RESUMEN
In studying the interaction between bacteria and host cells in vitro, the latter are frequently cultured on commercially available biotic matrices such as Matrigel® or Geltrex®. To avoid contamination, host cells are commonly grown in the presence of antibiotics. However, we present here the finding that cell culture on such a matrix in the presence of antibiotics interferes with the outcome of subsequent infection experiments by virtue of diminished bacterial survival. By comparing outcomes for penicillin-susceptible and resistant strains of Staphylococcus aureus, we show that residual penicillin remains in the matrix despite the antibiotics' withdrawal from culture. Hence, the use of antibiotics should be avoided in this context.
Asunto(s)
Antibacterianos/análisis , Antibacterianos/farmacología , Medios de Cultivo/química , Penicilinas/análisis , Penicilinas/farmacología , Staphylococcus aureus/efectos de los fármacos , Animales , Bovinos , Técnicas de Cultivo de Célula , Células Cultivadas , Células Epiteliales/microbiología , Humanos , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/microbiología , Sarcoma Experimental/química , Staphylococcus aureus/crecimiento & desarrolloRESUMEN
Four monoamine-monoamide dithiol (MAMA) ligands containing two or one nitroimidazole moieties were synthesized and labeled with (99m)Tc (labeling yield > 95%). The proposed structures of (99m)Tc-complexes are identified by comparison with analogous Re-MAMA complexes. (99m)Tc-MAMA complexes show better physicochemical characters than (99m)TcO-(PnAO-1-(2-nitroimidazole)). Reduction potentials of nitro groups of the rhenium complexes are within the range for bioreductive compounds. As expected, biodistribution studies demonstrate that the 2-nitroimidazole complex shows better tumor-to-tissue ratios than 4-nitroimidazole analog for mononitroimidazole complexes, but not for MAMA-bisnitroimidazoles due to higher lipophilicity. Both the bisnitroimidazole compounds show rapider excretion, lower background activity in liver and higher tumor-to-tissue ratios than the mononitroimidazoles. Better biodistribution characteristic makes both the MAMA-bisnitroimidazole complexes, especially (99m)Tc-15, be potential tumor hypoxia marker.
Asunto(s)
Biomarcadores de Tumor/farmacocinética , Nitroimidazoles/química , Compuestos Organometálicos/farmacocinética , Renio/química , Sarcoma Experimental/química , Tecnecio/química , Animales , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/química , Química Física , Técnicas Electroquímicas , Ratones , Ratones Endogámicos , Estructura Molecular , Compuestos Organometálicos/sangre , Compuestos Organometálicos/química , Oxidación-Reducción , Ratas , Sarcoma Experimental/metabolismo , Distribución TisularRESUMEN
PURPOSE: To examine the pathophysiologic impact of treatment with combretastatin A4 phosphate (CA4P) in regions of tumors that ultimately either necrose or survive treatment with this agent. METHODS AND MATERIALS: Proliferation, perfusion, vessel density, and expression of vascular endothelial growth factor (VEGF) were analyzed in the KHT tumor model after treatment with CA4P. Analyses were conducted in the whole tumor and the tumor periphery. RESULTS: Perfusion in the tumor periphery decreased 4 h after treatment, but returned to baseline 20 h later. Whole-tumor perfusion also decreased 4 h after treatment, but did not return to baseline. Vessel density decreased in the tumor as a whole, but not in the tumor periphery. No significant effect on the expression of VEGF was observed, but a decrease in proliferation in the whole tumor and the periphery was noted. CONCLUSIONS: The present study shows that those areas of a tumor that survive treatment with CA4P are affected by CA4P exposure, though only transiently. The decrease in perfusion could negatively affect therapies utilizing the combination of CA4P and conventional anticancer agents by decreasing drug delivery and tissue oxygenation. These findings suggest that the timing of CA4P treatments when used in conjunction with conventional anticancer therapies should be considered carefully.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Antineoplásicos Fitogénicos/uso terapéutico , Neovascularización Patológica/tratamiento farmacológico , Sarcoma Experimental/tratamiento farmacológico , Estilbenos/uso terapéutico , Animales , Femenino , Ratones , Ratones Endogámicos C3H , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Sarcoma Experimental/irrigación sanguínea , Sarcoma Experimental/química , Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Malignant growth is associated with various patterns of interaction between tumor cells and those of the body immune system, interaction between Fas-receptor (Fas) and Fas-ligand (FasL) expression being one of them. These mechanisms were simulated in vitro using the main cell populations from murine hepatoma MH-22a, histiocytic sarcoma J-774 and their clonal lines obtained from cocultivation of tumor cells and syngenic splenocytes. Fas and FasL expression was identified by the RT-PCR method while apoptosis--by electrophoresis of low molecular DNA fractions and clonogenic survival.
