RESUMO
The effect of the chemotatic peptide, N-formylmethionylleucylphenylalanine (FMLP), on actin conformation in human neutrophils (PMN) was studied by flow cytometry using fluorescent 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin to quantitate cellular F-actin content. Uptake of NBD-phallacidin by fixed PMN was saturable and inhibited by fluid phase F-actin but not G-actin. Stimulation of PMN by greater than 1 nM FMLP resulted in a dose-dependent and reversible increase in F-actin in 70-95% of PMN by 30 s. The induced increase in F-actin was blocked by 30 microM cytochalasin B or by a t-BOC peptide that competitively inhibits FMLP binding. Under fluorescence microscopy, NBD-phallacidin stained, unstimulated PMN had faint homogeneous cytoplasmic fluorescence while cells exposed to FMLP for 30 s prior to NBD-phallacidin staining had accentuated subcortical fluorescence. In the continued presence of an initial stimulatory dose of FMLP, PMN could respond with increased F-actin content to the addition of an increased concentration of FMLP. Thus, FMLP binding to PMN induces a rapid transient conversion of unpolymerized actin to subcortical F-actin and repetitive stimulation of F-actin formation can be induced by increasing chemoattractant concentration. The directed movement of PMN in response to chemoattractant gradients may require similar rapid reversible changes in actin conformation.
Assuntos
Actinas/sangue , Quimiotaxia de Leucócito/efeitos dos fármacos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/fisiologia , Amanitinas , Citocalasina B/farmacologia , Citometria de Fluxo , Corantes Fluorescentes , Humanos , Neutrófilos/efeitos dos fármacos , Conformação ProteicaAssuntos
Técnicas de Transferência de Genes , Células-Tronco Hematopoéticas , Proteínas Luminescentes , Animais , Biomarcadores , Separação Celular , Terapia Genética/métodos , Proteínas de Fluorescência Verde , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Humanos , Macaca mulatta , Proteínas de MembranaRESUMO
Multiparameter flow cytometric measurements of the Ha-ras oncogene product, Ha-p21, versus DNA content were used to study the effect of prednisolone, sodium butyrate, and hyperosmolality on the expression of this gene during the cell cycle of HT-29, a human colonic carcinoma cell line. In control cells the expression of Ha-p21 was cell cycle dependent; it increased during G1 and remained approximately constant as cells traversed the S- and G2 + M phases. Two compartments of G1 cells, one expressing low (G1A) and the other (G1B) high levels of Ha-p21 could be identified. Cells grown with prednisolone (1.4-2.1 microM) expressed higher Ha-p21 levels than controls. Cell cycle analysis revealed that this effect was accompanied by a change in the distribution of cells in G1 phase: whereas the proportion of cells in G1A was reduced, that of cells in G1B was increased. The steroid had no detectable effect on cells in S and G2 + M. By contrast, sodium butyrate and hyperosmolality caused a marked decrease in Ha-p21 content. This reduction was not accompanied by any modification of the proportion of cells in the cell cycle compartments. These results would suggest that Ha-p21 is not likely to be a primary regulator of cell cycle progression in HT-29 cells.
