Biomedical subjects

Paul Dazin

Publications and source records attributed to Paul Dazin.

6 recordsLinked to original sources

PubMed2004

Squamous cell carcinoma cell aggregates escape suspension-induced, p53-mediated anoikis: fibronectin and integrin alphav mediate survival signals through focal adhesion kinase.

Resistance to anoikis, or apoptosis triggered by detachment from the extracellular matrix (ECM), lengthens the survival of malignant cells, facilitating reattachment and colonization of secondary sites. To examine the molecular mechanisms underlying resistance to anoikis in human oral squamous cell carcinoma (SCC) cells, we cultured human squamous carcinoma (HSC-3) cells in suspension on plates coated with poly-2-hydroxyethyl methacrylate, which blocks access to the ECM. Cells in suspension that formed multicellular aggregates had significantly lower levels of apoptosis than single cells. Aggregates, but not single cells, had high levels of fibronectin. Preincubation with a cyclic arginine-glycine-aspartic acid peptide or fibronectin-blocking antibody significantly increased anoikis. Single cells had markedly lower expression of the integrin alpha(v) receptor than aggregates. Blocking alpha(v) function with a blocking antibody or by transfection with an antisense oligonucleotide increased apoptosis and inhibited aggregation. In single cells but not aggregates, phosphorylation of the integrin-associated focal adhesion kinase (FAK) at tyrosine 397 was reduced, and p53 levels were increased. Apoptosis was increased by blocking FAK with an antisense oligonucleotide and reduced by blocking p53. These findings show that SCC cells escape suspension-induced anoikis by forming multicellular aggregates that avail themselves of fibronectin survival signals mediated by integrin alpha(v). Single cells in suspension that do not form aggregates undergo anoikis because of decreased FAK phosphorylation and increased p53 levels. Thus, SCC cells appear to use neighboring cells and the ECM molecule FN to promote the metastatic phenotype.

Animals↗

PubMed2004

Functional differences between integrin alpha4 and integrins alpha5/alphav in modulating the motility of human oral squamous carcinoma cells in response to the V region and heparin-binding domain of fibronectin.

The high-affinity heparin-binding domain and the V region of fibronectin (FN) mediate invasion and migration of human oral squamous cell carcinoma (SCC) cells. We investigated the role of the alpha4, alpha5, and alphav integrin receptors--which are central to mediating interactions with these domains of FN--in regulating SCC cell migration. SCC cells expressed the alpha4, alpha5, and alphav integrin subunits on their surface, although alpha4 expression was low. Treatment with recombinant FN proteins containing an alternatively spliced V region (V+) and either an unmutated (H+) or a mutated, nonfunctional high-affinity heparin-binding domain (H-) increased expression of alpha5 and alphav and cell motility. Antisense alpha5 or alphav oligonucleotides inhibited cell motility stimulated by FN proteins, as did blocking antibodies to alpha5 or alphav. Blocking antibodies to alpha5 increased alphav and alpha4 levels, and blocking antibodies to alphav increased the levels of alpha5 and alpha4, without increasing cell motility. In contrast, an antisense alpha4 oligonucleotide and alpha4-blocking antibodies increased cell motility, especially migration stimulated by V+H+ and V+H- FN proteins. alpha4-Blocking antibodies alone increased motility, probably by inducing alpha5 and alphav expression. Transfection with alpha4 cDNA decreased cell motility and alpha5 and alphav expression. Thus, the increased motility induced by the FN protein is probably mediated by alphav and alpha5, whereas alpha4 downregulates this process in a transdominant fashion.

Base Sequence↗

PubMed2003

FAK promotes organization of fibronectin matrix and fibrillar adhesions.

