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Biomedical subjects

P C Marchisio

Publications and source records attributed to P C Marchisio.

At least 37 records · Page 2Linked to original sources
PubMed1998

Altered expression of alpha 6 integrin subunit in oral squamous cell carcinoma and oral potentially malignant lesions.

The expression and distribution of integrin chains alpha 2, alpha 3, alpha 5, alpha 6, beta 1, beta 4, collagen type IV, laminin 1 and laminin 5 in oral squamous cell carcinoma (SCC) and oral keratoses with and without dysplasia (OL) have been studied by immunochemistry and western blotting. Focal interruptions of basement membrane protein staining were detected in SCC indicating a loss of continuity, whereas tumour nests were apparently completely surrounded by laminin 1, type IV collagen and laminin 5; the loss of basement membrane components in OL was found in only one specimen showing severe dysplasia. The localisation of integrins showed altered suprabasal and pericellular expression of the alpha 6 chain in all but one SCC, as well as in many OL samples, whereas the beta 4 subunit showed only a faint pericellular redistribution in SCC. In OL, beta 4 was often seen in a normal basally polarised distribution. Western blotting analysis confirmed that alpha 6 protein levels were abnormally high in cancerous lesions, whereas quantitative recovery of the beta 4 subunit in SCC was only minimal, suggesting a dissociation in the synthetic ratios of the two chains of the alpha 6 beta 4 heterodimer in SCC. Because alterations in the polarised expression of integrin alpha 6 beta 4 have been seen during epithelial tumour progression and wound healing, we suggest that the lack of restricted basal polarisation of alpha 6 could be an early but non-specific marker of oral malignancy, indicating that the generation of abnormal signals from the extracellular matrix may be involved.

Antigens, CD↗
PubMed1998

Protein phosphatase 1 delta is associated with focal adhesions.

In all mammalian cells protein phosphatase-1 (PP1) exists in three isoforms, defined as alpha, gamma 1 and delta. Immunofluorescence studies with isoform-specific antibodies indicated that delta, but not alpha or gamma 1, is enriched at focal adhesions in HeLa cells, fibroblasts, endothelial cells and keratinocytes. This was confirmed also by interference reflection microscopy, which indicated that PP1 delta was in areas of tight adhesion of the membrane to the extracellular matrix at sites where the microfilament cytoskeleton is organized. In all the cell types so far considered the PP1 delta in focal adhesions represented only a small aliquot of the total PP1 delta, which was predominantly localized to the nucleus. The association of PP1 delta to focal adhesions was confirmed by the co-immunoprecipitation of PP1 delta with the focal adhesion kinase pp125FAK and with the alpha v integrin. Comparison between the amount of PP1 delta associated with focal adhesion proteins and that of PP1 delta recovered in an anti-PP1 delta immunoprecipitate confirmed that only a minor amount of the enzyme was associated with the focal adhesions. Since some focal adhesion proteins are phosphorylated on Ser/Thr, it is likely that PP1 delta may be involved in the regulation of focal adhesion functions and particularly in the signaling pathway generated by cell-substratum adhesion.

3T3 Cells↗
PubMed1998

Fibronectin modulates the effects of HIV-1 Tat on the growth of murine Kaposi's sarcoma-like cells through the down-regulation of tyrosine phosphorylation.

HIV-1 Tat plays a role in the pathogenesis of Kaposi's sarcoma. We therefore investigated the effect of Tat on the growth of murine Kaposi's sarcoma-like spindle (TTB) cells derived from dermal lesions. We observed that Tat and a peptide corresponding to the carboxyl-terminal region (Tat65-80) containing an RGD sequence inhibit TTB cell proliferation only when cells are cultured on fibronectin. This inhibitory effect correlates with redistribution of the alpha(v) integrin subunit on the surface of TTB cells and with down-regulation of tyrosine phosphorylation of specific substrates due to an increased tyrosine phosphatase activity. Indeed, phenylarsine oxide, a potent inhibitor of phosphotyrosine phosphatases, prevented the effects of Tat on TTB cells. We therefore argue that the action of Tat on TTB cells is mediated by the RGD motif through an integrin-based cell signaling pathway involving the activity of phosphotyrosine phosphatase(s), which would lead to a decrease in the levels of phosphotyrosine-containing proteins, among which is erk-2/p42MAPK.

