RESUMEN

The location of oral leukoplakia correlates strongly with the probability of finding dysplastic or malignant alterations at biopsy. It is well established that early detection can dramatically improve the 5-year survival rates for oral squamous cell carcinomas. Since aneuploidy is predictive of future conversion to malignancy, we hypothesized that dysplastic lesions from high-risk sites (floor of mouth, tongue and lips) would exhibit greater aneuploidy than low-risk sites (palate, gingiva and buccal mucosa). Epithelial sections from 60 archival samples diagnosed as mild dysplasia (36 females, 20 males) from various high/low risk locations were stained with Blue Feulgen Stain for DNA Ploidy Analysis (Clarient, Aliso Viejo, CA) and ploidy was analyzed using a ChromaVision ACIS II (Clarient, ALiso Viejo, CA) Image cytometry system. A DNA histogram was generated using an image analyzing software that evaluated the amount of Feulgen stain which is proportional to the amount of nuclear DNA. An ANOVA analysis followed by the Student's't' test revealed significant differences between means (P

Asunto(s)

Aneuploidia , Carcinoma de Células Escamosas/patología , Leucoplasia/patología , Neoplasias de la Boca/patología , Boca/patología , Carcinoma de Células Escamosas/genética , ADN de Neoplasias/análisis , Femenino , Humanos , Citometría de Imagen , Leucoplasia/genética , Masculino , Neoplasias de la Boca/genética , Estudios Retrospectivos , Colorantes de Rosanilina

RESUMEN

Squamous cell carcinomas of the head and neck are known for their aggressive growth and propensity to metastasize. Invasion is facilitated by matrix metalloproteineases (MMPs). Tissue inhibitors of MMPs (TIMPs) negatively regulate MMP activity. MMP and TIMP expression in head and neck squamous cell carcinomas was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). qRT-PCR allows measurement of several mRNAs from as little as 4 microg of total cellular RNA. We measured MMP-1, MMP-2, MMP-9, and TIMP-1 expression in 8 specimens of primary tumors and adjacent normal tissue. MMP-1 was overexpressed in 6 of 8 tumors, and MMP-9 was overexpressed in 4 of 7 tumors. MMP-2 was expressed in 3 of 8 tumors and 3 of 8 normal samples. TIMP-1 was expressed in all specimens. This work demonstrates that qRT-PCR can be used to examine expression of specific mRNAs in clinical specimens. Therefore this method provides another tool for the molecular analysis of tumors.

Asunto(s)

Carcinoma de Células Escamosas/enzimología , Neoplasias Laríngeas/química , Metaloproteinasas de la Matriz/análisis , Neoplasias Faríngeas/química , Inhibidor Tisular de Metaloproteinasa-1/análisis , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Metaloproteinasa 1 de la Matriz/análisis , Metaloproteinasa 2 de la Matriz/análisis , Metaloproteinasa 9 de la Matriz/análisis , Persona de Mediana Edad , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

BACKGROUND: Integrins are cell surface receptors which, in part, mediate the adhesion of cells to the extracellular matrix. In addition to providing a molecular "glue" essential for tissue organization and survival, integrins are dynamic signaling molecules. Integrins allow normal, nontransformed cells to sense that they are adhered to the extracellular matrix, thus providing a cell survival signal. This signal allows cells to proliferate in the presence of growth factors and in some instances prevents apoptosis. Integrins also mediate cell migration as it occurs in normal processes such as angiogenesis, wound healing, immune system function, and development. Aberrances in the expression and function of integrins contribute to many disease states including cancer. RESULTS: Focal adhesion kinase (FAK) becomes phosphorylated and activated during integrin-mediated cell adhesion. Focal adhesion kinase is a signal transducer of integrins (and certain soluble growth factors). Cells derived from FAK -/- mouse embryos exhibit reduced migration relative to wild-type cells. Cells which overexpress FAK show increased migration relative to wild-type cells. Focal adhesion kinase promotes cell survival under certain in vitro conditions. Focal adhesion kinase is overexpressed in invasive and metastatic colon, breast, thyroid, and prostate cancers. Enhanced FAK immunostaining is detected in small populations of preinvasive (carcinoma in situ) oral cancers and in large populations of cells in invasive oral cancers. CONCLUSIONS: Focal adhesion kinase is probably not a classical oncogene but may be involved in the progression of cancer to invasion and metastasis. It is hypothesized that overexpression of FAK in subpopulations of tumor cells leads to populations of cells with a high propensity toward invasion and metastasis. Focal adhesion kinase would have a dual role in this regard: Overexpression of FAK leads to (1) increased cell migration and (2) increased cell survival under anchorage-independent conditions. Further work is needed to test this model and to determine whether FAK represents a viable target for anticancer therapy.

