RESUMO
The pancreatic acinar carcinoma established in rat by Reddy and Rao (1977, Science 198:78-80) demonstrates heterogeneity of cytodifferentiation ranging from cells containing abundant well-developed secretory granules to those with virtually none. We examined the synthesis intracellular transport and storage of secretory proteins in secretory granule-enriched (GEF) and secretory granule-deficient (GDF) subpopulations of neoplastic acinar cells separable by Percoll gradient centrifugation, to determine the secretory process in cells with distinctly different cytodifferentiation. The cells pulse-labeled with [3H]leucine for 3 min and chase incubated for up to 4 h were analyzed by quantitative electron microscope autoradiography. In GEF neoplastic cells, the results of grain counts and relative grain density estimates establish that the label moves successively from rough endoplasmic reticulum (RER) leads to the Golgi apparatus leads to post-Golgi vesicles (vacuoles or immature granules) leads to mature secretory granules, in a manner reminiscent of the secretory process in normal pancreatic acinar cells. The presence of approximately 40% of the label in association with secretory granules at 4 h postpulse indicates that GEF neoplastic cells retain (acquire) the essential regulatory controls of the secretory process. In GDF neoplastic acinar cells the drainage of label from RER is slower, but the peak label of approximately 20% in the Golgi apparatus is reached relatively rapidly (10 min postpulse). The movement of label from the Golgi to the post-Golgi vesicles is evident; further delineation of the secretory process in GDF neoplastic cells, however, was not possible due to lack of secretory granule differentiation. The movement of label from RER leads to the Golgi apparatus leads to the post-Golgi vesicles suggests that GDF neoplastic cells also synthesize secretory proteins, but to a lesser extent than the GEF cells. The reason(s) for the inability of GDF cells to concentrate and store exportable proteins remain to be elucidated.
Assuntos
Carcinoma/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Transporte Biológico , Carcinoma/patologia , Diferenciação Celular , Grânulos Citoplasmáticos/ultraestrutura , Complexo de Golgi/metabolismo , Neoplasias Pancreáticas/patologia , RatosRESUMO
This is a review of several new approaches developed at or adopted by the Cooperative Prostate Cancer Tissue Resource (CPCTR) to resolve issues involved in tissue microarray (TMA) construction and use. CPCTR developed the first needle biopsy TMA, allowing researchers to obtain 200 or more consecutive cancer sections from a single biopsy core. Using radiographs of original paraffin blocks to measure tissue thickness we developed a method to produce TMAs with a larger number of usable sections. The modular approach to plan TMA construction is also a novel concept wherein TMAs of different types, such as tumor grade TMAs, metastasis TMA and hormone refractory tumors TMA can be combined to form an ensemble of TMAs with expanded research utility, such as support for tumor progression studies. We also implemented an open access TMA Data Exchange Specification that allows TMA data to be organized in a self-describing XML document annotated with well-defined common data elements. It ensures inter-laboratory reproducibility because it offers information describing the preparation of TMA blocks and slides. There are many important aspects that may be missed by both beginners and experienced investigators in areas of TMA experimental design, human subjects protection, population sample size, selection of tumor areas to sample, strategies for saving tissues, choice of antibodies for immunohistochemistry, and TMA data management.
Assuntos
Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Projetos de Pesquisa , Análise Serial de Tecidos/métodos , Anticorpos/imunologia , Humanos , Masculino , Estadiamento de Neoplasias , Neoplasias da Próstata/genética , Análise Serial de Tecidos/estatística & dados numéricos , Preservação de TecidoRESUMO
Identifying the genetic differences between two organisms or cell types has been a major goal in modern biomedical research. Recently, we developed a novel methodology that can rapidly identify the differences between two populations of DNA. This method, termed 'differential subtraction chain' (DSC), is based on a novel 'negative amplification' strategy that converts (amplifiable) tester sequences to counterpart (unamplifiable) driver sequences. The result is a double exponential elimination of amplifiable sequences in the testers, while preserving the sequences in the testers that have no counterpart in the drivers. We applied this methodology to the genome of a glioblastoma cell line. A homozygous deletion was rapidly identified. We extended this technique to identifying the unique sequences in mRNA. Two CDC25 transgene fragments were quickly identified in a cdc25B transgenic mouse. We also applied this methodology to systems with profound differences in mRNA expression. In a 'prostate epithelia subtracting blood cells' DSC reaction, a sample of unique gene fragments which are absent in the prostate but present in the blood were identified. Lastly, we detected rare (1 virus/100 cells) Herpes simplex virus type 2 (HSV-2) sequences in a tissue culture, indicating good sensitivity of this methodology. Overall, DSC represents a fast, efficient and sensitive method for identifying differences in genomic DNA and mRNA and can be easily applied in a variety of biological systems.
