RESUMO
Evidence supporting a broad role for the inactivation of the p53 gene in human tumorigenesis has been provided by studies showing that the p53 gene is mutated in many human cancers. In this study, we report on the mutational status of the p53 gene in prostate cancer cells and provide functional evidence that the wild-type p53 gene may have a role in suppressing prostatic tumorigenesis. Sequence analysis of exons 5-8 of the p53 gene reveals that three of five prostate cancer cell lines (TSUPr-1, PC3, DU145) contain mutations which alter the amino acid sequence of this most highly conserved portion of the gene. One of two primary prostatic cancer specimens examined also contained a mutation in this region. Transfection of the wild-type p53 gene versus a mutated p53 gene into two cell lines with p53 mutations results in reduced colony formation. Wild-type p53 gene expression is apparently incompatible with continued growth of these tumor cells inasmuch as none of the colonies which formed after wild-type transfections retain the transfected p53 sequences. Immunocytochemical data indicate that prostate carcinoma cells expressing the transfected wild-type p53 gene are growth arrested because they exhibit a reduced level of thymidine incorporation into DNA. This study is the first report of p53 gene mutations in prostate cancer cells and suggests a functional role for the p53 gene in suppressing prostatic tumorigenesis.
Assuntos
Alelos , Amplificação de Genes/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes p53/genética , Mutação/genética , Neoplasias da Próstata/genética , Transfecção/genética , Sequência de Aminoácidos , Humanos , Masculino , Dados de Sequência MolecularRESUMO
The cadherins are a family of transmembrane glycoproteins responsible for calcium-dependent cell-cell adhesion. This adhesion is mediated by a group of cytoplasmic proteins, the catenins, which act inside the cell to couple the cadherin molecule to the microfilament cytoskeleton. Dysfunction of E-cadherin-dependent cell-cell adhesion has been demonstrated to contribute to the acquisition of invasive potential of malignant adenocarcinoma cells. The potential role of alterations of catenin expression in tumor cell invasion is largely unexplored. We have previously found that E-cadherin is frequently down-regulated in clinical samples of prostate cancer (Umbas, R., Schalken, J. A., Aalders, T. W., Carter, B. S., Karthaus, H. F. M., Schaafsma, H. E., Debruyne, F. M. J., and Isaacs, W. B. Cancer Res., 52: 5104-5109, 1992). In this study, we further investigate this adhesion system in both benign and malignant human prostate cells in culture. Using antibodies to E-cadherin and its cytoplasmic accessory protein, alpha-catenin, we find that 5 of 6 human prostate cancer cell lines have reduced or absent levels of one or the other or both of these molecules when compared to normal prostatic epithelial cells. Only the LNCaP prostate cancer cell line is indistinguishable from normal prostate epithelium with respect to its E-cadherin-alpha-catenin complement. Interestingly, the PC-3 line is characterized by the presence of E-cadherin, but the complete lack of alpha-catenin found at both the RNA and protein level. This lack of alpha-catenin gene expression is explained by Southern analysis, which reveals a homozygous deletion of a large portion of the alpha-catenin gene in PC-3 cells. This loss of alpha-catenin is functionally manifested by negligible Ca(2+)-dependent aggregation of these cells in vitro, when compared to LNCaP cells. These results confirm that E-cadherin-dependent cell-cell adhesion is frequently aberrant in prostate cancer cells, and suggest that in a subset of prostate cancers, this adhesion may be inactivated by loss of alpha-catenin rather than E-cadherin itself. Furthermore, these results demonstrate that mutational inactivation of the alpha-catenin gene is one mechanism responsible for the loss of normal cell-cell adhesion in prostate cancer.
