MCF-7 ADRr cells, both in the presence and in the absence of anti-estrogens. After 72 hr, higher doses induced an arrest of cells at the G₂/M phase. The same effect was visible when lower doses of ADR were combined with ICI 182,780 or TAM. In terms of cell-cycle-blocking activity ICI 182,780 was largely more effective than TAM. © 1996 Wiley-Liss, Inc.     

Bisphenol-A induces cellular transformation,aneuploidy and DNA adduct formation in cultured Syrian hamster embryo cells

Bisphenol-A (BP-A) is a major component of epoxy, polycarbonate and other resins. For an assessment of in vitro carcinogenicity and related activity of BP-A, the abilities of this compound to induce cellular transformation and genetic effects were examined simultaneously using the Syrian hamster embryo (SHE) cell model. Cellular growth was reduced by continuous treatment with BP-A at doses ≥100 μM. However, colony-forming efficiencies were not decreased significantly following treatment with up to 200 μM BP-A for 48 hr. Morphological transformation of SHE cells was induced by treatment of cells with BP-A at 50 to 200 μM for 48 hr. BP-A exhibited transforming activity at doses ≥50 μM but was less active than the benzo[α]pyrene used as a positive control. Over the dose range that resulted in cellular transformation, treatment of SHE cells with BP-A failed to induce gene mutations at the Na⁺/K⁺ ATPase locus or the hprt locus. No statistically significant numbers of chromosomal aberrations were detected in SHE cells treated with BP-A. However, treatment of cells with BP-A induced numerical chromosomal changes in the near diploid range at doses that induced cellular transformation. ³²P-Postlabeling analysis revealed that exposure of cells to BP-A also elicited DNA adduct formation in a dose-dependent fashion. Our results indicate that BP-A has cell-transforming and genotoxic activities in cultured mammalian cells and potential carcinogenic activity. Int. J. Cancer 75:290–294, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Comparative study on the induction of cytostasis and apoptosis by ICI 182,780 and tamoxifen in an estrogen receptor-negative ovarian cancer cell line

We have compared the effects of a broad range of clinically relevant concentrations (0.1 to 10 μM) of the steroidal pure anti-estrogen ICI 182,780 and the non-steroidal partial anti-estrogen tamoxifen (TAM) on cell proliferation and induction of apoptosis in the estrogen receptor (ER)-negative ovarian carcinoma cell line A2780. Cell proliferation was assessed by evaluating the number of viable cells, changes in cell-cycle distribution and cell replication rate; while apoptosis induction was assessed by examining nuclear morphological changes associated with apoptotic death and DNA cleavage into 300 and 50 kbp units (large DNA fragmentation) and into 180 bp units (internucleosomal DNA fragmentation). We provide evidence that 0.1 to 10 μM ICI 182,780 and TAM significantly inhibit the growth of A2780 cells in a dose-dependent fashion. Cytokinetic analysis revealed that only 10 μM TAM caused a significant blockade in G₁ and a diminished replication rate. Conversely, we show that 0.1 to 10 μM ICI 182,780 and TAM induce apoptosis in a dose-dependent fashion. The earliest recognizable apoptotic change induced by treating the cells with these 2 drugs was DNA cleavage into 300 and 50 kbp units. This started to be visible in adherent cells, implying that apoptosis induction by ICI 182,780 and TAM was not determined by the loss of cell–substrate interaction. A further degradation of 300 and 50 kbp DNA fragments occurred in cells that had lost their adhesion to the culture plate. We observed the ladder pattern typical of internucleosomal DNA cleavage by treating A2780 cells with the highest dose (10 μM) of ICI 182,780 and TAM. Lower concentrations of these 2 drugs (0.1 to 1 μM) did not produce such a pattern of DNA fragmentation. Typical features of apoptotic nuclei were detectable after both drug treatments. However, cells undergoing apoptosis induced by ICI 182,780 showed hyper-aggregation of chromatin, whereas TAM-treated cells preferentially exhibited chromatin clumping. Int. J. Cancer 76:47–54, 1998.© 1998 Wiley-Liss, Inc.     

