Steroid receptor-mediated cytotoxicity of an antiestrogen and an antiprogestin in breast cancer cells.

The antiproliferative and cytotoxic effects of 4-hydroxytamoxifen, an antiestrogen with a high affinity for the estrogen receptor, and of 17 beta-hydroxy-11 beta-(4-methylaminophenyl)-17-(1-propynyl)estra-4,9-dien-3- one-6-7 (RU486), an antiprogestin with a high affinity for the progestin receptor, have been studied on human breast cancer cell lines in culture. The number of dead cells was evaluated by several techniques (trypan blue stain exclusion, DNA cleavage, lactic dehydrogenase activity, morphological changes, and cloning efficiency in soft agar) and found to be increased both by the antiestrogen and the antiprogestin at concentrations correlating with the affinities for their respective receptors. This cytotoxic effect was prevented by the occupation of the respective receptors with estrogen and progestin and was not found in the estrogen receptor- and progestin receptor-negative MDA MB 231 and BT20 cell lines. The contrast between the ultrastructural modifications of chromatin and the integrity of mitochondria suggested that the antihormone-induced cell death was by apoptosis. We conclude that in addition to the receptor-mediated cytostatic activity and the nonspecific cytotoxic activity, antiestrogens trigger a third type of effect that we designate as "receptor-mediated cytotoxic." Similar conclusions can be drawn for the antiprogestin RU486, indicating moreover that the antihormone and antiproliferative activities of this drug are clearly dissociated. The mechanism of these receptor-mediated cytotoxic activities of antiestrogen and antiprogesterone is not known but does not seem to be explained entirely by the antihormone activity of these drugs.

RU486, a progestin and glucocorticoid antagonist, inhibits the growth of breast cancer cells via the progesterone receptor.

The progestin and glucocorticoid antagonist RU486 was tested on the growth of several cell lines in culture. RU486 inhibited the growth of two progesterone receptor (RP) positive human breast cancer cell lines (MCF7 and T47D). The antiproliferative effect was dose dependent and its magnitude correlated with the RP content of the tested cells (T47D greater than estradiol-primed MCF7 greater than withdrawn MCF7). Cell growth inhibition was not prevented by the addition of dexamethasone, dihydrotestosterone, or estradiol, but the cells were rescued by low concentrations of the progestin R5020. RU486 had no effect on the growth of two RP negative human breast cancer cell lines and a rat fibroblast cell line. Moreover, RU486 had no progestin agonist activity in T47D cells when evaluated by measuring the 35S-labeling of two progestin-regulated proteins with mol wts of 48,000 and 250,000, but it totally prevented the induction of these two proteins by R5020. In conclusion, RU486 selectively inhibited the growth of human breast cancer cell lines with unoccupied RP sites and its effect was correlated with the RP concentration of these cells. We propose that RU486 is a RP-targeted drug of potential utility in breast cancer treatment.

Antiestrogenic effect of R5020, a synthetic progestin in human breast cancer cells in culture.

To see whether progestins prevent estrogen action in breast cancer cells, we have studied in vitro the effect of R5020 on the cell growth and the synthesis of secreted proteins in T47D and R27 human breast cancer cells. While R5020 had no effect on cell growth when tested alone, it significantly inhibited the growth of both cell lines in the presence of estradiol (1 nM). The effect was most clear-cut after 10-12 days of treatment and was dose dependent, a half-maximal inhibition occurred with 1 nM R5020. R27, a cloned MCF7 variant resistant to Tamoxifen, remained responsive to R5020, which prevented the effect of 17 beta-estradiol (E2) and inhibited cell growth in the presence of Tamoxifen. This suggests that the two antiestrogens are acting through different mechanisms. Dihydrotestosterone and dexamethasone did not reproduce or inhibit the effect of R5020 on cell growth. R5020 was ineffective in a rat tumor cell line containing androgen and glucocorticoid receptors but lacking progesterone receptors and estrogen receptors. These results suggest that R5020 is probably acting via progesterone receptors rather than via the androgen or glucocorticoid receptors. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we have shown that R5020 specifically decreases the production of the 52K protein, a major protein released by R27 cells after E2 stimulation. We conclude that R5020 has an antiestrogenic activity on breast cancer cells in culture, since it prevents the stimulation of cell growth and protein synthesis by E2.

Metabolic conversion and growth effects of n-6 and n-3 polyunsaturated fatty acids in the T47D breast cancer cell line.

