[Show abstract][Hide abstract] ABSTRACT: We describe the development of a cell system for in vivo screening of inhibitors of the mevalonate pathway. To this aim, we have constructed a bicistronic mRNA, transcribed from a constitutive cytomegalovirus promoter, containing the Renilla reniformis luciferase RNA open reading frame sequence as first cistron and the Firefly luciferase RNA sequence as a second cistron. The intercistronic space is made of the R17 binding sequence of the bacteriophage R17 protein. A chimeric protein able to bind to a specific sequence in the hairpin and to induce internal ribosome entry in the RNA switches on translation of the second cistron. This chimeric protein is made up of the bacteriophage RNA binding domain (R17) fused to the ribosome recruitment core of the eIF-4G1 eukaryotic translation initiation factor and to the CAAX box of H-Ras addressing the protein to the plasma membrane where it is not efficient. Internal ribosome entry upstream of the Firefly cistron is therefore under the dependence of the mevalonate pathway inhibitors. Indeed, products that are able to inhibit protein farnesylation rescue the cytoplasmic location of the R17-eIF-4G-CAAX protein, which once more becomes a translation factor for the expression of the second cistron. To exemplify the system, the present work checks the ability of various antiestrogens to interfere with the mevalonate pathway. It seems that pure antiestrogen, able to selectively bind the estrogen receptor, is unable to switch on the second Firefly cistron although selective antiestrogen-binding-site ligands are able to do so.
[Show abstract][Hide abstract] ABSTRACT: We have previously shown that FTI-277, a farnesyl transferase inhibitor (FTI), enhances the efficacy of tamoxifen (Tam) in inhibiting the proliferation of the estrogen dependent MCF-7 cell line. As the cellular response to Tam is the result of an inhibition of both estrogen receptor-dependent and -independent pathways, we have used the estrogen receptor selective anti-estrogen ICI182,780 and N-pyrrolidine(-phenylmethyl-phenoxy)-ethanamine-HCl (PBPE), a selective ligand of anti-estrogen binding site (AEBS), to dissect out the mechanism(s) associated with the observed additivity resulting from combination treatment with FTI-277 and Tam. Moreover, for these studies, FTI-277 has been replaced by R115,777, a FTI currently in phase III clinical trials.
The quantitative sulphorhodamine B (SRB) colorimetric assay was used to determine the growth inhibitory effect of agents on MCF-7 cells. Dose response interactions between R115,777-Tam, R115,777-ICI182,780 and R115,777-PBPE were evaluated, at the IC50 point, using the isobologram method. Apoptotic cell death (DNA fragmentation, nucleus condensation and cytokeratin 18 cleavage) and inhibition of the mevalonate pathway (western blot) were also determined.
Combinations of the specific FTI R115,777 with either ICI182,780 or PBPE exhibit a synergistic effect on MCF-7 cell growth inhibition, while its combination with Tam is additive, as previously reported for FTI-277. Apoptosis is detected after treatment with combinations of R115,777 with either Tam or PBPE but not with ICI182,780, suggesting that each combination inhibits cell proliferation by different mechanisms. Even though the ER pathway has not yet been deciphered, it is shown here that the AEBS pathway is able to interfere with the mevalonate pathway at the level of protein farnesylation.
Overall, this work reveals that combinations of R115,777 with either selective ER ligands or a selective AEBS ligand are able to induce large increases in their anti-proliferative activities on MCF-7 cells. Moreover, these results suggest that it may be of definite interest to evaluate combinations of R115,777 with different anti-estrogens in the treatment of ER positive breast tumours. Based on these experimental data, such combinations may prove beneficial in different clinical scenarios or when used in specific sequences; studying the combination of R115,777 with ICI182,780 for early treatment and reserving combinations with either Tam or a selective AEBS ligand, such as BMS-217380-01, for more resistant disease.
Full-text · Article · Feb 2005 · Breast cancer research: BCR
[Show abstract][Hide abstract] ABSTRACT: From the MCF-7 cell line we have developed, a human mammary cancer cell subline with the same karyotype as the mother strain and named MCF-7(SF), able to grow in serum-free chemically defined medium. This cell subline was firstly used to analyze the effect of basic fibroblast growth factor (FGF-2) in estrogen-receptor-positive human breast cancer cells. FGF-2 like estradiol is able to increase cell proliferation and pS2 expression but was also found to inhibit progesterone receptor (PR) expression. The anti-estrogen tamoxifen partly counteracts the effects of FGF-2 and to discriminate between its two main mediators (estrogen receptor vs. anti-estrogen binding site, AEBS) we compare the efficacies of pure anti-estrogen (ICI 182,780) and AEBS ligand (PBPE). It appears that pure anti-estrogen counteracts cell growth and pS2 effects of FGF-2 since AEBS ligand inhibits the cell growth but has no activity on pS2 expression. Secondly, adding insulin (10(-6)M) in the culture medium induces a strong increase in cell proliferation, which then elicits an inhibitory effect of FGF-2 and addition of anti-estrogens, are less efficient to further decrease growth, since the effects of FGF-2 and anti-estrogens on pS2 expression are conserved.