Characterization of the effects of the novel non-steroidal antiestrogen EM-800 on basal and estrogen-induced proliferation of T-47D,ZR-75-1 and MCF-7 human breast cancer cells in vitro

Since estrogens play a predominant role in the development and growth of human breast cancer, antiestrogens represent a logical approach to the treatment of this disease. The present study compares the effects of the novel non-steroidal anti-estrogen EM-800 and related compounds with those of a series of anti-estrogens on basal and 17β-estradiol (E₂)-induced cell proliferation in human breast cancer cell lines. In the absence of added E₂, EM-800 and related compounds failed to change basal cell proliferation, thus showing the absence of intrinsic estrogenic activity in the ER-positive T-47D, ZR-75-1 and MCF-7 cell lines. The stimulation of T-47D cell proliferation induced by 0.1 nM E₂ was competitively blocked by a simultaneous incubation with EM-652, EM-800, OH-tamoxifen, OH-toremifene, ICI 182780, ICI 164384, droloxifene, tamoxifen and toremifene at apparent Ki values of 0.015, 0.011–0.017, 0.040–0.054, 0.043, 0.044, 0.243 and 0.735 nM, approx. 10 nM and >10 nM, respectively. Similar data were obtained in ZR-75-1 and/or MCF-7 cells. Moreover, EM-652 was 6-fold more potent than OH-Tamoxifen in inhibiting the proportion of cycling MCF-7 cells. Our data show that EM-800 and EM-652 are the most potent known antiestrogens in human breast cancer cells in vitro and that they are devoid of the estrogenic activity of OH-tamoxifen and droloxifene suggested by stimulation of cell growth in the absence of estrogens in ZR-75-1 and MCF-7 cells. Int. J. Cancer 73:104–112, 1997. © 1997 Wiley-Liss, Inc.     

Different points of action of retinoids and anti-estrogens in G1 phase identified in synchronized T-47D breast cancer cells

Both retinoids and anti-estrogens inhibit breast cancer cell proliferation with accumulation of cells in the G₁ phase of the cell cycle, but the effect of retinoids is delayed compared to that of anti-estrogens. To determine whether this temporal difference is due to a simple delay in the action of retinoids on a common site or to different sites of action within the G₁ phase, we studied the cell cycle effects of retinoic acid (RA) and the anti-estrogen ICI 164384 (ICI) in T-47D cells partially synchronized by mevalonic acid rescue of lovastatin-induced cell cycle arrest. We found that cells entering the cell cycle semi-synchronously after mevalonic acid rescue of lovastatin treatment were immediately susceptible to ICI but not RA. This suggests that RA may act at a point up-stream and ICI at a point down-stream of lovastatin action. Consistent with this, cells recommencing cell cycle progression after RA treatment were susceptible to the effects of lovastatin, while cells pre-treated with ICI then rescued with estradiol were not. In addition, cells rescued from cell cycle arrest induced by either RA, ICI or lovastatin entered S phase with the same kinetics. Our findings suggest, first, that within G₁, RA acts before and ICI acts after the point of lovastatin action and, second, that despite these differences in the initiation of cell cycle arrest, the final nature of the cell cycle arrest is similar. Hence, retinoids and anti-estrogens may be expected to target different cell cycle-regulatory molecules to initiate cell cycle arrest, while overcoming this arrest may be accomplished by the activation of a common molecular pathway. Int. J. Cancer, 70:291–296, 1997. © 1997 Wiley-Liss, Inc.     

