RESUMO
Altered cell and tissue differentiation is characteristic of premalignant lesions long before they become invasive and metastatic. One approach to controlling preneoplastic lesions is to block their expansion with non-toxic agents that suppress cell proliferation and induce apoptosis. Here, we show that ellagic acid, a natural, dietary phenolic antioxidant when given at 10(-5) M for 48 hours to colon cancer cells (SW 480), induced down regulation of insulin like growth factor IGF-II, activated p21(waf1/Cip1), mediated a cumulative effect on G1/S transition phase and caused apoptotic cell death. SW480 colon cancer cells expressed significant mRNA levels for the mitogenic insulin like growth factor (IGF-II). Collectively, these observations suggest that growth inhibition by ellagic acid is mediated by signaling pathways that mediate DNA damage, triggers p53, which in turn activates p21 and at the same time alters the growth factor expression, resulting in the down regulation of IGF-II.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Ácido Elágico/farmacologia , Fator de Crescimento Insulin-Like II/genética , Ciclo Celular , Divisão Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Neoplasias do Colo/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Ciclinas/genética , Fragmentação do DNA/efeitos dos fármacos , DNA de Neoplasias/análise , Regulação para Baixo , Fase G1 , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Fator de Crescimento Insulin-Like II/biossíntese , RNA Mensageiro/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND/PURPOSE: Recent studies have reported a high incidence of p53 mutations in anaplastic Wilms' tumors (WT). Restoration of the normal p53 state by current gene therapy techniques is thus an attractive potential mode of therapy for this tumor, which is poorly responsive to standard therapy. The purpose of this study is to determine whether gene delivery of normal p53 is possible and to characterize the subsequent effect of restoring the wild-type p53 state. METHODS: Anaplastic WT RM1 cells (mutant p53) were transduced with replication-deficient adenoviral vectors containing either the wild-type p53 gene (rAd-p53) or the gene encoding a green fluorescent protein (rAd-GFP). The transduction efficiency of adenovirus for RM1 cells was determined by flow cytometric analysis of rAd-GFP-transduced cells. The effect of p53 transduction on cell viability was evaluated using a colorimetric proliferation assay. Apoptosis was evaluated by labeling DNA breaks using a TUNEL assay (Apo-Direct kit). RESULTS: Cells treated with increasing concentrations of viral particles relative to tumor cells (multiplicity of infection-MOI) showed a dose-dependent increase in the number of cells transduced. Twenty-four hours after viral treatment, the percentage of cells transduced for MOIs of 10, 50, 100, and 500 was 29.5, 60.9, 74.6, and 92.4, respectively; at 48 hours the percentage of cells transduced increased to 70.8, 90.7, 93. 7, and 96.3, respectively. Viral treatment at an MOI of 50 reduced cell proliferation by 10% at 17 hours and 97% at 5 days; at an MOI of 100, the relative reduction in proliferation was 15% and 99.8%, respectively. When assayed, 30% of cells became apoptotic at an MOI of 50, and 48% at an MOI of 100. CONCLUSIONS: Highly efficient delivery of the p53 tumor suppressor gene by adenoviral vector to anaplastic WT is possible. Subsequent restoration of the normal p53 state results in reduced viability and increased apoptosis. Gene replacement of p53 may represent a novel therapeutic agent for anaplastic Wilms' tumors.