Asunto(s)

Moléculas de Adhesión Celular/fisiología , Neoplasias/enzimología , Proteínas Tirosina Quinasas/fisiología , Receptor de Insulina/fisiología , Transducción de Señal/fisiología , Animales , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Humanos , Integrinas/fisiología , Microscopía Fluorescente , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias/fisiopatología , Fosforilación

RESUMEN

HYPOTHESIS: Scatter factor (SF) is a pleiotropic growth factor that recently has been shown to induce epithelial cell proliferation, random motility, and invasion via interaction with its receptor, a tyrosine kinase encoded by the c-met proto-oncogene. Studies involving a variety of solid tumors have suggested that overexpression of the SF/c-met ligand-receptor pair is associated with the acquisition of a malignant phenotype. We hypothesize that SF and c-met are overexpressed in epithelial malignancies of the head and neck including squamous cell carcinoma (SCC) of the oral cavity. STUDY DESIGN: Immunohistochemical staining of randomly selected normal, dysplastic, and malignant oral tissues. METHODS: Formalin-fixed, paraffin-embedded tissues were obtained from the Department of Oral Pathology at Shands Hospital (University of Florida), Gainesville, Florida. Examples of mild dysplasia, severe dysplasia, well-differentiated SCC, moderately differentiated SCC, and poorly differentiated SCC were randomly selected from the dictated reports of one of two staff oral pathologists. Histologically normal margins of each specimen served as normal controls. The tissues were immunohistochemically stained using commercially available antibodies against SF and c-met. Appropriate negative controls were run with each batch to ensure staining specificity. Evaluation of staining intensity was carried out using a computerized image analysis system. A one-way analysis of variance (ANOVA) with pairwise multiple-comparison procedures (Fisher method) was used to analyze the data. RESULTS: Statistically significant differences (P < .0001) in the intensity of staining were noted between the malignant and normal and the malignant and dysplastic tissues for both SF and c-met. No differences were appreciated when staining of normal and dysplastic sections of the SF-stained tissue were compared. CONCLUSIONS: The results suggest that the SF/c-met ligand-receptor pair is overexpressed in SCC of the oral cavity.

Asunto(s)

Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Regulación Neoplásica de la Expresión Génica/genética , Factor de Crecimiento de Hepatocito/metabolismo , Neoplasias Orofaríngeas/metabolismo , Neoplasias Orofaríngeas/patología , Proteínas Proto-Oncogénicas c-met/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Análisis de Varianza , Carcinoma de Células Escamosas/genética , Técnicas de Cultivo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proto-Oncogenes Mas

RESUMEN

BACKGROUND: Evidence suggests that focal adhesion kinase (FAK) is involved in the pathogenesis of certain cancers. Focal adhesion kinase is overexpressed in invasive and metastatic cancers of the breast, colon, thyroid, and prostate. The objective of this study was to determine the presence and cellular distribution of FAK in oral cancer and determine whether there is a difference in FAK expression in preinvasive and invasive oral cancers. METHODS: Immunohistochemistry was used to detect FAK expression in 20 archival oral cancer specimens. RESULTS: Focal adhesion kinase immunoreactivity was detected in all specimens that were examined. In tumors, there was an increase in the intensity of FAK staining and in the percentage of FAK-positive cells. Preinvasive tumors had populations of cancer cells which stained more intensely than neighboring cancer cells. CONCLUSIONS: Focal adhesion kinase expression appears to be increased in invasive and preinvasive oral cancers. It is speculated that enhanced expression of FAK may contribute to the aggressive phenotype of oral cancers.

