RESUMO
The Wilms tumor gene (WT1) encodes a zinc-finger containing transcription factor present in primitive hematopoietic progenitor cells. WT1 is also highly expressed in most cases of acute myeloid leukemia. Moreover, WT1 can interfere with induced differentiation of leukemic cell lines. These data suggest a function of WT1 in the maintenance of a primitive phenotype and a role in leukemogenesis by interfering with differentiation, prompting us to investigate its function in human hematopoietic progenitor cells. By retroviral transfer, human CD34(+) cord blood progenitor cells were transduced with a vector encoding either of two splicing variants of WT1, with or without the KTS insert in the zinc-finger domain, linked to expression of green fluorescent protein (GFP) via an internal ribosomal entry site. When compared to cells transduced with vector containing GFP only, WT1 expressing cells showed strongly reduced colony formation in methylcellulose and inhibited proliferation in suspension culture, with no apparent reduction in viability. Cell cycle phase distribution was not affected by WT1 expression. No signs of impaired differentiation, as judged by the surface markers CD11b, CD14 and glycophorin were detected. In contrast to the results with human CD34(+) progenitor cells, the proliferation of murine bone marrow cells was not significantly affected by WT1, consistent with previous data. We conclude that forced expression of WT1 in highly enriched human hematopoietic progenitor cells leads to strong anti-proliferative effects but is compatible with induced maturation of these cells.
Assuntos
Antígenos CD34/sangue , Divisão Celular/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Proteínas WT1/genética , Animais , Técnicas de Cultura de Células , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Sangue Fetal/citologia , Expressão Gênica/fisiologia , Vetores Genéticos , Humanos , Camundongos , Células Progenitoras Mieloides/citologia , Células Progenitoras Mieloides/efeitos dos fármacos , Retroviridae/genética , Transdução GenéticaRESUMO
The p53 tumor suppressor protein can induce both apoptosis and cell cycle arrest. Moreover, we and others have shown previously that p53 is a potent mediator of differentiation. For example, expression of ptsp53, a temperature-inducible form of p53, induces differentiation of leukemic monoblastic U-937 cells. The functions of p53 have for long been believed to be dependent on the transactivating capacity of p53. However, recent data show that both p53-induced cell cycle arrest and apoptosis can be induced independently of p53-mediated transcriptional activation, indicating alternative pathways for p53-induced apoptosis and cell cycle arrest. The bcl-2 proto-oncogene contributes to the development of certain malignancies, probably by inhibition of apoptosis. Interestingly, Bcl-2 has been shown to inhibit p53-mediated apoptosis as well as p53-mediated transcriptional activation. Asking whether Bcl-2 would interfere with the p53-mediated differentiation of U-937 cells, we stably transfected bcl-2 to U-937 cells inducibly expressing p53. Although the established Bcl-2-expressing clones were resistant to p53-mediated apoptosis, we did not observe any interference of Bcl-2 with the p53-mediated differentiation, suggesting separable pathways for p53 in mediating apoptosis and differentiation of U-937 cells. Neither did expression of Bcl-2 interfere with p53-induced expression of endogenous p21, suggesting that p53-induced differentiation might be dependent on the transcriptional activity of p53. To further investigate whether the p53-mediated differentiation of U-937 cells depends on the transcriptional activity of p53, we overexpressed transactivation-deficient p53, a transcriptionally inactive p53 mutant in these cells. However, in contrast to the effects of wild-type p53, expression of trans-activation-deficient p53 did neither induce signs of apoptosis nor of differentiation in U-937 cells. Our results indicate that the transcriptional activity of p53 is essential both for p53-mediated apoptosis and differentiation of U-937 cells.
Assuntos
Apoptose , Ciclo Celular , Diferenciação Celular , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular , Colecalciferol/farmacologia , Células Clonais/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Ciclinas/metabolismo , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Integrina alfaXbeta2/metabolismo , Cinética , Mutação/genética , Regiões Promotoras Genéticas/genética , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Temperatura , Ativação Transcricional , Transfecção , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Células U937RESUMO
Most often the investment in electronic patient records (EPR) is decided upon on the basis of descriptions of all the advantages this new technology will bring. We argue that an exchange of a paper-based patient record (PPR) with an EPR is an efficient way to automate manual routine work and distribute information but the real benefits from EPR depend on how clinicians use an EPR application to improve the quality of care. We introduce the 'price to pay' concept in order to illustrate that it takes a human effort to create better health care and that clinicians therefore play a very important role when implementing EPR systems.
