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To test our hypothesis that the HA binding peptide (HABP/tachyplesin) may be a new anti-neurofibromatosis agent, we utilized phage display approach to define its binding target. The results demonstrated that 1) HABP/tachyplesin the binds to collagen-like domain of Clq, a key component in the complement pathway; 2) HABP/tachyplesin appears to activate the classical complement cascade, since it triggered several down-stream events including the cleavage and deposition of C4 and C3 and the formation of C5b-9; 3) The HABP/tachyplesin binding to Clq and activation of classical complement cascade can be blocked if the tumor cells are treated with hyaluronidase or a large excess of hyaluronan, indicating that hyaluronan or related glycosarninoglycans were involved in this process; 4) Treatment of tumor cells with HABP/tachyplesin and serum results in an increase in membrane permeability as indicated by the ability of FITC-dextran to enter the cytoplasm; 5) The combination of HABP/tachyplesin and human serum can markedly inhibit the proliferation of tumor cells and this effect is attenuate if the serum is heat-inactivated or if hyaluronidase is added. This represents a new anti-tumor mechanism. We will continue to examine the action mechanism of HABP and-to test if the in vitro anti-tumor effect of HA binding peptide can be translated in vivo against the cell growth of neurofibromatosis.

To test our hypothesis that the HA binding peptide may be a new anti-neurofibromatosis agent via inducing apoptosis, we have proposed to focus on three aims: 1) To examine the anti-tumor effect of synthetic HA binding peptide on malignant neurofibromatosis cells; 2) To examine the anti-tumor effect of genetically expressed targeted HA binding peptide; 3) To examine the effect of targeted HA binding peptide on molecules involved in apoptosis. The results of first year study indicated that: 1) large scale synthesized HA binding peptide did possess HA binding activity; 2) synthetic HA binding peptide exerted an anti-tumor effect of on ST88-14 NFl cells; 3) HA binding peptide could bind to Bcl-2/Bcl-x(L), which may be one of the mechanisms by which HA binding peptide inhibits ST88-14 NFl cells; and 4) the cells transfected with expression vector carrying cDNA of HABP could express this peptide. The results of this year study demonstrated that: 1) HA binding peptide is capable of reducing the level of phosphorylated ERK1; 2) HABP reduces the level of cell cycle related molecules, such as cyclin B1 and cdc 2; 3) HABP binds to Bcl-2 in vivo and induces apoptosis; and 4) the effort has been made to set up the model system of NFl tumor exnograft in mice for test the effect of HABP in vivo. In the next year, we will continue to examine the action mechanism of HABP and to test if the in vitro anti-tumor effect of HA binding peptide can be translated in vivo against the cell growth of neurofibromatosis.

To test our hypothesis that the hyaluronan (HA) binding proteins (HABP) from cartilage is a new category of anti-tumor agents, we have proposed three aims: (1) To examine the effect of HABP on the tumor growth of prostate cancer cell lines; (2) To examine the effect of link protein and aggrecan on the tumor growth of prostate cancer; (3) To examine the possible anti-angiogenesis effect of HABPs. In past year, we have successfully finished the following works: (1) cloned 1,065 bp of cDNA cording for full length of human cartilage link protein; (2) inserted this cDNA into pcDNA3 expression vector; (3) stablely transfected into tumor cell lines; (4) examined the inhibitory effect of link protein on growth of endothelial cells; (5) demonstrated that the link protein could inhibit the tumor growth in vivo. The significance of these studies are (1) it determines that the link protein is an anti-tumor element in cartilage; (2) the cDNA cloned in this study may be used for gene therapy., The original document contains color images. All DTIC reproductions will be in black and white.

To test our hypothesis that the HA binding peptide may be a new anti-neurofibromatosis agent via inducing apoptosis, we have proposed to focus on three aims: 1) To examine the anti-tumor effect of synthetic HA binding peptide on malignant neurofibromatosis cells; 2) To examine the anti-tumor effect of genetically expressed targeted HA binding peptide; 3) To examine the effect of targeted HA binding peptide on molecules involved in apoptosis. In the past first year, we have finished the following tasks: 1) chemical synthesis HA binding peptide and control peptide in a large scale; 2) identification of its HA binding activity; 3) characterization of anti-tumor activity of HA binding peptide; 4) study of the effect of HA binding peptide on molecules involved in cell programmed death; and 5) construction of mammalian expression vector for HA binding peptide. The results of study indicated that: 1) large scale synthesized HA binding peptide did possess HA binding activity; 2) synthetic HA binding peptide exerted an anti-tumor effect of on ST88-14 NF1 cells; 3) HA binding peptide could bind to Bcl-2/Bcl-x(sub L), the critical anti-apoptosis factors, which may be one of the mechanisms by which HA binding peptide inhibits ST88-14 NF1 cells; 4) the cells transfected with expression vector carrying cDNA of HA binding peptide could express this peptide as evidenced by Western blotting. In the next year, we will use the synthetic HA binding peptide and newly constructed expression vector to test if the in vitro anti-tumor effect of HA binding peptide can be translated in vivo against the cell growth of neurofibromatosis., The original document contains color images. All DTIC reproductions will be in black and white.