Asunto(s)

Carcinoma in Situ/metabolismo , Carcinoma de Células Escamosas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Neoplasias de la Boca/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptor de Insulina/metabolismo , Anticuerpos Monoclonales , Carcinoma in Situ/patología , Carcinoma de Células Escamosas/patología , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Humanos , Inmunohistoquímica , Neoplasias de la Boca/patología

RESUMEN

The rootwood of Aeschynomene mimosifolia Vatke (Leguminosae) has yielded a new neoflavonoid, mimosifoliol (1), and an unusual C16-styrylcycloheptenone derivative, mimosifolenone (2). The structures of these compounds were determined on the basis of spectral analysis. Compound 1 demonstrated weak activity in DNA-strand scission assay, while compound 2 was found to be inactive. Mimosifoliol (1) was inactive toward several human cell lines, while 2 was moderately active against the KB cell line.

Asunto(s)

Acrilatos/aislamiento & purificación , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/farmacología , Cicloheptanos/aislamiento & purificación , Guayacol/análogos & derivados , Raíces de Plantas/química , Plantas Medicinales/química , Estirenos/aislamiento & purificación , Acrilatos/farmacología , Cicloheptanos/farmacología , Daño del ADN , Ensayos de Selección de Medicamentos Antitumorales , Guayacol/aislamiento & purificación , Guayacol/farmacología , Humanos , Células KB , Extractos Vegetales/química , Estirenos/farmacología , Células Tumorales Cultivadas , Zimbabwe

RESUMEN

Over the last few years, it has become clear that cell adhesion receptors function in signal transduction processes leading to the regulation of cell growth and differentiation. Signal transduction by both integrins and CAMs has been shown to involve activation of tyrosine kinases, while CAM signaling in neural cells involves G proteins as well. In the case of integrins, some of the downstream signaling events intersect with the Ras pathway, particularly the activation of MAP kinases. In fibroblasts, integrin mediated anchorage to the substratum regulates cell cycle traverse, while in epithelial cells, loss of anchorage can trigger programmed cell death. In many cell types, but particularly monocytic cells, integrin ligation has a profound impact on gene expression. Preliminary evidence also implicates CAMs and selectins in gene regulation. A consistent theme in signal transduction mediated by adhesion receptors concerns the role of the cytoskeleton. Integrin mediated signaling processes are interrupted by cytoskeletal disassembly. Identification of the APC and neurofibromatosis type 2 tumor suppressors suggest that cytoskeletal complexes also play a key role in signaling by cadherins and CD44, respectively. Thus, signaling by cell adhesion receptors may involve aspects that impinge on previously known signaling pathways including the RTK/Ras pathway and serpentine receptor/G protein pathways. However, novel aspects of signal transduction involving cytoskeletal assemblies may also be critical.

Asunto(s)

Cadherinas/fisiología , Moléculas de Adhesión Celular/fisiología , Integrinas/fisiología , Transducción de Señal , Animales , Proteínas Portadoras/fisiología , Citoesqueleto/fisiología , Regulación de la Expresión Génica , Humanos , Receptores de Hialuranos , Selectina L , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de Superficie Celular/fisiología , Receptores Mensajeros de Linfocitos/fisiología

RESUMEN

The focal adhesion kinase (FAK) gene encodes a tyrosine kinase (p125FAK) thought to be involved in signal transduction pathways used in cell adhesion, motility, and anchorage-independent growth. Because alterations in these cellular processes occur in tumor invasion and metastasis, we studied the protein expression of FAK in a variety of human tumors and found that in the 119 samples studied, increased levels of p125FAK correlated with the invasive potential of a tumor. By comparing FAK expression in tumors with normal tissue from the same patient, we found that p125FAK was significantly elevated in 17 (100%) of 17 invasive and metastatic colonic lesions and in 22 (88%) of 25 invasive and metastatic breast tumors. Additional studies of FAK expression in 13 high grade sarcomas showed high levels in all samples compared to benign, noninvasive mesenchymal specimens. Furthermore, FAK protein levels were elevated in preinvasive lesions, such as large (> 2 cm) colonic villous adenomas, whereas noninvasive, yet hypercellular, neoplastic tissues such as parathyroid and hepatocellular adenomas did not overexpress FAK. These data provide evidence that both epithelial and mesenchymal tumor progression are accompanied by increased p125FAK expression and suggest that the level of FAK expression might be a marker for the invasive potential of a tumor.

