Research of recombinant human RNase fused protein on angiogenesis and tumor growth

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Study 1 : To minimize the side effects, we made a new angiogenic inhibitor which consists of only physiologically active materials. We have fused a pancreatic-type ribonuclease (RNase) gene to human basic fibroblast growth factor (bFGF), and studied the inhibitory effect of the fused protein on angiogenesis and tumor growth in vitro and in vivo. Inhibitory effects on in vitro angiogenesis were evaluated by an assay using fragments of human placental blood vessels (Brown, 1996). In an in vitro study, angiogenesis index calculated in wells with the fused protein was 9% of the control and the fused protein inhibited angiogenesis dose dependently. In an in vivo study, A431 cells were injected into the subcutaneous layer of SCID mice and 1.45 mg of the fused protein was injected around the tumor every day for 3 weeks. The estimated tumor weight in the fused protein injection group (n=4, 474+/-73 mg) was lighter than that in the control group (1161+/-220 mg)(P=0.04). Effects of the fuse prot … More ein on tumor growth was also observed. These results suggest that the RNase-FGF fused protein is a candidate for a new angiogenic inhibitor.Study 2 : Human lymphocytes isolated from healthy histoincompatible donors were used in mixed lymphocyte cultures or were stimulated with phytohemaggulutinin (PHA) to promote IL-2R alfa expression. MJ, an HTLV-1 infected malignant T-cell line overexpressing IL-2Ralfa, and IL-2Ralfa-negative cell lines MOLT 4F and MT-1 were used as controls. Bovine RNaseA was chemically conjugated to 7G7B6, a monoclonal antibody to the alfa-chain of human IL-2 receptors, and several concentrations of the conjugates were added to the lymphocyte cultures. Inhibition of protein synthesis was measured as percent 3H-thymidine incorporation in 24 hours. 7G7B6-RNaseA dose-dependently inhibited protein synthesis in PHA-stimulated human lymphocytes at an IC50 of 2 X 10-7M, whereas RNase alone and RNase plus antibody had no inhibitory effect. 7G7B6-RNaseA also dose-dependently inhibited human mixed lymphocyte reaction at an IC50 of 2 X 10-6M, whereas RNase alone caused no inhibition. The conjugate also inhibited protein synthesis in MJ cells, a cell line that is infected with HTLV-1 and overexpresses the high-affinity IL-2 receptor, at an IC50 of 5 X 10-7M.However the conjugate had no inhibitory effect on IL-2 receptor non-expressing human T-cell lymphoblastic leukemia cell lines MOLT4F or MT-1. 7G7B6-RNaseA can inhibit protein synthesis in antigen-or mitogen-stimulated lymphocytes overexpressing high-affinity IL-2 receptors, and it may be useful as a safer therapy than conventional chemotherapies or immunotoxins for transplant rejection, certain lymphocyte malignancies, and other IL-2R-associated diseases, because it is composed of a mammalian cytotoxic enzyme. Less