RESUMEN
Previously, two men were cured of HIV-1 through CCR5Δ32 homozygous (CCR5Δ32/Δ32) allogeneic adult stem cell transplant. We report the first remission and possible HIV-1 cure in a mixed-race woman who received a CCR5Δ32/Δ32 haplo-cord transplant (cord blood cells combined with haploidentical stem cells from an adult) to treat acute myeloid leukemia (AML). Peripheral blood chimerism was 100% CCR5Δ32/Δ32 cord blood by week 14 post-transplant and persisted through 4.8 years of follow-up. Immune reconstitution was associated with (1) loss of detectable replication-competent HIV-1 reservoirs, (2) loss of HIV-1-specific immune responses, (3) in vitro resistance to X4 and R5 laboratory variants, including pre-transplant autologous latent reservoir isolates, and (4) 18 months of HIV-1 control with aviremia, off antiretroviral therapy, starting at 37 months post-transplant. CCR5Δ32/Δ32 haplo-cord transplant achieved remission and a possible HIV-1 cure for a person of diverse ancestry, living with HIV-1, who required a stem cell transplant for acute leukemia.
Asunto(s)
Trasplante de Células Madre de Sangre del Cordón Umbilical , Infecciones por VIH , VIH-1 , Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide Aguda , Masculino , Adulto , Femenino , Humanos , Sangre Fetal , Leucemia Mieloide Aguda/terapiaRESUMEN
Estrogen plays a critical role in development and maintenance of female reproductive and mammary tissues, but is also involved in maintenance of cardiovascular, skeletal, and neural function. Although it is widely accepted that the estrogen-occupied receptor mediates its effects by interacting with estrogen response elements (EREs) residing in target genes, a number of estrogen-responsive genes contain no identifiable ERE. To understand how estrogen-responsive genes lacking EREs but containing activator protein 1 (AP-1) and Sp1 sites respond to hormone treatment, we have identified four discrete regions of the human progesterone receptor gene that contain AP-1 or Sp1 sites and examined their abilities to modulate transcription in the presence of 17 beta-estradiol, ICI 182,780, tamoxifen, raloxifene, genistein, or daidzein. Transient cotransfection assays demonstrated that ER alpha was a more potent activator of transcription than ER beta in bone, uterine, and mammary cells. The Sp1-containing promoters were substantially more potent transcriptional enhancers than the AP-1-containing promoters, but a 1.5-kb region of the human progesterone receptor gene containing both AP-1 and Sp1 sites was the most hormone-responsive promoter tested. The ability of ligands to modulate transcription of AP-1- or Sp1-containing promoters was dependent on cell context, but the expression of AP-1 or Sp1 proteins was not necessarily related to transcriptional response. Taken together, these studies have helped to delineate the roles of ER alpha and ER beta in modulating transcription of genes containing AP-1 and Sp1 sites and define the effects of widely used, pharmacologic agents in target cells with distinct cellular environments.
Asunto(s)
Receptor alfa de Estrógeno/genética , Receptor beta de Estrógeno/genética , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/genética , Factor de Transcripción AP-1/genética , Activación Transcripcional , Sitios de Unión/genética , Línea Celular Tumoral , Femenino , Humanos , Ligandos , Especificidad de Órganos , Transcripción GenéticaRESUMEN
The progesterone receptor (PR) gene is regulated by estrogen in normal reproductive tissues and in MCF-7 human breast cancer cells. Although it is generally thought that estrogen responsiveness is mediated by interaction of the ligand-occupied estrogen receptor (ER) with estrogen response elements (EREs) in target genes, the human progesterone receptor (PR) gene lacks a palindromic ERE. Promoter A of the PR gene does, however, contain an ERE half site upstream of two adjacent Sp1 sites from +571 to +595, the +571 ERE/Sp1 site. We have examined the individual contributions of the ERE half site and the two Sp1 sites in regulating estrogen responsiveness. Transient transfection assays demonstrated that both Sp1 sites were critical for estrogen-mediated activation of the PR gene. Interestingly, rather than decreasing transcription, mutations in the ERE half site increased transcription substantially suggesting that this site plays a role in limiting transcription. Chromatin immunoprecipitation assays demonstrated that Sp1 was associated with the +571 ERE/Sp1 site in the endogenous PR gene in the absence and in the presence of estrogen, but that ERalpha was only associated with this region of the PR gene after MCF-7 cells had been treated with estrogen. Our studies provide evidence that effective regulation of transcription through the +571 ERE/Sp1 site requires the binding of ERalpha and Sp1 to their respective cis elements and the appropriate interaction of ERalpha and Sp1 with other coregulatory proteins and transcription factors.