Asunto(s)

Moléculas de Adhesión Celular/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Adenofibroma/metabolismo , Western Blotting , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Humanos , Leiomioma/metabolismo , Lipoma/metabolismo , Invasividad Neoplásica , Proteínas Proto-Oncogénicas c-abl/metabolismo , Sarcoma/metabolismo

RESUMEN

Integrin-mediated cell adhesion, or cross-linking of integrins using antibodies, often results in the enhanced tyrosine phosphorylation of certain intracellular proteins, suggesting that integrins may play a role in signal transduction processes. In fibroblasts, platelets, and carcinoma cells, a novel tyrosine kinase termed pp125FAK has been implicated in integrin-mediated tyrosine phosphorylation. In some cell types, integrin ligation or cell adhesion has also been shown to result in the increased expression of certain genes. Although it seems reasonable to hypothesize that integrin-mediated tyrosine phosphorylation and integrin-mediated gene induction are related, until now, there has been no direct evidence supporting this hypothesis. In the current report, we explore the relationship between integrin-mediated tyrosine phosphorylation and gene induction in human monocytes. We demonstrate that monocyte adherence to tissue culture dishes or to extracellular matrix proteins is followed by a rapid and profound increase in tyrosine phosphorylation, with the predominant phosphorylated component being a protein of 76 kD (pp76). Tyrosine phosphorylation of pp76 and other monocyte proteins can also be triggered by incubation of monocytes with antibodies to the integrin beta 1 subunit, or by F(ab')2 fragments of such antibodies, but not by F(ab) fragments. The ligation of beta 1 integrins with antibodies or F(ab')2 fragments also induces the expression of immediate-early (IE) genes such as IL-1 beta. When adhering monocytes are treated with the tyrosine kinase inhibitors genistein or herbimycin, both phosphorylation of pp76 and induction of IL-1 beta message are blocked in a dose-dependent fashion. Similarly, treatment with genistein or herbimycin can block tyrosine phosphorylation of pp76 and IL-1 beta message induction mediated by ligation of beta 1 integrin with antibodies. These observations suggest that protein tyrosine phosphorylation is an important aspect of integrin-mediated IE gene induction in monocytes. The cytoplasmic tyrosine kinase pp125FAK, although important in integrin signaling in other cell types, seems not to play a role in monocytes because this protein could not be detected in these cells.

Asunto(s)

Regulación de la Expresión Génica/fisiología , Genes Inmediatos-Precoces , Integrinas/fisiología , Monocitos/fisiología , Fosfoproteínas/biosíntesis , Tirosina/metabolismo , Benzoquinonas , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Genisteína , Humanos , Interleucina-1/biosíntesis , Isoflavonas/farmacología , Lactamas Macrocíclicas , Monocitos/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Quinonas/farmacología , Rifabutina/análogos & derivados , Activación Transcripcional