Animals↗
PubMed1997

Isolation of a novel beta4 integrin-binding protein (p27(BBP)) highly expressed in epithelial cells.

The integrin beta4 has a long cytodomain necessary for hemidesmosome formation. A yeast two-hybrid screen using beta4 cytodomain uncovered a protein called p27(BBP) that represents a beta4 interactor. Both in yeast and in vitro, p27(BBP) binds the two NH2-terminal fibronectin type III modules of beta4, a region required for signaling and hemidesmosome formation. Sequence analysis of p27(BBP) revealed that p27(BBP) was not previously known and has no homology with any isolated mammalian protein, but 85% identical to a yeast gene product of unknown function. Expression studies by Northern analysis and in situ hybridization showed that, in vivo, p27(BBP) mRNA is highly expressed in epithelia and proliferating embryonic epithelial cells. An antibody raised against p27(BBP) COOH-terminal domain showed that all beta4-containing epithelial cell lines expressed p27(BBP). The p27(BBP) protein is insoluble and present in the intermediate filament pool. Furthermore, subcellular fractionation indicated the presence of p27(BBP) both in the cytoplasm and in the nucleus. Confocal analysis of cultured cells showed that part of p27(BBP) immunoreactivity was both nuclear and in the membrane closely apposed to beta4. These results suggest that the p27(BBP) is an in vivo interactor of beta4, possibly linking beta4 to the intermediate filament cytoskeleton.

Amino Acid Sequence↗
PubMed1997

Topography and biological role of integrins in human skin.

We report the topography of integrins in the human epidermis and in cultured human keratinocytes. Both in situ and in vitro beta 1 integrins are exposed at the cell-cell adhesion interface while beta 4 is located on the basal membrane in contact with the basal lamina. Such defined sorting identifies discrete cell membrane domains that may be involved in defining, building up, and maintaining epithelial cell polarity. The distribution of integrins is deeply altered in hyperproliferative states like those occurring in several experimental conditions and in epidermal diseases.

Basement Membrane↗
PubMed1997

Chromogranin A fragments modulate cell adhesion. Identification and characterization of a pro-adhesive domain.

Although several functions have been suggested for chromogranin A, a glycoprotein secreted by many neuroendocrine cells, the physiological role of this protein and of its proteolytic fragments has not been established. We have found that mixtures of chromogranin A fragments can inhibit fibroblast adhesion. The anti-adhesive activity was converted into pro-adhesive activity by limited trypsin treatment. Pro-adhesive effects were observed also with recombinant N-terminal fragments corresponding to residues 1-78 and 1-115 and with a synthetic peptide encompassing the residues 7-57. These fragments induced adhesion and spreading of fibroblasts on plates coated with collagen I or IV, laminin, fetal calf serum (FCS) but not on bovine serum albumin. The long incubation time required for adhesion assays (4 h) and the FCS requirements for optimal adhesion suggest that the adhesive activity is likely indirect and requires other proteins present in the FCS or made by the cells. These findings suggest that chromogranin A and its fragments could play a role in the regulation of cell adhesion. Since chromogranin A is concentrated and stored within granules and rapidly released by neuroendocrine cells and neurons after an appropriate stimulus, this protein could be important for the local control of cell adhesion by stimulated cells.

3T3 Cells↗
PubMed1997

Circulating fragments of myelin-associated alpha 6 beta 4 integrin in Guillain-Barré syndrome.

