ABSTRACT
Os antígenos cancer-testis (CT) são proteínas imunogênicas expressas em tecido gametogênico e em diferentes tipos de tumor, sendo considerados candidatos promissores para a imunoterapia do câncer. Entretanto, pouco se sabe sobre a função desses antígenos na tumorigênese. Em 2006, identificamos CTSP-1 como um novo antígeno CT, frequentemente expresso em vários tumores. Nesse trabalho, investigamos a função de CTSP-1 por meio da identificação de proteínas expressas em tumores de próstata e que são capazes de interagir fisicamente com esse antígeno. Demonstramos que CTSP-1 interage com a proteína CTCF em ensaios de duplo-híbrido em leveduras, pulldown e de co-localização e, em seguida, analisamos o impacto da superexpressão de CTSP-1 no controle da expressão de genes CT mediada por CTCF e na progressão do ciclo celular. Utilizando o CT NY-ESO-1 como modelo, demonstramos que a superexpressão de CTSP-1 não altera os níveis endógenos de NY-ESO-1 na linhagem celular tumoral H1299. Por outro lado, observamos que a superexpressão de CTSP-1 48h após as transfecções em H1299 induz um bloqueio do ciclo em G0/G1, reduzindo a capacidade clonogênica dessas células por um mecanismo dependente dos níveis de expressão de CTSP-1. Resultados semelhantes não foram observados em ensaios com clones superexpressando CTSP-1 estavelmente, o que sugere que eles tenham se originado de células que conseguiram escapar do bloqueio em G0/G1. Resultados preliminares sugerem que a redução da capacidade clonogênica das células H1299 que superexpressam CTSP-1 48h após as tansfecções não está associada à ocorrência de morte por apoptose
Cancer-testis (CT) antigens are immunogenic proteins expressed in gametogenic tissues and in different histological types of tumors, being considered promising candidates for cancer immunotherapy. However, little is known about their role in tumorigenesis. In 2006, we identified CTSP-1 as a novel CT antigen, frequently expressed in different types of tumors. In this work, we investigated the functional role of CTSP-1 through the identification of proteins expressed in prostate tumors and that physically interact with this tumor antigen. We demonstrate that CTSP-1 interacts with the CTCF protein using the yeast two-hybrid system, pulldown and co-localization assays and have further analyzed the impact of CTSP-1 overexpression on the expression of CT genes mediated by CTCF and on the cell cycle progression. Using the CT antigen NY-ESO-1 as a model, we showed that the CTSP-1 overexpression does not alter the endogenous levels of NY-ESO-1 in the tumor cell line H1299. On the other hand, we observed that the overexpression of CTSP-1 in H1299 cells 48h after the transfections induces a cell cycle arrest in G0/G1 and reduces the clonogenic capacity of these cells by a mechanism dependent on the CTSP-1 expression levels. Similar results were not observed for cell clones stably overexpressing CTSP-1, suggesting that these clones have arisen from cells that managed to escape cell cycle arrest in G0/G1. Preliminary results suggest that the reduced clonogenic capacity of H1299 cells expressing CTSP-1 and analyzed 48h after the transfections is not associated with cell death by apoptosis
Subject(s)
Testicular Neoplasms/pathology , Antigens, Tumor-Associated, Carbohydrate , Apoptosis/physiology , Colony-Forming Units Assay/methods , Two-Hybrid System Techniques/instrumentationABSTRACT
Several major vascular tissues, such as the aorta-gonad-mesonephros región (AGM), yolk sac, and fetal liver have been confirmed to possess hematopoietic function. Recently, the placenta has been demonstrated as another hematopoietic organ. However, it is not conclusive whether the placenta possesses hematopoietic ability. Therefore, we undertook a series of experiments to study the hematopoietic functions of placenta. Fetal blood circulation in the placenta is difficult to be eliminated and its interference in the study of placental hematopoiesis is inevitable. With the application of placental flushing, fetal blood contained in the placenta was eliminated. We then made the further study of placental hematopoiesis after the El2.5 placenta was flushed. Our studies showed that placental cells expressing Sca-1, CD117 and CD34 were mainly restricted to the embryonic vessels of E12.5 placenta. The results of fluorescence activated cell sorter (FACs) analysis and colony forming cells (CFC) assay demonstrated that both placenta and placental blood contained hematopoietic stem/progenitor cells (HS/PCs), including CFU-GMs, CFU-GEMMs, BFU-Es, and HPP-CFCs. The frequency of HS/PCs in the placenta was 2-3 times that of placental blood. Therefore, it is necessary to clear placental blood out of the placenta in the studies of the hematopoietic potential of placenta. The placenta still possessed the hematopoietic potential after the fetal blood is flushed out. These observations provide further evidences that the placenta is a hematopoietic organ, as has been proposed for other embryonic hematopoietic sites.