Assuntos
Caderinas/análise , Proteínas do Citoesqueleto/genética , Deleção de Genes , Neoplasias da Próstata/metabolismo , Adesão Celular , Humanos , Masculino , Peso Molecular , Testes de Precipitina , Neoplasias da Próstata/genética , RNA Mensageiro/análise , Células Tumorais Cultivadas , alfa CateninaRESUMO
Research into molecular and genetic mechanisms underlying familial prostate cancer would be greatly advanced by in vitro models of prostate tumor cells representing primary tumors. We have successfully established an immortalized human prostate epithelial cell culture derived from primary tumors of familial prostate cancer patients with telomerase. The actively proliferating early-passaged 957E cells were transduced through infection with a retrovirus expressing the human telomerase catalytic subunit, human telomerase reverse transcriptase (hTERT). A high level of telomerase activity was detected in 957E/hTERT cells, but not in 957E cells. 957E/hTERT cells are currently growing well at passage 40, whereas 957E cells senesced at passage 5. 957E/hTERT cells exhibit epithelial morphology. Expression of an androgen-regulated prostate specific homeobox gene NKX3.1 and an epithelial cell-specific cytokeratin 8, but not prostate specific antigen or androgen receptor, was detected in 957E/hTERT cells. Prostatic stem cell antigen and p16 were also expressed in this line. 957E/hTERT cells showed growth inhibition when exposed to retinoic acid and transforming growth factor beta1, potent inhibitors of prostate epithelial cell growth. Chromosome analysis showed that the 957E/hTERT cell line (passage 10) was near diploid human male (XY), with most chromosome counts in the 44-46 range. However, there was random loss of chromosomes 8, 13, X, Y, and alteration in chromosome 4q. The late passage 957E/hTERT cell line (passage 32) was karyologically similar to the early passage 957E/hTERT cell line (passage 10) and also had the same alteration of 4q observed in the early passage 957E/hTERT cell line (passage 10) as well as a trisomy of chromosome 20. The well-characterized human cancer lines derived from such patients will be useful for the identification and characterization of prostate cancer susceptibility genes. This is the first documented case of an established human prostate cancer cell line from primary tumor of a familial prostate cancer patient.
Assuntos
Adenocarcinoma/genética , Neoplasias da Próstata/genética , Células Tumorais Cultivadas , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Adulto , Divisão Celular/efeitos dos fármacos , Proteínas de Ligação a DNA , Inibidores do Crescimento/farmacologia , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/genética , Telomerase/metabolismo , Transdução Genética , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1 , Tretinoína/farmacologiaRESUMO
Critical aspects of the biology and molecular basis for prostate malignancy remain poorly understood. To reveal fundamental differences between benign and malignant growth of prostate cells, we performed gene expression profiling of primary human prostate cancer and benign prostatic hyperplasia (BPH) using cDNA microarrays consisting of 6500 human genes. Frozen prostate specimens were processed to facilitate extraction of RNA from regions of tissue enriched in either benign or malignant epithelial cell growth within a given specimen. Gene expression in each of the 16 prostate cancer and nine BPH specimens was compared with a common reference to generate normalized measures for each gene across all of the samples. Using an analysis of complete pairwise comparisons of expression profiles among all of the samples, we observed clearly discernable patterns of overall gene expression that differentiated prostate cancer from BPH. Further analysis of the data identified 210 genes with statistically significant differences in expression between prostate cancer and BPH. These genes include many not recognized previously as differentially expressed in prostate cancer and BPH, including hepsin, which codes for a transmembrane serine protease. This study reveals for the first time that significant and widespread differences in gene expression patterns exist between benign and malignant growth of the prostate gland. Gene expression analysis of prostate tissues should help to disclose the molecular mechanisms underlying prostate malignant growth and identify molecular markers for diagnostic, prognostic, and therapeutic use.
Assuntos
Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Família Multigênica , Hiperplasia Prostática/genética , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologiaRESUMO
Cadherins are a family of calcium-dependent, cell-cell adhesion molecules that play an important morphoregulatory role in a wide variety of tissues. Alterations in cadherin function have been implicated in tumor progression in a number of adenocarcinomas. Despite the increasing number of new cadherins identified, little is known about cadherins in normal renal tissue and renal carcinomas. A novel cadherin transcript, cadherin-6, was recently described to be present in renal cancer cell lines and fetal kidney, but no data on protein expression nor tissue localization has been reported. In this study, we demonstrate that the expression of cadherin-6 is restricted to the proximal tubule epithelium. This finding is critical because these cells give rise to the majority of neoplasms of this organ. Furthermore we demonstrate typical cadherin features of cadherin-6, including cytoplasmic binding to alpha- and beta-catenin. We present data of cadherin-6 expression in a series of 32 primary renal cell cancers. Cadherin-6 expression tended to vary with histology in these samples. Whereas the majority of renal cell cancers with histology-associated poor prognosis (i.e., high grade clear cell carcinomas and sarcomatoid renal tumors) show aberrant expression of cadherin-6, in tumors with a favorable prognosis (i.e., low grade clear cell carcinomas and papillary cancers), normal cadherin-6 expression was predominant. Overall, these findings demonstrate specific expression of cadherin-6 in the proximal renal tubules in normal human kidney and suggest that alterations of cadherin-6 expression are associated with progression of renal cell carcinoma.