Full-text · Article · Mar 2003 · Biochemical Pharmacology
[Show abstract][Hide abstract] ABSTRACT: We show here that the two antagonists ICI 182 780, a pure estrogen antagonist, and 4-hydroxy-tamoxifen, a selective estrogen receptor modulator (SERM) have distinct effects on TFF1 (formerly pS2) gene chromatin structure and transcription. Indeed, ICI 182 780 decreased both the intensity of the hormone-dependent DNase I hypersensitive site pS2 HS-1 and transcription of the pS2 gene whereas 4-hydroxy-tamoxifen (OH-Tam) increased the intensity of pS2-HS1 and had no effect on pS2 gene transcription. Interestingly, these differential effects are associated with different fates of ERalpha following the two treatments: The ERalpha-OH-Tam complex was retained in the nucleus more efficiently than the ERalpha-estradiol complex. In contrast, ICI 182 780 provoked a rapid relocation of ERalpha complex to an insoluble nuclear fraction, followed by its degradation. Taken together, these data suggest that regulating the amount of ERalpha in the nucleus is a major way of action of estrogen antagonists with respect to chromatin remodeling and transcriptional control.
No preview · Article · Nov 2002 · Biochimica et Biophysica Acta
[Show abstract][Hide abstract] ABSTRACT: In eucaryotes, DNA packaging into nucleosomes and its organization in a chromatin fiber generate constraints for all processes involving DNA, such as DNA-replication, -repair, -recombination, and -transcription. Transient changes in chromatin structure allow overcoming these constraints with different requirements in regions where processes described above are initiated. Mechanisms involved in chromatin dynamics are complex. Multiprotein complexes which can contain histone-acetyltransferase, -deacetylase, -methyltransferase or -kinase activities are targeted by regulatory factors to precise regions of the genome. These enzymes have been shown to modify histone-tails within specific nucleosomes. Post-translational modifications of histone-tails constitute a code that is thought to contribute to the nucleosome or to the chromatin fiber remodeling, either directly, or through the recruitment of other proteins. Other multiprotein complexes, such as ATP-dependent remodeling complexes, play an essential role in chromatin fiber dynamics allowing nucleosome sliding and redistribution on the DNA. We will focus here on the chromatin structure and its consequences for DNA damaging, replication, repair, and transcription and we will discuss the mechanisms of chromatin remodeling.
[Show abstract][Hide abstract] ABSTRACT: Antiestrogen resistance is frequently observed in patients after longterm treatment with tamoxifen, a nonsteroidal antiestrogen widely used for endocrine therapy of breast cancer. In vitro studies in resistant cells showed that the expression of natural estrogen-responsive genes is frequently altered. Using MVLN cells, an MCF-7-derived cell model, we previously demonstrated that 4-hydroxytamoxifen (OHT) treatment irreversibly inactivated an estrogen-regulated chimeric luciferase response by a direct effect of the drug and not through a cell selection process (E. Badia et al., Cancer Res., 54: 5860-5866, 1994). In the present study, we present tamoxifen-resistant but still estrogen-dependent clones isolated after long-term treatment of MVLN cells with OHT and show that progesterone receptor (PR) expression was irreversibly decreased in some of these clones, whereas the PRA:PRB ratio of residual PR remained unchanged. The irreversible inactivation of both chimeric luciferase gene and PR gene expression was associated with the disappearance of DNase 1-hypersensitive sites. In the case of the chimeric gene, at least one of these sites was close to the estrogen responsive element. Genomic sequencing analysis of a clone with very low PR content did not reveal any methylation on CpG dinucleotides or any mutation in the PR gene promoter region. In all of the resistant clones tested and independently of their PR content, estrogen receptor expression was only lowered by half and remained functional, whereas pS2 expression was not modified. We also observed that the residual luciferase activity level (1-2%) of the MVLN clones, the luciferase expression of which had been irreversibly inactivated, was raised 4-fold by trichostatin A treatment. We conclude that long-term OHT treatment may modify the chromatin structure and thus could contribute to differentially silencing natural target genes.