Targeted disruption of the focal adhesion kinase (FAK) gene in mice is lethal at embryonic day 8.5 (E8.5). Vascular defects in FAK-/- mice result from the inability of FAK-deficient endothelial cells to organize themselves into vascular network. We found that, although fibronectin (FN) levels were similar, its organization was less fibrillar in both FAK-/- endothelial cells and mesoderm of E8.5 FAK-/- embryos, as well as in mouse embryonic fibroblasts isolated from mutant embryos. FAK catalytic activity, proline-rich domains, and location in focal contacts were all required for proper allocation and patterning of FN matrix. Cells lacking FAK in focal adhesions fail to translocate supramolecular complexes of integrin-bound FN and focal adhesion proteins along actin filaments to form mature fibrillar adhesions. Taken together, our data suggest that proper FN allocation and organization are dependent on FAK-mediated remodeling of focal adhesions.

Animals↗

PubMed2003

Intracellular retention is a common characteristic of childhood obesity-associated MC4R mutations.

Heterozygous mutations in the coding region of the serpentine Melanocortin 4 receptor are the most common genetic cause of human obesity described to date. There are still conflicting data regarding the overall prevalence of such mutations in obesity and limited information is available on the functional defects caused by most obesity-associated MC4R mutations. We report here the screening for mutations in the coding region of the MC4R of a new cohort of 172 patients presenting with severe childhood obesity and a family history of obesity. Three heterozygous MC4R mutations (Ser127Leu, Ala244Glu and Pro299His) were found in three patients of this cohort (1.74%), confirming that such mutations are implicated in a significant number of childhood obesity cases. A functional analysis of these mutant receptors, in addition to 11 other childhood obesity-associated MC4R mutations, indicates that they all alter the activation of the receptor by the endogenous agonist alpha-MSH. To further examine the functional defects caused by childhood obesity-associated MC4R mutations, we developed a novel, sensitive technique to quantitatively analyze the effect of a mutation on MC4R cell surface expression. Using this method we analyzed the cell surface expression of all the 14 described childhood obesity-associated MC4R missense mutations. We demonstrate that 81.3% of childhood obesity-associated heterozygous MC4R mutations lead to intracellular retention of the receptor. This result has implications for the potential pharmacologic rescue of childhood obesity-associated MC4R mutations and for the treatment of patients presenting with this condition.

Amino Acid Sequence↗

PubMed2002

The hypertrophic response in C2C12 myoblasts recruits the G1 cell cycle machinery.

Hypertrophy occurs in postmitotic muscle as an adaptive response to various physiological and pathological stresses. Studies in vascular smooth muscle cells and primary cardiomyocytes suggest that angiotensin II-mediated hypertrophy activates signaling pathways associated with cell proliferation. Regulation of cyclin-dependent kinase (Cdk)-cyclin activities is essential to cell size control in lower eukaryotes, yet their role in the hypertrophic response in muscle is incompletely understood. We describe an in vitro model of hypertrophy in C2C12 skeletal myoblasts and demonstrate that induction of hypertrophy involves transient activation of Cdk4, subsequent phosphorylation of Rb, and release of HDAC1 from the Rb inhibitory complex. We also demonstrate that E2F-1 becomes transcriptionally active yet remains associated with Rb. We propose a model whereby partial inactivation of the Rb complex leads to derepression of a subset of E2F-1 targets necessary for cell growth without division during hypertrophy.

Angiotensin II↗

PubMed2001

The heparin-binding domain and V region of fibronectin regulate apoptosis by suppression of p53 and c-myc in human primary cells.

In apoptosis the tumor suppressor p53 and the c-myc proto-oncogene are usually up-regulated. We show a novel alternative pathway of apoptosis in human primary cells that is mediated by transcriptionally dependent decreases in p53 and c-Myc and decreases in p21. This pathway is regulated by the alternatively spliced V region and high-affinity heparin-binding domain of fibronectin. Requirements for c-Myc, p53, and p21 signals in maintaining survival and for their decreases in inducing apoptosis were demonstrated by the ability of p53, c-Myc, and p21 ectopic expression to rescue this apoptotic phenotype, and the ability of p53-deficient and c-myc antisense conditions to trigger a faster rate of apoptosis.

Apoptosis↗