RESUMO
Trypanosoma brucei is lysed by tumor necrosis factor-alpha (TNF-alpha) in a dose-dependent way, involving specific binding of the cytokine to a trypanosomal glycoprotein present in the flagellar pocket of the parasite. TNF-alpha-gold particles are endocytosed via coated pits and vesicles and are directed towards lysosome-like digestive organelles. The specific uptake of the cytokine by the parasite results in a developmentally regulated loss of osmoregulatory capacity. TNF-alpha specific lysis is prevented when lysis assays are performed at a temperature <26 degrees C, despite uptake of the cytokine. Inhibition of lysis is also observed when a lysosomotropic agent is added during the first 2 h of incubation. Both monomorphic and pleomorphic trypanosomes are lysed but only when isolated during the peak of parasitaemia. Lysis is not observed with early infection stage parasites or procyclic (insect-specific) forms. Anti-TNF-alpha treatment of T. brucei-infected mice reveals a dramatic increase in parasitaemia in the blood circulation, the spleen, the lymph nodes, and the peritoneal cavity. These data suggest that in the mammalian host, TNF-alpha is involved in the growth control of T. brucei.
Assuntos
Trypanosoma brucei brucei/crescimento & desenvolvimento , Fator de Necrose Tumoral alfa/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Sítios de Ligação , Concentração de Íons de Hidrogênio , Lisossomos/metabolismo , Camundongos , Microscopia Eletrônica , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Proteínas de Protozoários/metabolismo , Temperatura , Fator de Necrose Tumoral alfa/farmacologia , Equilíbrio HidroeletrolíticoRESUMO
Blood platelets can interact with bacteria, possibly leading to platelet activation, cytokine and microparticle release and immune signalling. Besides, bacteria can also affect the platelet RNA content. We investigated the impact of non-pathogenic K12 and pathogenic O18:K1 Escherichia (E.) coli strains on platelet activation, RNA expression patterns, and selected proteins. Depending on bacteria concentration, contact of platelets with E. coli K12 lead to an increase of P-selectin (24-51.3%), CD63 (15.9-24.3%), PAC-1 (3.8-14.9%) and bound fibrinogen (22.4-39%) on the surface. E. coli O18:K1 did not affect these markers. Sequencing analysis of total RNA showed that E. coli K12 caused a significant concentration change of 103 spliced mRNAs, of which 74 decreased. For the RNAs of HMBS (logFC = +5.73), ATP2C1 (logFC = -3.13) and LRCH4 (logFC = -4.07) changes were detectable by thromboSeq and Tuxedo pipelines. By Western blot we observed the conversion of HMBS protein from a 47 kDA to 40 kDa product by E. coli K12, O18:K1 and by purified lipopolysaccharide. While ATP2C1 protein was released from platelets, E. coli either reduced the secretion or broke down the released protein making it undetectable by antibodies. Our results demonstrate that different E. coli strains influence activation, RNA and protein levels differently which may affect platelet-bacteria crosstalk.
Assuntos
Plaquetas/metabolismo , ATPases Transportadoras de Cálcio/genética , Escherichia coli K12/genética , Proteínas do Tecido Nervoso/genética , Uroporfirinogênio III Sintetase/genética , Antígenos de Bactérias/genética , ATPases Transportadoras de Cálcio/sangue , Infecções por Escherichia coli/sangue , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Escherichia coli K12/patogenicidade , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Lipopolissacarídeos/genética , Selectina-P/genética , Ativação Plaquetária/genética , RNA/sangue , RNA/genética , Análise de Sequência de RNA , Tetraspanina 30/genéticaRESUMO
The aim of our study was to investigate whether the level of actin polymerization plays a role in the motile and tissue infiltrating behavior of malignant lymphoma cells. For a panel of cell lines derived from the murine BW5147 T-cell lymphoma, we had previously shown a correlation between experimental metastasis formation and in vitro monolayer invasion. We have analyzed the motility and the F-actin content of six nonmetastatic, noninvasive (meta-inv-) and five metastatic, invasive (meta+inv+) variants of BW5147. Fourier analysis of cell contours was used to quantify shape changes of cells. All meta+inv+ lines rapidly protruded and retracted pseudopodia, whereas only one of the six meta-inv- lines showed this type of motility. Flow cytometry of cells stained with fluorescein-labeled phalloidin showed that the motile meta+inv+ cell lines have a higher F-actin content than their nonmotile meta-inv- counterparts. The results indicate that in lymphoma cells a high level of actin polymerization is a prerequisite for the formation of pseudopodia, which in turn are necessary for infiltration of the cells into tissues, and eventually for efficient metastasis formation. A corollary of this conclusion is that regulation of actin polymerization is a possible target for intervention aimed at moderating the spread of malignant lymphoma.