The incorporation and conversion of polyunsaturated fatty acids (PUFA) of n-3 and n-6 families were examined in the T47D breast cancer cell line in parallel with their effects on cell proliferation. In low serum-containing medium, PUFA exerted differential growth effects, depending both on their affiliation and unsaturation degree. The study of PUFA processing suggested that T47D cells are deficient in delta 6 and delta 4-desaturation activities whereas they can process to delta 5-desaturation. Thus, the PUFA growth effect on T47D cells appeared to be associated with a lack of desaturation.

The antiproliferative effect of tamoxifen in breast cancer cells: mediation by the estrogen receptor.

The effects of tamoxifen (Tam) and its 4-hydroxylated metabolite (OH-Tam) on the growth of two human breast cancer cell lines ( MCF7 and BT20 ) were evaluated by fluorometric DNA assay. The effects of the antiestrogens were dependent upon their concentrations and the nature of the cells. At concentrations below 4 microM, the degree of inhibition was related to their relative affinities for the estrogen receptor and was totally reversed by estradiol in MCF7 cells. No inhibition was observed in the estrogen receptor negative cell line BT20 . This supports and extends the idea that the antiproliferative effect of Tam at these concentrations is mediated by the estrogen receptor even in the absence of measurable estradiol concentration. At concentrations greater than 4 microM, Tam was cytotoxic on MCF7 and BT20 mammary cell lines within 2 days of treatment. The cytotoxic effect was irreversible and was not prevented by occupation of the estrogen receptor with estradiol, suggesting that it was not mediated by the estrogen receptor. The cytotoxicity of the triphenylethylene drugs, however, has some specificity since it was not observed in a fibroblast rat cell line ( 49F ) or in the two mammary cell lines with similar high concentrations of estradiol and diethylstilbestrol.

Regulation of gene expression by insulin in adipose cells: opposite effects on adipsin and glycerophosphate dehydrogenase genes.

Insulin is known to play the role of a positive effector both in vitro on the adipose conversion process and in vivo on the fatty acid synthesis and esterification processes in adipose tissue. The effects of insulin on the expression of two genes activated during adipose conversion, glycerol-3-phosphate dehydrogenase (GPDH) and adipsin genes, have been investigated in 3T3 F442A adipose cells. Within a physiological range of concentrations, insulin exerts opposite effects on the levels of GPDH (EC50 approximately 0.2 nM) and adipsin (EC50 approximately 1 nM) mRNAs. Its negative effect on the abundance of adipsin mRNA involves primarily a rapid inhibition of the transcriptional rate (less than 2 h). Its positive effect on the abundance of GPDH mRNA is due to a stimulation of the transcriptional rate accompanied by a delayed stabilization of GPDH mRNA. In addition, insulin exerts a specific effect on the length of the poly(A) tract of the adipsin mRNA. These results show that a single mechanism for the regulation of adipose-related genes by insulin can be excluded but rather suggest a complex phenomenon in which various levels of regulation take place.

Retinoic acid suppresses insulin-induced cell growth and cyclin D1 gene expression in human breast cancer cells.

We examined the effects of all-trans retinoic acid (RA) on the insulin-induced cell growth, cell cycle progression and cyclin D1 gene expression in breast cancer cells. RA exerted a dose-dependent growth inhibition on insulin-induced proliferation in T47D and MCF-7 hormone-dependent cell lines, whereas MDA-MB231 hormone-independent cells were not affected. The RA antagonism of insulin growth effect was associated with an inhibition of cell cycle progression and a suppression of insulin-induced cyclin D1 mRNA. The effect of RA on cyclin D1 mRNA was dose-dependent and was observed within 5 h of treatment when insulin response was maximal.

Wetting and molecular orientation of 8CB on silicon substrates

The wetting properties of 8CB ( 4(')-n-octyl-4-cyanobiphenyl) on silicon wafers have been studied with scanning polarization force microscopy (SPFM). Layer-by-layer spreading of 8CB droplets is observed. With the help of the surface potential mapping capability of SPFM, we found that the molecular dipole of the first monolayer of 8CB is parallel to the surface. A layer of nearly vertical molecular dimers on top of the monolayer has an associated surface potential of 40 mV, which is attributed to a distortion of the dimer. The dimer distortion propagates to the subsequent smectic bilayers, producing an additional 7 mV potential increase in the second layer, 2 mV on the third, and approximately 1 mV on the fourth.

Liquid-crystal-solid interface structure at the antiferroelectric-ferroelectric phase transition.