Asunto(s)
Apoptosis , Histiocitos , Neoplasias Hepáticas Experimentales/química , Glicoproteínas de Membrana/análisis , Sarcoma Experimental/química , Bazo/citología , Factores de Necrosis Tumoral/análisis , Receptor fas/análisis , Animales , Línea Celular Tumoral , Células Clonales , Técnicas de Cocultivo , ADN de Neoplasias/análisis , Electroforesis , Proteína Ligando Fas , Regulación Neoplásica de la Expresión Génica , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We performed a detailed investigation of N-glycan structures on BM-40 purified from different sources including human bone, human platelets, mouse Engelbreth-Holm-Swarm (EHS) tumor, and human BM-40 recombinantly expressed in 293 and osteosarcoma cells. These preparations were digested with endoglycosidases and N-glycans were further characterized by sequential exoglycosidase digestion and high-performance liquid chromatography (HPLC) analyses. Bone BM-40 carries high-mannose structures as well as biantennary complex type N-glycans, whereas the protein from platelets and 293 cells has exclusively bi- and triantennary complex type structures. BM-40 derived from the EHS tumor carries biantennary complex type and additional hybrid structures. Using the osteosarcoma-derived MHH-ES1 cell line we successfully expressed a recombinant BM-40 that bears at least in part the bone-specific high-mannose N-glycosylation in addition to complex type and hybrid structures. Using chromatography on Concanavalin-A Sepharose, we further purified a fraction enriched in high-mannose structures. This array of differentially glycosylated BM-40 proteins was assayed by surface plasmon resonance measurements to investigate the binding to collagen I. BM-40 carrying high-mannose structures binds collagen I with higher affinity, suggesting that differentially glycosylated forms may have different functional roles in vivo.
Asunto(s)
Colágeno/química , Glicósido Hidrolasas/química , Osteonectina/química , Osteonectina/aislamiento & purificación , Polisacáridos/química , Animales , Plaquetas/química , Huesos/química , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Colágeno/metabolismo , Glicosilación , Humanos , Manosa/química , Ratones , Osteonectina/metabolismo , Unión Proteica , Sarcoma Experimental/química , Resonancia por Plasmón de SuperficieRESUMEN
Past studies have shown that carbohydrate residues reactive with the Griffonia simplicifolia isolectin B4 (GS I-B4) are present on the surface of highly-malignant murine sarcoma cells but are lacking or expressed in much lower amounts on the surface of low-malignant cells isolated from the same parent tumors (Am J Pathol 111: 27; J Nat Cancer Inst 71: 1281). In the present study it is shown that an antibody which recognizes the trisaccharide Galalpha1-3Galbeta1-4GlcNAc- is reactive with the highly-malignant cells but is non-reactive with the low-malignant cells. Further studies show that the high-malignant cells not only bind GS 1-B4 but also bind Evonymus europaea lectin (which like GS I-B4 recognizes terminal galactose in alpha1-3 linkage) and Erythina crystagalli lectin (which recognizes sub-terminal galactose in the beta1-4 linkage--e.g., Galbeta1-4GlcNAc). In contrast, the low malignant cells bind Erythina crystagalli lectin as efficiently as the high malignant cells but do not bind (or bind much smaller amounts of) either GS I-B4 or Evonymus europaea lectin. The present studies also show that there is no significant difference between high- and low-malignant cells in expression of alpha-galactosidase activity. In contrast, the high-malignant cells express high levels of alpha-galactosyl transferase activity while this enzyme is virtually undetectable in low-malignant cells. Taken together, these studies indicate that differential expression of a single monosaccharide residue distinguishes high- and low-malignant murine sarcoma cells. These studies also identify a mechanism to account for surface carbohydrate differences between the high- and low-malignant cells.
Asunto(s)
Antígenos de Neoplasias/análisis , Péptidos Catiónicos Antimicrobianos , Biomarcadores de Tumor/análisis , Lectinas de Plantas , Sarcoma Experimental/química , Trisacáridos/análisis , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor/inmunología , Biomarcadores de Tumor/metabolismo , Northern Blotting , Conformación de Carbohidratos , Secuencia de Carbohidratos , Galactosiltransferasas/análisis , Galactosiltransferasas/genética , Lectinas/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Invasividad Neoplásica , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/genética , Proteínas de Plantas/farmacología , ARN Mensajero/análisis , ARN Neoplásico/análisis , Sarcoma Experimental/patología , Trisacáridos/inmunología , Trisacáridos/metabolismo , alfa-Galactosidasa/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: Increasing arterial oxygen partial pressure (pO2) by breathing hyperoxic gases is an effective means of improving tumor oxygenation, although the efficacy of adding CO2 to the inspiratory gas has been discussed controversially. This study aimed at analyzing the impact of different inspiratory CO2 fractions on the time course of oxygenation and perfusion changes in experimental tumors during and after inspiratory hyperoxia. MATERIAL AND METHODS: Perfusion and oxygenation of rat DS-sarcomas were studied during spontaneous breathing of pure oxygen or hyperoxic gas mixtures containing different CO2 fractions (1, 2.5 or 5%). Red blood cell (RBC) flux was assessed as a measure of tumor perfusion using the laser Doppler technique and temporal changes in mean tumor pO2 were measured polarographically. RESULTS: Mean tumor pO2 increased 3.6-fold with pure oxygen, approx. 3.3-fold when 1 or 2.5% CO2 was added and 2.7-fold during carbogen breathing. RBC flux also increased by 25-30% with all gases. With pure oxygen and with 1% CO2 (+99% O2), perfusion changes paralleled those of the mean arterial blood pressure whereas with higher CO2 fractions, a decrease in resistance to flow was observed. The differences found with the various gas mixtures were more pronounced after the end of hyperoxia. With pure oxygen, perfusion immediately returned to pretreatment values whereas with higher CO2 fractions perfusion remained elevated for at least 30 min. CONCLUSIONS: Higher inspiratory CO2 fractions (2.5 or 5%) lead to a prolonged improvement of tumor perfusion after the end of inspiratory hyperoxia when compared with pure oxygen breathing. Since no principal differences in oxygenation and perfusion were seen between the gases containing 2.5 and 5% CO2, the former may be preferable for inspiratory hyperoxia.