Assuntos
DNA/genética , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , Animais , DNA Viral/análise , Deleção de Genes , Genoma , Herpesvirus Humano 2/genética , Homozigoto , Humanos , Camundongos , Camundongos Transgênicos , Células Tumorais CultivadasRESUMO
Prostate-specific antigen (PSA) has been demonstrated to release the active form of insulin-like growth factor I in vitro (P. Cohen et al., J. Clin. Endocrinol. & Metab., 75: 1046-1053, 1992; P. Cohen et al., J. Clin. Endocrinol. & Metab., 79: 1410-1415, 1994; P. Cohen et al., Horm. Metab. Res., 26: 81-84, 1994) and has significant mitogenic activity on osteoblast cells, fibroblasts, and other cultured cells (C. S. Killian et al., Biochem. Biophys. Res. Commun., 192: 940-947, 1993). Recently, PSA has been found not only in prostate tissues but also in breast, colon, ovarian, and other tissues (E. P. Diamandis and H. Yu, J. Clin. Endocrinol. & Metab., 80: 1515-1517, 1995; E. P. Diamandis and H. Yu, Clin. Chem., 41: 204-210, 1995; A. Clements and A. Mukhtar, J. Clin. Endocrinol. & Metab., 78: 1536-1539, 1994). Therefore, PSA has been proposed as a candidate growth factor, cytokine, or growth factor regulator. In this setting, knowing how to manipulate or block the secretion of PSA by the prostate cancer cells could be a useful approach to controlling the progression of human prostate cancers. Using metabolic labeling experiments, we have studied the biosynthesis and secretion of PSA in LNCaP cells. We have also examined the effects of DTT, tunicamycin, 1-deoxymannojirimycin, pilocarpine, and testosterone on PSA biosynthesis and secretion. The results indicate that the secretion of PSA in LNCaP cells is constitutive instead of regulated and that the disruption of intramolecular disulfide bonds affects the transport of PSA from the endoplasmic reticulum to the Golgi apparatus. The biosynthesis of PSA is potentiated by testosterone and inhibited by brefeldin A and DTT. These results will help us understand PSA biosynthesis and secretion in human prostate cancers.
Assuntos
Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/metabolismo , Antígeno Prostático Específico/biossíntese , Antígeno Prostático Específico/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , 1-Desoxinojirimicina/farmacologia , Transporte Biológico/efeitos dos fármacos , Brefeldina A , Cloroquina/farmacologia , Ciclopentanos/farmacologia , Di-Hidrotestosterona/farmacologia , Ditiotreitol/farmacologia , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Humanos , Masculino , Proteínas de Neoplasias/química , Proteínas de Neoplasias/efeitos dos fármacos , Pilocarpina/farmacologia , Testes de Precipitina , Antígeno Prostático Específico/química , Antígeno Prostático Específico/efeitos dos fármacos , Saponinas/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Tunicamicina/farmacologiaRESUMO
The early diagnosis of bladder cancer is central to the effective treatment of the disease. Presently, there are no methods available to easily and specifically identify the presence of bladder cancer cells. The prevailing method for the detection of bladder cancer is the identification of bladder cancer cells by morphological examination of exfoliated cells or biopsy material by a pathologist. A hallmark of the malignant or transformed phenotype is an abnormal nuclear shape, the presence of multiple nucleoli, and altered patterns of chromatin organization. Nuclear structural alterations are so prevalent in cancer cells that they are commonly used as markers of transformation for many types of cancer. Nuclear shape is determined by the nuclear matrix, the dynamic skeleton of the nucleus. The nuclear matrix is the structural component of the nucleus that determines nuclear morphology, organizes the DNA in a three-dimensional fashion that is tissue specific, and has a central role in the regulation of a number of nuclear processes, including the regulation of DNA replication and gene expression. Previous investigations into prostate and breast cancer have revealed that nuclear matrix protein (NMP) composition undergoes alterations with transformation and that the nuclear matrix can serve as a marker for the malignant phenotype. In this study, we have identified NMPs with which it is possible to differentiate human bladder tumors from normal bladder epithelial cells. We examined the NMP composition of 17 matched tumor and normal samples from patients undergoing surgery for bladder cancer. We have identified six proteins present in all tumor samples that are not present in the corresponding normal samples and three proteins that are unique to the normal bladder tissues in comparison with the tumor samples. Five of the six bladder cancer-associated proteins were also identified in three human bladder cancer cells lines examined (253j, UMUC-2, and T24). Therefore, we have demonstrated that nuclear matrix composition is able to differentiate bladder cancer from normal bladder tissue and may provide useful tools for early detection and recurrence of the disease. Importantly, these markers may provide valuable tools for cytopathological screening for bladder carcinoma.