Guillain-Barré-Strohl syndrome (GBS) is an acute peripheral neuropathy causing reversible myelin damage. alpha 6 beta 4 is a laminin receptor of Schwann cells and myelin. Along with myelin breakdown, alpha 6 beta 4 immunoreactivity might be detected in patients' sera and provide a marker for monitoring GBS course. MAbs to beta 4 and alpha 6 were used in an ELISA test to detect protein in GBS serum samples as in normal individuals. In 66% GBS patients, alpha 6 beta 4 immunoreactivity was detected while controls were negative. The level of beta 4 was followed in different patients and found to fluctuate, always being positive in at least one sample. Treatment lowered immunoreactivity in two beta 4-positive GBS sera. Then, circulating alpha 6 beta 4 fragments represent a novel marker of extensive peripheral myelin damage and may be used to validate clinical diagnosis of GBS, evaluate its course and activity.

Acute Disease↗
PubMed1997

[Adhesion molecules in squamous cell carcinoma of the larynx: possible indication of prognosis].

Thirty patients with laryngeal tumors were divided into two groups on the basis of whether clinical and pathological features indicated good or bad prognosis. Samples of each tumor group were selected and examined by immunohistochemistry using mAbs, raised against integrin chains (beta 1, beta 4, alpha 2, alpha 3, alpha 6) and their ligands laminin 1 and 5, collagen type IV, two fibronectin isoforms (ED-A and ED-B) and two isoforms of tenascin known to be associated with neoplasm. Controls were provided by samples of tumor-free laryngeal mucosa removed during the surgical procedure. The normal topographical integrin pattern and the continuity of the basement membrane components was altered in both groups but the extent of these changes was significantly greater in those tumors with poor prognosis. Therefore, the groups could easily and reliably be distinguished by simply observing their immunohistochemical features. It is suggested that performing immunohistochemical analysis on biopsies may aid in early diagnosis as well as in adopting the proper therapeutic strategy to follow for these tumors. The above molecules may become one of the diagnostic tools available for head and neck surgical pathologists.

Adult↗
PubMed1996

Platelet-activating factor (PAF) induces the early tyrosine phosphorylation of focal adhesion kinase (p125FAK) in human endothelial cells.

Platelet-activating factor (PAF) is a potent activator of angiogenesis and controls the motility and the shape of vascular endothelium. The mechanism(s) whereby PAF exerts its action are in part known. Here we report that the biological active (R)PAF enantiomer administrated to cultured endothelial cells induces the early phosphorylation in tyrosine residues of focal adhesion kinase (p125FAX) and paxillin, two molecules involved in the early signaling and cytoskeleton assembly in cells that undergo integrin-mediated adhesion or are challenged by neuropeptides or lysophosphatidic acid. The phenomenon is rapidly turned on, lasts for a few minutes and is adhesion-independent indicating that the chain of events induced by (R)PAF, including p125FAK activation, precedes adhesion. The inhibitory effect of WEB2086, a PAF receptor antagonist, and the lack of activity exerted by the (S)PAF enantiomer, indicate that (R)PAF-mediated p125FAK activation, is PAF receptor-dependent. Calphostin C, an inhibitor of protein kinase C blocks the effect of (R)PAF on p125FAK phosphorylation suggesting that protein kinase C activation is up-stream the activation of this tyrosine kinase. When endothelial cells are exposed to a substratum that allows adhesion and spreading. (R)PAF-stimulated cells, change their adhesive phenotype and start migrating. Inhibitors of tyrosine kinases, like 3-(1,4,-dihydroxytetralyl) methylen-2-oxindole and herbimycin A, reduce the cells migration, the transendothelial flux of albumin and the enhancement of p125FAK activity induced by (R)PAF. The observation that increased tyrosine phosphorylation of p125FAK and its ensuring association with focal adhesion occurs rapidly upon (R)PAF challenge indicates that this signaling molecule has a primary and independent role also in the signaling cascade initiated by (R)PAF.

Amino Acid Sequence↗
PubMed1996

Hypertension-associated point mutations in the adducin alpha and beta subunits affect actin cytoskeleton and ion transport.