Subject(s)
Animals , Female , Mice , Pregnancy , Cell Separation/methods , Colony-Forming Units Assay/methods , Fetal Blood/cytology , Hematopoietic Stem Cells/cytology , Placenta/cytologyABSTRACT
The total number of CD34+ cells is the most relevant clinical parameter when selecting human umbilical cord blood (HUCB) for transplantation. The objective of the present study was to compare the two most commonly used CD34+ cell quantification methods (ISHAGE protocol and ProCount - BD) and analyze the CD34+ bright cells whose 7-amino actinomycin D (7AAD) analysis suggests are apoptotic or dead cells. Twenty-six HUCB samples obtained at the Placental Blood Program of New York Blood Center were evaluated. The absolute numbers of CD34+ cells evaluated by the ISHAGE (with exclusion of 7AAD+ cells) and ProCount (with exclusion of CD34+ bright cells) were determined. Using the ISHAGE protocol we found 35.6 ± 19.4 CD34+ cells/æL and with the ProCount method we found 36.6 ± 23.2 CD34+ cells/æL. With the ProCount method, CD34+ bright cell counts were 9.3 ± 8.2 cells/æL. CD34+ bright and regular cells were individually analyzed by the ISHAGE protocol. Only about 1.8 percent of the bright CD34+ cells are alive, whereas a small part (19.0 percent) is undergoing apoptosis and most of them (79.2 percent) are dead cells. Our study showed that the two methods produced similar results and that 7AAD is important to exclude CD34 bright cells. These results will be of value to assist in the correct counting of CD34+ cells and to choose the best HUCB unit for transplantation, i.e., the unit with the greatest number of potentially viable stem cells for the reconstitution of bone marrow. This increases the likelihood of success of the transplant and, therefore, the survival of the patient.
Subject(s)
Humans , /blood , Blood Cell Count/methods , Colony-Forming Units Assay/methods , Fetal Blood/cytology , Hematopoietic Stem Cells/cytology , Blood Banks , Cell Survival , Dactinomycin/analogs & derivatives , Flow Cytometry , Fluorescent Dyes , Reproducibility of ResultsABSTRACT
A detecçäo de progenitores hemopoiéticos da linhagem eritróide, BFU-E e CFU-E, envolveu a cultura de células mononucleares hemopoiéticas obtidas de medula óssea, sangue periférico e sangue de cordäo umbilical em meio de IMDM, suplementado com antibióticos, soro bovino fetal 25 porcento, soroalbumina bovina 1 porcento, meio condicionado da linhagem celular 5637 (como fonte exogena de fatores de crescimento) 1 porcento e adiçäo de eritropoietina humana recombinante em matriz semi-sólida (metilcelulose). Usando-se a técnica, populaçöes distintas de células progenitoras de linhagem eritróide puderam ser detectadas e definidas em sua linhagem pela capacidade de orientar colônias de aspecto característico, após um período de incubaçäo de 14-20 dias, à 37§C, em atmosfera úmida, com 5-19 porcento de CO2. Os tipos de colônias de BFU-E e CFU-E observados foram de tamanho variado, apresentado coloraçäo vermelha em tons variáveis. Células hemopoiéticas constituintes da linhagem eritróide, em diferentes graus de maturaçäo, foram morfologicamente identificadas após coloraçäo panóptica. A frequência de colônias de BFU-E e CFU-E observada, em nossos estudos foram, respectivamente: 33 mais ou menos 4,4 e 30,3 mais ou menos 5,4 para medula óssea; 12,7 mais ou menos 4,3 e 9,3 e 9,3 mais ou menos 1,5 para sangue periférico; 26,6 mais ou menos 8,7 e 25,6 mais ou menos 11 para sangue de cordäo umbilical