Monensin-induced inhibition of cell spreading in normal and dystrophic human fibroblasts.

Pizzey, John; Witkowski, Jan; Jones, Gareth E.

doi:10.1073/pnas.81.15.4960

Cited by 14 publications

(2 citation statements)

References 40 publications

(40 reference statements)

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“…We suggest that this may be a consequence of the known effects (68) of monensin on inhibiting the translocation of nascent glycoproteins which may be involved in recognition and adhesion at the cell surface. We have previously shown that only 30 min exposure to 0.5 IxM monensin is sufficient to reduce cell-substratum adhesive interactions in human skin fibroblasts (52,53).…”

Section: Discussionmentioning

confidence: 99%

Requirements for the Ca2+-independent component in the initial intercellular adhesion of C2 myoblasts.

Pizzey¹,

Jones²,

Walsh³

1988

The Journal of Cell Biology

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Abstract. Using a sensitive and quantitative adhesion assay, we have studied the initial stages of the intercellular adhesion of the C2 mouse myoblast line. After dissociation in low levels of trypsin in EDTA, C2 cells can rapidly reaggregate by Ca2+-independent mechanisms to form large multicellular aggregates. If cells are allowed to recover from dissociation by incubation in defined media, this adhesive system is augmented by a Ca2÷-dependent mechanism with maximum recovery seen after 4 h incubation. The Ca2÷-independent adhesion system is inhibited by preincubation of cell monolayers with cycloheximide before dissociation. Aggregation is also reduced after exposure to monensin, implicating a role for surface-translocated glycoproteins in this mechanism of adhesion. In coaggregation experiments using C2 myoblasts and 3T3 fibroblasts in which the Ca2÷-dependent adhesion system was inactivated, no adhesive specificity between the two cell types was seen. Although synthetic peptides containing the RGD sequence are known to inhibit cell-substratum adhesion in various cell types, incubation of C2 myoblasts with the integrin-binding tetrapeptide, RGDS, greatly stimulated the Ca2÷-independent aggregation of these cells while control analogs had no effect. These results show that a Ca2÷-independent mechanism alone is sufficient to allow for the rapid formation of multicellular aggregates in a mouse myoblast line, and that many of the requirements and perturbants of the Ca2÷-independent system of intercellular myoblast adhesion are similar to those of the Ca2+-dependent adhesion mechanisms. MYOGENESIS in vivo is a complex cellular process initially involving a series of cell-cell interactions whereby myoblasts fuse to form multinucleate myotubes (38, 77). Of fundamental importance in this process, before fusion, are the mechanisms involved in myoblast recognition and adhesion (36). Indeed, myoblast recognition can be thought of as a primary stage of myogenesis, and aggregation studies using cultured myoblasts present a convenient model for studying many of these early interactions (28,36,37). From the use of myoblast cultures, much has been learnt about the conditions required for myoblast adhesion. For example, the existence of two independent, noncomplementing adhesive systems has been identified (21, 36) and a role for glycoprotein secretion has also been suggested (9, 35). Although myogenesis begins with myoblast recognition and adhesion, these events themselves are multistep phenomena (19). Initially, interactions probably occur between surface-associated ligands and their corresponding receptors (42). These interactions may involve homophilic binding, as in neural cell adhesion molecule binding to neural cell adhesion molecule (58), or heterophilic binding, as with integrin-ligand binding (8). The integrins (57) are a family of structurally related receptors which bind many adhesive glycoproteins that contain the tripeptide arginineglycine-aspartic acid (RGD) as their cell recognition site. Such proteins include...

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Section: Discussionmentioning

confidence: 99%

Requirements for the Ca2+-independent component in the initial intercellular adhesion of C2 myoblasts.