Asunto(s)
Dióxido de Carbono/farmacología , Oxígeno/farmacología , Sarcoma Experimental/irrigación sanguínea , Animales , Dióxido de Carbono/sangre , Hipercapnia/sangre , Hiperoxia/sangre , Flujometría por Láser-Doppler , Masculino , Oxígeno/análisis , Oxígeno/sangre , Consumo de Oxígeno , Terapia por Inhalación de Oxígeno , Perfusión , Ratas , Ratas Sprague-Dawley , Flujo Sanguíneo Regional/efectos de los fármacos , Sarcoma Experimental/químicaRESUMEN
Telomerase is a specialized ribonucleoprotein enzyme complex which prevents the loss of the telomere. The activity of telomerase can be up- and down-regulated by various oxidative stresses but the effect of physical exercise is not known, whereas the modifying effect of cancer on telomerase activity is well documented. In the first study, we investigated the effect of mild and strenuous exercise training on telomerase activity, assessed by a PCR ELISA kit. No alteration in telomerase activity was detected. In the second investigation, solid sarcoma cells were transplanted to control, exercise trained or exercise trained and still exercising mice. On the 16th day after the transplantation, the size of tumors in the exercise trained group was 72% and in the exercising group 57% (P < 0.05) of that in the controls. Telomerase activity and 8-hydroxy-2'-deoxyguanosine levels in the liver were not significantly altered by exercise and/or sarcoma. We conclude that mild and strenuous exercise training does not significantly affect the activity of telomerase in the systems studied. Exercise training during sarcoma significantly retards the development of tumors and could possibly serve as a positive adjunct to treatment.
Asunto(s)
Desoxiguanosina/análogos & derivados , Neoplasias Hepáticas Experimentales/enzimología , Hígado/enzimología , Músculo Esquelético/enzimología , Condicionamiento Físico Animal/fisiología , Esfuerzo Físico/fisiología , Sarcoma Experimental/enzimología , Estrés Fisiológico/enzimología , Telomerasa/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Cruzamientos Genéticos , Daño del ADN , Desoxiguanosina/análisis , Femenino , Neoplasias Hepáticas Experimentales/química , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Trasplante de Neoplasias , Estrés Oxidativo , Ratas , Ratas Wistar , Sarcoma Experimental/químicaRESUMEN
Analysis of chromosome rearrangements in tumors is an important means for revealing genetic pathways underlying tumorigenesis and tumor progression. In five of 17 DMBA-induced rat sarcomas, cytogenetic analysis had disclosed homogeneously staining regions (hsrs), which are generally accepted to be cytogenetic signs of gene amplification. Using comparative genomic hybridization (CGH), regional increases in DNA copy number of the proximal part of rat chromosome (RNO) 4 were detected in four of the tumors harboring hsrs. Amplification of the Hgfr/Met oncogene, located at RNO4q21.2, was detected by fluorescence in situ hybridization (FISH) in five tumors. In four of them, a number of flanking genes located in the close vicinity of Hgfr/Met, including Cav1 (q21.1), Wnt2 (q21.2-q21.3), and Cftr (q21.3), also were amplified, though amplification was seen in a lower fraction of the cells than was Hgfr/Met amplification. In the fifth tumor (BL150T), Hgfr/Met was amplified in all cells and was the sole amplified gene of those tested. In addition, the Hgfr/Met FISH signals in BL150T were tightly clustered and formed compact and intense spots compared with the signals seen in the other four tumors. Application of the free chromatin FISH technique to BL150T showed that the genomic Hgfr/Met probe stained the extended chromatin fibers of up to 1.5 Mb with an almost uninterrupted signal, indicating that the BL150T amplicon was build up solely of Hgfr/Met gene sequences. Our results suggest that the Hgfr/Met oncogene is the primary target for amplification in a subset of rat DMBA sarcomas.