Assuntos
Genes p53/genética , Neoplasias Renais/genética , Proteína Supressora de Tumor p53/genética , Tumor de Wilms/genética , Animais , Apoptose , Western Blotting , Citometria de Fluxo , Terapia Genética , Vetores Genéticos , Humanos , Marcação In Situ das Extremidades Cortadas , Neoplasias Renais/terapia , Camundongos , Camundongos Nus , Recombinação Genética , Transdução Genética , Transgenes/genética , Células Tumorais Cultivadas , Tumor de Wilms/terapiaRESUMO
The protein RPE65 has an important role in retinoid processing and/or retinoid transport in the eye. Retinoids are involved in cell differentiation, embryogenesis and carcinogenesis. Since the kidney is known as an important site for retinoid metabolism, the expression of RPE65 in normal kidney and transformed kidney cells has been examined. The RPE65 mRNA was detected in transformed kidney cell lines including the human embryonic kidney cell line HEK293 and the African green monkey kidney cell lines COS-1 and COS-7 by reverse transcription PCR. In contrast, it was not detected in human primary kidney cells or monkey kidney tissues under the same PCR conditions. The RPE65 protein was also identified in COS-7 and HEK293 cells by Western blot analysis using a monoclonal antibody to RPE65, but not in the primary kidney cells or kidney tissues. The RPE65 cDNA containing the full-length encoding region was amplified from HEK293 and COS-7 cells. DNA sequencing showed that the RPE65 cDNA from HEK293 cells is identical to the RPE65 cDNA from the human retinal pigment epithelium. The RPE65 from COS-7 cells shares 98 and 99% sequence identity with human RPE65 at the nucleotide and amino acid levels, respectively. Moreover, the RPE65 mRNA was detected in three out of four renal tumor cultures analyzed including congenital mesoblastic nephroma and clear cell sarcoma of the kidney. These results demonstrated that transformed kidney cells express this retinoid processing protein, suggesting that these transformed cells may have an alternative retinoid metabolism not present in normal kidney cells.
Assuntos
Proteínas/genética , Transcrição Gênica , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Sequência de Bases , Células COS , Proteínas de Transporte , Bovinos , Linhagem Celular Transformada , Chlorocebus aethiops , DNA Complementar , Proteínas do Olho , Humanos , Rim , Dados de Sequência Molecular , Biossíntese de Proteínas , Proteínas/análise , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Urodelos , cis-trans-IsomerasesRESUMO
Ellagic acid is a phenolic compound present in fruits and nuts including raspberries, strawberries and walnuts. It is known to inhibit certain carcinogen-induced cancers and may have other chemopreventive properties. The effects of ellagic acid on cell cycle events and apoptosis were studied in cervical carcinoma (CaSki) cells. We found that ellagic acid at a concentration of 10(-5) M induced G arrest within 48 h, inhibited overall cell growth and induced apoptosis in CaSki cells after 72 h of treatment. Activation of the cdk inhibitory protein p21 by ellagic acid suggests a role for ellagic acid in cell cycle regulation of cancer cells.
Assuntos
Apoptose , Ciclinas/biossíntese , Ácido Elágico/farmacologia , Inibidores Enzimáticos/metabolismo , Fase G1/efeitos dos fármacos , Neoplasias/metabolismo , Proteína Supressora de Tumor p53/biossíntese , Western Blotting , Divisão Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21 , DNA/biossíntese , Fragmentação do DNA , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Anaplastic Wilms' tumors are commonly believed to be rare forms of progression, driven by p53 mutations, of the more common classical Wilms' tumor or nephroblastoma Contrary to classical Wilms' tumors, anaplastic tumors traditionally tend to metastasize, to be drug-resistant and to have a poor prognosis. The Bcl-2 gene product protects cells from programmed cell death, and its over-expression has been proposed to be tumorigenic and to mediate resistance to therapy. Because Bcl-2 has been reported to be transcriptionally repressed by p53, using immuno-histochemistry and mRNA analyses, we have examined Bcl-2 expression in a panel of 10 classical and 3 anaplastic nephroblastomas in which the p53 status had been previously analyzed. We found that classical Wilms' tumors expressed significant amounts of Bcl-2 mRNA and protein, whereas anaplastic tumors did not, regardless of p53 mutations. However, because mortality occurred both among patients with classical and among those with anaplastic tumors, which had divergent Bcl-2 expression, analysis of variance failed to demonstrate prognostic Bcl-2 significance. Therefore, we examined the expression of the Bcl-X and Bax genes, which are known to synergize and antagonize Bcl-2, respectively. With the exception of anaplastic tumor W17, the monotony of Bcl-X and Bax mRNA levels did not suggest that the expression of these apoptosis-regulating genes could have a role in the prognosis of nephroblastoma. In addition to the standard 2.7-kb Bcl-X(L) mRNA, W17 expressed a 3.5-kb mRNA species which had the same coding capacity for Bcl-X(L) as the 2.7-kb mRNA. Western analysis demonstrated that W17 had the highest level of Bcl-X(L) protein, suggesting that Bcl-X(L) over-expression could play a part in the development of anaplasia in rare Wilms' tumor cases without affecting prognosis.