Assuntos
Alfabetização Digital , Sistemas Computadorizados de Registros Médicos , Motivação , Humanos , Automação de Escritório , Inovação Organizacional , Terminologia como AssuntoRESUMO
The identification of novel tumour-associated antigens (TAAs) is pivotal for progression in the fields of tumour immunotherapy and diagnosis. In the present study, we have developed, based on flow cytometric evaluation and use of a mini-library composed of specific antibody clones linked to different antibiotic resistance markers, methods for positive and subtractive selection of phage antibodies employing intact cells as the antigen source. An scFv phage library (2.7 x 10(7)) was constructed from a primate (Macaca fascicularis) immunised with pooled human colon carcinomas. This library was selected for 3 rounds by binding to Colo 205 colon adenocarcinoma cells and proteolytic elution followed by phage amplification. Several antibodies reactive with colon carcinomas and with restricted reactivity to a few epithelial normal tissues were identified by immunohistochemistry. One clone, A3 scFv, recognised an epitope that was homogeneously expressed in 11/11 of colon and 4/4 pancreatic carcinomas studied and in normal tissue restricted to subtypes of epithelia in the gastrointestinal tract. The A3 scFv had an apparent overall affinity approximately 100-fold higher than an A3 Fab, suggesting binding of scFv homodimers. The cell surface density of the A3 epitope, calculated on the basis of Fab binding, was exceptionally high, approaching 3 million per cell. We also demonstrate efficient T-cell-mediated killing of colon cancer cells coated with A3 scFv fused to the low MHC class II binding superantigen mutant SEA(D227A). The identified A3 molecule thus represents a TAA with properties that suggest its use for immunotherapy of colon and pancreatic cancer.
Assuntos
Anticorpos Antineoplásicos/imunologia , Neoplasias do Colo/imunologia , Neoplasias Pancreáticas/imunologia , Biblioteca de Peptídeos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/biossíntese , Anticorpos Antineoplásicos/genética , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/imunologia , Bacteriófagos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/terapia , Sistema Digestório/imunologia , Epitélio/imunologia , Citometria de Fluxo , Humanos , Fragmentos de Imunoglobulinas/imunologia , Imunoterapia , Macaca fascicularis , Dados de Sequência Molecular , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/terapia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Superantígenos/genética , Superantígenos/imunologia , Linfócitos T/imunologia , Células Tumorais CultivadasRESUMO
The tumor suppressor gene p53 can mediate both apoptosis and cell cycle arrest. In addition, p53 also influences differentiation. To further characterize the differentiation inducing properties of p53, we overexpressed a temperature-inducible p53 mutant (ptsp53Val135) in the erythroleukemia cell line K562. The results show that wild-type p53 and hemin synergistically induce erythroid differentiation of K562 cells, indicating that p53 plays a role in the molecular regulation of differentiation. However, wild-type p53 did not affect phorbol 12-myristate 13-acetate-dependent appearance of the megakaryocyte-related cell surface antigens CD9 and CD61, suggesting that p53 does not generally affect phenotypic modulation. The cyclin-dependent kinase inhibitor p21, a transcriptional target of p53, halts the cell cycle in G1 and has also been implicated in the regulation of differentiation and apoptosis. However, transiently overexpressed p21 did neither induce differentiation nor affect the cell cycle distribution or viability of K562 cells, suggesting that targets downstream of p53 other than p21 are critical for the p53-mediated differentiation response.