Assuntos
Ciclo Celular , Neoplasias do Colo/fisiopatologia , Proteínas de Ligação ao GTP/metabolismo , Oncogenes , Proteínas Proto-Oncogênicas/metabolismo , Butiratos/farmacologia , Ácido Butírico , Linhagem Celular , Neoplasias do Colo/patologia , DNA de Neoplasias/análise , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração Osmolar , Prednisolona/farmacologiaRESUMO
Agents such as thapsigargin and endothelin elevate intracellular calcium levels by a combination of calcium release from intracellular stores and calcium influx across the plasma membrane; however, the relative contribution of influx vs. release in modulating calcium-dependent gene expression is not as well understood in nonexcitable cells as in excitable cells. In this report we have been able to separate thapsigargin-induced elevation of intracellular calcium into release and influx components, using carboxyamido-triazole (CAI), a known inhibitor of calcium influx with antiproliferative activity against a number of human carcinomas, to selectively inhibit influx without affecting release. The results of these experiments indicate that the ability of thapsigargin to induce calcium-dependent gene expression in nonexcitable cells is dependent on the induction of calcium influx, presumably through store-operated calcium channels. CAI treatment specifically inhibited thapsigargin- or endothelin-stimulated expression from the c-fos promoter in Rat-1 cells and in epithelial cell lines derived from ovary and breast. Use of the VL30 model system confirmed the ability of CAI to inhibit calcium-dependent gene expression and further demonstrated that the ability of elevated calcium to synergize with other signaling pathways required close temporal coupling. In addition to inhibiting endothelin-induced calcium influx, CAI treatment also resulted in a partial inhibition of IP3 production and calcium release. CAI treatment also blocked the increase in ERK1 kinase activity observed in response to either endothelin or thapsigargin, suggesting a role for calcium influx in the activation of mitogen-activated protein kinase pathways.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Cálcio/fisiologia , Endotelina-1/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes fos/efeitos dos fármacos , Tapsigargina/farmacologia , Triazóis/farmacologia , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Endotelina-1/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Genes Reporter/efeitos dos fármacos , Genes Reporter/genética , Genes fos/genética , Imidazóis/farmacologia , Ratos , Tapsigargina/antagonistas & inibidoresRESUMO
To evaluate the ability of replication-deficient, recombinant adenovirus vectors to transfer genes to human tumor cells in vivo, adenovirus vectors containing the Escherichia coli lacZ (Ad.RSV beta gal) gene (coding for beta-galactosidase; used as a cell marker for gene transfer) or the human alpha 1-antitrypsin (Ad-alpha 1AT) cDNA (used as an example of a secreted protein) were administered intraperitoneally to nude mice with human malignant mesothelioma cell (H-MESO-1) malignant ascites. Preliminary in vitro studies showed that both vectors effectively transferred genes to H-MESO-1 cells. Tumor cells recovered from ascites of animals intraperitoneally administered a control adenovirus revealed no evidence of beta-galactosidase (beta-gal) activity 3 or 14 days later. In contrast, beta-gal activity was detected at the same time points in tumor cells from animals receiving intraperitoneal Ad.RSV beta gal. Flow cytometric quantification of beta-gal activity in recovered cells showed < 3% beta-gal-positive cells in animals administered control virus, but in animals administered intraperitoneal Ad.RSV beta gal there was a mean of 71 +/- 18% positive cells at 3 days and 56 +/- 27% at 14 days. Human alpha 1AT was not detected by enzyme-linked immunosorbent assay (ELISA) in ascites of animals receiving a control virus; however, in ascites of animals administered Ad-alpha 1AT, 21,000 +/- 3,800 ng/ml of human alpha 1AT was detected at 3 days and 4,900 +/- 1,700 ng/ml at 14 days. These data demonstrate that replication-deficient recombinant adenovirus vectors can be used to transfer genes to malignant cells in vivo and suggest a new strategy for genetic modification for antitumor therapy.
Assuntos
Adenovírus Humanos/genética , Vírus Defeituosos/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Proteínas de Membrana/biossíntese , Mesotelioma/patologia , Proteínas Recombinantes de Fusão/biossíntese , alfa 1-Antitripsina/biossíntese , beta-Galactosidase/biossíntese , Animais , Ascite , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Regulador de Condutância Transmembrana em Fibrose Cística , Escherichia coli , Feminino , Humanos , Injeções Intraperitoneais , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , alfa 1-Antitripsina/genética , beta-Galactosidase/genéticaRESUMO
This report concerns the study of the relationship between protein expression and the cell cycle in exponentially proliferating benign and malignant human prostate epithelial cells in short-term cultures. Multiparameter flow cytometric measurements were performed to correlate the expression of prostate-specific acid phosphatase, epithelial membrane antigen and epitectin with cell cycle progression. The expression of the three proteins was heterogeneous in G1 cells. The early post-mitotic cells exhibited the lowest levels when compared with late G1 cells, wherein the expression was many times greater. There was no further increase as the cells progressed through S and G2 + M. These findings, corroborating prior observations in other systems, suggest the possibility that the levels of the proteins studied increase during the G1 phase of the cell cycle and drop during or immediately after cytokinesis. As an alternate explanation, the heterogeneity of protein expression characteristic of G1 cells may be due, at least in part, to an asymmetric apportionment of cell constituents at mitosis.
