RESUMEN
The microtubule-dependent molecular motor KIF23 (Kinesin family member 23) is one of two components of the centralspindlin complex assembled during late stages of mitosis. Formation of this complex is known as an essential step for cytokinesis. Here, we identified KIF23 as a new transcriptional target gene of the tumor suppressor protein p53. We showed that p53 reduces expression of KIF23 on the mRNA as well as the protein level in different cell types. Promoter reporter assays revealed that this repression results from downregulation of KIF23 promoter activity. CDK inhibitor p21(WAF1/CIP1) was shown to be necessary to mediate p53-dependent repression. Furthermore, we identified the highly conserved cell cycle genes homology region (CHR) in the KIF23 promoter to be strictly required for p53-dependent repression as well as for cell cycle-dependent expression of KIF23. Cell cycle- and p53-dependent regulation of KIF23 appeared to be controlled by differential binding of DREAM and MMB complexes to the CHR element. With this study, we describe a new mechanism for transcriptional regulation of KIF23. Considering the strongly supporting function of KIF23 in cytokinesis, its p53-dependent repression may contribute to the prevention of uncontrolled cell growth.
Asunto(s)
Silenciador del Gen , Proteínas de Interacción con los Canales Kv/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Oncogénicas v-myb/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Ciclo Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación de la Expresión Génica , Células HCT116 , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Células 3T3 NIH , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidad Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Elementos de Respuesta , Transcripción GenéticaRESUMEN
Successful pregnancy requires a functionally normal blastocyst encountering a receptive maternal endometrium. Interestingly, the cell cycle regulator and tumor suppressor p53 has been reported to support reproduction in mice by regulating the expression of the leukemia inhibitory factor gene in the maternal endometrium. However, in humans the hormonal system orchestrating successful pregnancy is considerably different from rodents. Particularly, the primate-specific dimeric glycoprotein hormone human chorionic gonadotropin (hCG) is essential for blastocyst implantation and maintenance of early human pregnancy. Here we provide evidence that p53 selectively induces expression of the hCGbeta7 (CGB7) gene. None of the other CGB genes was found to be regulated by p53. We show that expression of the CGB7 gene is upregulated upon p53 induction in human HFF, HCT116 and DLD1 cells as well as in cell preparations enriched in human primary first-trimester trophoblasts. The increase in CGB7 levels upon doxorubicin treatment is lost after siRNA-directed knockdown of p53. Furthermore, we describe CGB7 as a direct transcriptional target gene of p53 by identifying a p53-responsive element in the CGB7 promoter using reporter assays, electrophoretic mobility shift assays and chromatin immunoprecipitations. With these results we provide a new link between p53 transcriptional activity and human reproduction.
Asunto(s)
Gonadotropina Coriónica Humana de Subunidad beta/genética , Proteína p53 Supresora de Tumor/fisiología , Secuencia de Bases , Sitios de Unión , Gonadotropina Coriónica Humana de Subunidad beta/metabolismo , Doxorrubicina/farmacología , Femenino , Humanos , Datos de Secuencia Molecular , Embarazo , Interferencia de ARN , Elementos de Respuesta , Alineación de Secuencia , Trofoblastos/metabolismo , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Regulación hacia ArribaRESUMEN
The objective of this study was to evaluate the role of the fimbriated end and nonfimbriated epithelium of fallopian tubes with regard to p53 signature, tubal intraepithelial lesions in transition (TILT), and serous tubal in-situ carcinoma (STIC) in cases of different kinds of serous pelvic cancer. This study immunohistochemically evaluated (by Ki-67 and p53 staining) the presence of p53 signature, TILT lesions, and STIC in 14 consecutive cases of prophylactic salpingo-oophorectomy in women with BRCA-1/2 mutation (bilateral salpingo-oophorectomy), 11 cases of macroscopically inconspicuous adnexae of patients with primary contralateral tubal cancer (TC), 9 cases of primary peritoneal cancer (PPC), and 10 cases of serous ovarian borderline tumors, evaluating the fallopian tubes (using the Sectioning and Extensively Examining the FIMbria protocol), ovarian surface epithelium, and ovarian cortical inclusion cysts. The frequencies of p53 signature, TILT, and STIC were 35.7%, 7.1%, and 0% in cases of prophylactic surgery, 18.2%, 9.1%, and 18.2% in TC, and 11.1%, 0%, and 33.3% in PPC. These precursor lesions were missed during the initial routine screening and were found in the fimbriated end of the fallopian tubes in 94%. In 1 case of PPC, staining for p53 was negative in STIC. The studied adnexal tissue of serous ovarian borderline tumor and ovarian cortical inclusion cysts of all cases showed no alterations according to p53 signature, TILT, or STIC. STIC and p53 signature as precursor lesions of pelvic serous cancer were seen in macroscopically inconspicuous contralateral fallopian tubes in unilateral TC, in patients with elective bilateral salpingo-oophorectomy, and in patients affected by PPC. Therefore, we propose the complete processing of adnexal tissue and the use of step sectioning to establish the correct diagnosis. Immunohistochemistry for p53 and ki-67 may aid in the diagnosis, but is not necessary for routine investigation.
