RESUMO
The reliable analysis of the cell cycle status has become increasingly relevant for scientific and clinical work, especially for the determination of tumor cell growth. One established method to characterize the proliferation activity of cells is the analysis of the Ki-67 protein. Ki-67 is expressed in the nucleus during the whole cell cycle except for the G0 phase. Several different protocols exist for the examination of the Ki-67 protein in tissue and cell culture, but most of them are defined for human cells. For the analysis of the Ki-67 protein in murine tissue and cell culture there is a variety of protocols existing which recommend different fixation and permeabilization reagents or special kits. In this study, we established a reliable protocol for Ki-67 staining in murine cells and tissue based on PFA fixation, which can be used not only for flow cytometry but also for immunofluorescence microscopy analysis. We tested our protocol successfully with three different Ki-67 anti-mouse antibodies in cell culture, regenerating liver tissue and mouse melanoma tumor to demonstrate the general applicability.
Assuntos
Proliferação de Células/fisiologia , Antígeno Ki-67/análise , Coloração e Rotulagem/métodos , Animais , Divisão Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citometria de Fluxo/métodos , Humanos , Antígeno Ki-67/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência/métodos , Células Tumorais CultivadasRESUMO
While the development of cerebellar granule and Purkinje neurons has been extensively studied, little is known about the developmental mechanisms that lead to the generation and diversification of inhibitory GABAergic interneurons of the cerebellar cortex. To address this issue, we compared gene expression in complete, early postnatal murine cerebella to that in cerebella from which immature inhibitory interneurons and their precursors had been stripped based on their expression of green fluorescent protein (GFP) from the Pax2 locus. We identified some 300 candidate genes selectively enriched within immature cerebellar cortical inhibitory interneurons and/or their precursors, many of which were also expressed in their adult descendants and/or the embryonic cerebellar ventricular epithelium that gives rise to these cells. None of the genes identified, among them Tcfap2alpha, Tcfap2beta, Lbxcor1 and Lbx1, was cell-type specific. Rather, gene expression, and also splicing, changed dynamically during development and rather reflects stage of differentiation than lineage. Consistently, cluster analysis of transcriptional regulators and genes specific for adult cerebellar GABAergic cells does not suggest a hierarchical lineage relationship or an early commitment of subtypes of cerebellar cortical inhibitory interneurons. Together, these data support the notion that diversification of cerebellar inhibitory interneurons is highly regulative and subject to local signaling to postmigratory precursors.
Assuntos
Córtex Cerebelar/crescimento & desenvolvimento , Impressões Digitais de DNA , Regulação da Expressão Gênica no Desenvolvimento/genética , Interneurônios/metabolismo , Inibição Neural/genética , Células-Tronco/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Córtex Cerebelar/citologia , Análise por Conglomerados , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator de Transcrição PAX2/genéticaRESUMO
Synovial sarcomas account for 5-10% of all soft tissue sarcomas and the majority of synovial sarcomas display characteristic t(X;18) translocations that result in enhanced transcription of the insulin-like growth factor-2 (IGF-2) gene. IGF-2 is an essential fetal mitogen involved in the pathogenesis of different tumours, leading to cellular proliferation and inhibition of apoptosis. Here we asked whether activation of IGF signalling is of functional importance in synovial sarcomas. We screened human synovial sarcomas for expression of IGF-2 and the phosphorylated IGF-1 receptor (IGF-1R), which mainly mediates the proliferative and anti-apoptotic effects of IGF-2. Since both the phosphatidylinositol 3'-kinase (PI3K)-AKT pathway and the MAPK signalling cascade are known to be involved in the transmission of IGF-1R signals, expression of phosphorylated (p)-AKT and p-p44/42 MAPK was additionally assessed. All tumours expressed IGF-2 and 78% showed an activated IGF-1R. All tumours were found to express p-AKT and 92% showed expression of activated p44/42 MAPK. To analyse the functional and potential therapeutic relevance of IGF-1R signalling, synovial sarcoma cell lines were treated with the IGF-1R inhibitor NVP-AEW541. Growth was impaired by the IGF-1R antagonist, which was consistently accompanied by a dose-dependent reduction of phosphorylation of AKT and p44/42 MAPK. Functionally, inhibition of the receptor led to increased apoptosis and diminished mitotic activity. Concurrent exposure of selected cells to NVP-AEW541 and conventional chemotherapeutic agents resulted in positive interactions. Finally, synovial sarcoma cell migration was found to be dependent on signals transmitted by the IGF-1R. In summary, our data show that the IGF-1R might represent a promising therapeutic target in synovial sarcomas.