Assuntos
Próstata/citologia , Biossíntese de Proteínas , Fosfatase Ácida/biossíntese , Antígenos Glicosídicos Associados a Tumores/biossíntese , Ciclo Celular , Divisão Celular , Células Cultivadas , Células Epiteliais , Epitélio/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Cinética , Masculino , Glicoproteínas de Membrana/biossíntese , Mucina-1 , Próstata/metabolismo , Neoplasias da Próstata , Células Tumorais CultivadasRESUMO
The use of antibodies permits the study of oncogene product expression in cells and tissues. However, quantitation of the levels of expression in immunohistochemical preparations is beset by difficulties, and the available scoring system provide semiquantitative data at best. Here we describe the use of computer-assisted image analysis for determination of oncoprotein levels in a model system and compare the results with those generated by flow cytometric analysis. The oncogene products measured are located in the nucleus (c-myc p62 and c-fos p55), the inner surface of the membrane (c-ras p21), and both sides of the membrane (c-erbB-2 p185). In each instance, both analytic modalities yielded concordant results. Our data indicate that computer-assisted image analysis is a useful tool for quantitating cell components in immunohistochemical preparations.
Assuntos
Neoplasias da Mama/análise , Proteínas Oncogênicas/análise , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular , Citometria de Fluxo , Expressão Gênica , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Proteínas Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fos , Proteínas Proto-Oncogênicas c-myc , Proteínas Proto-Oncogênicas p21(ras) , Receptor ErbB-2 , Células Tumorais Cultivadas/análise , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologiaRESUMO
Fundamental principles of flow cytometry with emphasis on DNA measurements and cell cycle analysis in human cells and tissues are summarized. Some of the pitfalls of cell preparation techniques and histogram interpretation are discussed at length. While consensus has been reached for some organs and tumors that DNA quantitation by flow cytometry (or image cytometry) may be of prognostic value, for most cancers studied to date the information remains incomplete. Thoroughly lacking are well-structured prospective studies because retrospective studies, while suggestive, may not necessarily be of the same value. Potential usefulness of other tumor markers is briefly discussed. Many fundamental questions concerning definitions of "diploid" and "aneuploid" tumors have not been satisfactorily settled. While the goal of "objective measurements" is worthy of further pursuit, the interpretation of results is often highly subjective. The biologic reasons for behavioral differences between diploid and aneuploid tumors are still totally obscure.
Assuntos
Fracionamento Celular/métodos , Separação Celular/métodos , DNA de Neoplasias/análise , Citometria de Fluxo/métodos , Neoplasias/diagnóstico , Ciclo Celular , Apresentação de Dados , Humanos , Ploidias , Prognóstico , Coloração e Rotulagem/métodosRESUMO
A survey of over 225 recent studies examining the relationship between the flow cytometric DNA analysis of solid tumors and clinical prognosis indicates that criteria used to classify DNA histograms are variable and often inconsistent with the recommendations proposed by the Convention on Nomenclature for DNA Cytometry. Numerous reports not only lack unambiguous descriptions of the histogram features used to differentiate diploid from aneuploid DNA distributions, but also inadequately describe the technical aspects of data acquisition, standardization, and inclusion or exclusion of subpopulations by gating. In many cases, the coefficient of variation of the diploid and aneuploid G0/1 peaks, which would allow an assessment of histogram quality, is not reported. Because of the differences in DNA histogram interpretation, extrapolation of the results among laboratories may be difficult and is probably not reliable. This review summarizes the criteria that have been used to classify the DNA histograms and illustrates the effects of these different classifiers on DNA ploidy analysis and clinical conclusions.
Assuntos
DNA de Neoplasias/análise , Citometria de Fluxo , Neoplasias/genética , Ploidias , HumanosAssuntos
Neoplasias/terapia , Transdução de Sinais , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Cálcio/fisiologia , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Ensaios Clínicos como Assunto , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/fisiologia , Substâncias de Crescimento/fisiologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/efeitos dos fármacos , Camundongos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/fisiologia , Neoplasias/fisiopatologia , Oncogenes , Fosfatidilinositol 3-Quinases , Fosfatidilinositol Diacilglicerol-Liase , Diester Fosfórico Hidrolases/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/fisiologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/fisiologia , Transdução de Sinais/efeitos dos fármacos , Triazóis/farmacologia , Triazóis/uso terapêuticoRESUMO
Debris compensation is an important variable affecting S-phase fraction (SPF) analysis in flow cytometric DNA histograms. The SPF was estimated in fresh frozen breast carcinomas using the following four debris subtraction algorithms: modeling debris as an exponential curve (EXP); the incorporation of nuclei cut a single time into the exponential moel (EXP-SC); the random cutting of nuclei into multiple pieces of varying sizes (MC); and a combination of both nuclear cutting models (SC-MC). Comparison of SPF estimates indicated that the various debris subtraction models yielded differences in SPF, with SPF values obtained using the exponential model having considerable variation compared to SPF estimates from histograms where debris was modeled by algorithms based on nuclear slicing and fragmentation. However, SPF estimates could be affected by initial placement of the nuclear debris boundaries, the coefficient of variation of the G0/1 peak, and the relative amount of debris. Using the ratio of the height of the G0/1 peak to the height of the debris between the chicken red blood cells (CRBC) and G0/1 peaks as an objective measurement of nuclear debris, debris compensation was necessary in diploid DNA histograms where this ratio was as low as 1.5%. Taken in the context of SPF prognostic cutoff levels, variation in debris models and boundaries can change the classification of cases with borderline SPF levels into the poor prognostic high SPF categories, thereby making the comparison of SPF values derived from different studies difficult.