Asunto(s)
Carcinoma in Situ/patología , Cistadenocarcinoma Seroso/patología , Neoplasias de las Trompas Uterinas/patología , Neoplasias Ováricas/patología , Neoplasias Peritoneales/patología , Proteína p53 Supresora de Tumor/biosíntesis , Adulto , Carcinoma in Situ/genética , Carcinoma in Situ/metabolismo , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Neoplasias de las Trompas Uterinas/genética , Neoplasias de las Trompas Uterinas/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Antígeno Ki-67/biosíntesis , Persona de Mediana Edad , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Peritoneales/genética , Neoplasias Peritoneales/metabolismo , Lesiones Precancerosas/genética , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Regulatory T cells (Treg) expand during pregnancy and are present at the fetal-maternal interface at very early stages in pregnancy. The migration mechanisms of Treg to the pregnant uterus are still unclear. Human chorionic gonadotropin (hCG) is secreted by the blastocyst immediately after fertilization and has chemoattractant properties. Therefore, we sought to analyze whether hCG secreted by early trophoblasts attracts Treg to the uterus and hence contributes to maternal tolerance toward the fetus. Decidua and placenta tissue samples from patients having spontaneous abortions or ectopic pregnancies were employed to evaluate Treg and hCG levels. Age-matched samples from normal pregnant women served as controls. We further performed in vitro studies with primary first trimester trophoblast cells and a choriocarcinoma cell line (JEG-3) aiming to evaluate the ability of secreted hCG to attract Treg. Patients having miscarriages or ectopic pregnancy presented significantly decreased hCG mRNA and protein levels associated with decreased Foxp3, neuropilin-1, IL-10, and TGF-beta mRNA levels as compared with normal pregnant women. Using migration assays we demonstrated that Treg were attracted by hCG-producing trophoblasts or choriocarcinoma cells. Treg migration toward cells transfected with hCG expression vectors confirmed the chemoattractant ability of hCG. Our data clearly show that hCG produced by trophoblasts attracts Treg to the fetal-maternal interface. High hCG levels at very early pregnancy stages ensure Treg to migrate to the site of contact between paternal Ags and maternal immune cells and to orchestrate immune tolerance toward the fetus.
Asunto(s)
Movimiento Celular/inmunología , Gonadotropina Coriónica/fisiología , Intercambio Materno-Fetal/inmunología , Primer Trimestre del Embarazo , Segundo Trimestre del Embarazo , Linfocitos T Reguladores/inmunología , Aborto Espontáneo/inmunología , Aborto Espontáneo/metabolismo , Adulto , Línea Celular Tumoral , Células Cultivadas , Gonadotropina Coriónica/deficiencia , Gonadotropina Coriónica/genética , Técnicas de Cocultivo , Femenino , Células HCT116 , Humanos , Embarazo , Primer Trimestre del Embarazo/inmunología , Segundo Trimestre del Embarazo/inmunología , Embarazo Ectópico/inmunología , Embarazo Ectópico/metabolismo , Linfocitos T Reguladores/citologíaRESUMEN
The receptor for hyaluronan-mediated motility RHAMM exerts different functions in the cell as well as on the cell membrane. RHAMM can be exported to the cell surface where it binds hyaluronic acid (HA) and interacts with the HA receptor CD44. Processes like cell motility, wound healing and invasion are modulated by RHAMM. Intracellularly, RHAMM is associated with the cytoskeleton, microtubules, centrosomes and the mitotic spindle. It participates in the control of mitotic spindle stability and integrity. Furthermore, RHAMM is overexpressed in several cancer tissues. We found that RHAMM expression is differentially regulated during the cell cycle and that cell cycle-dependent synthesis of RHAMM is controlled by its promoter on the transcriptional level. RHAMM protein levels follow mRNA expression in the early phases of the cell cycle. However, they already peak in S phase and decrease before the maximum of RHAMM mRNA expression is reached in G(2)/M. Furthermore, RHAMM expression is downregulated by the tumor suppressor p53. This regulation is observed in a transgenic p53-inducible cell system as well as in cells with wild-type p53 treated with nutlin-3, doxorubicin or paclitaxel. Reporter assays demonstrated that the repression by p53 is regulated on the transcriptional level by the RHAMM promoter also comprising the first exon and the first intron of the gene. In general, our data support a role of RHAMM already in S phase. Additionally, p53-dependent downregulation is consistent with an oncogenic function of RHAMM and the recently reported tumor-suppressive function of CD44 transcriptional repression by p53.