RESUMEN

The purpose of this study was to explore the functional role of the cytoplasmic domain of the alpha subunit of the alpha 5/beta 1 integrin, a fibronectin receptor. Mutant CHO cells that express very low levels of endogenous hamster alpha 5 subunit (CHO clone B2) were transfected with an expression vector containing full-length or truncated human alpha 5 cDNAs to form chimeric human alpha 5/hamster beta 1 integrins. Three transfectants were examined: B2a27 expresses a full-length human alpha 5 subunit with 27 amino acids in the cytoplasmic domain; B2a10 expresses an alpha 5 with a 17-amino acid cytoplasmic truncation; B2a1 expresses an alpha 5 with a 26-amino acid truncation. Levels of alpha 5/beta 1 surface expression in B2a27 and B2a10 cells were similar to that in wild type CHO cells. The expression of alpha 5/beta 1 in B2a1 cells was less, amounting to 15-20% of WT levels, despite message levels that were three to five times greater than those of B2a27. The transfectants were used to examine the role of the alpha 5 cytoplasmic domain in cell adhesion, cell motility, cytoskeletal organization, and integrin-mediated tyrosine phosphorylation. The adhesion characteristics of B2a27 and B2a10 cells on fibronectin substrata were similar to each other and to wild type CHO cells. B2a1 cells displayed slight reductions in the strength and rate of adhesion to fibronectin. Cell motility in the presence of fibronectin was similar for B2a27, B2a10, and wild type CHO cells, while the B2a1 cells were substantially less motile. Comparable degrees of cell spreading and extensive organization of actin filaments were observed for B2a27, B2a10, and wild type CHO cells on fibronectin substrata. The B2a1 cells spread to a lesser degree, and some organization of actin was observed; the untransfected B2 cells remained round on fibronectin substrata and showed no actin reorganization. Since the reduced motility and cell spreading observed in the B2a1 cells might be due either to reduced surface expression of alpha 5/beta 1 or to the truncation in the alpha 5 cytoplasmic domain, we used flow cytometric cell sorting to select populations of B2a1 and B2a27 cells expressing similar levels of cell surface alpha 5. The deficits in spreading and motility were present in B2a1 cells expressing high levels of alpha 5. Thus the region of the alpha 5 cytoplasmic domain adjacent to the membrane seems to play an important role in cytoskeletal organization and cell motility. We also examined whether alpha subunit truncation would affect integrin-mediated tyrosine phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)

Asunto(s)

Antígenos CD/metabolismo , Receptores de Fibronectina/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Adhesión Celular , Movimiento Celular , Células Clonales , Cricetinae , Fibronectinas/metabolismo , Citometría de Flujo , Variación Genética , Vectores Genéticos , Humanos , Integrina alfa5 , Cinética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Fosfoproteínas/análisis , Fosfoproteínas/metabolismo , Fosfotirosina , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Receptores de Fibronectina/biosíntesis , Receptores de Fibronectina/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Tirosina/análogos & derivados , Tirosina/análisis

RESUMEN

Two 18-mer peptides were synthesized. Peptide E117 had the same amino acid sequence as that in the region 110-127 of human growth hormone [hGH (110-127)]. In the second peptide (L117), E117 was replaced with leucine. Biological activity of the peptides was assessed in cultured Nb2 lymphoma cells. L117 stimulated cell division and DNA synthesis in a concentration-dependent manner. Maximum stimulation was equivalent to that noted with native hGH although L117 was much less potent (L117; EC50 approximately 1 microM vs. hGH; EC50 approximately 20 pM). No demonstrable activity was found with peptide E117. These results suggest that the amphipathic helical structure of hGH (110-127) is crucial for biological activity.

Asunto(s)

División Celular/efectos de los fármacos , Hormona del Crecimiento/análogos & derivados , Hormona del Crecimiento/farmacología , Fragmentos de Péptidos/farmacología , Secuencia de Aminoácidos , Animales , ADN de Neoplasias/biosíntesis , Hormona del Crecimiento/química , Humanos , Linfoma de Células T , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Conformación Proteica , Estructura Secundaria de Proteína , Ratas , Relación Estructura-Actividad , Células Tumorales Cultivadas

RESUMEN

We have recently shown that changes in tyrosine phosphorylation of a 130-kDa protein(s) (pp130) may be involved in integrin signaling (Kornberg, L., Earp, H.S., Turner, C., Prokop, and Juliano, R. L. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8392-8396). One component of the pp130 protein complex reacts with an antibody generated against p125fak, which is a focal contact-associated tyrosine kinase (Schaller, M.D., Borgman, C. A., Cobb, B. S., Vines, R. R., Reynolds, A. B., and Parsons, J. T. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5192-5196). Both antibody-mediated integrin clustering and adhesion of KB cells to fibronectin leads to increased tyrosine phosphorylation of p125fak. The phosphorylation of p125fak is coincident with adhesion of cells to fibronectin and is maximal prior to cell spreading. Tyrosine phosphorylation of p125fak is induced when KB cells are allowed to adhere to fibronectin, collagen type IV, or laminin, but is not induced on polylysine. When KB cells are subjected to indirect immunofluorescence microscopy, p125fak colocalizes with talin in focal contacts. These data provide additional evidence that tyrosine kinases are involved in integrin signaling.