Assuntos
Glicoproteínas de Membrana , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Antígenos CD/metabolismo , Benzidinas/metabolismo , Western Blotting , Ciclo Celular , Morte Celular , Diferenciação Celular , Separação Celular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Citometria de Fluxo , Vetores Genéticos , Hemina/metabolismo , Hemina/farmacologia , Hemoglobinas/metabolismo , Humanos , Integrina beta3 , Células K562 , Camundongos , Mutagênese , Fenótipo , Fosforilação , Glicoproteínas da Membrana de Plaquetas/metabolismo , Testes de Precipitina , Temperatura , Acetato de Tetradecanoilforbol/farmacologia , Tetraspanina 29 , Fatores de Tempo , TransfecçãoRESUMO
The Wilms' tumor gene (WT1) encodes a transcription factor of the zinc finger type. A high expression of WT1 has been detected in a range of acute leukemias, and WT1 is downregulated during induced differentiation of some leukemic cell lines. Overexpression of WT1 in some myeloid cell lines confers resistance to differentiation induction. These observations suggest that a high WT1 expression in hematopoietic cells is incompatible with differentiation. In this study, each of the four different isoforms of WT1 was constitutively overexpressed in the leukemic cell line K562. K562 cells express endogenous WT1, which is downregulated as a response to induced differentiation along the erythroid and megakaryocytic pathways. We now demonstrate that a forced exogenous expression of the four different isoforms of WT1 in K562 does not affect the differentiation response, as judged by accumulation of hemoglobin in response to hemin or the expression of megakaryocytic cell surface markers in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). We conclude that downregulation of WT1 during induced differentiation of K562 cells is not a prerequisite for erythroid or megakaryocytic differentiation of these cells.
Assuntos
Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Células Precursoras Eritroides/patologia , Regulação Neoplásica da Expressão Gênica , Leucemia/patologia , Megacariócitos/patologia , Fatores de Transcrição/genética , Diferenciação Celular/efeitos dos fármacos , Expressão Gênica , Hemina/farmacologia , Humanos , Células K562 , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Proteínas WT1RESUMO
Most often the investment in Electronic Patient Records (EPR) is decided upon on the basis of descriptions of all the advantages this new technology will bring. However, most of these advantages from EPR's are only obtainable if the clinicians are motivated to invest their knowledge and resources in this implementation process. This paper proposes a simple method to qualify the decision making process by introducing the "price to pay" concept.
Assuntos
Alfabetização Digital , Sistemas Computadorizados de Registros Médicos , Motivação , Humanos , Automação de Escritório , Estatística como AssuntoRESUMO
The Wilms tumor gene, WT1, encodes a zinc-finger DNA binding protein which is thought to function as a tissue specific transcription factor, regulating cell growth and differentiation. High expression of WT1 has been detected in a range of acute leukemias. To elucidate a role for WT1 in leukemogenesis, we transfected the monoblastic cell line U937, which lacks detectable levels of endogenous WT1, with two isoforms of WT1. We showed that, in contrast to U937 control cells, cells constitutively expressing either of the isoforms, WT1(-KTS) or WT1(+KTS), did not respond to differentiation induction by retinoic acid or vitamin D3, as judged by the capacity to reduce nitro blue tetrazolium and morphology. Although U937 cells expressing WT1 were hampered in their ability to differentiate on incubation with retinoic acid and vitamin D3, the induced G1/G0-accumulation was similar to differentiating control cells treated with inducers. Furthermore, distinct effects on the maturation process were indicated by downregulation of the myeloid cell surface makers CD13 and CD15, while the upregulation of CD14 and CD11c on WT1 transfected cells was similar to control cells upon incubation with retinoic acid and vitamin D3. Taken together our results demonstrate that a constitutive expression of WT1 in the leukemic cell line U937 leads to impairment of differentiation responses, indicating that a high expression of WT1 can contribute to the differentiation block of acute leukemia.