Assuntos
Adenocarcinoma/patologia , Neoplasias da Mama/patologia , DNA de Neoplasias/análise , Fase S , Adenocarcinoma/genética , Algoritmos , Aneuploidia , Neoplasias da Mama/genética , Feminino , Citometria de Fluxo , Humanos , Estudos Prospectivos , Fase de Repouso do Ciclo CelularRESUMO
The specificity and sensitivity of the monoclonal antibody Ki-67 in identifying proliferating cell compartments was tested with the human promyelocytic leukemia cell line HL-60 using multi-parameter flow cytometry. While correlated measurements of DNA content and Ki-67 immunofluorescence indicated that the antigen was present in all phases of the cell cycle, reactivity with the antibody was highest in proliferating S and G2+M cells. The analysis of the BrdU content of cells sorted on the basis of reactivity with Ki-67 showed a correlation between Ki-67 reactivity and BrdU uptake. In HL-60 cells induced to differentiate with dimethyl sulfoxide (DMSO), the loss of reactivity with Ki-67 paralleled the exit of cells from the cell cycle. This was not observed in DMSO-resistant HL-60 cells. These results validate the usefulness of the Ki-67 antibody for determining the proliferative stage of mammalian cells in culture.
Assuntos
Anticorpos Monoclonais , Ciclo Celular , Leucemia Promielocítica Aguda/patologia , Bromodesoxiuridina/metabolismo , Compartimento Celular , Diferenciação Celular , Linhagem Celular , DNA de Neoplasias/análise , Dimetil Sulfóxido/farmacologia , Citometria de Fluxo , Imunofluorescência , HumanosRESUMO
DNA ploidy and cell cycle phases of benign human colonic epithelium peripheral to adenocarcinoma were analyzed by flow cytometry in 188 prospective cases. Human colonic epithelium was shown to be diploid with a mean DNA index (DI) of 1.01. The G0G1 compartment accounted for nearly 93% of the cells with the remainder in the S and G2+M phases. Parallel [3H]thymidine uptake on selected cases confirmed the relatively low proliferative activity of colonic mucosa. The DNA index and the cell cycle compartments exhibited no correlation to the ploidy, Dukes' stage, size, and anatomical location of the corresponding malignant tumors. Approximately 25% of the benign samples possessed DI values outside of the diploid range (defined as the mean +/- sd). Analysis of these apparently hypo- (less than 0.92) and hyper- (greater than 1.09) diploid, histologically normal samples in terms of cell cycle kinetics and their relationship to Dukes' stage, location, distance, and the ploidy of the tumor showed no correlation. The only characteristic differentiating these "aberrant" samples from diploid benign tissue was variation in DI possibly due to differences in fluorochrome binding or accessibility to DNA. Whereas these results indicate some degree of variability in the DNA content of benign colonic epithelia, neither DI nor cell cycle kinetics appear to be affected by the presence of a malignant tumor and are not representative of either the ploidy or pathologic stage of the corresponding colonic carcinoma.
Assuntos
Adenocarcinoma/patologia , Colo/patologia , Neoplasias do Colo/patologia , DNA de Neoplasias/análise , Aneuploidia , Ciclo Celular , Colo/análise , Diploide , Epitélio/análise , Epitélio/patologia , Feminino , Citometria de Fluxo , Humanos , Interfase , Cinética , Masculino , Estadiamento de Neoplasias , Ploidias , Prognóstico , Estudos ProspectivosRESUMO
The basic principles of flow cytometry with special emphasis to cell cycle analysis and DNA ploidy measurements are described. The concept of separation of various subcompartments of cell cycle, distinguished by simultaneous determinations of DNA versus RNA and DNA versus protein contents is presented. Based on DNA ploidy patterns, human tumors were divided into two groups designated as diploid range and non-diploid (aneuploid). Since it has been proposed that diploid range tumors have generally better prognoses than non-diploid tumors, the value of DNA ploidy patterns as a prognostic factor is discussed in major groups of human cancers.