Assuntos
Caderinas/metabolismo , Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Túbulos Renais/metabolismo , Transativadores , Caderinas/imunologia , Carcinoma de Células Renais/diagnóstico , Proteínas do Citoesqueleto/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Immunoblotting , Imuno-Histoquímica , Neoplasias Renais/diagnóstico , Prognóstico , Distribuição Tecidual , Células Tumorais Cultivadas , alfa Catenina , beta CateninaRESUMO
Considerable evidence now exists to support an important role for the E-cadherin-mediated cell-cell adhesion pathway as a suppressor of the invasive phenotype in adenocarcinoma cells. Previous studies have found that this pathway is frequently aberrant in prostate cancers, particularly those that are likely to metastasize. In this study, we report on the effects of re-establishment of this pathway in a prostate cancer cell line, PC-3, in which this adhesion system is dysfunctional by virtue of a deletion of the gene that codes for alpha-catenin, an E-cadherin-associated protein necessary for normal E-cadherin function. Re-expression of alpha-catenin was accomplished either by transfection of PC-3 cells with a copy of the alpha-catenin cDNA under the control of a heterologous promoter or by microcell-mediated transfer of chromosome 5, which contains the alpha-catenin gene and its normal regulatory elements. In both cases, re-expression of alpha-catenin is associated with a similar, dramatic alteration in cell morphology, whereby extensive cell-cell contact is observed. In the case of transfection of the cDNA, this expression is only transient, because the transfected cells either cease to proliferate or, more commonly, revert to the parental phenotype with concomitant cessation of alpha-catenin expression. In contrast, cells containing one or more copies of microcell-transferred chromosome 5 express alpha-catenin in a stable manner and continue to proliferate. Upon injection into nude mice, these latter cells are no longer tumorigenic, or form only slowly growing tumors with greatly extended doubling times when compared to the parental PC-3 cells. During passage in culture, clones that contain only one transferred copy of chromosome 5 reproducibly revert to the parental phenotype. This reversion is associated with loss of the chromosome 5 region containing the alpha-catenin gene and consequent loss of alpha-catenin expression, as well as re-emergence of tumorigenicity. Transfer of chromosome 5 into prostate cancer cells that are E-cadherin negative does not result in either morphological transformation or suppression of tumorigenicity, suggesting that these effects of alpha-catenin expression are dependent upon concomitant expression of E-cadherin. These data demonstrate the tumor suppressive ability of chromosome 5 in the PC-3 prostate cancer cells and suggest that re-expression of alpha-catenin with resultant restoration of E-cadherin function plays a critical role in this process.