Assuntos
Neoplasias Renais/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Tumor de Wilms/metabolismo , Northern Blotting , Criança , Pré-Escolar , Humanos , Lactente , Neoplasias Renais/patologia , Prognóstico , RNA Mensageiro/metabolismo , Tumor de Wilms/patologia , Proteína X Associada a bcl-2 , Proteína bcl-XRESUMO
Bcl-2 is present in a cytoplasmic distribution in cells that express high levels of this oncoprotein. In contrast, using immunocytochemistry in cells expressing low levels of bcl-2, such as KB human carcinoma cells, we and others have shown that bcl-2 is present on the surface of mitotic chromosomes. However, monoclonal antibodies reactive with an epitope representing amino acids 41-54 of the bcl-2 sequence did not detect bcl-2 in other phases of the cell cycle. This study extended those earlier findings to determine if bcl-2 was expressed as a cyclin or if this pattern was an artifact of immunocytochemistry. Immunofluorescence studies in several other human cell lines showed the same mitotic distribution of bcl-2. Other studies using flow cytometry also showed selective mitotic phase detection of bcl-2. A comparison of available commercial antibodies showed that, in spite of reactivity with denatured bcl-2 on Western blots, clear reactivity with bcl-2 in fixed cells was found only with those reactive with the (a.a. 41-54) epitope. With RNase protection and Western blot analyses, cells synchronized at various stages of the cell cycle showed constant levels of bcl-2 mRNA and protein. Analysis of bcl-2 using Western blots showed a band with the same apparent molecular weight as that seen in comparison with authentic bcl-2 overexpressed in the cytoplasm. The retention of bcl-2 on chromosomes in unfixed, permeabilized preparations was influenced by protease treatment, phosphate, and pH. Studies using isolated chromosomes showed that much of the bcl-2 in these cells was attached to chromosomes in mitosis, had the expected molecular weight, and was phosphorylated in the same manner as that seen in whole-cell extracts. These results show that bcl-2 is not a cyclin and that the bcl-2 localized on chromosomes is the same molecule seen by immunoblotting. These results suggest that the reactive (a.a. 41-54) epitope present in bcl-2 is somehow modified or masked in interphase.
Assuntos
Cromossomos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Animais , Anticorpos Monoclonais , Western Blotting , Ciclo Celular , Células Cultivadas , Cricetinae , Citoplasma/metabolismo , Epitopos/imunologia , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Camundongos , Mitose , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , RNA Mensageiro/biossíntese , RatosRESUMO
We have previously shown, by immunofluorescence of fixed cells, that bcl-2 is found only in mitotic chromosomes in KB cultured human tumor cells expressing low levels of this oncoprotein. However, other studies showed that bcl-2 did not change its levels during the cell cycle when analyzed using Western blots. In this study we analyzed the distribution of bcl-2 during interphase, the point at which it is undetectable by immunofluorescence, using biochemical extraction, immunoprecipitation, and cell fractionation with Western blots. Interestingly, when carefully examined by immunofluorescence in fixed cells, the earliest point in the cell cycle showing bcl-2 localization was early G2, in which bcl-2 could be found within the intact nucleus. In spite of showing no immunofluorescence reaction in fixed interphase cells, immunoprecipitation of gentle detergent extracts showed that bcl-2 from interphase cells reacted readily with the antibody used (#124) after extraction. However, immunoprecipitation using anti-bcl-2 followed by Western blots using anti-Bax showed that, unlike overexpressing cells, this bcl-2 was not complexed with Bax. Classical cell fractionation methods were used to separate nuclei from cytosol and cell membranes. Surprisingly, these experiments clearly showed that essentially all of the bcl-2 in interphase KB cells was present in the nucleus. Therefore, the lack of reaction in fixed cells with anti-bcl-2 antibody reflects either a masking or a conformational change of the reactive epitope in bcl-2 present within the nucleus. By correlation, this change may be related to the phosphorylation of bcl-2 that occurs just before mitosis. The nature of this novel yet highly conserved nuclear form of bcl-2 and the understanding of its function will require further study.