Asunto(s)

Moléculas de Adhesión Celular/metabolismo , Adhesión Celular , Integrinas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Western Blotting , Moléculas de Adhesión Celular/aislamiento & purificación , Células Cultivadas , Embrión de Pollo , Electroforesis en Gel de Poliacrilamida , Fibroblastos/fisiología , Fibronectinas/metabolismo , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Humanos , Células KB , Cinética , Peso Molecular , Fosforilación , Proteínas Tirosina Quinasas/aislamiento & purificación , Transducción de Señal

Asunto(s)

Matriz Extracelular/fisiología , Integrinas/fisiología , Proteínas Tirosina Quinasas/fisiología , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Ligandos , Datos de Secuencia Molecular , Fosforilación

RESUMEN

The integrin family of cell adhesion receptors mediates many of the interactions between cells and the extracellular matrix. Because the extracellular matrix has profound influences on cell behavior, it seems likely that integrins transduce biochemical signals across the cell membrane. The nature of these putative signals has, thus far, remained elusive. Antibody-mediated clustering of integrin receptors was used to mimic the integrin clustering process that occurs during formation of adhesive contacts. Human epidermal carcinoma (KB) cells were incubated with an anti-beta 1 integrin monoclonal antibody for 30 min on ice followed by incubation at 37 degrees C with anti-rat IgG. This treatment, which induced integrin clustering, stimulated the phosphorylation on tyrosine residues of a 115- to 130-kDa complex of proteins termed pp130. When integrins were clustered in the presence of the phosphatase inhibitor sodium orthovanadate, pp130 showed a substantial increase in phosphorylation compared to the case in which integrins were clustered in the absence of vanadate. Maximal pp130 phosphorylation was observed 10-20 min after initiation of integrin clustering in the absence of vanadate or after 5-10 min in its presence. These time courses roughly parallel the formation of integrin clusters on the cell surface as observed by fluorescence microscopy. pp130 phosphorylation depended on the amount of anti-integrin antibody present. Additionally, the tyrosine phosphorylation of pp130 showed specificity since it was stimulated by antibodies to the integrin alpha 3 and beta 1 subunits but not by antibodies to other integrin alpha subunits or to nonintegrin cell surface proteins. Immunoprecipitation experiments clearly demonstrated that pp130 is not itself a beta 1 integrin. It is postulated, therefore, that the integrin-stimulated tyrosine phosphorylation of pp130 may reflect part of an important signal transduction process between the extracellular matrix and the cell interior.

Asunto(s)

Integrinas/fisiología , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Complejo Antígeno-Anticuerpo , Línea Celular , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas In Vitro , Peso Molecular , Fosfoproteínas/química , Fosforilación , Fosfotirosina , Agregación de Receptores , Factores de Tiempo , Tirosina/análogos & derivados , Tirosina/metabolismo

RESUMEN

Lactogenic hormones are potent mitogenic agents in the Nb2 rat lymphoma cell line. Because selective phosphorylation/dephosphorylation of specific proteins by many polypeptide hormones appears to be important in regulating cell growth, the effect of lactogenic hormones on protein phosphorylation was studied in Nb2 cells. Human growth hormone (hGH) promoted the phosphorylation of many proteins (2- to 3-fold) and an Mr 29,000 species (pp29) (greater than or equal to 10-fold). In growth-arrested cells phosphorylation of pp29 peaked 4 h after addition of hGH whereas that of other phosphoproteins increased steadily. Ovine prolactin, another potent mitogenic hormone, caused a concentration-dependent increase in pp29 phosphorylation with an EC50 = 10-20 pM. Other agents which are not mitogenic in this cell line, i.e., 12-O-tetradecanoyl-phorbol-13-acetate (200 nM), 8-Br-cAMP (1 mM), and 8-Br-cGMP (1 mM), did not affect pp29 phosphorylation. Dexamethasone (100 nM), which inhibits lactogen-stimulated growth, reduced hGH-stimulated pp29 phosphorylation to control levels. Fractionation of detergent-treated extracts of hGH-treated cells showed that pp29 is associated with the ribosomal fraction. Taken together, these results show that pp29 phosphorylation is selective and is related to cell growth.