The adducin heterodimer is a protein affecting the assembly of the actin-based cytoskeleton. Point mutations in rat adducin alpha (F316Y) and beta (Q529R) subunits are involved in a form of rat primary hypertension (MHS) associated with faster kidney tubular ion transport. A role for adducin in human primary hypertension has also been suggested. By studying the interaction of actin with purified normal and mutated adducin in a cell-free system and the actin assembly in rat kidney epithelial cells (NRK-52E) transfected with mutated rat adducin cDNA, we show that the adducin isoforms differentially modulate: (a) actin assembly both in a cell-free system and within transfected cells; (b) topography of alpha V integrin together with focal contact proteins; and (c) Na-K pump activity at V(max) (faster with the mutated isoforms, 1281 +/- 90 vs 841 +/- 30 nmol K/h.mg pt., P < 0.0001). This co-modulation suggests a role for adducin in the constitutive capacity of the epithelia both to transport ions and to expose adhesion molecules. These findings may also lead to the understanding of the relation between adducin polymorphism and blood pressure and to the development of new approaches to the study of hypertension-associated organ damage.

Actins↗
PubMed1996

Changes in integrin and E-cadherin expression in neoplastic versus normal thyroid tissue.

UNLABELLED: BACK: The functional organization of polarized epithelia depends mostly on adhesion molecules belonging to the integrin and cadherin families. These molecules either recognize basement membrane components, such as laminins, or form intercellular junctions via homotypic interactions. Such tissue organization is often disrupted upon neoplastic transformation, and the resulting loss of functional polarization and cell cohesion might be a prerequisite for the invasive and metastatic behavior of carcinomas. PURPOSE: We studied modifications on thyroid adhesive mechanisms at various stages of neoplastic progression in terms of adhesion molecule expression, topography, and functional regulation by tyrosine kinases. Starting from this working hypothesis, we sought to identify one or more biological markers that would be suggestive of malignant transformation and poorer prognosis and that could be developed as a reliable indicator(s) in early diagnostic steps. METHODS: The study was carried out on both surgical samples and the corresponding fine-needle aspiration biopsy smears (numbers of specimens collected: 19 adenomas, seven follicular carcinomas, 13 papilary carcinomas, and 39 normal tissues). Immunohistochemistry of tissue sections and smears and immuno-precipitation and western blot analysis of protein extracts were done with a battery of monoclonal and polyclonal antibodies. Northern blotting was performed on RNA extracts from frozen tissue samples and use of an integrin subunit beta4 complementary DNA probe. RESULTS: Our findings can be summarized as follows: 1) In normal thyroid cells, the cooperative role of integrin alpha6beta4 and laminin 5/kalinin in hemidesmosome-mediated adhesion adhesion is missing, and recognition of the basal lamina occurs via integrin alpha3beta1 and laminin 1 and/or 2 (this pattern being maintained in adenomas but altered in carcinomas regardless of their histotype or differentiation grade); 2) only in carcinomas with clinical and/or histologic aggressiveness do neoexpression of integrin subunit beta4 and loss of laminin 2/merosin occur, indicating de novo assembly of integrin alpha6beta4; 3) pericellular redistribution and cytoskeletal disconnection of the E-cadherin-catenin complex occur; and 4) basal E-cadherin tyrosine phosphorylation decreases in carcinomas as compared with that in normal and adenomatous tissue. CONCLUSION: The malignant progression of thyroid tumors involves marked rearrangement of cell-basement membrane and cell-cell adhesion molecules and changes in their cytoskeleton linkage. These rearrangements are also easily and reproducibly detected on fine-needle aspiration biopsy smears. IMPLICATIONS: Immunodetection of adhesion molecules in sections and/or fine-needle smears may complement the toolbox of thyroid surgical pathologists; it may expand the possibilities of achieving a correct early diagnosis of thyroid tumors and of gaining some prognostic information on thyroid tumors.

Adenocarcinoma, Follicular↗
PubMed1996

Integrin synthesis and utilization in cultured human osteoblasts.