Asunto(s)
9,10-Dimetil-1,2-benzantraceno , Cromatina/genética , Amplificación de Genes/genética , Hibridación Fluorescente in Situ , Proteínas Proto-Oncogénicas c-met/genética , Proto-Oncogenes , Sarcoma Experimental/inducido químicamente , Sarcoma Experimental/genética , Animales , Mapeo Cromosómico , Ratas , Ratas Endogámicas BN , Ratas Long-Evans , Sarcoma Experimental/química , Coloración y EtiquetadoRESUMEN
BACKGROUND AND OBJECTIVE: MS-2 fibrosarcoma implanted in BALB-CDF1 mice was investigated by frequency and time domain measurements of the autofluorescence (AF) radiation emitted upon excitation by a N(2) laser beam (337.1 nm). STUDY DESIGN/MATERIALS AND METHODS: AF spectra were obtained by using a spectrograph, a multichannel plate and an optical multichannel analyzer for the steady state detection. Time-resolved spectra were performed by means of a monochromator, a photomultiplier, and a digital signal analyzer. RESULTS: Spectral measurements show that the autofluorescence intensity of pathologic tissue is lower than that of healthy one in the 400- to 500- spectral region. In the same spectral range, we found the fluorescence decay to be the sum of a fast and a slow component. The lifetime of the fast component of tumoral tissue is significantly lower than that of healthy samples. CONCLUSION: Frequency and time domain measurements used in combination show that MS2-fibrosarcoma is characterized by the probable presence of the free form of NADH.
Asunto(s)
Fibrosarcoma/diagnóstico , Rayos Láser , Sarcoma Experimental/diagnóstico , Espectrometría de Fluorescencia , Animales , Fibrosarcoma/química , Fibrosarcoma/metabolismo , Ratones , Ratones Endogámicos BALB C , NAD , Sarcoma Experimental/química , Sarcoma Experimental/metabolismoRESUMEN
Astrocytes produce laminin, a key extracellular matrix guidance molecule in the developing brain. Laminin is bound to transmembrane receptors on the surface of astrocytes known as integrins, which are, in turn, bound to the microfilament meshwork inside the astrocyte. Previous studies have shown that T4 regulates the pattern of integrin distribution in astrocytes by modulating the organization of the microfilaments. In this study, the effect of thyroid hormone on the secretion and topology of laminin in astrocytes was examined. Linear arrays of secreted laminin were observed on the surface of the T4-treated astrocytes within 10 h after seeding the cells onto poly-D-lysine-coated coverslips and became an organized meshwork by 24 h. In contrast, little if any laminin was identified on the surface of either hormone-deficient or T3-treated cells until 36 h after seeding and then was restricted to punctate deposits. Secretion of laminin into the medium by hormone-deficient and T3-treated cells was significantly greater than that by T4-treated cells. Conversely, deposition of laminin into the extracellular matrix was significantly greater in T4-treated cells than in hormone-deficient and T3-treated cells. Thyroid hormone had no effect on the production of laminin by astrocytes. These data show that T4 regulates the extracellular deposition and organization of laminin on the surface of astrocytes and provide a mechanism by which this morphogenic hormone can influence neuronal migration and axonal projection in the developing brain.
Asunto(s)
Astrocitos/química , Matriz Extracelular/química , Laminina/análisis , Hormonas Tiroideas/farmacología , Animales , Animales Recién Nacidos , Astrocitos/metabolismo , Astrocitos/ultraestructura , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Matriz Extracelular/ultraestructura , Inmunohistoquímica , Técnicas de Inmunoadsorción , Laminina/metabolismo , Ratones , Microscopía Fluorescente , Ratas , Sarcoma Experimental/química , Tiroxina/farmacología , Triyodotironina/farmacologíaRESUMEN
BACKGROUND: Uterine endometrial stromal sarcoma is a rare neoplasm with a morphology that closely resembles that of the proliferative endometrial stroma. To understand its pathologic characteristics, we established a novel cell line, MaMi, from a primary culture of an endometrial stromal sarcoma obtained from a 65-year-old Japanese woman. METHODS: We observed the morphology of MaMi cells and performed immunohistochemical analysis on the primary tumor and transplants in nude mice. Prolactin, insulin-like growth factor-binding protein-1, interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, intercellular adhesion molecule-1, E-selectin, vascular cell adhesion molecule-1, and fibronectin production in the culture medium of MaMi cells were also examined. RESULTS: MaMi cells were shown to exhibit a fibroblast-like morphology in vitro, and they adopted a more elongated appearance in response to 12-O-tetradecanoyl phorbol-13-acetate (TPA). On injection into nude mice, the cells gave rise to subcutaneous tumors. Immunohistologically, both the primary tumor and MaMi cell-induced tumors stained positively with antibodies to neuron-specific enolase or vimentin. MaMi cells constitutively produced IL-6, IL-8, and monocyte chemoattractant protein-1 in vitro. Interleukin-1beta, (100 pmol/L), tumor necrosis factor-alpha (1 nmol/L), and lipopolysaccharide (1 microg/mL) each increased the release of IL-6, IL-8, and monocyte chemoattractant protein-1 by MaMi cells. TPA (10 nmol/L) also stimulated the production of IL-6 and IL-8 by these cells, but inhibited that of monocyte chemoattractant protein-1. CONCLUSIONS: We demonstrated that MaMi cells closely resemble proliferative endometrial stromal cells not only morphologically, but also functionally. This cell line may prove valuable in understanding the role of cytokines produced by tumor cells in the pathogenesis of endometrial stromal sarcoma and may also be useful as an in vitro model of functioning endometrial stromal cells.