Assuntos
Núcleo Celular/metabolismo , Cromossomos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Western Blotting , Ciclo Celular , Humanos , Interfase , Microscopia de Fluorescência , Proteínas Proto-Oncogênicas/metabolismo , Frações Subcelulares/metabolismo , Células Tumorais Cultivadas , Proteína X Associada a bcl-2RESUMO
One reason for the failure of chemotherapy is the overexpression of the multidrug resistance gene, MDR1. The product of this gene is the multidrug transporter P-glycoprotein, an ATP-dependent pump that extrudes drugs from the cytoplasm. Some tumors inherently express P-glycoprotein, whereas others acquire the ability to do so after exposure to certain chemotherapeutic agents, often by the mechanism of gene amplification. Classical Wilms' tumors (nephroblastoma) typically respond to therapy and have a good prognosis. On the contrary, anaplastic Wilms' tumors are generally refractory to chemotherapy. These anaplastic variants are rare (4.5% of all Wilms' tumors reported in the United States), aggressive, and often fatal forms of tumor, which are commonly thought to result from the progression of classical Wilms' tumors. To investigate the basis for this differential response to therapy, we examined a number of classical and anaplastic Wilms' tumors for the expression of the MDR1 gene by immunohistochemical and mRNA analysis. Classical Wilms' tumors consistently did not express P-glycoprotein except in areas of tubular differentiation, as in normal kidney. Similarly, two of three anaplastic tumors failed to show P-glycoprotein expression. In contrast, cultured cells derived from a third anaplastic tumor, W4, exhibited strong P-glycoprotein expression and were drug resistant in vitro. Southern analysis revealed that W4 cells contained a single copy of the MDR1 gene per haploid genome similar to normal cells, demonstrating that the overexpression of MDR1 was not caused by gene amplification. Transcriptional activation of the MDR1 gene would be in keeping with the concept that p53 might act as a transcriptional repressor of the MDR1 gene.
Assuntos
Anaplasia/patologia , Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes MDR/efeitos dos fármacos , Tumor de Wilms/genética , Tumor de Wilms/patologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Anaplasia/genética , Northern Blotting , Southern Blotting , Humanos , Imuno-Histoquímica , Rim/metabolismo , Rim/patologia , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Tumor de Wilms/metabolismoRESUMO
All-trans-retinoic acid (RA) has been used to suppress growth of malignant cells and induce epithelial differentiation. We investigated whether RA had a similar effect on Wilms' tumor, a childhood tumor of the kidney that arises from the undifferentiated metanephric blastema. W13 cells, a cell line derived from a blastemal Wilms' tumor, were exposed to RA (10(-9)-10(-5) M) and its effects on cell proliferation, gene expression, and differentiation were examined. Treatment of W13 cells with RA resulted in a dose-dependent suppression of growth. Changes in expression of selected genes were determined by Northern analysis. After 24 h, there was a marked dose-dependent down-regulation of N-myc mRNA as well as up-regulation of insulin-like growth factor-II (IGF-II) mRNA. [125I]IGF-II ligand blotting of conditioned medium from RA-treated cultures revealed a dramatic alteration in the pattern of expression of insulin-like growth factor binding proteins (IGFBPs). Examination of RA-treated W13 cultures by light and electron microscopy did not reveal appreciable morphological changes. We conclude that RA inhibits growth and alters gene expression of W13 cells without inducing epithelial differentiation. The modulation of expression of IGF-II, IGFBP, and N-myc may play a role in RA-induced growth suppression of Wilms' tumor cells.
Assuntos
Tretinoína/uso terapêutico , Tumor de Wilms/classificação , Tumor de Wilms/tratamento farmacológico , Diferenciação Celular/efeitos dos fármacos , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/antagonistas & inibidores , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/biossíntese , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Fator de Crescimento Insulin-Like II/antagonistas & inibidores , Fator de Crescimento Insulin-Like II/biossíntese , Fator de Crescimento Insulin-Like II/genética , Neoplasias Renais/classificação , Neoplasias Renais/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/análise , Células Tumorais CultivadasRESUMO
The high-affinity interleukin 2 (IL-2) receptor is a heterotrimer consisting of alpha, beta, and gamma subunits. We examined the concentration of subunit mRNA for each of the three protein subunits on human hematopoietic cell lines, human peripheral blood mononuclear cells, and murine fibroblasts transfected with cDNAs encoding the human IL-2 receptor subunits. In most cultured hematopoietic cells, there was abundant gamma subunit message. In contrast, there was variable expression of both alpha and beta subunit message. Sensitivity of cells to the diphtheria fusion toxin DAB389IL-2 was not related to expression of any single IL-2 receptor subunit mRNA. Rather, the greatest sensitivity was observed for cells possessing all three subunit mRNAs. Cells displaying beta and gamma subunit mRNA showed a reduced but significant sensitivity to the fusion toxin. In contrast, cells with alpha and gamma subunit mRNA, but missing the beta subunit mRNA, were insensitive to DAB389IL-2. The data correlate with the requirement for an intermediate or a high-affinity receptor for cell intoxication. A critical concentration of the beta subunit may be required for toxin internalization and killing.