Assuntos
Genes do Tumor de Wilms , Leucemia/genética , Monócitos/citologia , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular , Linhagem Celular , Sobrevivência Celular , Colecalciferol/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Humanos , Integrina alfaXbeta2/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Transfecção , Tretinoína/farmacologiaRESUMO
Estramustine-binding protein (EMBP) is a M(r) 46,000 heterodimeric protein originally isolated from prostatic tissue. It has a demonstrated high affinity for, and selective binding of, estramustine, which is a derivative of 17 beta-estradiol and nornitrogen mustard with antimitotic activity. In this study, we have analysed the expression of an EMBP-like protein in astrocytoma specimens. Immunohistochemistry revealed a pronounced reactivity for EMBP in astrocytoma grades III-IV as well as in metastatic prostatic adenocarcinoma used as positive control. In astrocytoma grades I-II, the expression was weak. The EMBP-like protein was quantified by radioimmunoassay in astrocytoma tumor tissue with higher concentrations in malignant astrocytoma, grades III-IV, compared to grades I-II tumors. Western immunoblotting of immunoaffinity purified EMBP-like protein under nonreducing conditions revealed an immunoreactivity corresponding to M(r) 138,000 and 200,000, indicating a different structure of EMBP in astrocytoma compared to prostatic tissue. Specific binding and the presence of saturable binding sites for 3H-labeled estramustine were demonstrated in astrocytoma tissues expressing EMBP-like protein. Scatchard plot analysis showed a Kd at approximately 30 nM, which suggests a binding affinity for estramustine in the same range as previously reported for EMBP in the prostate. Moreover, the number of estramustine binding sites/g tumor as calculated from the Scatchard plots was well correlated with the EMBP levels determined in the radioimmunoassay. In conclusion, an EMBP-like protein is expressed in astrocytoma. This protein may be responsible for the specific binding of estramustine in the tumor tissue. Whether this specific binding of estramustine is of importance for the cytotoxic effect in glioma cells remains to be evaluated.
Assuntos
Astrocitoma/química , Neoplasias Encefálicas/química , Proteínas de Transporte/análise , Estramustina/metabolismo , Glioblastoma/química , Proteínas Secretadas pela Próstata , Astrocitoma/metabolismo , Western Blotting , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Humanos , Imuno-Histoquímica , RadioimunoensaioRESUMO
The monoclonal antibody C215 (IgG2a) was obtained by the immunization of BALB/c mice with the human colon adenocarcinoma cell line COLO 205 and used in the targeting of colorectal carcinomas. The partial characterization and purification of the C215 target molecule from solubilized COLO 205 membranes indicated that it is an integral membrane glycoprotein of the non-mucin type. The denatured antigen appeared as a major 40-kDa form in Western blots after SDS-polyacrylamide gel electrophoresis and migrated as a monomeric 36-kDa species after the reductive cleavage of intramolecular disulfide bridges. Using a five-step procedure, the antigen was purified 4,300-fold from COLO 205 tumors raised in nude mice to a homogeneity of 95% when assessed by capillary electrophoresis. Removal of N-linked carbohydrate by peptide:N-glycosidase treatment did not affect the visualization of the purified antigen in immunoblots but resulted in a faster migration in the SDS gels. The amino acid sequence was partially determined. Seventeen contiguous NH2-terminal amino acids were identified and coincided exactly with residues 82-98 of the GA733-2 protein cloned by Szala et al. (Szala, S., Froehlich, M., Scollon, M., Kasai, Y., Steplewske, Z., Koprowski, H., and Linnenback, A. J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3542-3546). Therefore, the predicted amino acid sequence of this protein was used to prepare overlapping synthetic peptides that cover the entire extracellular domain in order to identify the C215 epitope. A likely epitope, close to the NH2 terminus and corresponding to the first distinct hydrophilic stretch after the putative signal sequence, was identified in a peptide enzyme-linked immunosorbent assay. Moreover, GA733-2 cDNA was used for the cloning of the C215 protein from COLO 205 cells and the subsequent transfection to K36.16 mouse T cell leukemia cells. The transfected cells were C215 reactive in fluorescence-activated cell sorter analysis, and a 42 kDa band was visualized in Western blots under both non-reducing and reducing conditions. Our findings indicate a close relationship between the C215 antigen and other members of the GA-733 family, some of which are currently being used as targets in clinical trials with monoclonal antibodies. The mammalian expression system described here will enable further studies into the biological role of this protein and the construction of animal models in order to develop optimal therapeutic strategies.