Assuntos
Biomarcadores Tumorais/análise , Proteínas Nucleares/análise , Neoplasias da Bexiga Urinária/química , Antígenos Nucleares , Humanos , Peso Molecular , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/diagnósticoRESUMO
Previous studies demonstrated that CD44 is a metastasis suppressor gene for prostate cancer and that the expression of CD44 both at mRNA and protein levels is down-regulated during prostate cancer progression, with down-regulation being correlated with higher tumor grade, aneuploidy, and distant metastasis. In this study, we evaluated DNA hypermethylation as a potential mechanism accompanying this decreased CD44 expression in human prostate cancer. Nucleotide sequence analysis revealed a CpG island in the CD44 transcriptional regulatory region. We found that cytosine methylation of CD44 promoter occurs in CD44-negative prostate cancer cell line (i.e., LNCaP) but not in prostate cancer cell lines (i.e., TSU, PC3, and DU145) expressing this gene. In addition, we examined methylation status of CD44 in 84 matched normal and cancer prostate specimens. Hypermethylation of the 5' CpG island of CD44 gene was observed in 31 of 40 primary prostate cancer specimens, 3 of 4 distant organ site metastases obtained at autopsy from men who died of prostate cancer, and 4 of the 40 matched normal tissues. These results demonstrated that methylation of the 5' CpG island of CD44 gene is closely associated with transcriptional inactivation, resulting in a decreased expression of CD44 in human prostate cancer.
Assuntos
Adenocarcinoma/genética , Ilhas de CpG , Metilação de DNA , DNA de Neoplasias/química , Regulação Neoplásica da Expressão Gênica , Receptores de Hialuronatos/genética , Metástase Neoplásica/genética , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , Transcrição Gênica , Adenocarcinoma/patologia , Adenocarcinoma/secundário , Progressão da Doença , Humanos , Masculino , Prostatectomia , Neoplasias da Próstata/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
We have identified previously six nuclear matrix proteins (NMPs) that are bladder cancer specific. In this study, we analyzed the expression of one of these proteins, BLCA-4, in bladder tumors and normal bladder tissue. We also examined the appearance of BLCA-4 in the urine as a biomarker for bladder cancer. BLCA-4 was isolated from nuclear matrix preparations of bladder tumors, and its peptide sequence was determined. The antibodies generated against the resulting BLCA-4 peptides were then used to detect its presence in immunoblots and in urine samples by immunoassay. We analyzed tissue samples of bladder tumor and normal donor bladders and urine obtained from 51 normal individuals and 54 patients with pathologically confirmed bladder cancer. The BLCA-4 peptide sequences do not resemble any known human protein sequences. On immunoblot analysis, BLCA-4 expression was detectable in tumor and normal tissues from patients with bladder cancer but not in any of the normal bladder tissue obtained from organ donors. Using a prospectively determined cutoff level of 13 A (absorbance) units/microg protein, all 51 normal individuals tested were negative for BLCA-4 expression, whereas 53 of 55 samples from patients with bladder cancer were positive. These results suggest that BLCA-4 is present throughout the bladder in both the tumor and morphologically normal areas in bladder cancer patients. BLCA-4 is a very sensitive (96.4%) and specific (100%) marker for bladder cancer. BLCA-4 is a bladder cancer-specific marker that can be detected using a urine-based assay and can be used in the diagnosis of bladder cancer.
