|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1994 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1993 : ¥1,100,000 (Direct Cost : ¥1,100,000)
1. Purification of AMF derived from Gc-4 PF fibrosarcoma cells.
We have previously demonstrated that protein-free culture of Gc-4 PF cells is associated with high response to autocrine motility factor (AMF) and with high expression of its receptor, gp78. Two motile activities, acidic and basic AMFs with regard to binding features on ion exchange chromatography, were purified from the cultured medium by sequential DEAE anion-exchange, CM cation-exchange, and gel-filtration chromatographies. However, the two AMFs have a similar size of 55kDa and 65kDa under nonreducing and reducing conditions, respectively, with same pI of 6.5. The stimulated motility of both AMFs was inhibited by the pertussis toxin, but not by Streptomyces hyaluronidase. These two AMFs significantly stimulated the lung colonizing properties of the self-producing Gc-4 PF cells by 1.5-fold. These results suggest that both acidic and basic AMFs may correspond to the previously reported AMF.
2.Mechanism of AMF-gp78 signal tr
preincubation of Dunn osteosarcoma cells with both staurosporine and genistein resulted in a significant decrease in the stimulated motility in dose- and time-dependent manners, while H-8 had a negligible effect upon the stimulated motility. These data suggest that both kinase C and tyrosine kinase play a role in AMF-stimulated cell motility, while protein kinase A may not be required for the stimulation. Furthermore, a motility inhibitor, invasion-inhibiting factor 2 (IIF-2) suppressed the cell motility stimulated by AMF.
3.Expression of AMF and its receptor in patients with malignant tumor and rheumatoid arthritis (RA).
Among 118 patients with colorectal cancer, the incidence of gp78 positivity was correlated with tumor progression as reflected by histologic type, lymph node metastasis, vessel invasion, and tumor stage. The results indicate the expression of gp78 may be of prognostic value in patients with colorectal cancer.
We demonstrated the motility stimulating activity in the synovial fluid from three patients with RA.The ability of this chemokinetic activity to block binding of the monoclonal antibody to the receptor for AMF was demonstrated on immunoblots. These results suggest that AMF is expressed in rheumatoid synovial fluids.
So far we could not obtain the hybridoma cells secreting monoclonal antibody against AMF.The studies are in progress now. Less