Assuntos
Caderinas/fisiologia , Cromossomos Humanos Par 5 , Proteínas do Citoesqueleto/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Animais , Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Divisão Celular/fisiologia , Proteínas do Citoesqueleto/fisiologia , DNA/genética , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Nus , Fenótipo , Transfecção , Células Tumorais Cultivadas , alfa CateninaRESUMO
Inactivation of the p53 gene has been implicated in prostate cancer progression. To determine the role of p53 inactivation in the progression of clinical prostatic carcinomas, we assessed 67 tumors derived from patients with clinically localized disease for chromosome 17p and p53 gene allelic loss, p53 gene mutations using single-strand conformational polymorphism and direct sequencing, and p53 protein expression using immunohistochemical staining. Of 55 informative tumors, 10 demonstrated loss of 17p or the p53 gene; however, only a single tumor had a mutation in its remaining p53 allele. Significant p53 overexpression was observed in 2 of 38 tumors, and 9 others had faint staining of a few nuclei ( < 1%). p53 overexpression occurred in no informative tumor with allelic loss or mutation. In a 1-7-year follow-up, positive immunohistochemical staining did not confer an increased risk of recurrence (risk of recurrence, 0.86, P = 0.78), whereas allelic loss of chromosome 17p appeared to be highly correlated with recurrence (risk of recurrence, 3.7, P = 0.003). In an unrelated group of 42 patients with metastatic prostate cancer, p53 overexpression was found in 26 tumors (62%), and 15(36%) had high grade staining. Neither the presence nor the degree of expression correlated with time to progression or time to death. This series suggests that p53 gene inactivation is rare in primary prostatic tumors, not essential to the development of prostate cancer metastases, and of limited use as a prognostic marker in patients with primary or metastatic disease. Another gene or genes on chromosome 17p may be involved in prostate cancer progression.
Assuntos
Genes p53 , Mutação , Neoplasias da Próstata/genética , Sequência de Bases , Deleção Cromossômica , Cromossomos Humanos Par 17 , Humanos , Imuno-Histoquímica , Masculino , Dados de Sequência Molecular , Metástase Neoplásica , Polimorfismo Conformacional de Fita Simples , Proteína Supressora de Tumor p53/análiseRESUMO
Cyclooxygenase-2 (COX-2) is the inducible isoform of the rate-limiting enzymes that convert arachidonic acid to proinflammatory prostaglandins as well as a primary target for nonsteroidal anti-inflammatory drugs. Accumulating evidence suggests that up-regulation of COX-2 is associated with carcinogenesis in multiple organ systems including the large bowel, lung, breast, and prostate. In this report, we examine the expression of COX-2 protein and mRNA in prostate tissue containing various lesions and in prostate cancer cell lines. In the cell lines, LNCaP, DU145, PC-3, and TSU, COX-2 protein expression was undetectable under basal conditions but could be induced transiently by phorbol ester treatment in PC-3 and TSU cells, but not in DU145 and LNCaP cells. Immunohistochemical analysis of 144 human prostate cancer cases suggested that, in contrast to several previous reports, there was no consistent overexpression of COX-2 in established prostate cancer or high-grade prostatic intraepithelial neoplasia, as compared with adjacent normal prostate tissue. Positive staining was seen only in scattered cells (<1%) in both tumor and normal tissue regions but was much more consistently observed in areas of proliferative inflammatory atrophy, lesions that have been implicated in prostatic carcinogenesis. Staining was also seen at times in macrophages. Western blotting and quantitative RT-PCR analyses confirmed these patterns of expression. These results suggest that if nonsteroidal anti-inflammatory drugs are indeed chemopreventive and/or chemotherapeutic for prostate cancer, their effects are likely to be mediated by modulating COX-2 activity in non-PCa cells (either inflammatory cells or atrophic epithelial cells) or by affecting a COX-2-independent pathway.
Assuntos
Isoenzimas/biossíntese , Prostaglandina-Endoperóxido Sintases/biossíntese , Próstata/patologia , Neoplasias da Próstata/enzimologia , Atrofia/enzimologia , Western Blotting , Ciclo-Oxigenase 2 , Progressão da Doença , Epitélio/enzimologia , Epitélio/patologia , Humanos , Imuno-Histoquímica , Isoenzimas/genética , Masculino , Proteínas de Membrana , Prostaglandina-Endoperóxido Sintases/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/enzimologia , Células Estromais/patologia , Células Tumorais Cultivadas , Regulação para CimaRESUMO
A number of genetic changes have been documented in prostate cancer, ranging from allelic loss to point mutations and changes in DNA methylation patterns (summarized in Fig 1). To date, the most consistent changes are those of allelic loss events, with the majority of tumors examined showing loss of alleles from at least one chromosomal arm. The short arm of chromosome 8, followed by the long arm of chromosome 16 appear to be the most frequent regions of loss, suggesting the presence of novel tumor suppressor genes. Deletions of one copy of the Rb and p53 genes are less frequent as are mutations of the p53 gene, and accumulating evidence suggests the presence of an additional tumor suppressor gene on chromosome 17p, which is frequently inactivated in prostate cancer. Alterations in the E-cadherin/alpha catenin mediated cell-cell adhesion mechanism appear to be present in almost half of all prostate cancers, and may be critical to the acquisition of metastatic potential of aggressive prostate cancers. Finally, altered DNA methylation patterns have been found in the majority of prostate cancers examined, suggesting widespread alterations in methylation-modulated gene expression. The presence of multiple changes in these tumors is consistent with the multistep nature of the transformation process. Finally, efforts to identify prostate cancer susceptibility loci are underway and will hopefully elucidate critical early events in prostatic carcinogenesis.
