Objective-Telomerase plays a major role in the control of replicative capacity, a critical property for successful angiogenesis and maintenance of endothelial integrity. In this study, we examined the relationship between telomerase activity and endothelial cell proliferation as well as the regulation of this enzyme by fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-A (VEGF). Methods and Results-Telomerase was repressed in endothelial cells freshly derived from intact endothelium, whereas activity was present during logarithmic growth in culture. In cultured human umbilical vein endothelial cells (HUVECs), mRNA levels of hTERT-the catalytic subunit of telomerase-and enzyme activity decreased reversibly on induction of quiescence. Treatment of quiescent HUVECs with FGF-2 restored telomerase activity in a time-and dose-dependent manner, whereas VEGF had no such effect, although both factors induced comparable mitogenic responses. FGF-2, but not VEGF, upregulated the mRNA levels for hTERT and for the hTERT gene transactivation factor Sp1. Key Words: telomerase Ⅲ fibroblast growth factor-2 Ⅲ endothelial cell Ⅲ Sp1 Ⅲ senescence I nduction of angiogenesis is a novel therapeutic strategy presently being evaluated for the treatment of human ischemic vascular disease. The success of this strategy depends on an adequate proliferative response of vascular endothelial cells at the site of ischemia. 1 Cellular replicative potential is in part limited by the integrity of telomeres, the physical ends of chromosomal DNA. 2 Telomeric DNA shortens by Ϸ50 to 200 base pairs with each round of cell division as a consequence of the inability of conventional DNA polymerases to replicate the 3Ј termini of the template strands. A large body of evidence indicates that critical shortening of one or more telomeres activates a DNA damage checkpoint that blocks additional replication and leads to cell senescence. However, more recently, the integrity of the T-loop-a higher-order structure formed at the end of the telomere by telomeric DNA and specialized proteins-rather than telomere length, per se, has been implicated as a major determinant of senescence. 3,4 In many actively proliferating cells, maintenance of replicative capacity and chromosome stability requires the activity of telomerase, a specialized reverse-transcriptase that adds hexanucleotide repeats of the sequence TTAGGG to the 3Ј ends of nuclear DNA, thus counteracting telomeric erosion. 3 Studies in various immortalized cell lines 5 and normal human lymphocytes 6 have shown that like other enzymes involved in chromosomal DNA synthesis, 7 telomerase activity levels are differentially regulated during periods of proliferation and quiescence. Human telomerase consists of at least 3 subunits. Of these, the RNA component hTR, which provides the template for telomere synthesis, and the telomeraseassociated protein hTEP1 are constitutively expressed. 8,9 The third component, human telomerase reverse transcriptase (hTERT), which contains the catalytic activit...
Coffin-Lowry syndrome (CLS) is an X-linked disorder characterized by facial dysmorphism, digit abnormalities and severe psychomotor retardation. CLS had previously been mapped to Xp22.2. Recently, mutations in the ribosomal S6 kinase (Rsk-2) gene were shown to be associated with CLS. We have tested five unrelated individuals with CLS for mutations in nine exons of Rsk-2 using Single Strand Conformation Polymorphism (SSCP) analysis. Two patients had the same missense mutation (C340T), which causes an arginine to tryptophan change (R114W). This mutation falls just outside the N-terminal ATP-binding site in a highly conserved region of the protein and may lead to structural changes since tryptophan has an aromatic side chain whereas arginine is a 5 carbon basic amino acid. The third patient also had a missense mutation (G2186A) resulting in an arginine to glutamine change (R729Q).
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