Assuntos
Toxina Diftérica/toxicidade , Células-Tronco Hematopoéticas/metabolismo , Imunossupressores/toxicidade , Imunotoxinas/toxicidade , Interleucina-2/toxicidade , Receptores de Interleucina-2/metabolismo , Animais , Antineoplásicos/toxicidade , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão , Transfecção , Células Tumorais CultivadasRESUMO
The anaplastic variant of Wilms' tumor is regarded as the result of tumor progression of the more common classic Wilms' tumor. Anaplasia is rare and occurs in only 4.5% of tumors. Three anaplastic Wilms' tumors in our collection were examined in comparison with 10 classic Wilms' tumors for p53 expression by immunohistochemical techniques and Northern blot analysis, and their p53 gene structure was determined by single-stranded conformation polymorphism and sequence analysis. All classic tumors contained a wild-type p53 gene and expressed marginal levels of protein as expected for normal p53. In contrast, three out of three anaplastic tumors demonstrated evidence of p53 alterations consistent with a role of p53 in tumor progression. One of the anaplastic mutants (W4) did not express protein or p53 mRNA. Its apparently normal immunophenotype would have disguised the mutated nature of p53, which was detected only by mRNA and sequence analysis. The second anaplastic mutant (W16) contained normal levels of p53 mRNA, but overexpressed the protein in a fashion typical of mutated p53. The same immunophenotype was displayed by fixed primary tissue of the third anaplastic tumor (W17), but p53 mutation could not be confirmed for lack of frozen primary material. The present study emphasizes the importance of p53 function in the anaplastic progression of Wilms' tumor and the risk of error in assessing normal p53 function using a single methodology.
Assuntos
Genes p53 , RNA Mensageiro/biossíntese , Tumor de Wilms/genética , Tumor de Wilms/imunologia , Adolescente , Adulto , Sequência de Bases , Criança , Pré-Escolar , Regulação Neoplásica da Expressão Gênica , Genótipo , Humanos , Imunofenotipagem , Lactente , Dados de Sequência Molecular , Tumor de Wilms/patologiaRESUMO
The Wilms tumor suppressor gene WT1 encodes a developmentally regulated transcription factor that is mutated in a subset of embryonal tumors. To test its functional properties, we developed osteosarcoma cell lines expressing WT1 under an inducible tetracycline-regulated promoter. Induction of WT1 resulted in programmed cell death. This effect, which was differentially mediated by the alternative splicing variants of WT1, was independent of p53. WT1-mediated apoptosis was associated with reduced synthesis of the epidermal growth factor receptor (EGFR), but not of other postulated WT1-target genes, and it was abrogated by constitutive expression of EGFR. WT1 repressed transcription from the EGFR promoter, binding to two TC-rich repeat sequences. In the developing kidney, EGFR expression in renal precursor cells declined with the onset of WT1 expression. Repression of EGFR and induction of apoptosis by mechanism that may contribute to its critical role in normal kidney development and to the immortalization of tumor cells with inactivated WT1 alleles.