Assuntos
Neoplasias da Próstata/genética , Caderinas/genética , Caderinas/fisiologia , Linhagem Celular Transformada , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/fisiologia , DNA de Neoplasias , Expressão Gênica , Genes do Retinoblastoma , Genes p53 , Genes ras , Heterozigoto , Humanos , Masculino , Metilação , Oncogenes , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , alfa CateninaRESUMO
Blotting and probing of DNA, RNA and proteins after electrophoresis is a powerful technique for the study of the structure and function of biomolecules. Key factors in successful blotting experiments are efficiency of transfer, maintenance of the resolution obtained during gel electrophoresis, accuracy of the probes used and sensitivity of the detection method. We have recently developed a system for the high performance resolution of DNA with 10-fold greater capacity for sample loads than agarose or polyacrylamide. In the present study, we describe conditions for the rapid (less than one hour) and quantitative electrotransfer of DNA in the 100-23,000-base pair range, with subsequent conditions for probing of transfer membranes using radioactive or biotinylated probes. Our results suggest complete maintenance of the high-resolution characteristic of HydroLink gel electrophoresis and potentially increased sensitivity due to the high loading capacity in HydroLink gel electrophoresis.
Assuntos
DNA/análise , Eletroforese em Gel de Ágar/métodos , Polímeros , Autorradiografia , Biotina , Sondas de DNA , Etídio , Géis , Concentração de Íons de Hidrogênio , Desnaturação de Ácido Nucleico , Nylons , Radioisótopos de FósforoRESUMO
Novel polymers have been prepared for high performance electrophoretic separations of double-stranded DNA (dsDNA). These materials are part of a family of HydroLink high performance electrophoresis polymers. A comparison of the resolving capabilities of dsDNA HydroLink gels to agarose and polyacrylamide separations has been described in an accompanying paper. In this study, we demonstrate that dsDNA HydroLink gels possess ten times the loading capacity of comparable polyacrylamide or agarose gels without compromise to resolution or biological integrity of the separated DNA. A simplified procedure for recovery of separated components is also described.
Assuntos
DNA/isolamento & purificação , Eletroforese/métodos , Polímeros , Composição de Bases , Eletroforese em Gel de Ágar/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Géis , Indicadores e Reagentes , Peso MolecularRESUMO
HydroLink materials represent a novel family of gels composed of unique polymer matrices. The applications of HydroLink to molecular biology and, specifically, to DNA technology have been carefully investigated. Our results indicate that the HydroLink matrix developed for double-stranded DNA (dsDNA) is an excellent tool for electrophoretic separations in fixed electric fields. Excellent linear resolution from 100 to 5000 base pairs is easily achieved with good resolution albeit non-linear from 6000 to 23000 base pairs. The broad range of separation in addition to increased mechanical strength of dsDNA HydroLink represents a distinct advantage over other matrices currently used in DNA electrophoretic analysis.