Assuntos
Actinas/análise , Linfoma de Células T/patologia , Metástase Neoplásica , Animais , Movimento Celular , Camundongos , Camundongos Endogâmicos AKR , Invasividade Neoplásica , Células Tumorais CultivadasRESUMO
Dipyridamole (DPD), a widely used anti-thrombotic agent, inhibits in vitro directional migration of 10T1/2 mouse embryo cells in a dose dependent way. The inhibition is detectable at DPD concentrations comparable to those in human plasma after oral intake of the drug. Using a model for quantitative shape change analysis, it is demonstrated that DPD inhibits pseudopodal activity and stabilizes the outlines of 10T1/2 cells. The inhibition of pseudopodal activity is thought to be involved in the reduced migratory capacity and the decreased area occupied by DPD treated cells. Coomassie blue staining of detergent-extracted cells shows that stress fibre patterns in 10T1/2 cells are altered by DPD. In particular, highly regular, polygonal networks, reminiscent of those described in spreading cells, appear after DPD treatment. It is suggested that interference of DPD with the normal organization of the actin microfilament system accounts for its effects on spreading, motility, and growth of cultured cells, and possibly for part of its in vivo anti-thrombotic activity as well.
Assuntos
Movimento Celular/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Dipiridamol/farmacologia , Animais , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Cinética , Camundongos , Microscopia de Interferência , Microscopia de Contraste de FaseRESUMO
C3 exoenzyme from Clostridium botulinum ADP-ribosylates the small GTP-binding protein Rho with a high specificity. The use of C3 has shown that Rho-mediated signaling is involved in the regulation of actin-dependent processes in various cell types. In order to investigate the role of Rho-proteins in lymphocyte crawling, we have analyzed the effects of C3 on a T-cell line derived from the murine BW5147 lymphoma. Pretreatment of the lymphoma cells with C3, in conditions where Rho was actually ADP-ribosylated, strongly inhibited the characteristic shape changes resulting from extension and retraction of pseudopodia. Concomitantly, invasion of the cells through a monolayer of fibroblast-like cells was also inhibited. C3-treatment did not affect the total F-actin content of the cells, as measured by flow cytometry of cells stained with phalloidin. Yet, microscopical observation revealed that the accumulations of F-actin, which were seen in the pseudopodia of untreated cells, were absent after treatment with C3. This suggests that C3 may affect actin polymerization locally. The inhibitory effect of C3 on invasion was not restricted to the murine BW5147 lymphoma cell line, as it occurred also with CCRF-CEM, a human T-cell lymphoma line. Our results demonstrate that invasion-bound motility of lymphocytes depends on a Rho-mediated signal transduction pathway.