Asunto(s)
Quimiocina CCL2/biosíntesis , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/patología , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Sarcoma/metabolismo , Sarcoma/patología , Anciano , Animales , División Celular , Quimiocina CCL2/análisis , Selectina E/análisis , Neoplasias Endometriales/genética , Femenino , Fibronectinas/análisis , Humanos , Inmunohistoquímica , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Molécula 1 de Adhesión Intercelular/análisis , Interleucina-6/análisis , Interleucina-8/análisis , Cariotipificación , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Prolactina/análisis , Sarcoma/genética , Sarcoma Experimental/química , Sarcoma Experimental/patología , Células del Estroma/química , Células del Estroma/metabolismo , Células del Estroma/patología , Células Tumorales Cultivadas , Molécula 1 de Adhesión Celular Vascular/análisisRESUMEN
We have recently isolated R7, a spontaneous Moloney murine sarcoma virus (MoMuSV) 124 variant. Molecular cloning and sequence analysis showed that, relative to MoMuSV 124, R7 has an extra repeat in each enhancer and a truncated mos gene in frame with the truncated gag coding sequence. This report presents a detailed study on the pathology induced by R7. R7 induced not only sarcomas with well developed angiomatous components but also brain lesions. Brain lesions were observed in all less-than-48-hour-old BALB/c mice inoculated with greater than 2 x 10(5) R7 focus-forming units (FFUs). R7 was detected in all brains examined by day 9 after inoculation, and brain lesions were observed in two of four mice examined by day 14 after inoculation. Light microscopy of brains revealed that approximately 15% of the lesions were unenclosed blood pools of varying sizes containing red blood cells and inflammatory cells spreading into surrounding brain tissues. The remainder of the brain lesions had tumor cells. These lesions ranged from a few enlarged vascular endothelial cells intermixed with blood cells to large circumscribed lesions consisting of well developed tangled masses of vessels surrounded by blood pools. Activated astrocytes surrounded and infiltrated the tumors. In addition, the thymus of R7-infected mice regressed significantly and precipitously due to apoptosis (especially of cortical thymocytes) at the end stage of the disease.
Asunto(s)
Neoplasias Encefálicas/patología , Hemorragia Cerebral/patología , Virus del Sarcoma Murino de Moloney/patogenicidad , Infecciones por Retroviridae/patología , Sarcoma Experimental/patología , Infecciones Tumorales por Virus/patología , Animales , Neoplasias Encefálicas/química , Neoplasias Encefálicas/virología , Células Cultivadas , Hemorragia Cerebral/virología , Factor VIII/análisis , Proteína Ácida Fibrilar de la Glía/análisis , Hemangioendotelioma/química , Hemangioendotelioma/patología , Hemangioendotelioma/virología , Inmunohistoquímica , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos BALB C , Virus del Sarcoma Murino de Moloney/genética , Virus del Sarcoma Murino de Moloney/aislamiento & purificación , Mutación , Tamaño de los Órganos , Reacción en Cadena de la Polimerasa , Infecciones por Retroviridae/virología , Sarcoma Experimental/química , Sarcoma Experimental/virología , Neoplasias del Bazo/química , Neoplasias del Bazo/patología , Neoplasias del Bazo/virología , Neoplasias del Timo/química , Neoplasias del Timo/patología , Neoplasias del Timo/virología , Factores de Tiempo , Infecciones Tumorales por Virus/virologíaRESUMEN
Mouse uterine tumors were examined for genetic alterations in the ras proto-oncogene and p53 tumor suppressor gene and for other biologically relevant immunohistochemical markers that may increase our understanding of the events that occur in uterine cancer. Fourteen dimethylhydrazine (DMH)-induced uterine sarcomas, including 3 primary malignant fibrous histiocytomas (MFH), 7 transplanted MFH, 3 stromal sarcomas, and 1 undifferentiated sarcoma, were first screened by immunohistochemistry for p53 missense mutations, followed by single strand conformation polymorphism analysis and DNA sequencing for the identification of point mutations. There was 100% correlation between p53 protein immunopositivity and subsequent detection of p53 mutations in DMH-induced malignant fibrous histiocytomas. All MFH had a characteristic p53 G:C-->A:T transition mutation, consistent with O6-methylguanine mispairing with thymine, the most common DNA lesion caused by alkylating agents. DMH-induced uterine MFH with p53 mutations also had a higher proliferative rate (qualitatively evaluated by immunohistochemical detection of proliferating cell nuclear antigen) when compared with other DMH-induced sarcomas. Uterine sarcomas were further evaluated for biological end points, such as estrogen receptor and desmin. Neoplastic cells from stromal sarcomas (SS), undifferentiated sarcomas (US), and MFH did not stain for desmin. The estrogen receptor was detected in normal uteri and a small portion of MFH, SS, and US. Our data suggest that DMH-induced uterine sarcomas are not consistent with smooth muscle cell origin and that a subset of these tumors, specifically DMH-induced malignant fibrous histiocytomas, have unique p53 G:C-->A:T transitions and a high proliferative rate.