[Show abstract][Hide abstract] ABSTRACT: Chromatin restricts the accessibility of DNA to regulatory factors; its remodeling over the regulatory regions contributes to the control of gene expression. An increasing number of evidence links defects in chromatin remodeling machinery and cancer. Our aim is to elucidate the role of chromatin structure in the control of the expression of hormone-induced genes in breast cell lines estrogen-dependent or -independent for growth. Mammary tumor growth is controlled by steroid hormones via their nuclear receptor and by growth factors via tyrosine kinase receptors. 50 % of these tumors elude to hormonal control. This limits the anti-estrogen therapy. As a model, we have analyzed in several cell lines the chromatin organization of the regulatory regions of two genes, pS2 that is associated with a good prognostic, and cathepsin D (catD) that is a bad prognostic marker. The expression of the two genes is estrogen-regulated in estrogen-dependent cell line MCF7. In contrast in the hormone-independent cell line MDA MB 231, pS2 is not expressed and catD is constitutively expressed. Within the regulatory regions of pS2 gene, we have localized two regions that undergo a hormone-dependent change in chromatin structure in MCF7 cells but not in MDA MB 231. The lack of chromatin remodeling in MDA MB 231 cells is not due to the absence of expression of the estrogen receptor in the cell line. The expression of pS2 gene can be correlated with chromatin remodeling over the regulatory regions of pS2 gene. In contrast catD regulatory regions did not display hormone-dependent changes in chromatin structure, suggesting that hormone regulation takes place within regions with a constitutively open chromatin structure.
No preview · Article · Jun 2000 · Annales d Endocrinologie
[Show abstract][Hide abstract] ABSTRACT: Chromatin restricts the accessibility of DNA to regulatory factors; its remodelling over the regulatory regions contributes to the control of gene expression. An increasing number of evidence links defects in chromatin remodelling machinery and cancer. Our aim is to elucidate the role of chromatin structure in the control of the expression of hormone-induced genes in breast cell lines oestrogen-dependent or -independent for growth. Mammary tumour growth is controlled by steroid hormones via their nuclear receptor and by growth factors via tyrosine kinase receptors. 50% of these tumours elude to hormonal control. This limits the anti-oestrogen therapy. As a model, we have analysed in several cell lines the chromatin organisation of the regulatory regions of two genes, pS2 that is associated with a good prognostic, and cathepsin D (catD) that is a bad prognostic marker. The expression of the two genes is oestrogen-regulated in oestrogen-dependent cell line MCF7. In contrast in the hormone-independent cell line MDA MB 231, pS2 is not expressed and catD is constitutively expressed. Within the regulatory regions of pS2 gene, we have localised two regions that undergo a hormone-dependent change in chromatin structure in MCF7 cells but not in MDA MB 231 and that can be correlated with gene expression. In contrast catD regulatory regions did not display hormone-dependent changes in chromatin structure, suggesting that hormone regulation takes place within regions with a constitutively open chromatin structure.
No preview · Article · Feb 2000 · Advances in Experimental Medicine and Biology
[Show abstract][Hide abstract] ABSTRACT: We have compared the DNase I hypersensitivity of the regulatory region of two estrogen-regulated genes, pS2 and cathepsin D in hormone-dependent and -independent breast carcinoma cell lines. This strategy allowed the identification of two important control regions, one in pS2 and the other in cathepsin D genes. In the hormone-dependent MCF7 cell line, within the pS2 gene 5'-flanking region, we detected two major DNase I hypersensitive sites, induced by estrogens and/or IGFI: pS2-HS1, located in the proximal promoter and pS2-HS4, located -10.5 Kb from the CAP site, within a region that has not been cloned. The presence of these two DNase I hypersensitive sites correlates with pS2 expression. Interestingly in MCF7 cells, estrogens and IGFI induced indistinguishable chromatin structural changes over the pS2 regulatory region, suggesting that the two transduction-pathways converge to a unique chromatin target. In two cell lines that do not express pS2, MDA MB 231, a hormone-independent cell line that lacks the estrogen receptor alpha, and HE5, a cell line derived from MDA MB 231 by transfection that expresses estrogen receptor alpha, there was only one hormone-independent DNase I hypersensitive site. This site, pS2-HS2, was located immediately upstream of pS2-HS1. In MCF7 cells, two major DNase I hypersensitive sites were present in the 5'-flanking sequences of the cathepsin D gene, which is regulated by estrogens in these cells. These sites, catD-HS2 and catD-HS3, located at positions -2.3 Kb and -3.45 Kb, respectively, were both hormone-independent. A much weaker site, catD-HS1, covered the proximal promoter. In MDA MB 231 cells, that express cathepsin D constitutively, we detected an additional strong hormone-independent DNase I hypersensitive site, catD-HS4, located at position -4.3 Kb. This region might control the constitutive over-expression of cathepsin D in hormone-independent breast cancer cells. All together, these data demonstrate that a local reorganization of the chromatin structure over pS2 and cathepsin D promoters accompanies the establishment of the hormone-independent phenotype of the cells.