Assuntos
Apoptose/fisiologia , Proteínas de Ligação a DNA/fisiologia , Receptores ErbB/biossíntese , Genes do Tumor de Wilms/genética , Proteínas Imediatamente Precoces , Fatores de Transcrição/fisiologia , Processamento Alternativo , Animais , Animais Recém-Nascidos , Sequência de Bases , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce , Humanos , Rim/embriologia , Rim/metabolismo , Dados de Sequência Molecular , Osteossarcoma , Regiões Promotoras Genéticas/genética , Ratos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/fisiologia , Proteínas WT1RESUMO
The Wilms' tumor suppressor gene WT1 encodes a zinc finger transcription factor, whose expression inhibits the growth of the RM1 Wilms' tumor cell line. Transient transfection of WT1 constructs into 3T3 or 293 cells results in transcriptional repression of a number of cotransfected promoters containing the early growth response gene 1 consensus sequence. We now show that WT1 has properties of a transcriptional activator in RM1 cells, an effect that may be associated with the presence of a mutated p53 gene in these cells. Stable transfection of wild-type WT1 into RM1 cells results in induction of endogenous insulin-like growth factor 2 (IGF2) but not of other previously postulated WT1-target genes. The induction of IGF2 is dramatically enhanced by WT1 mutants encoding an altered transactivation domain. We conclude that IGF2 is a potentially physiological target gene for WT1 and that its induction may contribute to the growth-stimulating effects of WT1 variants.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Fator de Crescimento Insulin-Like II/genética , Fatores de Transcrição/fisiologia , Tumor de Wilms/metabolismo , Sequência de Bases , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Ativação Transcricional , Células Tumorais Cultivadas , Proteínas WT1 , Dedos de ZincoRESUMO
Human Wilms' tumor (WT) expresses insulin-like growth factor (IGF) II and its cognate receptor, type 1 IGF receptor, forming a self-stimulating "autocrine loop." The biological activity of IGF-II is modulated by a class of soluble receptors called IGF binding proteins (IGFBP). To determine if IGFBP play a role in the biology of WT, extracts of nude mouse heterotransplants of three blastemal WT were examined for the ability to bind radiolabeled IGF-II by ligand blot analysis. [125I]IGF-II bound to a protein of M(r) 35 kDa. To confirm that this binding protein was being expressed by the tumor itself and not background from the host, tumor explants were prepared in cell culture. Conditioned culture media from blastemal WT cell cultures were found to contain the 35-kDa IGFBP. This secreted binding protein was identified as IGFBP-2 by screening for reactivity to characterized IGFBP antisera. Total RNA from primary WT or WT cells in culture was examined for expression of IGFBP-2 mRNA using an RNase protection assay. All three WT expressed IGFBP-2 mRNA. These data suggest a role for IGFBP-2 in the IGF-II-dependent growth of Wilms' tumor and in the developing kidney.
Assuntos
Proteínas de Transporte/análise , Neoplasias Renais/química , Tumor de Wilms/química , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Humanos , Immunoblotting , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina , Fator de Crescimento Insulin-Like II/análise , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/fisiologia , Radioisótopos do Iodo , Neoplasias Renais/patologia , Neoplasias Renais/fisiopatologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores de Somatomedina/análise , Receptores de Somatomedina/genética , Receptores de Somatomedina/fisiologia , Células Tumorais Cultivadas/patologia , Tumor de Wilms/patologia , Tumor de Wilms/fisiopatologiaRESUMO
Wilms' tumor, or nephroblastoma, is a developmental malignancy of the kidney that affects approximately 1 in 10,000 children between 1 and 6 years of age. Typically, the histology of nephroblastoma reveals a disorganized renal developmental process showing blastema and epithelia randomly interspersed in varying amounts of stroma. This developmental disruption is associated with the loss of function of the tumor suppressor gene WT-1. This gene, located on chromosome 11 at band p13, codes for a zinc finger protein that may act as a transcriptional repressor. Familial cases of Wilms' tumor fit Knudson's "two hit" model, according to which a germ line mutation of one WT-1 allele predisposes to the tumor while an additional somatic mutation of the other allele causes malignant transformation. Originally proposed for retinoblastoma, this model defines the nature of the tumor suppressor gene as a gene that is tumorigenic when inactivated. However, not all Wilms' tumor cases fit this model because the majority of Wilms' tumors do not show a mutation of WT-1. For Wilms' tumor, the loss of tumor suppression appears to be more complex than for retinoblastoma. Some of the mechanisms recognized to date involve dominant negative WT-1 mutations, interaction of the WT-1 gene product with other mutated transcription factors such as p53, loss of imprinting, and mutations of other tumor suppressor genes at 11p15 or other loci. Although classic Wilms' tumor is associated with good prognosis (85% survival), its anaplastic form is often fatal. Despite the plethora of knowledge gained in recent years, Wilms' tumor remains the center of attention for further investigation because it offers opportunities for studying normal kidney development, for understanding the molecular basis for clinically important anaplastic forms, as well as for elucidating the molecular mechanisms of tumor suppressor genes. To facilitate this task, Wilms' tumor heterotransplants have been established in nude mice. This provides an indefinite source of tumor tissue and a means to test their growth properties in response to drug treatments or molecular genetic manipulations. Furthermore, the establishment of stable Wilms' tumor cell lines is essential to investigating further the molecular basis of tumorigenesis using recombinant DNA technology.