Assuntos
ADP Ribose Transferases/metabolismo , Adenosina Difosfato Ribose/metabolismo , Toxinas Botulínicas , Proteínas de Ligação ao GTP/metabolismo , Linfoma de Células T/metabolismo , Linfoma de Células T/patologia , Proteínas de Membrana/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , ADP Ribose Transferases/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Clostridium botulinum/enzimologia , Humanos , Linfoma de Células T/fisiopatologia , Camundongos , Invasividade Neoplásica , Transdução de Sinais , Linfócitos T/fisiologia , Células Tumorais Cultivadas , Proteína rhoB de Ligação ao GTPRESUMO
In order to investigate the role of microfilaments in the crawling movements of lymphoid cells, we have analyzed the effects of botulinum C2 toxin and of cytochalasin D (cytoD) on the actin cytoskeleton and on the motility of a BW5147 T-lymphoma-derived cell line. Actin was ADP-ribosylated by C2 toxin in the living cells, and this resulted in a time and dose-dependent disappearance of F-actin, as assessed by staining with labeled phalloidin. CytoD did not affect the amount of polymerized actin, but rather changed its distribution from a diffuse peripheral network to focal accumulations on one side of the cell. Both treatments affected the motility of the lymphoma cells in two assay systems. Fourier analysis was used to quantify shape changes performed by the cells. C2 toxin as well as CytoD caused the cessation of pseudopodal protrusion. Invasion of the lymphoma cells through a monolayer of fibroblast-like cells was also inhibited by the treatments, in a dose-dependent way. C2 toxin significantly inhibited invasion at concentrations at which only part of the actin pool had been ADP-ribosylated. We conclude that partial depolymerization, as well as disorganization, of the microfilament network impairs the active cellular deformations that are involved in the crawling movements of the lymphoma cells. From previous work, there is evidence to state that the monolayer invasion assay to some extent mimics tissue infiltration by hematopoietic cells. The present study is the first to analyze the role of actin polymerization in a model system that is relevant for the migration of lymphoid cells in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Actinas/análise , Toxinas Botulínicas/farmacologia , Citocalasina D/farmacologia , Linfoma de Células T/patologia , Actinas/antagonistas & inibidores , Animais , Movimento Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Humanos , Linfoma de Células T/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C3H , Invasividade Neoplásica , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
A simple monolayer invasion assay (MIA) was recently developed using confluent fibroblastic cells as a target and variants of the BW5147 murine T-cell lymphoma as invading cells. Metastatic variants were consistently invasive in the MIA whereas non-metastatic cells were not. In this paper it is reported that pertussis toxin (PT) treatment of a highly metastatic and invasive variant caused a marked delay of invasion in the MIA at concentrations from 50 pg/ml upwards. Surprisingly, PT treatment of the non-metastatic, non-invasive parental BW5147 cells induced a moderate but significant level of invasion. Morphometric analysis showed that PT provoked an increased pseudopodal activity in cells in which it also caused increased invasive potential, and a decreased motility in cells with decreased invasiveness. This finding strengthens the perception that invasive potential and the capability to perform shape changes are related characteristics in these lymphoma cells.
Assuntos
Linfoma/patologia , Invasividade Neoplásica , Toxina Pertussis , Fatores de Virulência de Bordetella/farmacologia , Animais , Relação Dose-Resposta a Droga , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C3HRESUMO
Organ-specific metastasis of tumour cells may result from selective invasion and growth or from selective host cell responses. The present study demonstrates how selective interactions with the host affect the metastatic pattern of two murine T cell hybridoma lines, derived from the BW 5147 thymoma. Upon intravenous inoculation into syngeneic mice BW-14 cells preferentially colonize the kidneys, whereas BW-19 cells metastasize mainly to the spleen and the liver. The organ-specific behaviour of the two cell lines appears to be determined by a differential interaction with the spleen microenvironment. Inoculation of BW-14 cells into splenectomized mice results in increased liver colonization, indicating a negative effect of the spleen on BW-14 tumour development in the liver. Macrophages are likely to be involved in this inhibition, since inoculation of BW-14 cells into macrophage-depleted mice also leads to increased liver and spleen metastasis. In contrast, inoculation of BW-19 cells into splenectomized mice results in decreased liver metastasis, which indicates that the spleen exerts a stimulating effect on BW-19 cells. Macrophages also appear to be involved in this stimulation, since macrophage depletion causes a similar decrease in liver and spleen colonization. Hence components of the splenic microenvironment, probably macrophages, exert inhibiting or stimulating activities on BW-14 or BW-19 cells respectively, thereby determining the subsequent liver or kidney colonization.
Assuntos
Linfoma de Células T/patologia , Metástase Neoplásica , Especificidade de Órgãos , Animais , Hibridomas , Neoplasias Renais/secundário , Neoplasias Hepáticas/secundário , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Esplenectomia , Neoplasias Esplênicas/secundário , Células Tumorais CultivadasRESUMO
Using an in vitro monolayer assay (MIA) we analyzed the invasive behaviour of a panel of B-cell hybridomas prepared by the fusion of non-invasive, non-metastatic NSO plasmacytoma cells and normal murine B-cells. Interaction of these hybridomas with fibroblast-like monolayers consisted mostly of adhesion on top of the monolayers, whereas only a fraction of these cells penetrated through the monolayer. This is in sharp contrast with the highly invasive properties displayed by T-cell hybridomas. Whereas T-cell hybridomas highly infiltrated monolayers of rat hepatocyte in vitro, B-cell hybridomas neither adhered to nor infiltrated hepatocyte monolayers. We found a good correlation between the degree of adhesion of B-cell hybridomas to fibroblast-like monolayers and their metastatic capabilities upon i.v. injection into syngeneic animals. Unlike T-cell hybridomas which formed diffuse metastasis in liver and spleen, B-cell hybridomas generated nodular metastatic lesions. . When normal LPS-stimulated B-lymphocytes were tested in the fibroblast-MIA, only part of the population infiltrated the monolayers. This again contrasts with T-lymphocytes where a majority of the cells penetrated through the monolayers. These results suggest that (i) B-lymphocytes express invasive properties, albeit to a lesser extent than T-lymphocytes, (ii) non-invasive B-lymphoma cells can acquire invasiveness following cell fusion with a normal B-cell, (iii) these invasive properties contribute to the malignancy of the hybridomas when tested in recipient animals.