Assuntos
Receptor IGF Tipo 1/fisiologia , Sarcoma Sinovial/metabolismo , Transdução de Sinais/fisiologia , Adulto , Antineoplásicos/farmacologia , Apoptose , Western Blotting/métodos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Imunofluorescência , Humanos , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Pirróis/farmacologia , RNA Mensageiro/análise , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sarcoma Sinovial/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
The inositol 5-phosphatase SHIP (SHIP-1) is a negative regulator of signal transduction in hematopoietic cells and targeted disruption of SHIP in mice leads to a myeloproliferative disorder. We analyzed the effects of SHIP on the human leukemia cell line Jurkat in which expression of endogenous SHIP protein is not detectable. Restoration of SHIP expression in Jurkat cells with an inducible expression system caused a 69% reduction of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and a 65% reduction of Akt kinase activity, which was associated with reduced phosphorylation of glycogen synthase kinase 3beta (GSK-3beta) (Ser-9) without changing the phosphorylation of Bad (Ser-136), FKHR (Ser-256) or MAPK (Thr-202/Tyr-204). SHIP-expressing Jurkat cells showed an increased transit time through the G1 phase of the cell cycle, but SHIP did not cause a complete cell cycle arrest or apoptosis. Extension of the G1 phase was associated with an increased stability of the cell cycle inhibitor p27(Kip1) and reduced phosphorylation of the retinoblastoma protein Rb at serine residue 780. Our data indicate that restoration of SHIP activity in a human leukemia cell line, which has lost expression of endogenous SHIP, downregulates constitutively activated phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling and leads to an increased transit time through the G1 phase of the cell cycle.
Assuntos
Fase G1 , Quinase 3 da Glicogênio Sintase/metabolismo , Leucemia/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Apoptose , Proteínas de Transporte/metabolismo , Inibidor de Quinase Dependente de Ciclina p27 , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Ativação Enzimática , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead , Glicogênio Sintase Quinase 3 beta , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Jurkat , Leucemia/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Fosforilação , Proteínas Proto-Oncogênicas c-akt , Proteína do Retinoblastoma , Fatores de Tempo , Fatores de Transcrição/metabolismo , Proteína de Morte Celular Associada a bcl , Domínios de Homologia de srcRESUMO
The occurrence of intracellularly and extracellularly generated free radicals during shock wave exposure on an experimental Siemens lithotripter was tested with the radical sensitive dyes hydroethidine and dichlorofluorescin (DCFH). DCFH, a nonfluorescent compound, is oxidised to dichlorfluorescein (DCF) by hydrogen peroxide in the presence of peroxidase. DCF green fluorescence intensity was used for fluorescence spectrometric measurement of hydrogen peroxide generated during shock-wave treatment of cell-free dye solutions. The fluorescence intensity of ethidium, the oxidised form of hydroethidine, was used for the flow-cytometric measurement of intracellular oxidising reagents present in RT4 tumour cells during shock-wave exposure. Changes in membrane permeability, which influence the intracellular content of ethidium, were controlled by counterstaining the cells with propidium iodide, an indicator for membrane integrity. We observed no increase in intracellular ethidium fluorescence intensity after shock-wave treatment of single cell suspensions and therefore no indication for shock-wave-induced intracellular free radicals.