Assuntos
DNA de Neoplasias/análise , Citometria de Fluxo/métodos , Neoplasias/análise , RNA Neoplásico/análise , Animais , Ciclo Celular , Feminino , Humanos , Masculino , Ploidias , PrognósticoRESUMO
It has been postulated that the expression of the product (p21) encoded by the ras genes may have a role in cell cycle events. Simultaneous multiparameter flow cytometry was used to measure the p21 content in relation to the cell cycle of several cancer cell lines of human origin. These studies revealed that p21 levels rise during the G1 phase of the cycle and remain approximately constant as cells traverse the S and G2 + M phases. The threshold level of p21 expression of S phase cells was used to divide the G1 cell population into cells with low (G1A) and high (G1B) p21 content. The p21 levels of G1B cells were approximately ten times higher than those of G1A cells. The validity of this subdivision was confirmed by synchronous measurements of RNA content and p21. Cells with low RNA content, hence in early part of G1 phase of the cell cycle, expressed low levels of p21, and cells with higher RNA content expressed higher levels of p21. These observations suggest that the levels of p21 are much lower at the onset of the cell cycle than at its end; hence a drop in p21 expression is likely to occur during or immediately after mitotic division.
Assuntos
Ciclo Celular , Oncogenes , Proteínas Proto-Oncogênicas/análise , Linhagem Celular , Separação Celular , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras)RESUMO
To better understand the changes that occur in cytoplasmic actin during cell movement, we studied the effect of inhibitors of cell movement on the molecular conformation of actin and its incorporation into the Triton-insoluble cytoskeleton of human neutrophils. The sulfhydryl reactive compound N-ethylmaleimide caused an increase in cellular F-actin as measured by uptake of the F-actin specific fluorescent probe 7-nitrobenz-2-oxadiazole-phallacidin. However, N-ethylmaleimide reduced the amount of actin associated with the Triton-insoluble cytoskeleton. Dithiobisnitrobenzoic acid, a sulfhydryl reagent that does not cross cell membranes efficiently, did not alter the F-actin content of neutrophils. The effect of N-ethylmaleimide was blocked by the presence of dithiothreitol, a donor of sulfhydryl groups. N-ethylmaleimide did not affect the polymerization of actin in a cell-free system. Cytochalasin B did not alter F-actin content of neutrophils but did decrease actin in cytoskeletons of resting neutrophils. Cytochalasin inhibited the increase in F-actin initiated by the chemoattractant N-formylmethionylleucylphenylalanine. We propose that N-ethylmaleimide blocks the stabilization of G-actin in cytoplasm, interferes with the incorporation of F-actin polymer into the cytoskeleton, and depolymerizes the cytoskeleton. In contrast cytochalasin stabilizes G-actin in the presence of chemotactic peptide. These data suggest that reversible conversion of G-actin to F-actin and incorporation of F-actin into the Triton-insoluble cytoskeleton are important for neutrophil movement.
Assuntos
Actinas/sangue , Citocalasina B/farmacologia , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Neutrófilos/metabolismo , Reagentes de Sulfidrila/farmacologia , Movimento Celular/efeitos dos fármacos , Ácido Ditionitrobenzoico/farmacologia , Ditiotreitol/farmacologia , Etilmaleimida/farmacologia , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Inibidores de Proteases/farmacologiaRESUMO
The DNA ploidy and cell-cycle distribution of three separate fresh tissue samples of 60 colorectal adenocarcinomas were analyzed by flow cytometry. DNA ploidy was concordant among the three samples in 38 cases (63.3%). In the remaining 22 cases (36.6%), the DNA histograms of two of the three multiple samples were similar; however, the ploidy of the third sample was discordant. No relationship was observed between Dukes' stage and histologic grade with concordance or discordance among samples. Thus, in about one third of the colonic carcinomas, a single sample showing either a diploid or diploid-cycling DNA histogram would not detect aneuploid DNA patterns. Comparison of scrapes and fine-needle aspirates of tumors as alternative sampling methods of tumors for DNA ploidy analysis indicated a strong association with the tumor ploidy (84% and 96%, respectively) only when the ploidy of the multiple samples was concordant. In about 25% of the cases, tumor scrapes and fine-needle aspirates did not correlate with the "most abnormal" ploidy observed in one of the three tissue samples. The data suggest that single or even double tissue samples may not show aneuploid DNA patterns in a substantial proportion of colorectal cancers.