Total internal reflection is used to probe the molecular organization at the surface of a tilted chiral smectic liquid crystal at temperatures in the vicinity of the bulk antiferroelectric-ferroelectric phase transition. Data are interpreted using an exact analytical solution of a real model for ferroelectric order at the surface. In the mixture T3, ferroelectric surface order is expelled with the bulk ferroelectric-antiferroelectric transition. The conditions for ferroelectric order at the surface of an antiferroelectric bulk are presented.

Stimulatory effect of arachidonic acid on T-47D human breast cancer cell growth is associated with enhancement of cyclin D1 mRNA expression.

Experimental and human studies have provided evidence that a high intake of n-6 polyunsaturated fatty acids stimulates mammary carcinogenesis. Arachidonic acid, an n-6fatty acid consumed in the diet or derived from dietary linoleic acid, is thought to play a key role in enhancement of mammary tumor development. In this study, we investigated the effects of arachidonic acid on T-47D breast cancer cell growth, cell cycle progression, and the expression of cyclin D1 mRNA. Our data show that arachidonic acid stimulated the growth of T-47D cells with a twofold stimulation at 5 microg/ml. This effect was associated with an increase in the proportion of cells in the S phase of the cell cycle and preceded by stimulation of the expression of cyclin D1 mRNA, with maximal induction at 5 microg/ml. Cyclin D1 mRNA levels were increased within two hours of treatment and were maximal at five hours. These results suggest that arachidonic acid may exert a stimulatory effect on breast cancer cell growth and that this effect possibly involves the induction of cyclin D1 gene expression leading to cell cycle progression.

Monoterpenes inhibit cell growth, cell cycle progression, and cyclin D1 gene expression in human breast cancer cell lines.

Monoterpenes are found in the essential oils of many commonly consumed fruits and vegetables. These compounds have been shown to exert chemopreventive and chemotherapeutic activities in mammary tumor models and represent a new class of breast cancer therapeutic agents. In this study, we investigated the effects of limonene and limonene-related monoterpenes, perillyl alcohol and perillic acid, on cell growth, cell cycle progression, and expression of cyclin D1 cell cycle-regulatory gene in T-47D, MCF-7, and MDA-MB-231 breast cancer cell lines. Our results revealed that limonene-related monoterpenes caused a dose-dependent inhibition of cell proliferation. Of the three monoterpenes tested, perillyl alcohol was the most potent and limonene was the least potent inhibitor of cell growth. The enantiomeric composition of limonene and perillyl alcohol did not interfere with their effect on cell growth. Sensitivity of breast cancer cell lines to monoterpenes was in the following order: T-47D > MCF-7 > MDA-MB-231. Growth inhibition induced by perillyl alcohol and perillic acid was associated with a fall in the proportion of cells in the S phase and an accumulation of cells in the G1 phase of the cell cycle. Finally, we showed that the effects of limonene-related monoterpenes on cell proliferation and cell cycle progression were preceded by a decrease in cyclin D1 mRNA levels.

Steroidal and nonsteroidal antiestrogens in breast cancer cells in culture.

The mode of action of two types of antiestrogens, tamoxifen and progestins, has been studied in the estrogen responsive cell lines, MCF7 and T47D, established from metastatic human breast cancer. (1) Non steroidal antiestrogens: We present evidence indicating that tamoxifen inhibits the growth of breast cancer cells via an interaction with the estrogen receptor (RE), which leads to a partial activation of the receptor and a dissociated effects on gene expression. At concentrations of less than 4 microM, effects of non steroidal antiestrogens are only observed when RE sites are available. At concentrations greater than 4 microM, an additional (cytotoxic?) effect of tamoxifen is observed which is not mediated by the RE. (2) Progestins: Direct antiestrogenic effect of progestins (R5020, progesterone) on breast cancer cells have been demonstrated. Three series of responses to R5020 are obtained: (a) A decreased cell proliferation (antiestrogenic and progestin specific effect). (b) A decreased production of total proteins in the culture medium (antiestrogenic effect). (c) The increased production of a 48,000 dalton protein which is released into the medium after treatment with several progestins (progesterone, medroxyprogesterone acetate, R5020) but not other steroids (specific progestin effect). These responses appear to be mediated by the progesterone receptor and are not observed in RP negative cell line (BT20). Even though these two types of antiestrogens inhibit cell proliferation via different receptors, a common final mechanism (decreased production of estrogen induced growth factors or increased production of antiestrogen induced inhibitory factor) is not excluded.