Assuntos
Carcinoma Papilar/metabolismo , Corantes Fluorescentes , Litotripsia/instrumentação , Fenantridinas , Neoplasias da Bexiga Urinária/metabolismo , Permeabilidade da Membrana Celular , Etídio/química , Espaço Extracelular/metabolismo , Citometria de Fluxo , Fluoresceínas , Radicais Livres/análise , Humanos , Peróxido de Hidrogênio/metabolismo , Líquido Intracelular/metabolismo , Oxirredução , Peroxidases/metabolismo , Propídio , Espalhamento de Radiação , Espectrometria de Fluorescência , Células Tumorais CultivadasRESUMO
Shock-wave-induced cell damage of suspended or pelleted bladder cancer cells was analysed with the flow cytometric propidium iodide (PI)/fluorescein diacetate assay, and electrical volume determination using the CASY 1 analyser system and growth curves. The CASY system revealed a smaller fraction of suspended RT4 cells with impaired membrane integrity than the flow cytometric assay. No differences were found for pelleted RT4 cells and suspended J82 cells. The discrepancies of the two viability assays indicated a different response of the cell membrane to shock waves which was dependent on the exposure system and the cell type. Growth curves indicated delayed cell death for suspended RT4 cells and exclusively immediate cell death for pelleted RT4 cells and suspended J82 cells. PI positive suspended RT4 cells were morphologically intact while pelleted RT4 cells and suspended J82 cells were mainly disrupted. From these data it can be concluded that intracellular or membrane alterations seem to be correlated with the occurrence of cavitational effects while cell disruption can likewise occur by the direct impact of the shock wave.
Assuntos
Carcinoma de Células de Transição/patologia , Ultrassonografia , Neoplasias da Bexiga Urinária/patologia , Contagem de Células , Morte Celular , Divisão Celular , Permeabilidade da Membrana Celular , Sobrevivência Celular , Citometria de Fluxo , Humanos , Microscopia de Fluorescência , Células Tumorais CultivadasRESUMO
Nanotechnology, the manufacture and use of structures and implements of around a few 100 nm in size, is becoming a key technology of the twenty-first century. An important element for the manufacture of nanoparticles is gold. Gold nanoparticles can be custom made and chemically modified in their size and form. Initial investigations have shown that they are physiologically non-hazardous. A potential application is in neovascular age-related macular degeneration. Gold nanoparticles of suitable dimensions introduced into newly forming blood vessels can be targeted and heated which selectively destroys these blood vessels. This principle has already been demonstrated in cultivated endothelial cells.
Assuntos
Nanotecnologia/métodos , Oftalmologia/métodos , Neovascularização de Coroide/patologia , Neovascularização de Coroide/terapia , Endotélio Vascular/patologia , Ouro , Humanos , Nanopartículas Metálicas , Microscopia Eletrônica , Degeneração Macular Exsudativa/patologia , Degeneração Macular Exsudativa/terapiaAssuntos
Citometria de Fluxo/métodos , Proteínas Luminescentes/genética , Plasmídeos/análise , Replicação do DNA/genética , Eletroporação , Genes Reporter/genética , Proteínas de Fluorescência Verde , Herpesvirus Humano 4/genética , Humanos , Plasmídeos/genética , Transfecção/genética , Células Tumorais CultivadasRESUMO
Whenever studied in a quantitative fashion, hypertonicity-induced cation channels (HICCs) are found to be the main mediators of regulatory volume increase. In most instances, these channels are either inhibited by amiloride (but insensitive to Gd3+ and flufenamate) or they are efficiently blocked by Gd3+ and flufenamate (but insensitive to amiloride). Of note, however, from two preparations so far a mixed type of pharmacology has also been reported. Whereas the ion selectivity of amiloride-sensitive HICCs has not been studied in much detail yet, amiloride-insensitive channels are either equally permeable to Na+, K+, Cs+ and Li+ but impermeable to N-methyl-D-glucamine (NMDG+) or they exhibit a permeability to Li+ and NMDG+ that amounts to some 50% when compared with that of Na+. Also in this respect, however, some peculiarities do exist. Concerning the actual molecular correlate, evidence was reported that HICCs may be related to the (amiloride-sensitive) epithelial Na+ channel and/or to transient receptor potential channels. Recent findings suggest that HICCs may contribute to cell proliferation, just as the K+ channels that are employed in regulatory volume decrease are mediators of the opposing process, i.e. apoptosis.