Assuntos
Biomarcadores Tumorais/análise , Proteínas Nucleares/análise , Neoplasias da Bexiga Urinária/química , Bexiga Urinária/química , Adulto , Idoso , Anticorpos , Feminino , Humanos , Immunoblotting , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Proteínas Nucleares/química , Valores de Referência , Doadores de Tecidos , Bexiga Urinária/citologia , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/cirurgiaRESUMO
Diclofenac sodium is a widely used enteric-coated nonsteroidal anti-inflammatory drug. We describe a woman with Hemoccult-positive stools and iron deficiency anemia who developed both a colonic ulcer and a "diaphragm-like" colonic stricture while taking enteric-coated diclofenac. These lesions were evident on colonoscopy but not on barium studies. Biopsy specimens of the ulcer and stricture revealed particulate matter that was indistinguishable from diclofenac pill fragments by electron microscopy. Discontinuation of diclofenac therapy resulted in resolution of anemia and Hemoccult-positive stools. We conclude that (1) enteric-coated diclofenac is associated with both colonic ulcers and diaphragm-like colonic strictures; (2) the pathophysiologic mechanism for the development of both ulcers and strictures may involve a direct action of diclofenac within these lesions; (3) colonoscopy may be superior to barium studies in evaluating patients receiving diclofenac who have iron deficiency anemia and/or Hemoccult-positive stools.
Assuntos
Doenças do Colo/induzido quimicamente , Diclofenaco/efeitos adversos , Anemia Hipocrômica/induzido quimicamente , Biópsia , Colo/patologia , Constrição Patológica/induzido quimicamente , Preparações de Ação Retardada , Diclofenaco/uso terapêutico , Feminino , Humanos , Microscopia Eletrônica , Pessoa de Meia-Idade , Sangue Oculto , Úlcera/induzido quimicamenteRESUMO
We have developed a ligand-specific method for the visualization, isolation, and biochemical characterization of cell surface and intracellular membranes mediating endocytic transport. Iron dextran particles (FeDex) bearing either covalently conjugated galactosyl bovine serum albumin (GalBSA/FeDex) or asialofetuin (ASF/FeDex) are bound by the asialoglycoprotein receptor (ASGP-R) of HepG2 cells and transported to lysosomes with kinetics indistinguishable from those of free GalBSA or ASF. FeDex particles, which have a 3 to 5 nm electron-dense colloidal iron core, can be visualized by electron microscopy. Following incubation of GalBSA/FeDex with HepG2 cells at 37 degrees C, FeDex particles are seen at the cell surface, in endosomes, and in lysosomes. Surface membrane and intracellular organelles bearing a sufficient number of FeDex particles can be efficiently isolated from disrupted cells by high gradient magnetic affinity chromatography (HIMAC). Plasma membranes and endosomal/lysosomal membranes isolated by HIMAC are 35 to 40-fold enriched for GalBSA/FeDex or ASF/FeDex relative to the postnuclear supernatant. Alkaline phosphodiesterase I (APDE) and galactosyltransferase are each enriched 8-fold in the plasma membrane fraction prepared by HIMAC whereas neither beta-galactosidase nor glucose-6-phosphatase are detected in this fraction. The intracellular membrane fraction, containing both endosomes and lysosomes, is enriched for galactosyltransferase and beta-galactosidase but not for APDE or glucose-6-phosphatase. Use of FeDex conjugates in conjunction with HIMAC provides an effective method for ligand-specific isolation of membranes and correlation of morphological and biochemical characteristics.
Assuntos
Fracionamento Celular/métodos , Cromatografia de Afinidade/métodos , Endossomos/química , Glicoconjugados , Complexo Ferro-Dextran , Lisossomos/química , Animais , Linhagem Celular , Endossomos/ultraestrutura , Galactosidases/análise , Galactosiltransferases/análise , Glucose-6-Fosfatase/análise , Humanos , Ligantes , Lisossomos/ultraestrutura , Fosfodiesterase I , Diester Fosfórico Hidrolases/análise , Soroalbumina BovinaRESUMO
Colloidal iron dextran particles bearing wheat germ agglutinin (WGA/FeDex) were bound by glycoconjugates expressed at the surface of HepG2 cells. Bound WGA/FeDex was internalized when cells were incubated at 37 degrees C and accumulated in intracellular structures which have the same buoyant density as the plasma membrane when examined on Percoll density gradients. The intracellular structures containing WGA/FeDex were identified as multivesicular bodies (MVB) by transmission electron microscopy. WGA/FeDex was not transported to lysosomes nor did it interfere with uptake and transport of GalBSA to lysosomes by the asialoglycoprotein receptor. WGA/FeDex was seen predominantly in non-coated invaginations at the cell surface, suggesting it may enter cells at a different site than GalBSA/FeDex. Highly enriched plasma membranes and MVBs containing superparamagnetic [125I]WGA/FeDex particles were prepared by high gradient magnetic affinity chromatography (HIMAC). Plasma membranes prepared by HIMAC were enriched 30-fold for [125I]WGA/FeDex, 15-fold for alkaline phosphodiesterase I, and 9-fold for galactosyltransferase relative to the crude post-nuclear homogenate and consisted entirely of plasmalemmal sheets. Intracellular structures containing WGA/FeDex were enriched 35-fold for [125I]WGA/FeDex, 10-fold for alkaline phosphodiesterase I, and 10-fold for galactosyltransferase but did not contain lysosomal beta-galactosidase. WGA/FeDex has a different ultimate destination in HepG2 cells than ligands internalized by the asialoglycoprotein receptor and can be used to obtain highly enriched plasma membranes and MVBs from cultured cells.