This study describes the adhesion of human osteoblasts, cultured in vitro, to proteins of the extracellular matrix, the biosynthesis of integrins, their topography and organization in focal contacts. The adhesion of osteoblasts to laminin, type I collagen, vitronectin and fibronectin was 77-100%, in 2 h and at 55 nM substrata concentration, and it was accompanied by spreading of the cells. Adhesion to fibronectin (FN), laminin (LN) and type I collagen (COL) was inhibited by antibodies to the beta 1 integrin and antibodies to the alpha 5 chain affected adhesion only to fibronectin. Using a panel of polyclonal antibodies against alpha 2, alpha 3, alpha 5, alpha v, beta 1 and beta 3 integrins we detected synthesis of alpha 3 beta 1, alpha 5 beta 1, alpha v beta 3, and an alpha v beta 1-like dimer by immunoprecipitation of metabolically labelled cell lysates. Studies of immunolocalization demonstrated the presence of the same integrins identified in lysates, plus alpha 4, alpha 1 and beta 5 subunits. In cells adhering in the presence of serum we showed organization of beta 3 and alpha v integrins in focal contacts. In cells adhering to fibronectin alpha 5 and beta 1 integrins were localized in focal contacts. In cells spread on laminin or type I collagen none of the integrins investigated was localized in focal contacts.

Adult↗
PubMed1996

Tubulointerstitial responses in the progression of glomerular diseases: albuminuria modulates alpha v beta 5 integrin.

Proteinuria represents one of the most unfavorable prognostic factors in the progression of nephropathies. Several lines of evidence support a role for proteinuria per se in the development of interstitial fibrosis, albeit the molecular mechanisms are still unknown. We investigated the potential role of integrins expressed on tubular cells in regulating the synthesis and organization of interstitial matrix or as mediators of tubulointerstitial damage in conditions mimicking the nephrotic milieu. Under basal conditions, cultured tubular cells highly expressed alpha 3 beta 1 and, at focal contacts, alpha v beta 3. In contrast, alpha v beta 5 was weakly and diffusely distributed all over the plasma membrane. Cultures on a variety of matrix substrates (fibronectin, laminin, collagen types I and IV, vitronectin, von Willebrand factor, fibrinogen) did not induce any phenotypic change in integrin expression by tubular cells. Conversely, the addition of albumin resulted in a highly increased membrane expression of beta 5, which was organized in typical focal contacts and was related to the dose of albumin added. Immunofluorescence, flow cytometry, immunoprecipitation and RT-PCR experiments argue for a complex mechanism that includes increased post-transcriptionally regulated protein synthesis, accelerated conversion of precursors to mature forms, and increased surface delivery to discrete adhesive structures. Up-regulation of the beta 5 chain in tubular cells was confirmed in 9 out of 11 kidney biopsies from proteinuric glomerulonephritides including membranous and focal sclerosing glomerulonephritis, while it was not expressed in nonproteinuric kidneys including five biopsy specimens. This is the first report indicating that proteinuria up-regulates the surface expression and distribution of a specific integrin chain on tubular cells. These observations suggest the participation of integrins in a hitherto unexplored mechanism of tubulo-interstitial responses to glomerular injury.

Albuminuria↗
PubMed1996

Significance of adhesion molecules as biological prognostic factor in locally advanced laryngeal squamous cell carcinomas.

Thirty laryngeal carcinomas were studied immunohistochemically in order to evaluate whether the expression and different distribution of adhesion molecules influence the clinical features and progression of the tumors. On the basis of clinical and pathological variables, two different groups were established: one with good and the other with poor prognosis. The patients were included in one of the two groups on the basis of prognostic factors previously studied by multivariate analysis (the validity of this choice was confirmed by the NED survival curves of the two groups). Different integrins, type I and V laminin and type IV collagen were evaluated by means of monoclonal antibodies in the tumoral specimens and in normal mucosa. Univariate statistical analysis was performed to evaluate differences between the two groups. The degree of expression and pattern of distribution were different in tumor compared with normal mucosa and significant differences were found between the good- and worst-prognosis tumors.

Antibodies, Monoclonal↗
PubMed1995

The alpha 3 beta 1 integrin is involved in melanoma cell migration and invasion.