Assuntos
Amilorida/farmacologia , Células Epiteliais/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Animais , Cátions , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular , Tamanho Celular , Células Epiteliais/efeitos dos fármacos , Humanos , Soluções Hipertônicas/metabolismo , Concentração Osmolar , Canais de Potássio/metabolismoRESUMO
The Ki-67 protein is a nuclear and nucleolar protein, which is tightly associated with somatic cell proliferation. Antibodies raised against the human Ki-67 protein paved the way for the immunohistological assessment of cell proliferation, particularly useful in numerous studies on the prognostic value of cell growth in clinical samples of human neoplasms. The primary structure revealed potential phosphorylation site for a range of essential kinases, PEST sequences, and a forkhead-associated domain, which are features present in a variety of cell-cycle-regulating proteins, but information about the position of the Ki-67 protein within the protein network that drives the cell cycle remained scarce. There is now evidence that posttranslational modifications based on phosphorylation by cdc2 kinase and PKC accompany the remarkable redistribution of the Ki-67 protein from the interior of the nucleus to the perichromosomal layer during mitosis and vice versa. The discovery of Ki-67 equivalents in other species is advantageous for a precise and cross-species investigation of the structural requirements for its yet unknown function. The recently published data add new pieces to the challenging puzzle of this multifaceted protein, which are waiting to be put together.
Assuntos
Antígeno Ki-67/fisiologia , Animais , Ciclo Celular , Humanos , Antígeno Ki-67/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Ki-67 is a nuclear protein present in all proliferating cells that are in the active part of the cell division, but not in resting cells. This feature is extensively used in tumor diagnostics to estimate the growth fraction of a given cell population. We now demonstrate that the spatial and temporal regulation of the Ki-67 protein during the cell cycle is associated with mitosis-specific phosphorylation. These posttranslational modifications of the Ki-67 protein are accompanied by a characteristic redistribution of the protein from the interior of the nucleus to the periphery of the condensed chromosomes and vice versa. Phosphorylation could be suppressed by activating cell-cycle checkpoints that control the entry into mitosis through the activity of the cyclin B/cdc2 complex. In vitro experiments confirm that the presence of the cdc2 kinase and its regulatory subunit cyclin B is required for the phosphorylation of the Ki-67 protein. We further demonstrated that the Ki-67 protein is a new member of the family of MPM-2 reactive phosphoproteins, which includes both structural and functional proteins that are necessary for the control and timing of mitosis. Phosphorylation and dephosphorylation of the Ki-67 protein are therefore controlled by key regulatory structures of the cell cycle and occur at two hallmark events within the cell cycle: the breakdown and the reorganization of the nucleus during mitosis.
Assuntos
Antígeno Ki-67/metabolismo , Metáfase/fisiologia , Prófase/fisiologia , Processamento de Proteína Pós-Traducional , Trifosfato de Adenosina/farmacologia , Anáfase/fisiologia , Anticorpos , Antineoplásicos/farmacologia , Proteína Quinase CDC2/metabolismo , Ciclina B/metabolismo , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Epitopos/imunologia , Epitopos/metabolismo , Citometria de Fluxo , Imunofluorescência , Fase G2/efeitos dos fármacos , Fase G2/fisiologia , Fase G2/efeitos da radiação , Células HeLa , Humanos , Antígeno Ki-67/análise , Antígeno Ki-67/imunologia , Nocodazol/farmacologia , Fosfoproteínas/imunologia , Fosfoproteínas/metabolismo , Radioisótopos de Fósforo , Fosforilação , Fase S/efeitos dos fármacos , Fase S/fisiologia , Estaurosporina/farmacologia , Timidina/antagonistas & inibidoresRESUMO
Flow cytometric multiparameter analysis of two proliferation associated antigens, Ki-67 and p120, was combined with cell cycle kinetic analysis, achieved by continuous labeling with 5-Bromodeoxyuridine (BrdUrd), followed by staining with Hoechst 33258 and 7-Aminoactinomycin D (7AAD). Exponential and plateau phase monolayer cultures of the human bladder carcinoma cell line J82 were examined. Resting cells, characterized by their absent BrdUrd incorporation, showed no reactivity with the MIB1 antibody, which was used for the detection of the Ki-67 antigen. Proliferating cells revealed a cell cycle phase dependent Ki-67 staining intensity, which was partially related to the time period spent in G1 after mitosis. In contrast to the Ki-67 antigen expression, no decrease in p120 immunofluorescence staining intensity of non-cycling cells could be observed. We could demonstrate that a dissection of the history of cell replication, obtained by the BrdUrd/Hoechst technique combined with a simultaneous immunofluorescence staining reveals detailed information, on a single cell level, about time dependent expression of proliferation associated antigens in all cell cycle compartments.