Asunto(s)
Estrógenos/metabolismo , Receptores de Progesterona/genética , Factor de Transcripción Sp1/metabolismo , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Cartilla de ADN , Receptor alfa de Estrógeno , Regulación de la Expresión Génica , Humanos , Ligandos , Plásmidos , Pruebas de Precipitina , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Transcripción GenéticaRESUMEN
The progesterone receptor (PR) gene is activated by estrogen in normal reproductive tissues and in MCF-7 human breast cancer cells. Although it is typically thought that estrogen responsiveness is mediated through estrogen response elements (EREs), the human PR gene lacks a palindromic ERE sequence. We have identified an activating protein-1 (AP-1) site at +745 in the human PR gene that bound purified Fos and Jun and formed a complex with Fos/Jun heterodimers present in MCF-7 nuclear extracts. Surprisingly, mutating the +745 AP-1 site in the context of a 1.5-kb region of the PR gene significantly enhanced estrogen receptor (ER) alpha-mediated transactivation, suggesting that the wild-type +745 AP-1 site plays a role in inhibiting PR gene expression in the presence of hormone. In support of this idea, transient transfection assays demonstrated that increasing levels of Fos and Jun repressed transcription of a reporter plasmid containing the +745 AP-1 site. Fos levels were transiently increased, ERalpha levels were decreased, and Jun was dephosphorylated after MCF-7 cells were treated with estrogen. Chromatin immunoprecipitation assays demonstrated that Jun was associated with the +745 AP-1 site in the endogenous PR gene in the presence and in the absence of estrogen, but that ERalpha and Fos were only associated with the +745 AP-1 site after estrogen treatment of MCF-7 cells. Our studies suggest that the human PR gene is regulated by multiple transcription factors and that the differential binding of these dynamically regulated trans-acting factors influences gene expression.
Asunto(s)
Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Receptores de Progesterona/genética , Factor de Transcripción AP-1/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Secuencia Conservada , Estradiol/farmacología , Receptor alfa de Estrógeno , Estrógenos/farmacología , Regulación de la Expresión Génica , Humanos , Ratones , Mutación , Fosforilación , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/aislamiento & purificación , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/aislamiento & purificación , Conejos , Ratas , Receptores de Estrógenos/efectos de los fármacos , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/efectos de los fármacos , Receptores de Progesterona/metabolismo , Elementos de Respuesta , Transcripción GenéticaRESUMEN
Estrogens exert their regulatory transcriptional effects, which can be stimulatory or repressive, at diverse gene sites via two estrogen receptors, ERalpha and ERbeta. Since these two ERs have different tissue distributions, ligands that have the capacity to selectively activate or inhibit these two ERs would be useful in elucidating the biology of these two receptors and might assist in the development of estrogen pharmaceuticals with improved tissue selectivity. We have developed several ligands that showed ERalpha or ERbeta selectivity at promoter-gene sites containing consensus estrogen response elements (EREs): ERalpha-selective agonist (propyl-pyrazole-triol (PPT)), ERalpha-selective antagonist (methyl-piperidino-pyrazole (MPP)), ERbeta-potency selective agonist (diarylpropionitrile (DPN)) and ERbeta-selective antagonist/ERalpha-agonist (R,R-tetrahydrochrysene (R,R-THC)). In this study, we have examined the activity of these compounds at a range of gene sites where ER stimulates gene expression through non-consensus EREs (complement C3), or multiple half-EREs (NHE-RF/EBP50), or by tethering to DNA via other proteins (TGF beta3 and progesterone receptor A/AP-1), and at gene sites where ER represses gene transcription (interleukin-6). At all of these genes, PPT showed full stimulation through ERalpha while displaying no agonism through ERbeta. MPP antagonized estradiol actions on gene transactivation and transrepression through ERalpha, with little or no effect on transcription mediated through ERbeta. DPN displayed subtype-selective agonism, being ca. 30-fold more potent through ERbeta. R,R-THC was a complete antagonist through ERbeta and displayed agonism through ERalpha, the level of which was promoter dependent. Because these ligands maintain their agonist or antagonist character and ER subtype-selectivity at gene sites of diverse nature, where estradiol is either stimulatory or inhibitory, these compounds should prove useful in elucidating the biological functions of ERalpha and ERbeta.