Assuntos
Linfócitos B/patologia , Comunicação Celular , Hibridomas/patologia , Metástase Neoplásica , Animais , Fibroblastos/patologia , Fígado/patologia , Camundongos , Camundongos Endogâmicos CBARESUMO
We have previously found that an increased tumorigenicity and spontaneous metastatic potential of BW5147-derived T lymphoma cells was associated with a decrease in major histocompatibility complex (MHC) class I H-2Kk antigen expression. This suggested that H-2Kk antigens may control the tumorigenic potential of BW T lymphoma cells. Our current experiments aimed to prove this association by specifically altering H-2Kk expression by gene transfection. Transfected cells expressing a high level of H-2Kk antigens were significantly less tumorigenic and metastatic after subcutaneous inoculation. However, there was selection in vivo for cells expressing a reduced level of H-2Kk antigens, which concomitantly led to an increased tumorigenicity. These data further confirmed the strong association between H-2Kk expression and tumorigenicity. We subsequently tested whether the immune system is implicated in this phenomenon by inoculating the H-2Kk transfectants into irradiated, immunocompromised recipients. Our results indicate that the reduced tumorigenicity of the BW H-2Kk transfectants is due to an immune rejection mechanism, mediated by CD8+ immune effector cells, as revealed by in vivo depletion experiments with anti-CD8 antibodies. Hence, we hereby demonstrated that H-2Kk antigens increased the immunogenicity of BW cells, via a CD8-dependent mechanism, which consequently reduced their tumorigenicity.
Assuntos
Antígenos H-2/fisiologia , Linfoma de Células T/patologia , Animais , Southern Blotting , Antígenos CD8/análise , Linhagem Celular , Antígenos H-2/análise , Antígenos H-2/genética , Depleção Linfocítica , Linfoma de Células T/imunologia , Camundongos , Metástase Neoplásica , Linfócitos T/fisiologia , TransfecçãoRESUMO
The cell adhesion molecule (CAM) L1 is involved in homotypic and heterotypic adhesion between neural cells. It has recently also been identified on leucocytes. We have investigated the expression of L1 on hematopoietic tumor cell lines and found that several tumors including the ESb-MP lymphoma are positive for L1. A potential role for L1 in spontaneous metastasis formation was examined using these cells. From wild-type (wt) L1high lymphoma cells we selected by a fluorescence-activated cell sorter (FACS) stable L1low expression variants. Syngeneic DBA/2 mice injected subcutaneously with L1low clones showed faster primary tumor growth, developed visceral metastases significantly faster and died earlier than animals carrying L1high wt cells. L1 high revertants from the L1low variants showed again a reduced metastatic capacity and a malignancy similar to the wt cells. Expression of L1 on the tumor variants and revertants correlated directly with their homotypic aggregation behaviour in vitro. L1 expression correlated negatively with metastatic capacity. These results suggest that L1 molecules may contribute to the overall malignant potential of the lymphoma cells, presumably by interfering with cell-cell interactions critical for tumor growth and dissemination.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Linfoma/patologia , Metástase Neoplásica , Animais , Agregação Celular , Divisão Celular , Glicosilação , Técnicas In Vitro , Complexo Antígeno L1 Leucocitário , Camundongos , Células Tumorais CultivadasRESUMO