Assuntos
Endossomos/metabolismo , Glicoconjugados/metabolismo , Complexo Ferro-Dextran/metabolismo , Aglutininas do Germe de Trigo/metabolismo , Animais , Sítios de Ligação , Transporte Biológico , Fracionamento Celular/métodos , Linhagem Celular , Membrana Celular/metabolismo , Cromatografia de Afinidade/métodos , Humanos , Ligantes , Lisossomos/metabolismo , Microscopia Eletrônica , Temperatura , Células Tumorais CultivadasRESUMO
Ten pancreatic secretory proteins have been demonstrated in differentiated pancreatic acinar carcinoma cells by the protein A-gold immunocytochemical approach. The high resolution of the technique has allowed for the localization of the different proteins in the cellular compartments involved in protein secretion: RER, Golgi and secretory granules. The quantitative evaluation of the labeling for amylase has demonstrated the presence of an increasing gradient in the intensity from the RER to the Golgi and to the secretory granules which may reflect the process of protein concentration along the secretory pathway. These results, together with those obtained using the pulse-labeling autoradiographic approach, demonstrate that differentiated acinar carcinoma cells are capable of processing secretory proteins. When intensities of labeling obtained for different proteins on acinar carcinoma cells were compared to those obtained on normal pancreatic acinar cells, major differences were observed for some proteins. In addition, studies performed on the pancreatic tissue of the tumor-bearing animals have shown the presence of morphological alterations in the acinar cells.
Assuntos
Carcinoma/enzimologia , Hidrolases/metabolismo , Pâncreas/enzimologia , Neoplasias Pancreáticas/enzimologia , Peptídeo Hidrolases/metabolismo , Animais , Carcinoma/ultraestrutura , Desoxirribonucleases/metabolismo , Histocitoquímica , Lipase/metabolismo , Masculino , Microscopia Eletrônica , Neoplasias Pancreáticas/ultraestrutura , Ratos , Ratos Endogâmicos F344 , Ribonucleases/metabolismo , alfa-Amilases/metabolismoRESUMO
The standard practice of tissue fixation in 10% formalin followed by embedding in paraffin wax preserves cellular morphology at the expense of availability and quality of DNA and RNA. The negative effect on cellular constituents results from a combination of extensive cross-linking and strand scission of DNA, RNA, and proteins induced by formaldehyde as well as RNA loss secondary to ubiquitous RNase activity and negative effects of high temperature exposure during paraffin melting, microscopic section collection, and tissue adherence to glass slides. An effective strategy to correlate cellular phenotype with molecular genotype involves microdissection of tissue sections based on specific histopathological features followed by genotyping of minute representative samples for specific underlying molecular alterations. Currently, this approach is limited to short-length polymerase chain reaction amplification (<250 bp) of DNA, due to the negative effects of standard tissue fixation and processing. To overcome this obstacle and permit both cellular morphology and nucleic acid content to be preserved to the fullest extent, we instituted a system of cold-temperature plastic resin embedding based on the use of the water-miscible methyl methacrylate polymer known as Immunobed (Polysciences, Warminster, PA). The system is simple, easy to adapt to clinical practice, and cost-effective. Immunobed tissue sections demonstrate a cellular appearance equivalent or even superior to that of standard tissue sections. Moreover, thin sectioning (0.5-1.0 microm thickness) renders ultrastructural evaluation feasible on plastic-embedded blocks. Tissue microdissection is readily performed, yielding high levels of long DNA and RNA for genomic and transcription-based correlative molecular analysis. We recommend the use of Immunobed or similar products for use in molecular anatomical pathology.