Assuntos
Antígenos de Neoplasias/análise , Dactinomicina/análogos & derivados , Citometria de Fluxo/métodos , Corantes Fluorescentes , Antígeno Ki-67/análise , Proteínas Nucleares/análise , Bisbenzimidazol , Bromodesoxiuridina , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Microscopia de Fluorescência , Células Tumorais Cultivadas , tRNA MetiltransferasesRESUMO
The mini-chromosome maintenance proteins (MCM), which are involved in the control of DNA replication, and the cyclin-dependent kinase inhibitors, such as p27/KIP1, represent two groups of proteins that are currently under investigation as diagnostic tumour markers. The expression of p27 and MCM3 was compared with the expression of the Ki-67 protein, an approved marker for proliferating cells, extensively used in histopathology and cancer research. The expression pattern of all three proteins was assessed on germinal centres and oral mucosa, which display a well-defined spatio-temporal organization. The expression of the p27 protein was closely related to differentiated cells, whereas MCM3 and Ki-67 were predominantly localized to the regions of proliferating cells. However, it is important to note that considerable numbers of cells that were growth-arrested, as confirmed by the absence of the Ki-67 protein, stained positive for the MCM3 protein. These results were verified in vitro using growth-arrested Swiss 3T3. The MCM3 protein is therefore expressed in cells that have ceased to proliferate, but are not terminally differentiated, according to the absence of p27 protein expression. In conclusion, a combined analysis of Ki-67, MCM3, and p27 protein expression may provide a more detailed insight into the cell proliferation and differentiation processes that determine individual tumour growth.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/fisiologia , Antígeno Ki-67/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Western Blotting , Divisão Celular/fisiologia , Proteínas de Ligação a DNA , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Componente 3 do Complexo de Manutenção de Minicromossomo , Proteínas NuclearesRESUMO
Flow cytometric multi-parameter analysis has proven to be a powerful tool to characterize subpopulations of cell suspensions, and is applied routinely in hematology. However, in studies of cancer where there is interest in defining phenotypic markers in conjunction with DNA content, this method has hardly been applied [6]. Our objective was to develop a methodology that extends previous investigations on relative and absolute quantitation of the epidermal growth factor receptor (EGFR) in dual parameter analysis in vitro on human bladder cancer cell lines [3]. In order to quantitate EGFR content in tumours and to relate it to DNA content, tumour selection, DNA-content, and EGFR-content measurements should be carried out simultaneously. Different fluorescent dyes were used to optimize DNA assessment and antibody staining, using a single laser instrument as a practical approach for clinical routine. In vitro cultures were used to validate the quality of tumour cell selection and antigen quantitation. Therefore, two urothelial tumour cell lines-lowly and highly differentiated-were incubated under different conditions: monolayer (ML), three-dimensional multi-cellular spheroids (MCS) and cocultures (COCU) with the fibroblast cell line NI were investigated and EGFR quantitation was related to S-phase fraction (SPF). Accurate determination of instrument settings allows simultaneous three-colour analysis with DNA assessment. Tumor cell selection based on staining with phycoerythrine (R-PE) against a highly expressed urothelial glycoprotein, detected with the antibody