Human rheumatoid synovial cells in culture secrete at least three related metalloproteinases that digest extracellular matrix macromolecules. One of them, termed matrix metalloproteinase 2 (MMP-2), has been purified as an inactive zymogen (proMMP-2). The final product is homogeneous on SDSjPAGE with M , = 72000 under reducing conditions. The NH2-terminal sequence of proMMP-2 is Ala-Pro-Ser-Pro-Ile-Ile-Lys-Phe-Pro-Gly-AspVal-Ala-Pro-Lys-Thr, which is identical to that of the so-called '72-kDa type IV collagenase/gelatinase'. The zymogen can be rapidly activated by 4-aminophenylmercuric acetate to an active form of MMP-2 with M , = 67 000, and the new NH2-terminal generated is Tyr-Asn-Phe-Phe-Pro-Arg-Lys-Pro-Lys-Trp-Asp-Lys-Asn-GlnIle. However, following 4-aminophenylmercuric acetate activation, MMP-2 is gradually inactivated by autolysis. Nine endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, neutrophil elastase, cathepsin G, matrix metalloproteinase 3, and thermolysin) were tested for their abilities to activate proMMP-2, but none had this ability. This contrasts with the proteolytic activation of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). The optimal activity of MMP-2 against azocoll is around pH 8.5, but about 50% of activity is retained at pH 6.5. Enzymic activity is inhibited by EDTA, 1,lO-phenanthroline or tissue inhibitor of metalloproteinases, but not by inhibitors of serine, cysteine or aspartic proteinases. MMP-2 digests gelatin, fibronectin, laminin, and collagen type V, and to a lesser extent type IV collagen, cartilage proteoglycan and elastin. Comparative studies on digestion of collagen types IV and V by MMP-2 and MMP-3 (stromelysin) indicate that MMP-3 degrades type IV collagen more readily than MMP-2, while MMP-2 digests type V collagen effectively. Biosynthetic studies of MMPs using cultured human rheumatoid synovial fibroblasts indicated that the production of both proMMP-1 and proMMP-3 is negligible but it is greatly enhanced by the treatment with rabbit-macrophage-conditioned medium, whereas the synthesis of proMMP-2 is constitutively expressed by these cells and is not significantly affected by the treatment. This suggests that the physiological and/or pathological role of MMP-2 and its site of action may be different from those of MMP-1 and MMP-3.Connective tissue cells in culture synthesize and secrete tissue collagenase (matrix matalloproteinase 1 ; EC 3.4.24.7) and at least two other matrix metalloproteinases (MMP-2 and MMP-3) that digest various components of the extracellular matrix. The deduced primary sequences of these three MMPs from their cDNA sequencing have indicated that they are structurally related [l -61, but the substrate specificities of these enzymes are distinct. MMP-1 digests collagen types I, I1 and I11 at specific sites and generates characteristic threequarter and one-quarter fragments of the native collagen molecules [7 -91. It has also been shown to digest collagen typesCorrespondence to H. Nagase,

The mechanism of activation of tissue procollagenase by matrix metalloproteinase 3 (MMP-3)/stromelysin was investigated by kinetic and sequence analyses. MMP-3 slowly activated procollagenase by cleavage of the Gln80-Phe81 bond to generate a fully active collagenase of Mr = 41,000. The specific collagenolytic activity of this species was 27,000 units/mg (1 unit = 1 microgram of collagen digested in 1 min at 37 degrees C). Treatment of procollagenase with plasmin or plasma kallikrein gave intermediates of Mr = 46,000. These intermediates underwent rapid autolytic activation, via cleaving the Thr64-Leu65 bond, to give a collagenase species of Mr = 43,000 that exhibited only about 15% of the maximal specific activity. Similarly, (4-aminophenyl)mercuric acetate (APMA) activated procollagenase by intramolecular cleavage of the Val67-Met68 bond to generate a collagenase species of Mr = 43,000, but with only about 25% of the maximal specific activity. Subsequent incubation of the 43,000-Mr species with MMP-3 resulted in rapid, full activation and generated the 41,000-Mr collagenase by cleaving the Gln80-Phe81 bond. In the case of the proteinase-generated 43,000-Mr species, the action of MMP-3 was approximately 24,000 times faster than that on the native procollagenase. This indicates that the removal of a portion of the propeptide of procollagenase induces conformational changes around the Gln80-Phe81 bond, rendering it readily susceptible to MMP-3 activation. Prolonged treatment of procollagenase with APMA in the absence of MMP-3 also generated a 41,000-Mr collagenase, but this species had only 40% of the full activity and contained Val82 and Leu83 as NH2 termini. Thus, cleavage of the Gln80-Phe81 bond by MMP-3 is crucial for the expression of full collagenase activity. These results suggest that the activation of procollagenase by MMP-3 is regulated by two pathways: one with direct, slow activation by MMP-3 and the other with rapid activation in conjunction with tissue and/or plasma proteinases. The latter event may explain an accelerated degradation of collagens under certain physiological and pathological conditions.