Assuntos
Neoplasias Renais/patologia , Tumor de Wilms/patologia , Animais , Criança , Pré-Escolar , Humanos , Lactente , Rim/embriologia , Neoplasias Renais/genética , Modelos Biológicos , Mutação , Transplante de Neoplasias , Tumor de Wilms/genéticaRESUMO
Clinically the anaplastic variant of Wilms' tumor differs from the classical Wilms' tumor by its poor prognosis. To begin to understand and characterize the distinctive biology of this rare form of Wilms' tumor, a study of the histology, ultrastructure, and mRNA expression was performed on the anaplastic tumor and its associated cell line. The anaplastic tumor generated mouse heterotransplants that were readily used to establish epithelial cell cultures. The epithelial cultures, in turn, produced tumors when reinjected into nude mice. Microscopic evaluation revealed that the anaplastic epithelial cells were less differentiated than their epithelial counterpart in classical Wilms' tumors. In general the molecular profile of the anaplastic tumor was more consistent with that of an epithelial-rich classic Wilms' tumor than with the classic triphasic Wilms' tumor. Unlike the classic triphasic Wilms' tumor that contains blastema, stroma, and epithelial tubules, the anaplastic tumor expressed only marginal levels of insulin-like growth factor 2 (IGF-2) mRNA and imperceptible levels of the Wilms' tumor gene (WT-1), Pax-2, and Pax-8 mRNA. In common with the classic Wilms' tumor, the anaplastic variant retained the expression of the N-myc gene while failing to express C-myc. A comparison of cultures derived from an epithelial-rich, classic Wilms' tumor and the anaplastic Wilm's tumor indicated that both lacked IGF-2 and WT-1 mRNA expression. However, the well-differentiated epithelial cell culture derived from the classic Wilms' tumor expressed C-myc, Pax-8, and Pax-2 mRNA, none of which were expressed by the anaplastic epithelial cells. Furthermore, the well-differentiated epithelial cell component failed to express N-myc, which was expressed by both the primary triphasic Wilms' tumor and the anaplastic tumor. Overall, the findings indicate that patterns of gene expression within a single component do not correlate with the aggressive clinical behavior of the anaplastic Wilms' tumor.
Assuntos
Neoplasias Renais/patologia , Tumor de Wilms/patologia , Anaplasia , Animais , Pré-Escolar , Humanos , Neoplasias Renais/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia Eletrônica , Transplante de Neoplasias , RNA Mensageiro/análise , RNA Neoplásico/análise , Células Tumorais Cultivadas , Tumor de Wilms/genéticaRESUMO
Humans have a complex expression of metallothionein (MT) genes which involves many MT isoforms encoded by a family of genes containing an upper limit of 12 possible functional genes, in contrast to most animals which have one or two functional MT genes. In the present study, human proximal tubule (HPT) cells were exposed to cadmium (Cd) to determine if these cultures might serve as a model system to study MT gene expression in the renal proximal tubule. Three independent isolates of HPT cells were shown to repeatably induce MT protein when exposed continually to a non-toxic dose of 1 microgram/ml of Cd administered as CdCl2. Accumulation of MT protein was noted within 3 h and persisted over the 16-day time course. The expression of mRNA for the MT-IIA, MT-IA, B, E, F and G genes was also assessed through 16 days of exposure to 1 microgram/ml of Cd versus control media. Of these, the mRNA for the MT-IIA, MT-IE, MT-IF and MT-IG genes were detected in the cells exposed to 1 microgram/ml of Cd. Overall, the results were supportive that the HPT cells can provide a valuable model system to study the regulation of MT gene expression as it applies to the human renal proximal tubule.