Uro5 or against cytokeratin appeared to be possible in FL2, using the fluorochrome combination fluorescein-isothiocyanate (FITC), R-PE and propidiumiodide (PI). Using this staining protocol, relative and absolute EGFR quantitation (quantum simply cellular beads) is shown to be accurate, when FITC is used for EGFR staining and measured in the green fluorescence channel (FL1). Using this colour combination EGFR content and SPF of tumour cells were compared in different growth states, and could be monitored reliably. In spite of a higher emission spectrum of 7-aminoactinomycin-D (7-AAD), this DNA stain provided no advantage over PI. Broad coefficients of variation (CV) were found when intact cells were stained, thus hindering accurate assessment of ploidy and S-phase fraction. Similarly, Syto-13, a DNA dye detected in FL1, could not be optimized for multi-parameter measurements. Although the emission maximum is at 520 nm, the spectrum is too wide to compensate fluorescence overlap in FL2 or FL3. Quantum Red (QR), used as a streptavidin conjugate in FL3, could not be combined with two other colours for DNA staining, since sufficient compensation was not obtainable when an argon ion laser is used. The coculture model allows verification of tumour cell selection and discrimination. The high differentiated tumour cell line RT4 shows an unambiguous correlation between EGFR content and S-phase fraction. The low differentiated tumour cell line J82 presents a similar pattern of post-transcriptional EGFR regulation with respect to culture condition, however, the S-phase fraction is basically unaffected.
Assuntos
Carcinoma/genética , Carcinoma/patologia , DNA de Neoplasias/análise , Receptores ErbB/análise , Citometria de Fluxo/métodos , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Humanos , Espectrometria de Fluorescência/métodos , Células Tumorais CultivadasRESUMO
The monoclonal antibody Ki-S2 binds to a recently characterized proliferation-specific protein, p100. To assess its distribution pattern under physiologic and pathologic conditions, we performed immunohistochemical analyses on an exhaustive spectrum of normal tissues, 624 miscellaneous solid cancers, and 95 hematologic malignancies, and compared the results with Ki-67 immunostaining on consecutive sections. In addition, Ki-S2 expression was related to the DNA content by dual parameter flow cytometric analysis in parallel with Ki-67 labeling using a human cancer cell line. Immunoreactivity was enhanced at the G1/S transition and persisted through G2 and M phase. After adequate antigen retrieval, the antibody was found to yield identical results on fresh and formalin-fixed, paraffin-embedded material. The antibody specifically labeled actively proliferating cells, which constitute a subset of the population recognized by Ki-67. In normal human tissues, Ki-S2 immunolabeling hardly ever exceeded 40% of the Ki-67+ cell fraction. Immunoreactive scores of the two antibodies exhibited a linear correlation, but statistically significant differences in the ratio of Ki-S2-positive to Ki-67-positive cells were nevertheless observed between different tissue types. In contrast, the ratio of Ki-S2 and Ki-67 immunoreactive scores varied widely in neoplastic cells and tissues, occasionally attaining a ratio of almost 1:1. This suggests that loss of growth regulatory mechanisms in malignant cells might result in an extreme reduction of the G1 phase fraction and thus in a significantly shorter doubling time. Therefore, antibody Ki-S2 is likely to allow a more precise evaluation of the cell fraction that will complete a division cycle and a more confident appraisal of the malignancy potential of a neoplastic process.