Asunto(s)
Genes p53/genética , Neoplasias Uterinas/genética , 1,2-Dimetilhidrazina , Animales , División Celular , Desmina/análisis , Femenino , Inmunohistoquímica , Ratones , Ratones Endogámicos CBA , Mutación Puntual , Polimorfismo Conformacional Retorcido-Simple , Antígeno Nuclear de Célula en Proliferación/análisis , Receptores de Estrógenos/análisis , Sarcoma Experimental/química , Sarcoma Experimental/genética , Sarcoma Experimental/patología , Proteína p53 Supresora de Tumor/análisis , Neoplasias Uterinas/inducido químicamente , Neoplasias Uterinas/química , Neoplasias Uterinas/patologíaRESUMEN
Islet amyloidosis is characterized by the deposition and accumulation of amylin in pancreatic beta-cells and is observed in 90% of patients with type 2 diabetes. Previous studies have also revealed the presence of the specific heparan sulfate proteoglycan, perlecan, colocalized to islet amyloid deposits, similar to perlecan's known involvement with other amyloid proteins. In the present study, perlecan purified from the Engelbreth-Holm-Swarm (EHS) tumor was used to define perlecan's interactions with amylin (i.e., islet amyloid polypeptide) and its effects on amylin fibril formation. Using a solid phase-binding immunoassay, human amylin, but not rat amylin, bound immobilized EHS perlecan with a single dissociation constant (Kd) = 2.75 x 10(-6) mol/l. The binding of human amylin to perlecan was similarly observed using perlecan heparan sulfate glycosaminoglycans (GAGs), and was completely abolished by 10 micromol/l heparin. Using thioflavin T fluorometry, Congo red staining, and electron microscopy methodology, intact perlecan was found to enhance amylin fibril formation in a dosage-dependent manner, with the majority of these effects attributed to the heparan sulfate GAG chains of perlecan. Other sulfated GAGs and related macromolecules were also effective in the enhancement of amylin fibril formation in the order of heparin > heparan sulfate > chondroitin-4-sulfate = dermatan sulfate = dextran sulfate > pentosan polysulfate, implicating the importance of the specific GAG/carbohydrate backbone. The sulfate content of heparin/heparan sulfate was also important for the enhancement of amylin fibril formation in the order of heparin > N-desulfated N-acetylated heparin > completely desulfated N-sulfated heparin > completely desulfated N-acetylated heparin. These studies suggest that the enhancement effects of perlecan on amylin fibril formation are mediated primarily by both specific GAG chain backbone and GAG sulfate content, and implicate perlecan as an important macromolecule that is likely involved in the pathogenesis of islet amyloidosis.