We examined resected specimens from 40 cases of advanced rectal cancer to determine the extent of microtubular cancer nests and undifferentiated cancer cells (budding). We investigated the relationship between this budding and lymphatic invasion (ly), venous invasion (v), and lymph node metastasis (n), respectively. Moreover, we examined the relationship between ly, budding, and n in the preoperative biopsy specimens of 112 patients, including those of the 40 cases mentioned above. The degree of budding, which was abundant in the actively invasive region, showed a strong correlation with the degree of ly and the existence of n in the resected specimens. Also, budding was recognized in a relatively large portion of the biopsy specimens (52 of 112 [46.4%]) and lymph node metastasis was found in 41 of 52 specimens (78.8%). In 57 specimens, neither ly nor budding was found, and 16 of these specimens (28.1%) had positive lymph nodes. These results implied that the degree of budding in the actively invasive region can be a great help in predicting the presence of n. The presence or absence of budding in preoperative biopsy specimens also can be an important factor (along with the degree of differentiation and ly) in estimating the probability of n.

603The precursor of matrix metalloproteinase 9 (proMMP-9), also known as '92 kDa progelatinase/type IV procollagenase', was purified from the conditioned medium of U937 monocytic leukaemia and HT1080 fibrosarcoma cell lines stimulated with phorbol 12-myristate 13-acetate. ProMMP-9 in these culture media is non-covalently complexed with the 29 kDa tissue inhibitor of metalloproteinases (TIMP), but free proMMP-9 was separated from the TIMP-proMMP-9 complex by chromatography on Green A Dyematrex gel. The final product was homogeneous on SDS/PAGE, with a molecular mass of 88 kDa without reduction and 92 kDa with reduction. Treatment of proMMP-9 with 4-aminophenylmercuric acetate converted the 88 kDa precursor into 80 kDa and 68 kDa forms. Gelatin-containing zymographic analysis showed zones of lysis associated with all three species. However, only the 68 kDa species was shown to be catalytically active by its ability to bind to a2-macroglobulin. In the presence of an equimolar amount of TIMP, only the 80 kDa species was generated by treatment with 4-aminophenylmercuric acetate, but no enzyme activity was detected. This indicates that TIMP binds to the 80 kDa intermediate and inhibits the generation of the active 68 kDa species. Eight endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, cathepsin G, neutrophil elastase and thermolysin) were tested for their ability to activate proMMP-9. Of them, trypsin was the most effective activator of proMMP-9. Only partial activation (10-30%) was observed with plasmin, cathepsin G and chymotrypsin. The active forms generated by trypsin were identified as 80 kDa, 74 kDa and 66 kDa by their abilities to bind to a2-macroglobulin. In the presence of an equimolar amount of TIMP, proMMP-9 was also converted into the same molecular-mass species by trypsin, but they were not proteolytically active. This suggests activated MMP-9 is inhibited by TIMP. Activated MMP-9 digested gelatin, type-V collagen, reduced carboxymethylated transferrin and, to a lesser extent, type-IV collagen and laminin A chain. The specific activity against gelatin was estimated to be 15000 units/mg (1 unit = 1 ,ug of gelatin degraded/min at 37°C) by titration with a2-macroglobulin. Comparative studies on digestion of gelatin and collagen types IV and V by MMP-9 and MMP-2 indicated that both enzymes degrade these substrates into similar fragments. However, the susceptibilities of laminin, fibronectin and reduced carboxymethylated transferrin to these two MMPs were sufficiently different to indicate differences in substrate specificities between these two closely related proteinases.

Background. The question of whether lymphatic permeation in rectal carcinoma is an index of prognosis remains controversial. Methods. A long term prospective study of lymphatic permeation of 288 rectal carcinomas was performed. The degree of lymphatic permeation was divided into four stages (Ly0 through Ly3). The median follow‐up for the surviving patients was 84 months (range, 23 to 151 months). Results. In patients with UICC Stage I or II disease, there was no correlation between the Ly0 and Ly1‐3 groups' recurrence or survival rates. In patients with Stage IIIa disease, the rate of postoperative recurrence was significantly higher in the Ly1 or Ly2 + Ly3 groups compared with the Ly0 group. The 10‐year survival and disease free survival rates in patients with Stage IIIa disease were both 90.0% in the Ly0 group, 63.6% and 62.5% in the Ly1 group, and 52.0% and 45.5% in the Ly2 + Ly3 groups, respectively. A significant difference was noted in the survival rates between the Ly0 and Ly2 + Ly3 groups (P < 0.05), and was noted in the disease free survival rates between the Ly0 and Ly1 (or Ly2 + Ly3) groups (P < 0.05, P < 0.005). In patients with Stage IIIb disease, there were trends toward higher recurrence, lower survival, or disease free survival rates in the Ly2 + Ly3 groups compared with the Ly1 group (P < 0.1). Conclusions. The degree of lymphatic permeation is an important prognostic factor in patients with Stage III disease, especially in those with Stage IIIa disease. It should be classified as a clinical subgroup.