Novel Mechanisms of Antiprogestin Action

Horwitz, Kathryn B.; Tung, Lin; Takimoto, Glenn S.

doi:10.3109/02841869609098493

Cited by 24 publications

(17 citation statements)

References 49 publications

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“…In the presence of cAMP, RU486 can exhibit inappropriate gene activation in T47D subline expressing PR isoform B but not in cells expressing isoform A (Sartovius et al, 1994). It was proposed that PR-B has a unique third transactivation domain that may confer agonist-like properties in the presence of antiprogestin, whereas PR isoform A exhibits transdorminant negative activity toward PR isoform B (Horwitz et al, 1995).…”

Section: Discussionmentioning

confidence: 99%

Demonstration of mixed properties of RU486 in progesterone receptor (PR)-transfected MDA-MB-231 cells: a model for studying the functions of progesterone analogues

Lin

et al. 2001

Br J Cancer

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Summary Progesterone antagonist RU486 (mifepristone) has been implicated for many anti-neoplastic and obstetrical applications. But the compound has demonstrated undesired agonist-like effect depending on cell, tissue and species studied. Using PR-transfected breast cancer cells MDA-MB-231, this report describes the similarities and differences between progesterone-and RU486-mediated effects on cell growth, cell differentiation and, at the molecular level, on the activation of p44/p42 MAP kinases (MAPK). Like progesterone, RU486 inhibited cells growth by arresting the cells in G0/G1 phase of the cell cycle. In contrast to progesterone that induced cell spreading, RU486 induced a multipolar, stellate morphology. RU486-treated cells showed no increase of stress fibers, nor was there any increase of focal adhesions as progesterone-treated cells did. Furthermore, despite of the fact that both compounds inhibited cell growth, RU486 significantly stimulated the activation of p44/p42 MAP kinases whereas progesterone markedly inhibited the activation. Nonetheless, the effects of RU486 were PR-mediated and RU486 was able to antagonize the effect of progesterone on cell growth and focal adhesion. In conclusion, RU486 can act not only as a progesterone antagonist, a progesterone agonist but also induced morphological and molecular changes that were distinct from progesterone-mediated effects in PR-transfected MDA-MB-231 cells. The non-progesterone-like effect of RU486 may be mediated through a pathway that is different from the progesterone-mediated pathway, or it is the result of a blockade of certain critical step(s) in the progesterone-mediated pathway. MATERIALS AND METHODS ChemicalsProgesterone was obtained from Sigma Chemical Co (St. Louis, MO USA). RU486 (mifepristone) and ZK98299 (onapristone) were from Siniwest Holdings, Inc. All tissue culture plastics and reagents were obtained from GIBCO BRL, Life Technologies, Inc. Cell cultureMDA-MB-231 cells were obtained from American Tissue Culture Collection (ATCC) in 1995 at passage 28. MDA-MB-231 cells were cloned using 96-well plates by plating 0.5 cell/well. Clone 2 (known as MDA-MB-231-CL2) was selected for transfection studies. All cells were routinely maintained in phenol-red containing Dulbecco's Modified Eagle Medium (DMEM) supplemented with 7.5% fetal calf serum (FCS), 2 mM glutamine and 40 mg/l gentamycin. TransfectionMDA-MB-231-CL2 cells were transfected with PR expression vectors hPR1 and hPR2 coding for PR isoform B and isoform A, respectively, in pSG5 plasmid (Kastner et al, 1990). Vector pBK-CMV (Stratagene) containing the neomycin-resistant gene was cotransfected with hPR1 and hPR2. Details of transfection were described previously (Lin et al, 1999). Cell growthCells (2 × 10 4 ) in their late exponential growth phase were seeded onto the 6-well plates in phenol red-free DMEM supplemented with 2 mM L-glutamine, 40 mg/l gentamycin and 5% dextran-coated charcoal (DCC)-treated FCS (Test Medium). Treatment of FCS with dextran-coated charcoal was to remove fro...

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Section: Discussionmentioning

confidence: 99%

Demonstration of mixed properties of RU486 in progesterone receptor (PR)-transfected MDA-MB-231 cells: a model for studying the functions of progesterone analogues

Lin

et al. 2001

Br J Cancer

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“…As a control, we performed the same co-IPs using extracts from T47DY cells that express low levels of both PR isoforms and found no significant signal (Supplemental Fig. S4B, lane 6; Horwitz et al 1995).…”

Section: Resultsmentioning

confidence: 99%

Unliganded progesterone receptor-mediated targeting of an RNA-containing repressive complex silences a subset of hormone-inducible genes

Vicent

Nacht²,

Zaurín³

et al. 2013

Genes Dev.