Assuntos
DNA/isolamento & purificação , Eletroforese/métodos , Polímeros , Composição de Bases , Eletroforese em Gel de Ágar/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Géis , Peso MolecularRESUMO
Serum and seminal plasma concentrations or activities of acid phosphatase (AP), prostate specific antigen (PSA), and canine prostate specific esterase (CPSE) were measured in normal dogs, dogs with benign prostatic hyperplasia (BPH), dogs with bacterial prostatitis, and dogs with prostatic carcinoma to determine if these assays would be of value in differentiating dogs with prostatic carcinoma from normal dogs, and dogs with other prostatic disorders. In addition, tissue sections of prostatic adenocarcinomas were stained with antiprostatic AP, anti-CPSE, and anti-PSA antibodies to determine if these would be suitable immunohistochemical markers of prostatic carcinoma. Prostate-specific antigen was not detected in canine serum or seminal plasma. Serum and seminal AP activities did not differ significantly between normal dogs and those with prostatic diseases, or among dogs with different prostatic disorders. Serum CPSE activities were significantly higher in dogs with BPH than in normal dogs. Mean serum CPSE activities in dogs with BPH, bacterial prostatitis, and prostatic carcinoma were not significantly different from each other. Slight to moderate immunohistochemical staining of canine prostatic adenocarcinomas was noted for prostatic AP and PSA; most tumors did not stain for CPSE. These results show that proteins of prostatic origin appear in the serum of dogs as a result of prostatic pathology, especially BPH. Canine prostatic adenocarcinoma does not appear to be associated with significant increases in CPSE or AP activities, possibly because of down-regulation of these enzymes by prostatic carcinoma cells. It is also possible that failure to detect significant differences resulted from limited statistical power for some groups and pairwise analyses because of the small number of dogs evaluated.
Assuntos
Fosfatase Ácida/metabolismo , Doenças do Cão/diagnóstico , Esterases/metabolismo , Antígeno Prostático Específico/análise , Doenças Prostáticas/veterinária , Análise de Variância , Animais , Biomarcadores , Doenças do Cão/sangue , Doenças do Cão/enzimologia , Doenças do Cão/microbiologia , Cães , Estudos de Avaliação como Assunto , Masculino , Doenças Prostáticas/diagnóstico , Doenças Prostáticas/enzimologia , Doenças Prostáticas/microbiologia , Hiperplasia Prostática/diagnóstico , Hiperplasia Prostática/veterinária , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/veterináriaRESUMO
A clearer definition of the molecular determinants that drive the development and progression of prostate cancer (PCa) is urgently needed. Efforts to map recurrent somatic deletions in the tumor genome, especially homozygous deletions (HODs), have provided important positional information in the search for cancer-causing genes. Analyzing HODs in the tumors of 244 patients from two independent cohorts and 22 PCa xenografts using high-resolution single-nucleotide polymorphism arrays, herein we report the identification of CHD1, a chromatin remodeler, as one of the most frequently homozygously deleted genes in PCa, second only to PTEN in this regard. The HODs observed in CHD1, including deletions affecting only internal exons of CHD1, were found to completely extinguish the expression of mRNA of this gene in PCa xenografts. Loss of this chromatin remodeler in clinical specimens is significantly associated with an increased number of additional chromosomal deletions, both hemi- and homozygous, especially on 2q, 5q and 6q. Together with the deletions observed in HEK293 cells stably transfected with CHD1 small hairpin RNA, these data suggest a causal relationship. Downregulation of Chd1 in mouse prostate epithelial cells caused dramatic morphological changes indicative of increased invasiveness, but did not result in transformation. Indicating a new role of CHD1, these findings collectively suggest that distinct CHD1-associated alterations of genomic structure evolve during and are required for the development of PCa.
Assuntos
Montagem e Desmontagem da Cromatina , DNA Helicases/genética , DNA Helicases/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Deleção de Genes , Neoplasias da Próstata/genética , Animais , Linhagem Celular , Regulação para Baixo , Células HEK293 , Homozigoto , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , PTEN Fosfo-Hidrolase/genética , Polimorfismo de Nucleotídeo Único , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Transplante HeterólogoRESUMO
We report the use of UDPgalactose:N-acetyl-D-glucosaminyl-glycopeptide 4-beta-D-galactosyl-transferase (EC 2.4.1.38), purified from bovine milk, to detect nonreducing terminal N-acetylglucosamine residues on glycoproteins immobilized on nitrocellulose by electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels. Soluble galactosyltransferase incorporates radiolabeled galactose from the substrate UDP-[6-3H]galactose into the appropriate immobilized acceptor with high specificity. Incorporation is proportional to substrate amount and is saturable with time. The kinetics of labeling are independent of substrate amount. Half-maximal incorporation occurs by 4 h and saturation occurs by 16 h. We have used galactosyltransferase as a probe (i) to verify the presence of nonreducing terminal N-acetylglucosamine residues in bovine rod outer segment membrane rhodopsin and in several glycoproteins in F9 murine teratocarcinoma cells and (ii) to detect previously reported endo-beta-N-acetylglucosaminidase activity in a commercial preparation of endoglycosidase F.