Asunto(s)
Amiloide/metabolismo , Glicosaminoglicanos/metabolismo , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/metabolismo , Proteoglicanos/metabolismo , Amiloide/química , Amiloide/fisiología , Animales , Benzotiazoles , Colorantes , Rojo Congo , Colorantes Fluorescentes , Fluorometría , Glicosaminoglicanos/química , Heparitina Sulfato/química , Heparitina Sulfato/aislamiento & purificación , Humanos , Inmunoensayo , Polipéptido Amiloide de los Islotes Pancreáticos , Microscopía Electrónica , Proteoglicanos/química , Proteoglicanos/aislamiento & purificación , Ratas , Sarcoma Experimental/química , Coloración y Etiquetado , TiazolesRESUMEN
Proliferation and differentiation of epithelial cells are thought to be regulated by soluble factors in extracellular fluid and insoluble components of the extracellular matrix. We have examined the combined effects of soluble factors and an extracellular matrix (EHS matrix) on DNA synthesis, cell proliferation, and surfactant protein gene expression in primary cultures of alveolar type II epithelial cells. Cells on EHS matrix cultured in DMEM containing insulin, cholera toxin, EGF, aFGF, 5% rat serum, and 15-fold concentrated bronchoalveolar lavage fluid (D-GM) formed larger aggregates than cells cultured on the same substratum in DMEM containing 5% rat serum (D-5). Cells cultured in D-GM on EHS matrix incorporated more [3H]-thymidine than cells on the same substratum in D-5, with an eight-fold increase seen on day 4 of culture. This increase in [3H]-thymidine incorporation was accompanied by a labeling index of greater than 65% of the cells. Cell counts showed that exposure of type II cells on EHS matrix to D-GM resulted in increased cell number on day 4 of culture. [3H]-thymidine autoradiography combined with immunostaining with anti-cytokeratin, anti-SP-A, and anti-vimentin antibodies demonstrated that the proliferating cells were epithelial cells that contained SP-A. Type II cells cultured on plastic in D-GM also showed increased [3H]-thymidine incorporation compared to cells cultured in D-5. The level of [3H]-thymidine incorporation by cells on plastic, however, was significantly less than that seen in cells cultured in the same medium on EHS matrix. Type II cells cultured on EHS matrix in D-GM had a decreased abundance of mRNAs for SP-A and SP-C than cells cultured on EHS matrix in D-5 as determined by Northern analysis. This inhibition was reversed by switching from D-GM to D-5 on day 4 and culturing the cells for an additional 4 days. In contrast, SP-B mRNA was increased in response to D-GM. This increase was not reversed by switching from D-GM to D-5 on day 4. These results suggest that the interaction of soluble factors and extracellular matrix components has a strong influence on type II cell proliferation, which were partially associated with the reversible inhibition of lung tissue-specific protein mRNAs. Their dynamic interplay among the type II cell, the extracellular matrix, and growth factors may determine multicellular functions and play an important role in normal lung development and in the repair of the lung epithelium following injury.
Asunto(s)
Factores Biológicos/farmacología , Líquido del Lavado Bronquioalveolar/química , Replicación del ADN , Células Epiteliales/efectos de los fármacos , Matriz Extracelular/fisiología , Proteolípidos/biosíntesis , Alveolos Pulmonares/citología , Surfactantes Pulmonares/biosíntesis , Animales , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Proteolípidos/genética , Proteína A Asociada a Surfactante Pulmonar , Proteínas Asociadas a Surfactante Pulmonar , Surfactantes Pulmonares/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Sarcoma Experimental/química , Organismos Libres de Patógenos EspecíficosRESUMEN
Perlecan is a modular heparan sulfate proteoglycan that is an intrinsic constituent of all basement membranes and extracellular matrices. Because of its strategic position and unique structure, perlecan has been implicated in modulating the activity of various growth factors required for normal development and tissue homeostasis. To gain insights into the potential function of perlecan in vivo, we examined the spatiotemporal distribution of its mRNA and protein core during murine embryogenesis. We utilized a new affinity-purified antibody that recognizes specifically the protein core of perlecan together with an in situ RT-PCR approach to perform a systematic analysis of perlecan expression and deposition during murine ontogeny. Perlecan appeared early (E10.5) in tissues of vasculogenesis including heart, pericardium, and major blood vessels. Its early expression coincided with the development of the cardiovascular system. Subsequently (E11-13), the greatest deposition of perlecan occurred within the developing cartilage, especially the cartilage undergoing endochondral ossification, where it remained elevated throughout all the developmental stages, and up to adulthood. Interestingly, the mRNA levels of perlecan were always higher in all the vascularized tissues, principally within endothelial cells, while chondrocytes displayed relatively low mRNA levels. This suggests a higher biosynthesis and turnover rates in the blood vessels vis-à-vis those of cartilaginous and other mesenchymal tissues. During later stages of development (E13-17.5) perlecan mRNA levels progressively increased and its expression correlated with the onset of tissue differentiation of various parenchymal organs including the developing kidneys, lungs, liver, spleen, and gastrointestinal tract. The central nervous system showed no perlecan expression with the exception of the calvaria and choroid plexus. Collectively, the results indicate that perlecan may play crucial roles not only in vasculogenesis but also in the maturation and maintenance of differentiated tissues, including cartilage.
Asunto(s)
Vasos Sanguíneos/embriología , Regulación del Desarrollo de la Expresión Génica , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/fisiología , Neovascularización Fisiológica/genética , Proteoglicanos/fisiología , Animales , Anticuerpos , Sistema Cardiovascular/embriología , Cartílago/embriología , Diferenciación Celular , Endotelio Vascular/embriología , Células Epiteliales/citología , Heparitina Sulfato/biosíntesis , Heparitina Sulfato/genética , Inmunohistoquímica , Hibridación in Situ , Mesodermo/citología , Ratones , Reacción en Cadena de la Polimerasa , Proteoglicanos/biosíntesis , Proteoglicanos/genética , Conejos , Sarcoma Experimental/químicaRESUMEN
Using electron immunocytochemistry, blood vessels in the normal rat liver and in 2 different animal models of liver metastases: (1) Hooded Lister rat with MC28 tumour, a sarcoma, and (2) nude rat with HT29 tumour, a carcinoma, were investigated for the presence of endothelin-1. In the normal livers, small subpopulations of vascular endothelial cells displayed discrete immunoreactivity for endothelin-1. In the livers with malignant tumours, there was a substantial increase in endothelin-1-immunoreactive endothelial cells in vessels located at the tumour periphery. In the controls, antibody to endothelin-1 also labelled sporadically some fibroblast/fibroblast-like cells associated with the blood vessels. In contrast, intense immunoreactivity for endothelin-1 was frequently associated with the tumour cells and/or fibroblast cells in both types of tumour examined.