Estrogenic and anti-estrogenic regulation of estrogen receptor in MCF-7 breast-cancer cells: Comparison of immunocytochemical data with biochemical measurements

Data from immunocytochemical assessment of estrogen receptor (ER) regulation in MCF-7 cells under estrogenic and anti-estrogenic stimulation were compared with those obtained by enzyme immunoassay (Abbott ER-EIA). Similar trends were observed, although ER level variations were less marked when assessed immunocytochemically. We confirmed reports of ER disappearance in the presence of estrogens (Es; E₂ and DES) and pure anti-estrogens (AEs; RU 58,668 and ICI 164,384) as well as its increase with partial AEs (4-OH-TAM and RU 39,119). E₂-induced ER down-regulation was partly blocked by actinomycin D (AMD), okadaic acid (OK) and cycloheximide (CHX) when assessed by these 2 methods. Down-regulation by pure AEs was not impeded by CHX, indicating that they operate differently from Es (i.e., transformation of ER to a form sensitive to constitutive degradation activity). In situ pre-labeling of the cells with [³H]TAZ indicated that all investigated ligands eliminate pre-existing ER through binding to newly synthetized receptors, since [³H]TAZ co-valently associates with ER; E₂ and RU 58,668 were more effective than 4-OH-TAM in this regard. CHX blocked ER disappearance even in the presence of pure AEs, which is in contrast to the data established with cells not pre-exposed to [³H]TAZ. Nuclear location of [³H]TAZ-ER complexes may explain this discrepancy, since pure AE-ER complexes were reported to be incapable of nuclear translocation. Int. J. Cancer 78:760–765, 1998. © 1998 Wiley-Liss, Inc.     

Human breast cancer cell lines resistant to pure anti-estrogens are sensitive to tamoxifen treatment

The pure steroidal anti-estrogens ICI 164,384 and ICI 182,780 are very potent growth inhibitors of the estrogen receptor-positive human breast cancer cell line MCF-7. However, long-term treatment of MCF-7 cells with 10^?7 M concentrations of these compounds results in selection of proliferating colonies of resistant cells. Our report describes 4 ICI 164,384- and 3 ICI 182,780-resistant MCF-7 sublines established after long-term treatment. Resistant sublines are estrogen receptor-positive, and all sublines have lost expression and estrogen inducibility of the progesterone receptor protein. Based on IC₅₀ concentrations, all tested resistant sublines had a reduced sensitivity to pure anti-estrogens on the order of 100- to 1000-fold compared with parent MCF-7 cells. All resistant cell lines have survived propagation for more than 15 subcultivations in the presence of 10^?7 M pure anti-estrogen. The MCF-7/182^R-6 subline has been tested for stability of resistance and appeared to be stably resistant after 13 weeks of propagation without the selective pressure of ICI 182,780. Cell lines resistant to the ICI 182,780 compound are cross-resistant to the ICI 164,384 compound and vice versa. However, the sublines resistant to pure anti-estrogens are sensitive to tamoxifen. Our results show that although the pure steroidal anti-estrogens are very potent growth inhibitors, they do not circumvent development of resistance. © 1995 Wiley-Liss, Inc.     

Involvement of genotoxic effects in the initiation of estrogen-induced cellular transformation: studies using Syrian hamster embryo cells treated with 17beta-estradiol and eight of its metabolites

To examine a direct involvement of genotoxic effects of estrogens in the initiation of hormonal carcinogenesis, the abilities of 17beta-estradiol (E2) and 8 of its metabolites to induce cellular transformation and genetic effects were studied using the Syrian hamster embryo (SHE) cell model. Treatment with E2, estrone (E1), 2-hydroxyestrone (2-OHE1), 4-hydroxyestrone (4-OHE1), 2-methoxyestrone (2-MeOE1), 16alpha-hydroxyestrone (16alpha-OHE1), 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2) or estriol (E3) for I to 3 days inhibited SHE cell growth in a concentration-dependent manner. Concentration-dependent increases in the frequency of morphological transformation in SHE cells were exhibited by treatment for 48 hr with each of all estrogens examined, except for E3. The transforming activities of the estrogens, determined by the induced transformation frequencies, were ranked as follows: 4-OHE1 > 2-OHE1 > 4-OHE2 > 2-OHE2 > or = E2 or E1 > 2-MeOE1 or 16alpha-OHE1 > E3. Somatic mutations in SHE cells at the Na+/K+ATPase and /or hprt loci were induced only when the cells were treated with 4-OHE1, 2-MeOE1 or 4-OHE2 for 48 hr. Some estrogen metabolites induced chromosome aberrations in SHE cells following treatment for 24 hr. The rank order of the clastogenic activities of the estrogens that induced chromosome aberrations was 4-OHE1 > 2-OHE1 or 4-OHE2 > 2-OHE2 > E1. Significant increases in the percentage of aneuploid cells in the near diploid range were exhibited in SHE cells treated for 48 hr or 72 hr with each of the estrogens, except for 4-OHE1 and E3. Our results indicate that the transforming activities of all estrogens tested correspond to at least one of the genotoxic effects by each estrogen, i.e., chromosome aberrations, aneuploidy or gene mutations, suggesting the possible involvement of genotoxicity in the initiation of estrogen-induced carcinogenesis.     