Locomotion of lymphocytes and other leukocytes is an essential feature of the immune system, and therefore the evaluation of the locomotor behaviour of a lymphocyte population is part of its functional analysis. Paradoxically, the locomotor status of leukocytes is usually assessed on the basis of static information, by counting the number of spherical versus non-spherical cells. In this paper we describe two methods for the measurement of shape changes in microscopic images of lymphoid cells. First we computed a simple shape change factor, coined incongruence factor, based on the degree of non-overlap of the contours of the cell at the beginning and at the end of a 1 min time interval. Second we have used Fourier analysis of the cell outline: a function describing the undulations of the cell outline is broken down into sinusoidal 'waves' of increasing frequency, each with its corresponding amplitude. The amplitude values for the first ten frequencies produced a satisfactory mathematical description of lymphoid cell shapes, and the change of these amplitudes over a 1 minute time interval produced a quantitative description of the shape alterations of the cells. We have used five approaches to evaluate the shape and shape changes in the following populations of mouse lymphoma cells: a constitutively low-motile T lymphoma cell line (BW5147), a high-motile hybridoma (BW-O-Li1) either on plastic or on a precultured fibroblast-like monolayer, BW-O-Li1 cells after penetration through the monolayer, and BW-O-Li1 cells after treatment with cytochalasin B. We compare the results from direct visual evaluation of cell shape, from computer assisted assessment of sphericity and from Fourier analysis of cell shape at one moment, with the two methods for quantitative shape change analysis. All approaches revealed a clear distinction between spherical low-motile populations, and non-spherical high-motile cells. Moreover, the incongruence factor proved to be a reliable single parameter of active cell deformation. In addition, the Fourier analysis of cell outlines produced useful measures of static shape and of dynamic shape change, at any user-defined level of accuracy.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Linfócitos/patologia , Linfoma de Células T/patologia , Animais , Movimento Celular , Tamanho Celular , Citocalasina B/farmacologia , Análise de Fourier , Linfoma de Células T/tratamento farmacológico , Camundongos , Células Tumorais CultivadasRESUMO
Pertussis toxin is known to elicit lymphocytosis in whooping cough patients and experimental animals, by blocking the extravasation of lymphocytes and stimulating their release from lymphoid organs such as the thymus. The mechanisms responsible for these unique effects of PT are not fully understood. The effect of pertussis toxin (PT) on the invasive behavior of human CCRF-CEM T lymphoma cells has been investigated with the use of a monolayer invasion assay (MIA). We had previously found that invasion of murine T lymphoma cells in this model system was correlated with their ability to extravasate and form metastases after i.v. injection in syngeneic animals. We now show that human CEM cells can also penetrate through a precultured confluent monolayer of murine 10T1/2 fibroblast-like cells within a few hours. In a quantitative MIA run over 24 h, PT at concentrations above 10(-14) M inhibited invasion of the CEM cells. In addition, PT stimulated the release ('evasion') of CEM cells that had invaded under the monolayer before the toxin was added. The A subunit of PT was totally inactive, the B subunit had a small residual effect, and reconstitution of the AB complex partially restored the activity. The invasion-inhibiting activity of two different holotoxin preparations and of the subunits perfectly matched their activity in the Chinese hamster ovary cell clustering assay, which is known to depend on a functional AB complex. We suggest that inhibition of monolayer invasion by PT can be used as an in vitro model system to investigate the cellular and molecular mechanisms underlying the lymphocytosis-promoting action of the toxin. Furthermore, the method is sufficiently sensitive to be used for titration of toxin activity. Our data indicate that the ADP-ribosylating activity of the A subunit is indeed required, and that the promotion of lymphocytosis is not elicited by the binding of the B subunit alone.
