The activities of extracellular signal-regulated kinases (ERK1/ERK2) is required for proliferation of several types of cells. The performed analysis showed stimulation of ERK's by fetal calf serum (FCS) or fibronectin in the C3H 10T1/2 cell cultures at logarithmic phase of growth. The ERKs activity was not stimulated in confluent cells. This could not be accounted for a partial down regulation of ERK since its level was stable in both types of cells regardless of their density and kind of stimulation. Searching for ERK upstream elements we studied the integrin receptor gene transcript by RT-PCR and focal adhesion kinase (FAK) by Western blotting and phosphorylation assays. It was found that FCS and fibronectin stimulated phosphorylating activity of FAK in the cells at the logarithmic phase of growth, but were inefficient in the confluent cells. RT-PCR showed the presence of α5 and β1 integrin transcripts, and p125FAK was at the same level regardless of the type of stimulation. These data indicate that the ability of FAK to be activated plays an important role in ERK regulation and, in consequence in proliferation and growth inhibition during confluence.
We studied the effect of the modification of focal adhesion kinase (FAK) on the growth, migration and adhesion of C3H 10T1/2 cells. Cells transfected with plasmid coding for antisense FAK displayed a low level of FAK protein. Interestingly, the transfected cells achieved a higher saturation density at confluence, and displayed reduced adhesion and enhanced emigration from a confluent layer of cells when stimulated with fibronectin. In conclusion, it can be postulated that FAK plays an important role in the mechanism of contact inhibition.
Five families with the Lesch-Nyhan syndrome (LNS) and two families with the Kelley-Seegmiller syndrome (KSS) were studied. Seven different mutations were identified. Two transitions, C526→ T (Prol76Ser) and G481→A (Ala161Thr), in patients with a milder form of hypoxanthine-guanine phosphoribo-syltransferase (HPRT) deficiency were detected. In patients with the Lesch-Nyhan syndrome two transitions, G569→A (Glyl90Glu) and C508→T (Arg170Ter), two transversions, C222→A (Phe74Leu) and C482→A (Ala161Glu), and a deletion of seven nucleotides (from A394 to G400) were observed. All except two of the identified mutations are novel. The C222→A substitution in exon III is located within one of the clusters of hot spots of the HPRT gene and has been previously described in four unrelated patients. The other recurrent mutation C508→T in exon VII has been reported in eight families.
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