Contrasting ability of antiestrogens to inhibit MCF-7 growth stimulated by estradiol or epidermal growth factor

A potential mechanism is described by which a growth factor may prevent the action of antiestrogens or reactivate the growth of hormone-responsive breast carcinoma in patients undergoing tamoxifen (TAM) treatment. Epidermal growth factor (EGF)-stimulated growth (10⁻⁸M EGF) was assayed in the MCF-7 breast cancer cell line in the presence of various concentrations (10⁻¹⁰ to 10⁻⁶M) of three antiestrogens, 4-hydroxytamoxifen (OH TAM), TAM and ICI 164384. In each case, the EGF-stimulated increases in DNA were not inhibited by the antiestrogen. OH TAM and ICI 164384 inhibited estradiol (E₂) stimulated cell proliferation in a dose-related fashion. However, in the presence of both E₂ and EGF, these two antiestrogens inhibited E₂ effects only; EGF promotion of growth was unaffected. Pretreatment of MCF-7 cells for 2 days with either OH TAM or ICI 164384 did not inhibit EGF-induced increases in cell proliferation. We propose that eventual antiestrogen therapeutic failure may be caused by the paracrine influences of growth factors from neighboring cells.     

Estrone sulfatase activity and effect of antiestrogens on transformation of estrone sulfate in hormone-dependent vs. independent human breast cancer cell lines

Summary The effect of the anti-estrogens ICI 164,384 and tamoxifen on the estradiol (E₂) concentration after incubation of estrone sulfate (E₁-S) with different hormone-dependent (MCF-7 and T-47D) and hormone-independent (MDA-MD-231 and MDA-MB-436) mammary cancer cells, as well as the estrone sulfatase activity in these various cell lines, are presented. The anti-estrogen ICI 164,384 decreased very significantly the concentration of E₂ after incubation of E₁-S with MCF-7 (control, mean ±SE: 100±24 pg/mg DNA; + ICI 164,384 [10^–6M]: 7±2 pg/mg DNA). This effect was much more intense than with tamoxifen. A similar effect was observed with T-47D cells. However, no significant effect was observed in the hormone-independent cells. In the intact cell, estrone sulfatase activity was very intense in the hormone-dependent cells, but very small in the hormone-independent cells. However, this activity became very strong after homogenization in the hormone-independent cells. The data suggest that estrone sulfate can play an important role on the bioavailability of E₂ in hormone-dependent breast cancer, and that understanding the control of estrone sulfatase activity can open new knowledge of the estrogen responses and new possibilities of therapeutic application in breast cancer.     

Gene Expression Profiling Identifies Lobe-Specific and Common Disruptions of Multiple Gene Networks in Testosterone-Supported, 17β-Estradiol- or Diethylstilbestrol-Induced Prostate Dysplasia in Noble Rats

The xenoestrogen diethylstilbestrol (DES) is commonly believed to mimic the action of the natural estrogen 17β-estradiol (E₂). To determine if these two estrogens exert similar actions in prostate carcinogenesis, we elevated circulating levels of estrogen in Noble (NBL) rats with E₂/DES-filled implants, while maintaining physiological levels of testosterone (T) in the animals with T-filled implants. The two estrogens induced dysplasia in a lobe-specific manner, with E₂ targeting only the lateral prostate (LP) and DES impacting only the ventral prostate (VP). Gene expression profiling identified distinct and common E₂-disrupted versus DES-disrupted gene networks in each lobe. More importantly, hierarchical clustering analyses revealed that T + E₂ treatment primarily affected the gene expression pattern in the LP, whereas T + DES treatment primarily affected the gene expression profile in the VP. Gene ontology analyses and pathway mapping suggest that the two hormone treatments disrupt unique and/or common cellular processes, including cell development, proliferation, motility, apoptosis, and estrogen signaling, which may be linked to dysplasia development in the rat prostate. These findings suggest that the effects of xenoestrogens and natural estrogens on the rat prostate are more divergent than previously suspected and that these differences may explain the lobe-specific carcinogenic actions of the hormones.     

Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture: the role of autophagy

Active cell death in hormone-dependent cells was studied usingcultured human mammary carcinoma cells (MCF-7) treated withthe anti-estrogens (AEs) tamoxifen (TAM), 4-hydroxy-tamoxifen(OH-TAM) or ICI 164 384 (10^–8–10^–5M) as amodel. The following results were obtained. (i) In untreatedMCF-7 cells a wave of replication occurred in the first 5 daysof culture. All three AEs caused a dose-dependent inhibitionof cell replication. (ii) TAM and OH-TAM at 10^–5 M, butnot ICI 164 384, caused lytic cell death (necrosis) within 24h, which was not inhibited by estradiol (10^–9–10^–6M).(iii) Lower concentrations of TAM or OH-TAM (up to 10^–6M)or ICI 164 384 induced a more gradual appearance of cell deathbeginning at day 3. This type of cell death was inhibited byestradiol (10^–9 M), indicating its active nature. (iv)Nuclei showed two distinct patterns of alternation: (a) apoptosis-likecondensation and fragmentation of chromatin to crescent massesabutting the nuclear envelope; (b) condensation of the chromatinto a single, pyknotic mass in the center of the nucleus, detachedfrom the nuclear envelope. Quantitative histological evaluationrevealed the predominance of pyknosis. (v) Biochemical DNA analysisrevealed that only a relatively small amount of the total DNAwas finally degraded into low molecular weight fragments (20kb and less). (vi) Active cell death, with both apoptotic andpyknotic nuclear morphology, was associated with extensive formationof autophagic vacuoles (AV). 3-Methyladenine, a known inhibitorof AV formation, partially prevented cell death as detectedby nuclear changes. (vii) ICI 164 384 was about 10 times moreeffective than TAM or OH-TAM at inhibiting DNA synthesis, buthad equal potency in inducing active cell death. It is concludedthat AEs have anti-proliferative and anti-survival effects onMCF-7 human mammary cancer cells in culture. These two effectsare under separate control because they differ by kinetics,dose dependence and sensitivity to the various AEs. Active celldeath in MCF-7 cells seems to be initiated by autophagy, incontrast to concepts of apoptosis, and thus corresponds to autophagic/lysosomalor type II death as previously defined. This may be importantbecause of biochemical and molecular differences between thesevarious subtypes of active cell death.     

Activation of 17{beta}-estradiol and estrone by dimethyldioxirane and inhibition of rat liver nuclear and nucleolar RNA synthesis in vitro

17ß-estradiol (E₂), estrone and diethylstilbestrol(DES) had no effect on nuclear and nucleolar RNA synthesis invitro. However, after reacting with dimethyldioxirane (DMDO),a versatile epoxide-forming oxidant, these estrogens were ableto inhibit and in a dose-dependent manner nuclear and nucleolarRNA synthesis in vitro. It was also found that the time requiredfor the maximal activation of these chemicals by DMDO varied:estrone, 10 min; E₂, 30 min; DES, 60 min. Tamoxifen (TAM) wasalso able to inhibit nuclear and nucleolar RNA synthesis ina dose-dependent manner, but the mechanism of this inhibitionwas more complex. Control experiments clearly indicated, unlikeE₂, estrone and DES, TAM per se was able to directly inhibitRNA synthesis in vitro. TAM after activation by DMDO was ableto further inhibit RNA synthesis contributing part of the totalobserved inhibition. These data show for the first time thatE₂, estrone, DES and TAM can be activated by DMDO and possiblyto epoxides. We propose that epoxidation of E₂ and estrone maybe the underlying mechanism of carcinogenesis for these estrogensin vivo.     