Pizzey¹,

Jones²,

Walsh³

1988

The Journal of Cell Biology

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“…DMD skin fibroblast populations in vitro express a variety of syndrome-specific cellbiological (6)(7)(8)(9)(10), and biochemical (11)(12)(13)(14)(15)(16)(17) abnormalities. Recent investigations into the protein turnover in DMD fibroblasts in vitro (18)(19)(20) (17) and from our laboratory (20) indicate a significant decrease in protein synthesis (17,20) and a significant increase in the degradation of short-and long-lived proteins in DMD fibroblast populations in vitro (20).…”

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confidence: 99%

Differential degradation of [35S]methionine polypeptides in Duchenne muscular dystrophy skin fibroblasts in vitro.

Rodemann¹,

Bayreuther²

1986

Proc. Natl. Acad. Sci. U.S.A.

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Rates of protein turnover have been measured in three normal and three Duchenne muscular dystrophy (DMD) skin fibroblast cell lines. Cell populations were analyzed at identical states with regard to cell number, state of topoinhibition, and cumulative population doublings (CPD). Net protein synthesis measured by the incorporation of [35SSmethionine in an 18-hr pulse was reduced by an average of 34%; degradation of total cellular protein measured after an 18-hr pulse with [35S]methionine and a 24-hr chase was enhanced by an average of 50% in DMD fibroblasts. Twodimensional gel electrophoresis analyses revealed that the breakdown of the majority of [35S]methionine polypeptides was markedly increased in DMD fibroblasts. Quantitative determinations of the differential degradation rates of 10 selected proteins in the tropomyosin region of two-dimensional gels were undertaken by scintillation counting and computer analyses. In three series of experiments, the degradation of the 10 proteins in DMD fibroblasts was enhanced by individual polypeptides between 12.0% and 151.2% as measured by scintillation counting or between 0.8% and 128% as determined by computer analyses.Duchenne muscular dystrophy (DMD) is an X-chromosomelinked recessive disorder, characterized by a progressive muscle degeneration in the primary organs of manifestation, the skeletal and cardiac muscles. The primary genetic defect of this disorder has not been defined; dystrophy of skeletal and cardiac muscles is considered to be the result of an abnormal protein metabolism (1-3). Analyses of the primary and/or secondary biochemical defects in degenerating muscle in DMD in vivo have been hampered by secondary changes-e.g., concomitant regeneration and degeneration of myoblasts, fibroblasts, and fat cells (1). The secondary changes make the selection of an appropriate reference base and of a representative control in vivo difficult.Numerous cellular and biochemical abnormalities have also been demonstrated in nerve, erythrocyte, and fibroblast cells in DMD patients (1, 4, 5). DMD skin fibroblast populations in vitro express a variety of syndrome-specific cellbiological (6-10), and biochemical (11-17) (20).In the present study, we analyzed net protein synthesis and degradation of total cellular protein and of individual proteins in DMD and normal skin fibroblasts by two-dimensional gel electrophoresis. We will provide evidence that in DMD skin fibroblasts the majority of proteins are affected by a decreased net synthesis and an enhanced degradation. Analyses ofthe degradation of individual [35S]methionine polypeptides in the tropomyosin region of two-dimensional gels revealed a significant polypeptide-specific increase in the rates of degradation in DMD fibroblasts. MATERIALS AND METHODSCell Cultures. Skin fibroblast populations of three male patients (6-15 yr old) with advanced to very advanced manifestations of the DMD syndrome and of three agematched normal male probands were established from skin biopsies as described elsewhere (14). Three DMD ...

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et al. 1986

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Monensin-induced inhibition of cell spreading in normal and dystrophic human fibroblasts.

Cited by 14 publications

References 40 publications

Requirements for the Ca2+-independent component in the initial intercellular adhesion of C2 myoblasts.

Requirements for the Ca2+-independent component in the initial intercellular adhesion of C2 myoblasts.

Differential degradation of [35S]methionine polypeptides in Duchenne muscular dystrophy skin fibroblasts in vitro.

Letters to the Editor

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