Association of apolipoprotein epsilon 4 allele with hypertriglyceridemia in obesity.

Hypertriglyceridemia is the most frequent lipid abnormality associated with obesity. Genetic polymorphism of apolipoprotein E (apoE) has been demonstrated to influence lipid levels. We wanted to assess the role of apoE alleles in the hypertriglyceridemias of the obese population. The apoE phenotypes and lipid status were investigated in a population of 172 obese French subjects. The frequencies of phenotypes E4/3, E4/4 and E4/2 were 29.7%, 8.1% and 2.1%, respectively, in a subgroup with triglycerides greater than or equal to 200 mg/dl (n = 37) versus 14.2%, 2.7% and 0.9% in the normolipidemic subgroup (p less than 0.005). The odds ratio of hypertriglyceridemia was 3.15 for obese subjects with epsilon 4; 27.7% of hypertriglyceridemias could be attributed to epsilon 4 allele. It is concluded that the genetic polymorphism of apoE modulates the effects of obesity on lipids and lipoproteins and that allele epsilon 4 increases the risk of obesity-induced hypertriglyceridemia.

Essential role of collagens for terminal differentiation of preadipocytes.

In order to study the role of collagens in the differentiation of TA1 preadipose cells in vitro, ethyl-3,4-dihydroxybenzoate (EDHB) was used as a specific inhibitor of collagen synthesis. The secretion of collagenous proteins only was severely decreased after exposure to EDHB, and this was accompanied by a decrease of differentiation as indicated by low activity levels of glycerophosphate dehydrogenase. The effect of EDHB was dose-dependent and also dependent upon the stage of cell differentiation. Northern-blot analysis show that EDHB addition to undifferentiated cells did not prevent the induction of A2COL6 gene, a marker of the preadipose state, but prevented the induction of the gene encoding for the adipocyte lipid binding protein and the modulation of the expression of the lipoprotein lipase gene which are both indicators of the adipose state. These results demonstrate that differentiation of preadipose cells into adipose cells requires active synthesis of collagens during the preadipose state.

Difference between R5020 and the antiprogestin RU486 in antiproliferative effects on human breast cancer cells.

We have compared the effects of the progestin R5020 and the antiprogestin RU486 on the growth of the MCF-7 and T47D breast cancer cell lines. Differences between the two compounds were demonstrated in several parameters. 1. Estradiol was required for the efficient inhibition of cell growth of both lines by R5020 but not by RU486. Therefore in the total absence of estrogen (phenol-red free medium), the effects of the two drugs on cell growth were dissociated, RU486 remaining inhibitory while R5020 was inactive. 2. The proteins secreted by cells were differently affected, since R5020 induced a 48K protein and decreased the production of the estrogen-regulated 52K protein, while RU486 had no effect on these two parameters. 3. The morphology of cells treated by R5020 was more altered in the presence of estradiol than in its absence, while that of cells treated by RU486 was not affected whether or not estradiol was present. 4. There was a greater reduction of estrogen receptor sites in MCF-7 cells produced by R5020 than by RU486. Even though the two drugs appear to act through the same progesterone receptor and to inhibit total protein secretion, it is likely that they exert their antiproliferative effects on cultured breast cancer cells by different mechanisms. R5020 antagonizes the stimulation produced by estradiol. RU486 by contrast exerts a more direct progesterone receptor mediated inhibitory effect requiring no synergism by estradiol and therefore does not act through a partial progestin activity.

Organization of cyanobiphenyl liquid crystal molecules in prewetting films spreading on silicon wafers.

We observe prewetting films of 8CB (4'-n-octyl-4-cyanobiphenyl) spreading at room temperature on silicon wafers by ellipsometry and x-ray reflectivity. Ellipsometry indicates the formation of a nondense monolayer spreading in front of a 45-A-thick film. X-ray reflectivity, performed using a ribbon geometry for the liquid crystal (LC) reservoir, allows us to determine the organization of the 8CB molecules in the homogenous film. It consists of a trilayer stacking with a smecticlike bilayer standing above a polar monolayer with tilted molecules. We show that the thickness of the bilayer is equal to the smectic periodicity in the bulk material and that the tilt angle of the molecules in contact with the solid surface is close to 60 degrees, in good agreement with second-harmonic generation studies reported by other groups. Such organization can be precisely determined using x-ray reflectivity because it induces a modulation of the electron density along the normal to the surface. Furthermore, a study of the ellispometric profile of a drop heated in the nematic phase, where we observe a complete spreading of the LC, shows the complex structuration of the LC close to the solid interface. In particular, the spreading behavior of the trilayer compared to the subsequent smecticlike bilayers indicates the existence of specific interaction between the trilayer and silicon wafer.