Assuntos
DNA/genética , Fígado/metabolismo , Inclusão em Plástico/métodos , RNA/genética , Temperatura Baixa , DNA Viral/genética , DNA Viral/isolamento & purificação , Genótipo , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Humanos , Fígado/virologia , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Pancreatic acinar carcinoma fragments, pulse-chase labeled with [3H]-leucine, responded to carbamylcholine chloride by increased secretion of [3H]leucine-labeled protein into external buffer medium. Secretion of labeled protein by the carcinoma fragments increased in concentration-dependent fashion between 10(-8) -10(-5) M carbamylcholine, was completely inhibited at 4 degrees C, and was accompanied by an equivalent increase in the secretion of preformed amylase. A maximally effective carbamylcholine concentration of 10(-5) M was observed for both carcinoma fragments and normal pancreas lobules. However, the maximal rate of protein secretion by the carcinoma fragments was only approximately one-fifth the rate determined for the normal pancreas lobules. This modest secretory response indicates a population of partially differentiated cells in the pancreatic acinar carcinoma which secrete less enzyme than fully differentiated adult pancreatic acinar cells. Secretory responsiveness can be utilized as a quantitative acinar cell response in studies on differentiation in pancreatic carcinoma.
Assuntos
Carbacol/farmacologia , Carcinoma/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , Amilases/metabolismo , Animais , Leucina , Masculino , Colagenase Microbiana/farmacologia , Neoplasias Experimentais/metabolismo , Ratos , Ratos Endogâmicos F344 , TrítioRESUMO
Deletions were constructed within a functional human immunodeficiency virus type 1 (HIV-1) proviral clone in order to assess the role of the envelope protein in virus particle formation. A graded exonuclease deletion technique was used to produce 12 clones with deletions of 175-308 nucleotides in the first conserved domain of envelope. This included 9 clones with frameshift deletions and 3 clones with in-frame deletions. Isogenic pairs of env deletion clones were produced with or without an additional deletion in the vif and vpr genes. Upon transfection, all clones produced virus particles, as determined by p24 antigen, reverse transcriptase, and sucrose gradient assays with conditioned media. Virus particles produced from clones with deletions in env or vif and vpr, or both regions, banded on sucrose gradients with a mobility similar to that of virus produced by the parental clone. The p24 gag capsid protein in the particles was resistant to trypsin, but the particles were disrupted by treatment with Triton X-100, suggesting the presence of a surrounding lipid bilayer. Furthermore, electron microscopic studies revealed both mature and immature virus particles derived from COS cells transfected with the env deletion clones. Cocultivation experiments with lymphoid cells and cells transfected with each of the env deletion clones demonstrated that the virus particles were noninfectious.
Assuntos
HIV-1/fisiologia , Proteínas dos Retroviridae/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Linhagem Celular , Centrifugação com Gradiente de Concentração , Deleção Cromossômica , Clonagem Molecular , Produtos do Gene gag/análise , Genes env , Genes vif , Genes vpr , Antígenos HIV/análise , Proteína do Núcleo p24 do HIV , HIV-1/genética , HIV-1/ultraestrutura , Humanos , Microscopia Eletrônica , Provírus/genética , Proteínas dos Retroviridae/genética , Transfecção , Proteínas do Core Viral/análise , Proteínas do Envelope Viral/genética , Replicação ViralRESUMO
The Pittsburgh Reference Library Alliance (RLA) represents a successful response by hospital laboratories to the new realities of medical economics and practice. By using informatics technology to integrate the laboratory resources of community hospitals and academic medical centers across western Pennsylvania, the RLA has created a large virtual laboratory that can compete for price with large national referral laboratories. More significantly, the combination of medical expertise, the ties to academic and community centers, and the regional medical database of the RLAs allows laboratory medicine to be practiced in a new proactive way. This should provide better and more cost-effective patient care. The success of the RLA is a model for regional cooperation in pathology and potentially in other medical specialties and demonstrates the importance of informatics in the future of medical practice.
