Ras activated ERK and PI3K pathways differentially affect directional movement of cultured fibroblasts.

BACKGROUND: Cell migration is essential in physiological and pathological processes, such as wound healing and metastasis formation. Ras involvement in these processes has been extensively demonstrated. This work attempts to characterize Ras regulation of the phenomena determining directional cell migration by separately analyzing the role of its principal effector pathways, MAPK and PI3K. METHODS: NIH3T3 and NIHRasV12 fibroblasts were followed in wound healing assays to study, in time and under a directional stimulus, cell migration both under standard conditions and in presence of MAPK and PI3K inhibitors. Several parameters, descriptive of specific aspects of cell motion, were evaluated by coupling dynamic microscopy with quantitative and statistical methods. Quantitative Western Blots coupled with immunofluorescence stainings, were used to evaluate ERK activation. RESULTS: Constitutive RasV12 activation confers to NIH3T3 the ability to close the wound faster. Neither increased cell proliferation nor higher speed explains the accelerated healing, but the increased directional migration drives the wound closure. Inhibition of ERK activation, which occurs immediately after wound, greatly blocks the directional migration, while inhibition of PI3K pathway reduces cell speed but does not prevent wound closure. CONCLUSION: Ras is greatly involved in determining and regulating directionality, ERK is its key effector for starting, driving and regulating directional movement.

Analysis and modelling of motility of cell populations with MotoCell.

BACKGROUND: Cell motility plays a central role in development, wound-healing and tumour invasion. Cultures of eukaryotic cells are a complex system where most cells move according to 'random' patterns, but may also be induced to a more coordinate migration by means of specific stimuli, such as the presence of chemical attractants or the introduction of a mechanical stimulus. Various tools have been developed that work by keeping track of the paths followed by specific objects and by performing statistical analysis on the recorded path data. The available tools include desktop applications or macros running within a commercial package, which address specific aspects of the process. RESULTS: An online application, MotoCell, was developed to evaluate the motility of cell populations maintained in various experimental conditions. Statistical analysis of cell behaviour consists of the evaluation of descriptive parameters such as average speed and angle, directional persistence, path vector length, calculated for the whole population as well as for each cell and for each step of the migration; in this way the behaviour of a whole cell population may be assessed as a whole or as a sum of individual entities. The directional movement of objects may be studied by eliminating the modulo effect in circular statistics analysis, able to evaluate linear dispersion coefficient (R) and angular dispersion (S) values together with average angles. A case study is provided where the system is used to characterize motility of RasV12 transformed NIH3T3 fibroblasts. CONCLUSION: Here we describe a comprehensive tool which takes care of all steps in cell motility analysis, including interactive cell tracking, path editing and statistical analysis of cell movement, all within a freely available online service. Although based on a standard web interface, the program is very fast and interactive and is immediately available to a large number of users, while exploiting the web approach in a very effective way. The ability to evaluate the behaviour of single cells allows to draw the attention on specific correlations, such as linearity of movement and deviation from the expected direction. In addition to population statistics, the analysis of single cells allows to group the cells into subpopulations, or even to evaluate the behaviour of each cell with respect to a variable reference, such as the direction of a wound or the position of the closest cell.

Search for genetic variants in the retinoid X receptor-gamma-gene by polymerase chain reaction-single-strand conformation polymorphism in patients with resistance to thyroid hormone without mutations in thyroid hormone receptor beta gene.

Resistance to thyroid hormone (RTH) is an inherited disease characterized by reduced tissue sensitivity to thyroid hormone. Approximately 90% of subjects with RTH have mutation in the thyroid hormone receptor beta (TRbeta) gene. Approximately 10% of subjects diagnosed as having RTH do not carry mutation in the TRbeta gene. A possible linkage was reported with the retinoid X receptor-gamma (RXR-gamma) gene in two families. The aim of this study is to search for mutation within the RXR-gamma gene in unrelated subjects with diagnosed RTH without mutations in the TRbeta gene. Four subjects with RTH were studied, and sequence variants in the RXR-gamma gene were searched by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). Analysis of all the 10 exons of the RXR-gamma gene, including intron-exon boundaries, promoter region and 3' untranslated region (UTR) reveled two variant bands in subjects II and III. Sequencing of these variants showed two single nucleotide polymorphisms (SNPs): 447C > T in exon 3 for patients II and IVS9 + 6A > G for patient III. Both SNPs were also present at high frequency in a group of normal subjects and in nonaffected relatives of subject III. In conclusion, in patients with RTH we have found two SNPs in the RXR-gamma gene; these SNPS are common in the general population, thus excluding a role for the RXR-gamma gene in these patients.

Endothelial cells overexpressing basic fibroblast growth factor (FGF-2) induce vascular tumors in immunodeficient mice.

Basic fibroblast growth factor (FGF-2) is expressed in vascular endothelium during tumor neovascularization and angioproliferative diseases, including vascular tumors and Kaposi's sarcoma (KS). We have investigated the in vivo biological consequences of endothelial cell activation by endogenous FGF-2 in a mouse aortic endothelial cell line transfected with a retroviral expression vector harboring a human FGF-2 cDNA and the neomycin resistance gene. FGF-2 transfectants, named pZipbFGF2-MAE cells, caused the rapid growth of highly vascularized, non-infiltrating tumors when injected in nude mice. In contrast, lesions grew poorly when cells were injected in immunocompetent syngeneic animals. Histologically, the tumors had the appearance of hemangioendothelioma with spindled areas resembling KS and with numerous CD31+ blood vessels and lacunae. Southern blot analysis of tumor DNA, as well as disaggregation of the lesion followed by in vitro cell culture, revealed that less than 10% of the cells in the tumor mass retain FGF-2 overexpression and neomycin resistance at 6-8 weeks post-injection. Nevertheless, in vitro G418 selection allowed the isolation from the tumor of a FGF-2-overexpressing cell population showing biochemical and biological characteristics similar to those of pZipbFGF2-MAE cells, including the capacity to originate vascular lesions when re-injected in nude mice. To evaluate the effect of angiostatic compounds on the growth and vascularization of pZipbFGF2-MAE cell-induced lesions, nude mice were treated weekly (100mg/kg, i.p.) with the angiostatic sulfonated distamycin A derivative 2,2'-(carbonyl-bis-[imino-N-methyl-4,2-pyrrole carbonyl-imino-{N-methyl-4,2-pyrrole}carbonylimino])-bis-(1,5-naphthalene) disulfonic acid (PNU 153429). The results demonstrate that PNU 153429 inhibits the growth of the lesions and causes a approximately 50% decrease in CD31+ microvessel density. In conclusion, the data indicate that FGF-2-overexpressing endothelial cells cause vascular lesions in immunodeficient mice which may represent a novel model for opportunistic vascular tumors suitable for the evaluation of angiostatic compounds.