Asunto(s)
Carcinoma/irrigación sanguínea , Endotelina-1/análisis , Neoplasias Hepáticas Experimentales/secundario , Sarcoma Experimental/irrigación sanguínea , Animales , Carcinoma/química , Endotelio Vascular/química , Fibroblastos/química , Hígado/irrigación sanguínea , Hígado/química , Neoplasias Hepáticas Experimentales/irrigación sanguínea , Neoplasias Hepáticas Experimentales/química , Masculino , Microscopía Inmunoelectrónica , Ratas , Ratas Endogámicas , Ratas Desnudas , Sarcoma Experimental/químicaRESUMEN
The sulfated HNK-1 carbohydrate present on glycolipids and on several neural recognition molecules has been shown to mediate the adhesion of murine small cerebellar neurons and astrocytes to the extracellular matrix molecule laminin-1. In this study, we characterized the binding of the HNK-1 carbohydrate to laminin-1 extracted from the Engelbreth-Holm-Swarm (EHS) sarcoma and distinguished it unequivocally from binding sites for other sulfated carbohydrates. Electron microscopic analysis of rotary shadowed complexes of laminin-1 and a HNK-1 neoglycoprotein revealed a major binding site on the G domain that comprises the C-terminal globule of the laminin alpha1 chain. The HNK-1 carbohydrate also interacted with placental laminin isoforms containing an alpha chain variant. It bound to the proteolytic laminin-1 fragment E8 comprising the domains G1-G3, but not to fragment E3 that carries the major heparin-binding site on domains G4-G5. No binding was observed to the short arm containing fragments E1XNd or P1. Binding studies with native or denatured laminin E8 fragments and proteolytic or recombinant fragments of the G domain localized the HNK-1 carbohydrate binding site to domain G2. The binding could be clearly distinguished from binding sites for other sulfated carbohydrates such as heparin and sulfatides. Further, the binding could not be abolished by reduction and alkylation or by urea treatment of laminin-1 and was independent of the native conformation of laminin-1 and of Ca2+. The G2 domain is also involved in the adhesion of HNK-1 carbohydrate expressing early postnatal cerebellar neurons and is different from heparin- and sulfatide-mediated cell adhesion to laminin-1. HNK-1 carbohydrate-mediated cell adhesion appears, however, to be dependent on the native conformation of laminin-1 indicating a more complex cellular recognition process.
Asunto(s)
Antígenos CD57/metabolismo , Adhesión Celular/fisiología , Glucolípidos/metabolismo , Laminina/metabolismo , Neuronas/fisiología , Animales , Astrocitos/fisiología , Sitios de Unión , Unión Competitiva , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Cerebrósidos/metabolismo , Glucolípidos/química , Glicoproteínas/metabolismo , Heparina/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Laminina/química , Ratones , Ratones Endogámicos ICR , Microscopía Electrónica , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Sarcoma Experimental/química , Albúmina Sérica Bovina/metabolismo , Sulfoglicoesfingolípidos/metabolismoRESUMEN
The biodistribution of two recently developed tumour markers, trimethylated (CP(Me)3) and trimethoxylated (CP(OMe)3) carotenoporphyrin, was investigated by means of laser-induced fluorescence (LIF) after i.v. injection into 38 tumour-bearing (MS-2 fibrosarcoma) female Balb/c mice. At 3, 24, 48 or 96 h after administration, the carotenoporphyrin fluorescence was measured in tumoral and peritumoral tissue, as well as in the abdominal, thoracic and cranial cavities. The fluorescence was induced by a nitrogen laser-pumped dye laser, emitting light at 425 nm, and analysed by a polychromator equipped with an image-intensified CCD camera. The fluorescence was evaluated at 490, 655 and 720 nm: the second and third wavelengths represent the carotenoporphyrin (CP)-related peaks, whereas the first one is close to the peak of the tissue autofluorescence. The tumour and the liver were the two tissue types showing the strongest carotenoporphyrin-related fluorescence, whereas the cerebral cortex and muscle consistently exhibited weak substance-related fluorescence. In most tissue types, the fluorescence intensities decreased over time. A few exceptions were observed, notably the liver, in which the intensity remained remarkably constant over the time period investigated.