Assuntos
Laboratórios Hospitalares/organização & administração , Modelos Organizacionais , Patologia Clínica/organização & administração , Centros Médicos Acadêmicos/economia , Centros Médicos Acadêmicos/organização & administração , Análise Custo-Benefício , Bases de Dados Factuais , Hospitais Comunitários/economia , Hospitais Comunitários/organização & administração , Humanos , Laboratórios Hospitalares/economia , Laboratórios Hospitalares/tendências , Informática Médica/economia , Informática Médica/organização & administração , Inovação Organizacional , Patologia Clínica/economia , Patologia Clínica/tendências , Pennsylvania , Guias de Prática Clínica como AssuntoRESUMO
OBJECTIVE: To develop and evaluate a diagnostic algorithm based on the alteration of mast cell and nerve fiber observed in bladder tissue of patients with interstitial cystitis (IC). MATERIALS AND METHODS: Non-IC samples from 6 control groups (N = 10, 10, 13, 2, 11, and 3, respectively) and nonclassic interstitial cystitis (NC-IC, N = 20) were stained with Giemsa stain in order to calculate the detrusor to mucosa mast cell ratio (DMMCR) using quantitative image analysis and morphometry (QIAM). Immunohistochemical staining for S-100 protein was also performed to quantify nerve fiber proliferation in the detrusor muscle of the bladder. RESULTS: The average DMMCR of NC-IC was 1.19. Bacille Calmette-Guérin (BCG) cystitis was 0.84 and microscopically normal bladder tissue from patients with bladder or prostate cancer was 0.45. No case of IC that we examined had a DMMCR < 0.5. The number and percentage area of nerve fibers in the detrusor in IC were increased compared to controls and BCG (IC, 2.01%; BCG, 0.95%; control, 1.3%). CONCLUSION: A diagnostic algorithm is proposed for IC based on the findings that indicate that: 1) if the DMMCR > 0.75, then IC is present; 2) if the DMMCR < 0.5, then IC is negative; and 3) if the DMMCR is between 0.5 and 0.75, a quantitative S-100 protein staining analysis can be employed to evaluate nerve fiber proliferation to detect those marginal cases of NC-IC. The findings of the study also suggest that a neuroimmune process or mediation may be involved in the pathogenesis of IC.
Assuntos
Algoritmos , Cistite Intersticial/patologia , Mastócitos/patologia , Fibras Nervosas/patologia , Humanos , Músculo Liso/patologiaRESUMO
OBJECTIVES: The relationship between perineural invasion and prognosis has been demonstrated to be poor in a number of malignancies. This has not been evaluated in the bladder. We performed a study to determine the occurrence of nodal metastases, extranodal metastases, and disease-free survival in patients with perineural invasion (PNI) and/or angiolymphatic invasion (ALI) in transitional cell carcinoma of the bladder (TCCB) from radical cystectomy specimens. METHODS: A retrospective review of 27 patients treated with radical cystectomy for TCCB was conducted. Comparisons were performed between three groups: PNI with or without ALI (PNI +/- ALI, 12 patients), ALI alone (8 patients), and a control group (no PNI or ALI) (7 patients). RESULTS: The mean patient age was 70 years (range 49 to 83). The overall median follow-up period was 11 months (range 1 to 32). PNI +/- ALI was predominantly found in Stage T3b disease (14 of 20 [70%] cases). The overall 1-year disease-free survival was 48%, 67%, and 83% for the PNI +/- ALI, ALI alone, and control groups, respectively. Nodal metastases (for all stages combined) were found in 6 of 12 (50%), 3 of 8 (38%), and 1 of 7 (14%) patients in the PNI +/- ALI, ALI alone, and control groups, respectively. Similarly, extranodal metastatic disease was found in 5 of 12 (42%), 4 of 8 (50%), and 1 of 7 (14%) patients in the PNI +/- ALI, ALI alone, and control groups, respectively. The percentage of deaths for the PNI +/- ALI, ALI only, and control groups were 33%, 50%, and 14%, respectively. CONCLUSIONS: In TCCB, perineural invasion with or without angiolymphatic invasion and angiolymphatic invasion alone are associated with a higher incidence of nodal and extranodal metastases and death.