Asunto(s)
Regulación hacia Abajo/efectos de los fármacos , Receptores de Estrógenos/agonistas , Receptores de Estrógenos/antagonistas & inhibidores , Elementos de Respuesta , Activación Transcripcional/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Estradiol/agonistas , Antagonistas de Estrógenos , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Compuestos Heterocíclicos/farmacología , Humanos , Ligandos , Regiones Promotoras Genéticas/genética , Relación Estructura-Actividad , TransfecciónRESUMEN
The progesterone receptor (PR) gene is induced by estrogen in reproductive and mammary tissues and in MCF-7 human breast cancer cells even though the human PR gene lacks an estrogen response element. We have identified a region from -80 to -34 in the PR gene that contains two Sp1 sites and confers estrogen responsiveness to a heterologous promoter in an estrogen and estrogen receptoralpha (ERalpha)-dependent manner. Sp1 present in MCF-7 nuclear extracts and purified Sp1 bind to and protect both Sp1 sites from DNase I cleavage, but the proximal Sp1 site is preferentially protected. Mutation of either Sp1 site decreases Sp1-DNA complex formation and ERalpha-mediated transactivation. ERalpha enhances Sp1 binding, but does not interact directly with the -80/-34 region. Our studies suggest that ERalpha confers estrogen responsiveness to the PR gene by enhancing Sp1 interaction with the Sp1 site in the -80/-34 region of the human PR gene.
Asunto(s)
Neoplasias de la Mama/genética , Regulación de la Expresión Génica , Receptores de Estrógenos/fisiología , Receptores de Progesterona/genética , Elementos de Respuesta/fisiología , Factor de Transcripción Sp1/fisiología , Sitios de Unión , Neoplasias de la Mama/metabolismo , Huella de ADN , Receptor alfa de Estrógeno , Estrógenos/metabolismo , Genes Reporteros , Humanos , Mutación , Regiones Promotoras Genéticas , Receptores de Progesterona/metabolismo , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
The progesterone receptor (PR) gene is activated by estrogen in MCF-7 human breast cancer cells. Although the human PR gene does not contain an estrogen response element (ERE), we have identified a putative activating protein-1 (AP-1) site at +90 in the PR gene that was hypersensitive to deoxyribonuclease I cleavage in genomic Southern analysis, bound purified Fos and Jun, formed a complex with Fos/Jun heterodimers present in MCF-7 nuclear extracts in gel mobility shift assays, and functioned as an estrogen-responsive enhancer in transient cotransfection assays. When the +90 AP-1 site was mutated in the context of the PR gene, estrogen responsiveness was significantly decreased. Purified estrogen receptor (ER) enhanced binding of Fos and Jun to the +90 AP-1 site and bound to an adjacent imperfect ERE half-site. Mutating this ERE half-site diminished the binding of ER, Fos, and Jun and decreased transcription. Chromatin immunoprecipitation assays demonstrated that the ER, Fos, and Jun were present at the +90 AP-1 site in the endogenous PR gene only after treatment of MCF-7 cells with estrogen. These studies suggest that the cooperative interaction of the ER with Fos and Jun proteins helps confer estrogen responsiveness to the endogenous PR gene.
Asunto(s)
Neoplasias de la Mama/metabolismo , Estrógenos/farmacología , Receptores de Estrógenos/fisiología , Receptores de Progesterona/genética , Elementos de Respuesta , Factor de Transcripción AP-1/fisiología , Sitios de Unión , Southern Blotting , Núcleo Celular/química , ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Estradiol/farmacología , Receptor alfa de Estrógeno , Humanos , Mutagénesis , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Transcripción AP-1/genética , Transfección , Células Tumorales CultivadasRESUMEN
Estrogen receptor (ER) activity can be modulated by the action of other nuclear receptors. To study whether ER activity is altered by orphan nuclear receptors that mediate the cellular response to xenobiotics, cross-talk between ER and constitutive androstane receptor (CAR), steroid and xenobiotic receptor, or peroxisome proliferator-activated receptor gamma was examined in HepG2 cells. Of these receptors, CAR substantially inhibited ER-mediated transcriptional activity of the vitellogenin B1 promoter as well as a synthetic estrogen responsive element (ERE)-containing promoter. Treatment with an agonist of CAR, 1,4-bis-(2-(3,5-dichloropyridoxyl))benzene, potentiated CAR-mediated transcriptional repression. In contrast, an antagonist of CAR, androstenol, alleviated the repression effect. Although CAR interacted with the ER in solution, CAR did not interact with the ER bound to the ERE. CAR/retinoid X receptor bound to the ERE but with much lower affinity than ER. Incremental amounts of CAR elicited a progressive reduction of the ER activity induced by the p160 coactivator glucocorticoid receptor interacting protein 1 (GRIP-1). In turn, increasing amounts of GRIP-1 progressively reversed the depression of ER activity by CAR. An agonist or antagonist of CAR potentiated or alleviated, respectively, the CAR-mediated repression of the GRIP-1-enhanced ER activity, which is consistent with the ability of theses ligands to increase or decrease, respectively, the interaction of CAR with GRIP-1. A CAR mutant that did not interact with GRIP-1 did not inhibit ER-mediated transactivation. Our data demonstrate that xenobiotic nuclear receptor CAR antagonizes ER-mediated transcriptional activity by squelching limiting amounts of p160 coactivator and imply that xenobiotics may influence ER function of female reproductive physiology, cell differentiation, tumorigenesis, and lipid metabolism.