Control of molecular orientation in electrostatically stabilized ferroelectric liquid crystals.

The continuously reorientable (XY-like) ferroelectric polarization density of a chiral smectic liquid crystal is shown experimentally to produce nearly complete screening of the applied electric field in an appropriate cell geometry. This screening, combined with the expulsion of polarization charge for large polarization materials, is shown to produce electrostatic control of the orientation of a uniform optic axis or polarization field.

Cloning and regulation of a mRNA specifically expressed in the preadipose state.

A cDNA library of Ob1771 preadipocytes was constructed, and a cDNA clone designated pOb24 was isolated by differential screening. The pOb24 mRNA, 6 kilobases in length, rose sharply in early differentiating Ob1771 and 3T3-F442A cells and decreased thereafter. In mouse adipose tissue, it was present at a high level in stromal-vascular cells (containing adipose precursor cells) and at a low level in mature adipocytes. Thus, pOb24 mRNA appears to be both in vitro and in vivo an unique marker of the preadipose state, i.e. of cell commitment during adipose cell differentiation. In contrast to glycerol-3-phosphate dehydrogenase mRNA, the emergence of pOb24 mRNA in Ob1771 cells required neither growth hormone or triiodothyronine as obligatory hormones nor insulin as a modulating hormone. Comparative studies of the expression of pOb24 and dihydrofolate reductase genes during the cell cycle suggest that arrest at the G1/S boundary was critical for the entry into the preadipose state. Tumor necrosis factor and transforming growth factor-beta were able to induce a large decrease of pOb24 mRNA level in growth-arrested Ob1771 cells. This decrease was shown to be only confined to early differentiating, glycerol-3-phosphate dehydrogenase negative cells as no decrease of pOb24 mRNA level was observed in glycerol-3-phosphate dehydrogenase positive cells. This result suggests that signals generated by tumor necrosis factor and transforming growth factor-beta have no effect on a commitment-related gene in late differentiated cells.

Cloning of alpha 2 chain of type VI collagen and expression during mouse development.

We have previously described the molecular cloning of a cDNA probe which detects a 6 kb mRNA termed pOb24. pOb24 mRNA appeared to be a marker of the preadipose state both in vitro and in vivo. A pOb24 genomic fragment was isolated and used to screen cDNA libraries in order to isolate the full-length pOb24 cDNA and to identify the corresponding protein. The screening yielded a new cDNA clone which detected a 3.7 kb mRNA species in addition to the 6 kb mRNA species. Sequences at the 3' end of the 6 kb and 3.7 kb mRNAs indicate that both mRNAs are generated from the same gene through the use of two different polyadenylation sites. The protein encoded by the 3.7 kb mRNA appeared to be homologous to the human alpha 2 chain of type VI collagen (A2COL6). The expression of the A2COL6 gene was not confined to adipose tissue; mRNA species can be detected in ovaries, adrenal glands and lungs but not in liver and skeletal muscle. The expression appeared specific for initial phase(s) of cell differentiation since it is parallel to that of the MyoD1 gene during muscle embryogenesis in vivo. In the myogenic C2C12 cell line, the A2COL6 gene exhibited the same regulation as MyoD1 and myogenin genes. These results indicate that A2COL6 gene expression is a marker of the preadipose state, but may also be a marker of other differentiation programmes such as that of muscle.

The adipocyte: relationships between proliferation and adipose cell differentiation.

The differentiation of adipose precursor cells can be divided into early and late events. Growth arrest at the G1/S boundary triggers the activation of early genes, i.e., pOb24 and lipoprotein lipase; the expression of both genes is primarily regulated at a transcriptional level. The expression of late markers, which lead to terminal differentiation and accumulation of neutral lipids, takes place after a limited number of mitoses of early-marker-expressing cells. Only terminal differentiation requires the presence of growth hormone and triiodothyronine as obligatory hormones and insulin as a modulating hormone, and results in the formation of triacylglycerol-filled, non-dividing cells. It appears that terminal differentiation involves the cyclic AMP pathway, the diacylglycerol pathway, and a third pathway triggered by insulinlike growth factor-I and insulin. It is thus proposed that a combination of mitogenic-adipogenic signals is required to trigger terminal differentiation of preadipose cells.