Antiestrogenic properties of keoxifene,trans-4-hydroxytamoxifen,and ICI 164384, a new steroidal antiestrogen,in ZR-75-1 human breast cancer cells

The agonistic/antagonistic properties of two non-steroidal antiestrogens, namelytrans-4-monohydroxy-tamoxifen (OH-TAM) and keoxifene (LY156758), and the new steroidal antiestrogen ICI164384, a 7-alkylamide derivative of estradiol (E₂), were assessed by measuring their effect on the proliferation of ZR·75-1 cells, an estrogen-responsive human breast cancer cell line. While subnanomolar concentrations of both OH-TAM and LY156758 had significant estrogenic stimulatory activity on cell growth in the absence of estrogens and higher concentrations were inhibitory, ICI164384 behaved exclusively as a growth inhibitor and more potently so than the two other compounds. The three antiestrogens had similar potency to inhibit the mitogenic effect of E₂ and at 300 nM, all antiproliferative effects were completely reversible by the estrogen. ICI164384 was a weaker competitor of³H-labeled E₂ or R2858 (moxestrol) uptake in intact ZR-75-1 cells in a 1-hour assay, partly because of a slower intracellular access to estrogen specific binding sites. Moreover, ICI164384 interacted in a rapidly (~ 6 h) reversible manner with estrogen-specific binding sites, while the non-steroidal antiestrogens induced a longer-acting (> 24 h) down-regulation of specific [³H]R2858 uptake. The present data indicate that, among the antiestrogens studied, ICI164384 is the only compound acting as a pure antiestrogen in ZR-75-1 breast cancer cells, while LY156758 and OH-TAM behave as antiestrogens endowed with partial agonistic activity in this system.     

Estrogen-induced anchorage-independence in human endometrial stromal cells

Estrogens are important etiologic agents for most gynecologic malignancies, and chronic exposure to estrogen that is unopposed by progestins conveys the greatest risk. Treatments with estrogen facilitate the process of malignant transformation in rodents, but relatively few studies of estrogen-induced carcinogenesis have been performed using human cells. Most malignancies in estrogen-responsive tissues arise from epithelial cells, but an increasing body of evidence emphasizes the role of stromal cells as mediators of the effects of estrogens on epithelial cells. Our studies were designed to assess estrogens as carcinogens for human endometrial stromal cells and to provide a basis for studies of the role of stroma in estrogen-induced carcinogenesis in humans. Acute treatments with the estrogens diethylstilbestrol (DES), 17β-estradiol E₂ and β-dienestrol enhance anchorage-independent proliferation (AIP) of SV40-immortalized human endometrial stromal cells in the rank order of DES > E₂ > β-dienestrol. The anti-estrogenic compound tamoxifen inhibits DES-induced AIP. The magnitude of DES-induced AIP increases with prolonged duration of treatment. After 11 months of chronic treatment with 0.1 nM DES, AIP was 20-fold higher than in vehicle-treated control cultures. Expression of the estrogen receptor was altered by treatments with DES in parallel with increased capacity for AIP. These conditionally immortal human endometrial stromal cells appear to be a good model for estrogen-induced transformation of human cells. © 1995 Wiley-Liss, Inc.     

Prevention of estrogen carcinogenesis in the hamster kidney by ethinylestradiol: some unique properties of a synthetic estrogen