Assuntos
Acetilglucosamina/análise , Galactosiltransferases , Glucosamina/análogos & derivados , Glicoproteínas/análise , Acetilglucosaminidase/análise , Animais , Bovinos , Linhagem Celular , Colódio , Glicosídeo Hidrolases/análise , Cinética , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Camundongos , Leite/enzimologia , Proteínas de Neoplasias/análise , Oxirredução , Rodopsina/análise , Solubilidade , TeratomaRESUMO
We have analyzed the ATPase and dATPase activities associated with the yeast DNA polymerase alpha complex. The ATPase/dATPase was primarily a single-stranded DNA-dependent ATPase. Analysis of the stimulatory effect of a large number of DNA substrates demonstrated that polynucleotides longer than 60 nucleotides (nts) had the maximal effect. The stimulation by oligonucleotides smaller than 60 nts, in general, decreased proportionally with decreased length of the oligomer. Poly- or oligopyrimidines were twice as stimulatory as the poly- or oligopurines of the same length. In addition to DNA, replication protein A (RP-A), a single-stranded DNA (ssDNA) binding protein, also stimulated the ATPase activity. Photo-cross-linking of the ATP binding component of the pol alpha complex to [alpha-32P]ATP at 0 degree C resulted in the exclusive labeling of a 90-kDa polypeptide. The labeling was inhibited by ATP and dATP but not by any other ribo- or deoxynucleotides, which suggest that the 90-kDa polypeptide is specific for ATP/dATP binding and possibly the active site for the ATPase/dATPase. We have also reported here a novel DNA unwinding activity associated with the multiprotein complex of DNA polymerase alpha. The complex was able to unwind M13mp19 ssDNA hybridized to an oligonucleotide (17-60 nucleotides long) with a protruding 3'-terminus. Regardless of the size of the duplex, the DNA unwinding was significantly stimulated by RP-A, while RP-A itself did not have any DNA unwinding activity. Consequently, it appeared that the DNA polymerase alpha complex possessed a putative RP-A-dependent helicase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Adenosina Trifosfatases/metabolismo , DNA Helicases/metabolismo , DNA Polimerase II/metabolismo , DNA de Cadeia Simples/farmacologia , Saccharomyces cerevisiae/enzimologia , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Cátions Bivalentes , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , DNA Polimerase II/isolamento & purificação , Cinética , Magnésio/farmacologia , Manganês/farmacologia , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/metabolismo , Fotoquímica , Poli dA-dT/metabolismo , Moldes GenéticosRESUMO
A method for successfully removing primary and secondary antibodies from nitrocellulose blots while preserving the originally immobilized polypeptides was developed. Polypeptides were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and electrophoretically transferred to nitrocellulose. Nonspecific binding sites were blocked with 5% (w/v) nonfat dried milk. After blots were reacted sequentially with antibodies directed against the antigen of interest and with radiolabeled secondary antibody, a 10-min wash in 5% (w/v) milk was required prior to drying and autoradiography. A 30-min incubation at 70 degrees C in 2% (w/v) sodium dodecyl sulfate containing 100 mM beta-mercaptoethanol quantitatively removed the antibodies and allowed reuse of the blot. A modification of this method similarly allowed reuse of Western blots when proteins were immobilized on nylon. Potential applications and limitations of this method are discussed.