Assuntos
Carcinoma de Células de Transição/secundário , Carcinoma de Células de Transição/cirurgia , Cistectomia , Neoplasias do Sistema Nervoso/secundário , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/cirurgia , Neoplasias Vasculares/secundário , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células de Transição/epidemiologia , Feminino , Seguimentos , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Neoplasias do Sistema Nervoso/epidemiologia , Prognóstico , Estudos Retrospectivos , Neoplasias Vasculares/epidemiologiaRESUMO
OBJECTIVES: Current primary prostate cell culture techniques use an overnight digestion or extensive media preparation. In this report, we describe a method for the culture of benign and neoplastic cells from human prostatectomy specimens that is rapid and contains no undefined factors in the medium. METHODS: Characterization of the human cultured prostate cells was performed using immunohistochemical methods and monoclonal antibodies AE1/AE3 and cytokeratin 8, as well as monoclonal antibodies against prostate-specific antigen (PSA). Polymerase chain reaction was used to measure the exclusive epithelial and stromal cell products, c-met and hepatocyte growth factor (HGF), respectively. Electron microscopy was performed to assess the cell junctions and morphologic features of epithelial cells. Optimum cell growth in different media was tested using a cell replication assay. RESULTS: Microscopic evidence revealed that the cells demonstrate typical epithelial morphology, with polyhedral cells forming tight junctions in a continuous monolayer. Desmosomes were present in electron micrographs of epithelial cells. The cultured epithelial cells described in this report also demonstrate positive cytokeratin staining. The epithelial cells reacted positively with PSA antibody, indicating that the cells retain their secretory role in cell culture for a limited period. Epithelial cells expressed the HGF receptor, c-met; stromal cells secreted HGF. Insulin, transferrin, and selenium increased the growth of cells in the chemically defined media, compared with minimum essential media (MEM) and Ham's F12. CONCLUSIONS: In summary, essentially pure cultures of prostate stromal or epithelial cells have been established using simple isolation and culture methods. These cells will be useful for the investigation of related growth factors, such as insulin-like growth factor I and insulin-like growth factor II, and in understanding the basis for stromal-epithelial cell interactions.
Assuntos
Hiperplasia Prostática/patologia , Neoplasias da Próstata/patologia , Células Cultivadas , Técnicas Citológicas , Epitélio/metabolismo , Epitélio/patologia , Fator de Crescimento de Hepatócito/biossíntese , Humanos , Imuno-Histoquímica , Masculino , Proteínas Proto-Oncogênicas c-met , Receptores Proteína Tirosina Quinases/biossíntese , Células Estromais/metabolismoRESUMO
OBJECTIVES: 1,25-Dihydroxyvitamin D can inhibit the proliferation of prostate cancer cells, but its clinical use is limited by hypercalcemia. We examined the effects of a "noncalcemic" vitamin D analogue, 1,25-Dihydroxy-16-ene-23-yne-cholecalciferol (16-23-D3), on the proliferation of human prostate cancer cells in a mouse model. METHODS: Twenty-four athymic nude mice were inoculated with human prostate carcinoma cells from the PC-3 cell line. Twelve mice (experimental group) received injections of 1.6 micrograms of 16-23-D3 on alternate days over a 22-day period. Twelve mice (control group) received sham injections. Tumor volumes, pathologic findings, and terminal serum calcium levels were compared between groups. RESULTS: The relative increase in tumor volume was significantly lower in the experimental than in the control group in the first interval following treatment (P < 0.01). Mean tumor volumes in the experimental group were approximately 15% smaller than in the control group. Serum calcium levels did not differ between groups. CONCLUSIONS: 16-23-D3 showed modest antiproliferative effects on prostate cancer cells in this model without evidence of drug-induced hypercalcemia. These findings support the concept that vitamin D analogues can inhibit the proliferation of human prostate cancer cells in vivo.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/uso terapêutico , Calcitriol/análogos & derivados , Neoplasias da Próstata/tratamento farmacológico , Adenocarcinoma/patologia , Animais , Antineoplásicos/farmacologia , Calcitriol/farmacologia , Calcitriol/uso terapêutico , Cálcio/sangue , Divisão Celular/efeitos dos fármacos , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Projetos Piloto , Neoplasias da Próstata/patologia , Células Tumorais Cultivadas/transplanteRESUMO
Our prospective study was designed to evaluate the routine use of frozen section of pelvic lymph node specimens prior to radical prostatectomy in patients with pre-biopsy prostate specific antigen (PSA) levels less than 20 ng/ml. Included in our study were 241 patients who had clinically localized disease on digital rectal examination, a negative preoperative metastatic work-up, and a pre-biopsy serum PSA of less than 20 ng/ml. If a palpable abnormality of pelvic lymph nodes was detected at the time of surgery, specimens were sent for frozen section analysis. Lymphatic specimens that were palpably normal were sent for permanent section only. Ten (4.1%) of the 241 patients had pelvic lymph node metastases demonstrated by permanent section. Of the patients with lymph node metastasis, 50% had Gleason scores of 8-10 on their needle biopsy specimens. None of these patients had frozen section analysis of their lymph nodes. Frozen section failed to detect lymph node metastasis in all cases analyzed. Routine frozen section analysis of pelvic lymphadenectomy specimens in patients with clinically localized prostate cancer, Gleason score 2-7 on the needle biopsy, and prebiopsy PSA of less than 20 ng/ml is unnecessary.