Assuntos
Adenocarcinoma/química , Neoplasias do Colo/química , DNA de Neoplasias/análise , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia por Agulha , Ciclo Celular/fisiologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , DNA de Neoplasias/genética , Feminino , Citometria de Fluxo , Variação Genética , Humanos , Masculino , Pessoa de Meia-Idade , PloidiasRESUMO
Computer-assisted image analysis was used to demonstrate in exponentially proliferating human tumor cells the uneven postmitotic apportionment of several oncogene-encoded proteins (ras p21; erbB-2 p185; fos p55; myc p62). This observation may provide the explanation for the high degree of heterogeneity of postmitotic cells and the asynchrony in cell cycle traverse of cultured cells.
Assuntos
Compartimento Celular , Mitose , Proteínas Proto-Oncogênicas/metabolismo , Ciclo Celular , Citometria de Fluxo , Humanos , Processamento de Imagem Assistida por Computador , Técnicas Imunoenzimáticas , Técnicas In Vitro , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptor ErbB-2 , Células Tumorais CultivadasRESUMO
During angiogenesis, endothelial cells react to stimulation with finely tuned signaling responses. The role of calcium-regulated signaling in angiogenesis has not been defined. This study investigated the calcium dependency of endothelial cell proliferation and invasion by using an inhibitor of ligand-stimulated calcium influx, CAI (carboxy-amidotriazole). Incubation with CAI significantly inhibited proliferation of human umbilical vein endothelial cells (HUVECs) in response to serum (IC50 = 1 microM) or basic fibroblast growth factor (FGF2; P2 < 0.005 at 10 microM). Statistically significant inhibition of HUVEC adhesion and motility to basement membrane proteins laminin, fibronectin, and type IV collagen was demonstrated (adhesion, P2 < 0.004-0.01; motility, P2 < 0.009-0.018). Marked inhibition of native and FGF2-induced gelatinase activity was shown by zymogram analysis and was confirmed by Northern blot analysis. CAI inhibited HUVEC tube formation on Matrigel and inhibited in vivo angiogenesis in the chicken chorioallantoic membrane assay, 67% at 20 microM and 56% at 10 microM compared with 16% for an inactive CAI analog or 9% for 0.1% dimethyl sulfoxide control. Incubation of HUVECs with CAI and/or FGF2 followed by immunoprecipitation with anti-phosphotyrosine antibody showed inhibition of FGF2-induced tyrosine phosphorylation of proteins in the range 110-150 kDa. These results suggest that calcium-regulated events are important in native and FGF2-stimulated HUVEC proliferation and invasion, perhaps through regulation of FGF2-induced phosphorylation events, and indicate a role for calcium in the regulation of angiogenesis in vivo.
Assuntos
Cálcio/fisiologia , Endotélio Vascular/fisiologia , Neovascularização Patológica , Triazóis , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Adesão Celular , Divisão Celular , Movimento Celular , Células Cultivadas , Colágeno , Combinação de Medicamentos , Endotélio Vascular/citologia , Proteínas da Matriz Extracelular/fisiologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Gelatinases/metabolismo , Humanos , Técnicas In Vitro , Laminina , Metaloproteinase 2 da Matriz , Metaloendopeptidases/metabolismo , Fosfotirosina , Proteoglicanas , Transdução de Sinais , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Specific mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), the most common autosomal recessive fatal genetic disease of Caucasians, result in the loss of epithelial cell adenosine 3',5'-cyclic-monophosphate (cAMP)-stimulated Cl- conductance. We show that the influx of a fluorescent dye, dihydrorhodamine 6G (dR6G), is increased in cells expressing human CFTR after retrovirus- and adenovirus-mediated gene transfer. dR6G influx is stimulated by cAMP and is inhibited by antagonists of cAMP action. Dye uptake is ATP-dependent and inhibited by Cl- removal or the addition of 10 mM SCN-. Increased staining is associated with functional activation of CFTR Cl- permeability. dR6G staining enables both the fluorescent assessment of CFTR function and the identification of successfully corrected cells after gene therapy.