Assuntos
Linfocitose/induzido quimicamente , Linfoma de Células T/patologia , Invasividade Neoplásica , Toxina Pertussis , Fatores de Virulência de Bordetella/farmacologia , Células Cultivadas , Fibroblastos , HumanosRESUMO
Phenothiazines inhibit the typical shape changes displayed by activated lymphocytes and thereby their migration through polycarbonate filters. The structure activity relationship of this effect is distinct from calmodulin inhibition. Our aim was to study this effect of phenothiazines on lymphocyte migration in an environment with living solid tissue cells. We assessed the effect of trifluoperazine and chlorpromazine (TFP and CP, two strong inhibitors of lymphocyte motility) and pimozide (PIM, a much weaker inhibitor of lymphocyte motility but a strong inhibitor of calmodulin) on invasion of human Molt-4 T-cells across precultured fibroblast monolayers. As expected invasion was inhibited by TFP and CP in the micromolar range that also inhibited motility. Surprisingly, PIM inhibited monolayer invasion at least as efficiently as TFP and CP (from 2.25 microM on). Preincubation of the monolayers or the lymphoid cells show that PIM exerted this novel invasion inhibiting effect on the monolayer. TFP and CP had a much weaker effect on the monolayer. Since these three compounds inhibit calmodulin in the same order, it is likely that this effect on the monolayer was caused by inhibition of a calmodulin-dependent pathway. KN-62, a specific inhibitor of calmodulin-dependent protein kinase II acted on the monolayer like PIM, whereas ML-7, a specific inhibitor of myosin regulatory light chain kinase, inhibited lymphoid cell motility like TFP and CP. In conclusion, invasion of T-cells across cellular monolayers is inhibited both by PIM and by phenothiazines like TFP and CP, but via distinct mechanisms: TFP and CP inhibit lymphocyte motility via a calmodulin independent pathway, whereas PIM impairs the monolayer's tolerance for invasion, most likely via a calmodulin and CamKII dependent pathway.
Assuntos
Calmodulina/antagonistas & inibidores , Movimento Celular/efeitos dos fármacos , Antagonistas de Dopamina/farmacologia , Linfócitos T/efeitos dos fármacos , Animais , Calmodulina/metabolismo , Células Cultivadas , Clorpromazina/farmacologia , Haloperidol/farmacologia , Humanos , Camundongos , Pimozida/farmacologia , Sulfonamidas/farmacologia , Linfócitos T/fisiologia , Trifluoperazina/farmacologia , Células Tumorais Cultivadas , Vasodilatadores/farmacologiaRESUMO
We have identified a novel 529bp fragment that is repeated 200- to 300-fold in the genome of Toxoplasma gondii. This 529bp fragment was utilised for the development of a very sensitive and specific PCR for diagnostic purposes, and a quantitative competitive-PCR for the evaluation of cyst numbers in the brains of chronically infected mice. The 529bp fragment was found in all 60 strains of T. gondii tested, and it discriminates DNA of T. gondii from that of other parasites. Toxoplasma gondii DNA was detected in amniotic fluid of patients, as well as in various tissues from infected mice. Polymerase chain reaction with the 529bp fragment was more sensitive than with the 35-copy B1 gene. For the quantitative competitive-PCR, a 410-bp competitor molecule was co-amplified with similar efficiency as the 529bp fragment. Quantitative competitive-PCR produced a linear relationship between the relative amounts of PCR product and the number of tachyzoites in the range of 10(2)-10(4) tachyzoites and 100-3000 tissue cysts. A highly significant correlation between visual counting of brain cysts and quantitative competitive-PCR was obtained in mice chronically infected with Toxoplasma. Thus, quantitative competitive-PCR with the 529bp fragment can be used as an alternative for the tedious visual counting of brain cysts in experimental animals. With the quantitative competitive-PCR, furthermore, we could confirm the copy number of the 529bp fragment in tachyzoites and estimate the number of bradyzoites per cyst.
Assuntos
DNA de Protozoário , Reação em Cadeia da Polimerase/métodos , Sequências Repetitivas de Ácido Nucleico , Toxoplasma/genética , Toxoplasmose/diagnóstico , Animais , Sequência de Bases , Encéfalo/parasitologia , Humanos , Dados de Sequência Molecular , Sensibilidade e Especificidade , Toxoplasmose Animal/diagnóstico , Toxoplasmose Cerebral/diagnósticoRESUMO
Monomeric (G), total (T) and filamentous (F) actin and the state of actin polymerisation (F:G) were determined and actin filaments were visualized in hepatoma Morris 5123 cells cultured in the presence of methotrexate (MTX) at various concentration. The exposure of the cells to this drug resulted in a decrease of total and polymerised actin in cytoplasm and in some changes in actin filament organization. This coincided with a decrease of the cells' ability to migrate through Matrigel coated filters and with inhibition of tumour formation after reimplantation of the methotrexate treated cells to experimental rats.