Assuntos
Colódio , Peptídeos/isolamento & purificação , Animais , Anticorpos/isolamento & purificação , Sítios de Ligação , Eletroforese em Gel de Poliacrilamida , Fígado/análise , Fígado/imunologia , Leite , Membrana Nuclear/análise , Membrana Nuclear/imunologia , Nylons , Peptídeos/imunologia , Ratos , Dodecilsulfato de SódioRESUMO
Recently, the large filamentous striated-muscle protein titin has been observed in non-muscle cells, and, in one instance, has been proposed to have a nuclear function as a chromosomal component contributing to structure and elasticity. In this study, we sought to further characterize the presumptive nuclear isoform of titin. Immunofluorescence microscopy with multiple titin-specific monoclonal antibodies shows localization to the nucleus in interphase cells and to the spindle machinery in mitotic cells in all cell types examined; localization to condensed chromosomes is not observed. An abundant 700-kDa phosphoprotein is the predominant species immunoprecipitated with these antibodies. Sequencing of peptide fragments of the immunopurified protein reveals identity to AHNAK, a nuclear phosphoprotein, an identification that was confirmed by Western blot analysis with antibodies to AHNAK and peptide fragmentation patterns. Sequence comparison suggests similarities between the repetitive heptad phi+/-phiP+/-phi+/- motif in AHNAK and the PEVK region of titin, potentially explaining the cross-reactivity observed between AHNAK antibodies and titin antibodies. Interestingly, although some AHNAK antibodies stain interphase nuclei, no evidence of mitotic spindle localization is seen, suggesting that the identity of the protein at the latter location is more closely related to titin than AHNAK. This concept is further supported by observations that cell lines not expressing AHNAK have similar antititin antibody localization to the mitotic spindle. We conclude that (1) multiple titin antibodies, particularly those recognizing the PEVK region, cross-react with AHNAK, and (2) the mitotic spindle staining observed with antititin antibodies is most likely due to the association of titin or a titin-like molecule with this structure.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Membrana/metabolismo , Mitose/fisiologia , Proteínas Musculares/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Quinases/metabolismo , Fuso Acromático/metabolismo , Motivos de Aminoácidos/imunologia , Sequência de Aminoácidos , Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Núcleo Celular/ultraestrutura , Conectina , Reações Cruzadas/imunologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Dados de Sequência Molecular , Peso Molecular , Proteínas Musculares/genética , Proteínas Musculares/imunologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Proteínas Quinases/genética , Proteínas Quinases/imunologia , Homologia de Sequência de Aminoácidos , Fuso Acromático/imunologia , Fuso Acromático/ultraestruturaRESUMO
BACKGROUND: E-cadherin and alpha-catenin are components of adherens junctions which mediate calcium-dependent, cell-cell adhesion in a homotypic manner. Both these molecules have been defined as useful tumor markers as their altered expression correlates with increased tumor aggressiveness and dedifferentiation. More recently, alterations of a third component of adherens junctions, beta-catenin, have been observed to play a role in several human cancers. Dysregulation of beta-catenin, either by direct mutation or by defects in interacting pathways/regulators, can result in its cytoplasmic accumulation and nuclear translocation. In the nucleus, beta-catenin forms a transcriptional complex capable of upregulating target genes, many of which encode proliferative factors. Given its oncogenic activity and connection to human cancer, we examined the beta-catenin gene and its expression in prostate cancer. METHODS: By single-stranded conformational polymorphism (SSCP) and DNA sequencing analyses, we screened exon 3 of beta-catenin from a panel of 81 primary tumors obtained at radical prostatectomy, 22 lymph node metastases from untreated patients, and a unique set of 61 metastatic tissues from 19 patients who died of hormone-refractory disease. RESULTS: We found putative activating mutations (missense and deletion) at a rate of 5% (7/138). One patient had the same 72 base pair deletion in each of nine separate metastases examined, indicating that this change was associated with a clonal population of metastatic cells. CONCLUSIONS: Immunohistological staining of mutation-positive tumors demonstrated beta-catenin accumulation and nuclear localization in a heterogeneous fashion. Consistent with this in vivo finding, our in vitro analyses demonstrate that certain mutations can result in increased beta-catenin nuclear activity in prostate cancer cell lines. These data implicate the beta-catenin signaling pathway in the development of a subset of prostate cancers.