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A close chromatin conformation precludes gene expression in eukaryotic cells. Genes activated by external cues have to overcome this repressive state by locally changing chromatin structure to a more open state. Although much is known about hormonal gene activation, how basal repression of regulated genes is targeted to the correct sites throughout the genome is not well understood. Here we report that in breast cancer cells, the unliganded progesterone receptor (PR) binds genomic sites and targets a repressive complex containing HP1g (heterochromatin protein 1g), LSD1 (lysine-specific demethylase 1), HDAC1/2, CoREST (corepressor for REST [RE1 {neuronal repressor element 1} silencing transcription factor]), KDM5B, and the RNA SRA (steroid receptor RNA activator) to 20% of hormone-inducible genes, keeping these genes silenced prior to hormone treatment. The complex is anchored via binding of HP1g to H3K9me3 (histone H3 tails trimethylated on Lys 9). SRA interacts with PR, HP1g, and LSD1, and its depletion compromises the loading of the repressive complex to target chromatinpromoting aberrant gene derepression. Upon hormonal treatment, the HP1g-LSD1 complex is displaced from these constitutively poorly expressed genes as a result of rapid phosphorylation of histone H3 at Ser 10 mediated by MSK1, which is recruited to the target sites by the activated PR. Displacement of the repressive complex enables the loading of coactivators needed for chromatin remodeling and activation of this set of genes, including genes involved in apoptosis and cell proliferation. These results highlight the importance of the unliganded PR in hormonal regulation of breast cancer cells.

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“…It has thus been speculated that expression of these ER variants may be altered during breast tumorigenesis and progression and may have a role in progression from hormone dependence to independence in breast cancer . This aspect of tumour progression consists of altered oestrogen signalling, the acquisition of resistance to the cytostatic effects of the anti-oestrogen tamoxifen and subsequently in the failure to respond to agents such progestins and probably antiprogestins (RU 486) (Horwitz et al, 1995). The apparent lower relative expression of D6-PR in normal breast samples compared to tumour tissues, as well as in high PR tumours compared to low PR tumours, is therefore of interest, since normal tissue, high PR tumours and low PR tumours may represent steps in tumour progression which correlate with increasing relative D6-PR expression.…”

Section: Discussionmentioning

confidence: 99%

Altered expression of exon 6 deleted progesterone receptor variant mRNA between normal human breast and breast tumour tissues

et al. 1999

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The progesterone receptor (PR) is an important prognostic marker in breast cancer as well as a marker of responsiveness to endocrine therapies. The presence of several exon-deleted PR variant mRNAs in both normal and neoplastic breast samples has recently been reported. Amongst them, a variant mRNA deleted in exon 6 (D6-PR mRNA) that if translated would encode a truncated PR-like protein missing the hormone binding domain and one of the transactivating domains of the wild-type PR protein. In order to determine whether changes in D6-PR variant expression could occur during tumorigenesis, its expression was investigated by reverse transcription and polymerase chain reaction in ten normal reduction mammoplasty samples, nine breast tumours with high PR levels (> 100 fmol mg −1 protein) and eight breast tumours with low PR levels (< 15 fmol mg −1 protein), as determined by ligand binding assay. The relative expression of D6-PR to wild-type PR mRNA was lower ( P < 0.01) in normal than in all tumour breast samples. Moreover, a trend to lower ( P < 0.1) relative D6-PR expression was observed in high PR tumours, compared to low PR tumours. These data suggest that increased expression of D6-PR occurs during tumorigenesis. © 1999 Cancer Research Campaign

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Novel Mechanisms of Antiprogestin Action

Cited by 24 publications

References 49 publications

Demonstration of mixed properties of RU486 in progesterone receptor (PR)-transfected MDA-MB-231 cells: a model for studying the functions of progesterone analogues

Demonstration of mixed properties of RU486 in progesterone receptor (PR)-transfected MDA-MB-231 cells: a model for studying the functions of progesterone analogues

Unliganded progesterone receptor-mediated targeting of an RNA-containing repressive complex silences a subset of hormone-inducible genes

Altered expression of exon 6 deleted progesterone receptor variant mRNA between normal human breast and breast tumour tissues

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