Assuntos
Cádmio/toxicidade , Cloretos/toxicidade , Expressão Gênica/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Metalotioneína/genética , RNA Mensageiro/metabolismo , Northern Blotting , Cádmio/metabolismo , Cloreto de Cádmio , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Expressão Gênica/genética , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Masculino , Metalotioneína/biossíntese , Pessoa de Meia-Idade , RNA Mensageiro/genética , Transcrição GênicaRESUMO
Wilms' tumor (WT) is an embryonal renal neoplasm with features resembling fetal kidney development. A family of genes potentially involved in WT induction is called the paired box (PAX) gene family. In this study we examined by Northern blot analysis the expression of several PAX genes in a variety of WTs and other childhood neoplasms. RNA was isolated from four primary WTs and 12 WTs propagated in nude mice (heterotransplant), as well as from a variety of other childhood renal and nonrenal embryonal tumors. RNA samples were electrophoretically separated in 1.2% agarose gels, transferred to nylon membranes, and hybridized to random primer-labeled PAX2, PAX8, and WT1 probes. Membranes were then exposed to x-ray films at -70 degrees C with intensifying screens. PAX2 and WT-1 expression were seen in all four primary WTs; PAX8 was seen in three of the four primary WTs. Of the 12 heterotransplant Wilms' tumors, PAX2, PAX8, and WT1 were concomitantly expressed in seven tumors. Another heterotransplant WT expressed WT1 alone. Expression of these three genes, with one exception, was not seen in the other childhood renal and nonrenal solid tumors. The PAX genes are transcriptional regulators; their protein products bind to specific DNA segments and control gene expression. Their role in the pathogenesis of Wilms' tumor and their interaction with WT1 are unclear. Elucidation of the functional significance of the PAX genes will provide important insights into not only the pathogenesis of WT but also the molecular control of the developing kidney.
Assuntos
Proteínas de Ligação a DNA/genética , Expressão Gênica , Genes do Tumor de Wilms , Neoplasias Renais/genética , Proteínas Nucleares , Transativadores/genética , Fatores de Transcrição/genética , Tumor de Wilms/genética , Animais , Northern Blotting , Genes Homeobox/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Hibridização de Ácido Nucleico , Fator de Transcrição PAX2 , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados , RNA Mensageiro/análise , RNA Neoplásico/análise , Células Tumorais CultivadasRESUMO
A human Wilms tumor cell line (RM1) was developed to test the tumor suppressor activity of WT1, a zinc finger transcription factor that is expressed in the developing human kidney and is mutationally inactivated in a subset of Wilms tumors. Transfection of each of four wild-type WT1 isoforms suppressed the growth of RM1 cells. The endogenous WT1 transcript in these cells was devoid of exon 2 sequences, a splicing alteration that was also detected in varying amounts in all Wilms tumors tested but not in normal kidney. Production of this abnormal transcript, which encodes a functionally altered protein, may represent a distinct mechanism for inactivating WT1 in Wilms tumors.
Assuntos
Proteínas de Ligação a DNA/genética , Genes do Tumor de Wilms/fisiologia , Tumor de Wilms/genética , Tumor de Wilms/patologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Divisão Celular/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/fisiologia , Genes do Tumor de Wilms/genética , Humanos , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Transplante de Neoplasias , RNA Mensageiro/genética , Células Tumorais Cultivadas , Proteínas WT1RESUMO
Despite significant improvement in the therapy for acute lymphoid leukemia (ALL) of childhood, approximately 30% of patients relapse. Unfortunately, since no successful treatment for recurrent disease has been developed, the majority of these patients die. Recently, we presented evidence consistent with the presence of a limited program of differentiation in B-precursor ALL that is reminiscent of normal B-cell development. We found that ALL cell populations consist of both a subpopulation of progenitors with the immunophenotype of normal B-cell precursors that has self-renewal capability and a second subpopulation with a more mature early B-cell immunophenotype that is without self-renewal capability but can proliferate to a limited extent. In our recent studies we were able to grow the progenitor cells in the ALL blast colony assay and establish their leukemic origin using the polymerase chain reaction. Our results suggest that these progenitors are the cells that sustain the disease. We hypothesize that these cells may remain quiescent, for a time, and either eventually die or regain proliferative capability and cause relapse. Further studies aimed both at detecting residual ALL and determining changes in their biology may provide an understanding of the mechanisms of relapse in this disease.