The VLA3 (alpha 3 beta 1) integrin receptor recognizes several ligands; however, the function of this integrin is still debated. Expression of VLA3 appears to be increased in malignant melanoma and correlates with the degree of dermal invasiveness. Here we have studied the role the alpha 3 integrin subunit in malignant melanoma cell migration and invasion into extracellular matrices. The 2/14 clone of the Me665/2 human melanoma cell line, which expresses high levels of VLA integrins, was highly migratory and invasive, while the low integrin expressing 2/56 clone showed limited migration and was not invasive. Antibodies to the beta 1 subunit inhibited adhesion, migration, and invasion of two different malignant melanoma cell lines, the 2/14 clone and A2058 cells, indicating a crucial role for VLA integrins in these phenomena. While anti-alpha 6 antibodies inhibited adhesion to laminin and anti-alpha 5 antibodies inhibited adhesion to fibronectin, antibodies to the alpha 3 subunit did not inhibit adhesion of these cells to laminin, fibronectin, or collagen i.v. In contrast, the P1B5 anti-alpha 3 antibodies were good inhibitors of the migration of these cells toward laminin, fibronectin, and collagen IV and also blocked invasion of these cells through a reconstituted basement membrane matrix (Matrigel). Another anti-alpha 3 antibody, F4, did not effect migration, while both the P1B5 and F4 antibodies induced cellular aggregation on Matrigel. Our data suggest a specific role for alpha 3 beta 1 in the migration and invasion of melanoma cells.

Antibodies, Monoclonal↗
PubMed1995

Transforming growth factor-beta 1 modulates beta 1 and beta 5 integrin receptors and induces the de novo expression of the alpha v beta 6 heterodimer in normal human keratinocytes: implications for wound healing.

The molecular mechanism underlying the promotion of wound healing by TGF-beta 1 is incompletely understood. We report that TGF-beta 1 regulates the regenerative/migratory phenotype of normal human keratinocytes by modulating their integrin receptor repertoire. In growing keratinocyte colonies but not in fully stratified cultured epidermis, TGF-beta 1: (a) strongly upregulates the expression of the fibronectin receptor alpha 5 beta 1, the vitronectin receptor alpha v beta 5, and the collagen receptor alpha 2 beta 1 by differentially modulating the synthesis of their alpha and beta subunits; (b) downregulates the multifunctional alpha 3 beta 1 heterodimer; (c) induces the de novo expression and surface exposure of the alpha v beta 6 fibronectin receptor; (d) stimulates keratinocyte migration toward fibronectin and vitronectin; (e) induces a marked perturbation of the general mechanism of polarized domain sorting of both beta 1 and beta 4 dimers; and (f) causes a pericellular redistribution of alpha v beta 5. These data suggest that alpha 5 beta 1, alpha v beta 6, and alpha v beta 5, not routinely used by keratinocytes resting on an intact basement membrane, act as "emergency" receptors, and uncover at least one of the molecular mechanisms responsible for the peculiar integrin expression in healing human wounds. Indeed, TGF-beta 1 reproduces the integrin expression pattern of keratinocytes located at the injury site, particularly of cells in the migrating epithelial tongue at the leading edge of the wound. Since these keratinocytes are inhibited in their proliferative capacity, these data might account for the apparent paradox of a TGF-beta 1-dependent stimulation of epidermal wound healing associated with a growth inhibitory effect on epithelial cells.

3T3 Cells↗
PubMed1995

Integrin expression in head and neck cancers.

Neoplastic transformation results in rearrangement of the cell membrane with a breakdown of the regular expression of adhesion molecules such as integrins. A relationship may exist between the intensity of this alteration, and the form it takes, and a tumour's ability to metastatise. We studied this possibility by investigating the epithelial integrin expression of 35 head and neck squamous cell carcinomas. Two types of positivity were observed: one associated with tumours with a better prognostic index, the other with those characterized by greater malignancy.

Carcinoma, Squamous Cell↗