Urokinase plasminogen activator (uPA) is thought to exert its effects on cell growth, adhesion, and migration by mechanisms involving proteolysis and interaction with its cell surface receptor (uPAR). The functional properties of uPA and the significance of its various domains for chemotactic activity were analyzed using human airway smooth muscle cells (hAWSMC). The wild-type uPA (r-uPAwt), inactive urokinase with single mutation (His 204 to Gln) (r-uPA H/Q ), urokinase with mutation of His 204 to Gln together with a deletion of growth factor-like domain (r-uPA H/Q -GFD), the catalytic domain of urokinase (r-uPA LMW ), and its kringle domain (r-KD) were expressed in Escherichia coli. We demonstrate that glycosylated uPA, r-uPAwt, r-uPA H/Q , and r-uPA H/Q -GFD elicited similar chemotactic effects. Half-maximal chemotaxis (EC 50 ) were apparent at approximately 2 nM with all the uPA variants. The kringle domain induced cell migration with an EC 50 of about 6 nM, whereas the denaturated r-KD and r-uPA LMW were without effect. R-uPAwt-induced chemotaxis was dependent on an association with uPAR and a uPA-kringle domain-binding site, determined using a monoclonal uPAR antibody to prevent the uPA-uPAR interaction, and a monoclonal antibody to the uPA-kringle domain. The binding of iodinated r-uPAwt with hAWSMC was due to interaction with a high affinity binding site on the uPAR, and a lower affinity binding site on an unidentified cell surface target, which was mediated exclusively through the kringle domain of urokinase. Specific binding of r-uPA H/Q -GFD to hAWSMC involved an interaction with a single site whose characteristics were similar to those of the low affinity site of r-uPAwt binding to hAWSMC. uPAR-deficient HEK 293 cells specifically bound r-uPAwt and r-uPA H/Q -GFD via a single, similar type of binding site. These cells migrated when stimulated by r-uPA H/Q -GFD and uPAwt, but not r-uPA LMW . HEK 293 cells transfected with the uPAR cDNA expressed two classes of sites that bound r-uPAwt; however, only a single site was responsible for the binding of r-uPA H/Q -GFD. Together, these findings indicate that uPA-induced chemotaxis is dependent on the binding of the uPA-kringle to the membrane surface of cells and the association of uPA with uPAR.
The imprinted H19 gene is highly expressed in human embryos, fetal tissues and is nearly completely shut off in adults. However, it is reexpressed in a number of tumors including bladder carcinoma, demonstrating that HI9 RNA is an oncofetal RNA. Tumors induced by injection of bladder carcinoma cell lines express H19 in contrast to the cells before injection. These observations support the notion of a positive correlation between HI9 expression and bladder carcinoma. Loss of imprinting of H19 and IGF-2 was observed in samples of human bladder carcinoma.
Increased interest in development of combined gene therapy emerges from results of recent clinical trials that indicate good safety yet unexpected low efficacy of “single-gene” administration. Multiple studies showed that vascular endothelial growth factor 165 aminoacid form (VEGF165) and hepatocyte growth factor (HGF) can be used for induction of angiogenesis in ischemic myocardium and skeletal muscle. Gene transfer system composed of a novel cytomegalovirus-based (CMV) plasmid vector and codon-optimized human VEGF165 and HGF genes combined with intramuscular low-voltage electroporation was developed and tested in vitro and in vivo . Studies in HEK293T cell culture, murine skeletal muscle explants and ELISA of tissue homogenates showed efficacy of constructed plasmids. Functional activity of angiogenic proteins secreted by HEK293T after transfection by induction of tube formation in human umbilical vein endothelial cell (HUVEC) culture. HUVEC cells were used for in vitro experiments to assay the putative signaling pathways to be responsible for combined administration effect one of which could be the ERK1/2 pathway. In vivo tests of VEGF165 and HGF genes co-transfer were conceived in mouse model of hind limb ischemia. Intramuscular administration of plasmid encoding either VEGF165 or HGF gene resulted in increased perfusion compared to empty vector administration. Mice injected with a mixture of two plasmids (VEGF165+HGF) showed significant increase in perfusion compared to single plasmid injection. These findings were supported by increased CD31+ capillary and SMA+ vessel density in animals that received combined VEGF165 and HGF gene therapy compared to single gene therapy. Results of the study suggest that co-transfer of VEGF and HGF genes renders a robust angiogenic effect in ischemic skeletal muscle and may present interest as a potential therapeutic combination for treatment of ischemic disorders.
Teratocarcinoma is a germ-line carcinoma giving rise to an embryoid tumor with structures derived from the three embryonic layers: mesoderm, endoderm, and ectoderm. Teratocarcinoma is widely used as an in vitro model system to study regulation of cell determination and differentiation during mammalian embryogenesis. Murine embryonic carcinoma (EC) PCC3 cells express insulin-like growth factor I (IGF-I) and its receptor, while all derivative tumor structures express IGF-I and IGF-II and their receptors. Therefore the system lends itself to dissect the role of these two growth factors during EC differentiation. With an episomal antisense strategy, we defme a role for IGF-I in tumorigenicity and evasion of immune surveillance. Antisense IGF-I EC transfectants are shown to elicit a curative anti-tumor immune response with tumor regression at distal sites. In contrast, IGF-II is shown to drive determination and differentiation in EC cells. Since IGF-I and IGF-U bind to type I receptor and antisense sequence used for IGF-II cannot form duplex with endogenous IGF-I tran-
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