Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a 29 kDa glycopro- tein, which regulates metalloproteinase activity, but is also known as erythroid potentiating activity (EPA). We have successfully generated a glycosylated recombinant tissue inhibitor of metalloproteinases (rTIMP- 1) in a baculovirus expression system for studies on the role of TIMP-1 in tumor progression and vascular invasion. The optimal time point for the production of TIMP-1 as a single 29 kDa molecule was 20 hours post- infection. Most of the TIMP-1 was present intracellularly in the recombinant baculovirus infected Sf9 insect cells. Partially purified rTIMP-1, was shown to have metalloproteinase inhibitory activity, both in a soluble collagenase assay and by reverse zymography. At 24 hours or later time points post-infection, rTIMP-1 was visualized under nonreducing conditions as a 66 immunoreactive band, which did not exhibit any metalloproteinase inhibitory activity. TIMP-1 expression was assessed by in situ hybridization and immunohistochemistry in carcinogenically-induced hepatocellular carcinomas (HCC) of cynomolgus monkeys. TIMP-1 mRNA was not detected in normal monkey livers. In the livers carrying HCC, the highest TIMP-1 expression was at the periphery of the tumor nodules, especially in the fibrous capsules. TIMP-1 mRNA was also detected in the microvasculature of the primary tumors and metastases. In contrast to the mRNA expression, TIMP-1 protein expression was low everywhere, which may reflect a rapid secretion of TIMP-1 into the blood stream. We have succeeded in generating large amounts of fully glycosylated, active rTIMP-1 which will be of major importance in defining the role of TIMP-1 in tumor invasion and neovascularization.