Assuntos
Antígenos de Diferenciação/metabolismo , Proteínas Nucleares/metabolismo , Anticorpos Monoclonais , Antígenos de Diferenciação/fisiologia , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Endonucleases , Citometria de Fluxo , Humanos , Imuno-Histoquímica/métodos , Cinética , Proteínas Nucleares/fisiologia , Distribuição TecidualRESUMO
Combining whole genome amplification (WGA) methods with novel laser-based microdissection techniques has made it possible to exploit recent progress in molecular knowledge of cancer development and progression. However, WGA of one or a few cells has not yet been optimized and systematically evaluated for samples routinely processed in tumor pathology. We therefore studied the value of established WGA protocols in comparison to an improved PEP (I-PEP) PCR method in defined numbers of flow-sorted and microdissected tumor cells obtained both from frozen as well as formalin-fixed and paraffin-embedded tissue sections. In addition, the feasibility of I-PEP-PCR for mutation analysis was tested using clusters of 50-100 unfixed tumor cells obtained by touch preparation of ten breast carcinomas by conventional sequencing of exon 7 and 8 of the p53 gene. Finally, immunocytochemically stained microdissected single disseminated tumor cells from bone marrow aspirates were investigated with respect to mutations in codon 12 of Ki-ras by restriction fragment length polymorphism (RFLP)-PCR after I-PEP-PCR. The modified I-PEP-PCR protocol was superior to the original PEP-PCR and DOP-PCR protocols concerning amplification of DNA from one cell (efficiency rate I-PEP-PCR 40% versus PEP-PCR 15% and DOP-PCR 30%) and five cells (efficiency rate I-PEP-PCR 100% versus PEP-PCR 33% and DOP-PCR 20%). Preamplification by I-PEP allowed 100% sequence accuracy in > 4000 sequenced base pairs and Ki-ras mutation detection in isolated single disseminated tumor cells. For reliable microsatellite analysis of I-PEP-preamplified DNA, at least 10 unfixed cells from fluorescence-activated cell sorting, 10 cells from frozen tissue, or at least 30 cells from formalin-fixed and paraffin-embedded tissue sections were required. Thus, I-PEP-PCR allowed multiple reliable microsatellite analyses suited for microsatellite instability and losses of heterozygosity and mutation analysis even at the single cell level, rendering this technique a powerful new tool for molecular analyses in diagnostic and experimental tumor pathology.
Assuntos
Análise Mutacional de DNA , Genoma , Neoplasias/genética , Reação em Cadeia da Polimerase/métodos , Neoplasias da Medula Óssea/genética , Neoplasias da Medula Óssea/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Separação Celular , Primers do DNA , DNA de Neoplasias/genética , Estudos de Viabilidade , Feminino , Genes ras/genética , Humanos , Perda de Heterozigosidade , Repetições de Microssatélites , Neoplasias/patologia , Inclusão em Parafina , Células Tumorais CultivadasRESUMO
Ischemic disorders of the heart can cause an irreversible loss of cardiomyocytes resulting in a substantial decrease of cardiac output. The therapy of choice is heart transplantation, a technique that is hampered by the low number of donor organs. In the present study, we describe the specific labeling, rapid but gentle purification and characterization of cardiomyocytes derived from mouse pluripotent embryonic stem (ES) cells. To isolate the subpopulation of ventricular-like cardiomyocytes, ES cells were stable transfected with the enhanced green fluorescent protein (EGFP) under transcriptional control of the ventricular-specific 2.1 kb myosin light chain-2v (MLC-2v) promoter and the 0.5 kb enhancer element of the cytomegalovirus (CMV(enh).). First fluorescent cells were detected at day 6 + 8 of differentiation within EBs. Four weeks after initiation of differentiation 25% of the cardiomyocyte population displayed fluorescence. Immunohistochemistry revealed the exclusive cardiomyogenic nature of EGFP-positive cells. This was further corroborated by electrophysiological studies where preferentially ventricular phenotypes, but no pacemaker-like cardiomyocytes, were detected among the EGFP-positive population. The enzymatic digestion of EBs, followed by Percoll gradient centrifugation and fluorescence-activated cell sorting, resulted in a 97% pure population of cardiomyocytes. Based on this study, ventricular-like cardiomyocytes can be generated in vitro from EBs and labeled using CMV(enh)./MLC-2v-driven marker genes facilitating an efficient purification. This method may become an important tool for future cell replacement therapy of ischemic cardiomyopathy especially after the proof of somatic differentiation of human ES cells in vitro.