Assuntos
Actinas/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Metotrexato/farmacologia , Animais , Antimetabólitos Antineoplásicos/farmacologia , Materiais Biocompatíveis/química , Movimento Celular , Colágeno/farmacologia , Citoplasma/metabolismo , Citosol/metabolismo , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Laminina/farmacologia , Microscopia de Fluorescência , Invasividade Neoplásica , Proteoglicanas/farmacologia , Ratos , Células Tumorais CultivadasRESUMO
Despite the wide clinical use of glucocorticoids in the chemotherapy of leukaemia and lymphoma, there have been limited efforts at understanding the effects of these hormones on metastasis formation. The purpose of this study was to investigate the effects of glucocorticoids on the tissue-infiltrating capability of lymphoid cells. Using an in vitro invasion assay, we found that dexamethasone, a synthetic glucocorticoid analogue, inhibited the invasion of a murine T-cell hybridoma through a monolayer of fibroblast-like cells. Even low doses of dexamethasone were effective at inhibiting cellular transmigration (EC50 = 0.4 nM). A maximal decrease was observed after an overnight culture in the presence of dexamethasone. The effect persisted for at least 24 h after removal of the drug and required the binding of the hormone to its intracellular glucocorticoid receptor. Our results suggest that the decreased invasiveness of dexamethasone-treated cells is not the consequence of reduced motility or deficient production of an autocrine factor required for cell migration. This in vitro study suggests that glucocorticoids may act to reduce dissemination of lymphoma cells in vivo.
Assuntos
Dexametasona/farmacologia , Metástase Neoplásica/prevenção & controle , Linfócitos T/efeitos dos fármacos , Animais , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Dexametasona/metabolismo , Fibroblastos/fisiologia , Molécula 1 de Adesão Intercelular/fisiologia , Camundongos , Camundongos Endogâmicos C3H , Linfócitos T/fisiologiaRESUMO
The invasive behaviour of 8 lymphoma cell lines were tested by an in vitro monolayer invasion assay. The metastatic cell lines (TAM 4D1.2, DCH10Sp, TAM 4D6.2, E4 and BWLi) were more invasive than their non-metastatic counterparts (TAS 5C4, BWO and DCH 10). There was a positive correlation between their invasiveness and the PGE1- and forskolin stimulated cellular cAMP levels. Invasiveness and basal cAMP levels could not be correlated. Pretreatment with pertussis toxin (50 ng/ml) for 24 hours provoked did not significantly affect the basal and PGE1-stimulated cAMP levels in all cells. Yet, the toxin catalysed the ADP-ribosylation of 40 kDa components in all cells and provoked a significant increase in the invasiveness of non-metastatic cell lines and a decrease in the invasiveness of metastatic cell lines. These data suggest that the invasiveness of T-lymphoma cell lines might be controlled by a complex interplay between different signal transducing pathways in the membrane, rather than by the intracellular level of cAMP.
Assuntos
AMP Cíclico/análise , Linfoma/patologia , Alprostadil/farmacologia , Animais , Colforsina/farmacologia , Linfoma/análise , Invasividade Neoplásica , Metástase Neoplásica , Toxina Pertussis , Linfócitos T , Células Tumorais Cultivadas , Fatores de Virulência de Bordetella/farmacologiaRESUMO
BW-O-Li1 murine T-lymphoma cells display target preference for invasion in two different in vitro models. In the precultured chick heart fragment (PHF) assay, BW-O-Li1 preferentially invaded into aggregates of MCF-7 mammary carcinoma cells rather than into PHFs. In a monolayer invasion assay (MIA), BW-O-Li1 cells preferentially invaded under 10 T1/2 mouse embryo cell monolayers rather than under MCF-7 monolayers. Thus, although BW-O-Li1 cells were perfectly able to invade into any of the targets presented, they migrated and accumulated preferentially in one of the targets when a choice was offered. We suggest that this in vitro 'homing' phenomenon can be exploited to investigate certain aspects of organ-specific metastasis.
Assuntos
Hibridomas/patologia , Linfoma de Células T/patologia , Invasividade Neoplásica , Animais , Movimento Celular , Galinhas , Feminino , Humanos , Camundongos , Miocárdio/patologia , Células Tumorais CultivadasRESUMO
The metastatic phenotype (M+) is determined by the expression of the invasive (I+) and growth (G+) phenotypes at various sites along the metastatic pathway. The G, I and M phenotypes are not confined to tumor cells and may be expressed by normal cells, such as leukocytes, under certain physiological conditions. The study of the I and M phenotypes of leukocytes may yield valuable information on common mechanisms, underlying invasion and metastasis of normal and malignant cells. In this review we summarize experimental approaches that were developed for the analysis of the I and M phenotypes of normal and malignant leukocytes.