Asunto(s)

Hormona del Crecimiento/farmacología , Lactógeno Placentario/farmacología , Prolactina/farmacología , Proteínas Ribosómicas/metabolismo , Animales , Cicloheximida/farmacología , Dexametasona/farmacología , Humanos , Linfoma , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Quinasas/farmacología , Ratas , Proteína S6 Ribosómica , Células Tumorales Cultivadas

RESUMEN

Rat lymphoma cells (Nb2) are exquisitely sensitive to lactogenic hormones and are an ideal system to study receptor-mediated signal transduction. The effect of human growth hormone (hGH) on macromolecular synthesis, intracellular cAMP concentrations and protein phosphorylation was investigated in Nb2 cells maintained in serum-free medium. hGH stimulated the incorporation of radiolabeled precursors into protein, RNA and DNA in a time-dependent manner. The concentration of hGH inducing half-maximal DNA synthesis was 11 pM, indicating that Nb2 cells cultured in serum-free medium maintain the same sensitivity to lactogen as cells in horse serum-containing medium. hGH over a period of 4 h had no effect on intracellular cAMP regardless of the presence or absence of isobutylmethylxanthine (IBMX). IBMX (250 microM), increased intracellular cAMP levels 2-fold indicating that the cAMP assay was sufficiently sensitive to detect relatively small changes in intracellular cAMP. Cyclic AMP had no effect on protein phosphorylation. However, hGH, prolactin and placental lactogen enhanced phosphorylation of many protein targets, as well as that of a specific protein (Mr = 29,000). Rat growth hormone, which is not mitogenic, had no effect on protein phosphorylation. These results suggest that lactogen-mediated Nb2 mitogenesis does not involve modulation of intracellular cAMP concentration and that cAMP-independent protein phosphorylation may play a role.

Asunto(s)

AMP Cíclico/fisiología , Hormona del Crecimiento/fisiología , Proteínas/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Animales , División Celular , Colforsina/farmacología , AMP Cíclico/metabolismo , ADN/biosíntesis , Fosforilación , Lactógeno Placentario/fisiología , Prolactina/fisiología , Biosíntesis de Proteínas , ARN/biosíntesis , Células Tumorales Cultivadas

RESUMEN

Using [1-14C]oleate-labelled autoclaved Escherichia coli as substrate, we demonstrate that many, but not all, commercial preparations of xanthine oxidase contain phospholipase A2 activity as a contaminant. Phospholipase A2 activity (64.3-545.6 nmol phospholipid hydrolyzed per min per mg protein) was optimal in the neutral to alkaline pH range, was Ca2+-dependent, and was unaffected by the addition of xanthine. Phospholipase A2 activity was totally inhibited by 1.0 mM EDTA while radical production by xanthine plus xanthine oxidase was unaffected by EDTA. Even chromatographically purified xanthine oxidase (Sigma Grade III) contained substantial phospholipase A2 activity (64.3 nmol/min per mg). Since the preparation of xanthine oxidase employs proteolytic digestion of milk or buttermilk by pancreatin, an extract of pancreas which is an organ rich in phospholipase A2 activity, we speculate that the contaminant phospholipase A2 is introduced by this treatment. Because xanthine oxidase is used extensively to study free radical-induced cell injury and membrane phospholipid alterations, the presence of a potent extracellular phospholipase A2 may have influenced previously published reports and such studies in the future should be interpreted with care.

Asunto(s)

Fosfolipasas A/análisis , Fosfolipasas/análisis , Xantina Oxidasa/análisis , Calcio/metabolismo , Radicales Libres , Fosfolipasas A2