Ethinylestradiol (EE) has evident paradoxical effects on cancer risk for human breast and hepatic cancer which parallel in some respects its effects on estrogen-induced neoplasms in the hamster kidney and liver. EE has been shown to be only weakly carcinogenic in the hamster kidney, but the most potent carcinogenic estrogen in the hamster liver following prolonged treatment. Unexpectedly, when EE and potent carcinogenic estrogens, such as diethylstilbestrol (DES), 17beta- estradiol (E2) and Moxestrol (MOX), are administered concomitantly, estrogen-induced carcinogenesis in the kidney is completely prevented. In studying this novel finding, we found that, compared with E2 exposure alone, EE at 0.05 and 1.0 nM significantly (P < 0.001) inhibited the rise in proliferation of cultured primary hamster proximal renal tubular (PRT) cells in the presence of E2 (1.0 nM). Consistent with these findings, combined EE + DES treatment for 5.0 months reduced hamster kidney c-myc, c-fos and c-jun RNA expression to 43, 37 and 52%, respectively, compared with levels observed after DES treatment alone. Interestingly, TAM + DES treatment for the same period also resulted in the same low level of RNA expression of these proto- oncogenes. c-MYC, c-FOS and c-JUN protein products were comparably reduced after either EE + DES or TAM + DES treatment. It appears that c- fos expression and c-FOS protein levels in the hamster kidney were more responsive to TAM inhibition. These data demonstrate that EE possesses unique anti-tumorigenic properties in vivo in the hamster kidney. Additionally, the observed anti-estrogen-like effect of EE on cell proliferation of cultured PRT cells suggests that EE may interfere critically with estrogen receptor (ER)-mediated mitogenic pathway(s) affected by potent carcinogenic estrogens, thus preventing subsequent gene dysregulation and, hence, tumor development. Based on competition studies, the differential binding of EE to hamster kidney ER relative to that of the other estrogens (E2, DES, MOX) appears not to contribute to the prevention of estrogen carcinogenesis at this organ site by EE.      

Randomized trial of diethylstilbestrol vs. tamoxifen in postmenopausal women with metastatic breast cancer. An updated analysis

One hundred fiftyone postmenopausal women with progressive metastatic breast cancer and no prior hormonal therapy were treated with either diethylstilbestrol (DES) or tamoxifen (TAM). One hundred fortythree eligible patients were followed until death or for a minimum of 14.1 years on the DES arm or 16.7 years on the TAM arm. The overall objective response was 42% for DES and 33% for TAM (p=0.31) and the median duration of response was 11.8 months for DES and 9.9 months for TAM (p=0.38). Duration of response and progressionfree survival were not found to be significantly different between DES and TAM (p=0.32 and 0.65, respectively). The median survival was 3.0 years for DES vs. 2.4 years for TAM. The 5year survival was 35% for the DES arm and 16% for the TAM arm. Survival was significantly better for women on DES than for women on TAM (adjusted p=0.039). Review of records did not show any difference in pattern of treatment failure or subsequent treatments in the DES and TAM arms.Treatment with DES was more commonly associated with toxicity such as nausea, edema, vaginal bleeding, and cardiac problems, whereas hot flashes were commonly seen with TAM therapy.The initial treatment with DES is associated with increased survival. The basis of this survival advantage is not known. TAM still is the preferred agent in the treatment of metastatic breast cancer, but this trial underscores the fact that estrogens have activity and remain in the armamentarium for treatment of selected patients with metastatic breast cancer.     

Combined effects of toremifene and paclitaxel on human breast cancer cell lines

Effects of toremifene (TOR) in combination with paclitaxel (TXL) on various human breast cancer cell lines were evaluated. TOR and TXL exhibited additive effects on estrogen receptor (ER)-positive cancer cell lines, MCF-7 and T-47D, and a sub-additive effect on a tamoxifen (TAM)-resistant line, T-47D/TAM. To all three ER-negative cancer cell lines, the combined treatment also showed additive effects on MDA-MB-134VI, MDA-MB-231 and MDA-MB-453. Furthermore, a synergistic effect was observed on a multi-drug resistant (MDR) line, Adr. This synergistic effect was more potent in the combination with TOR than that with TAM. The combined treatment increased intracellular TXL, and the accumulation by TOR was 1.5-fold that by TAM. Consequently, the ratio of G2M arrested cells was higher, with statistical significance, in the TOR combination than in the TAM combination. In addition, these synergistic effects in MDR cells were also observed in the combination of TXL with major clinical active metabolites, N-desmethyl-TOR (TOR-1) and 4-hydroxy-TOR (TOR-2). These results suggest that the combination therapy of TOR and TXL might be an effective clinical treatment for breast cancer patients.