Matrix metalloproteinase-9 (MMP-9) may play a critical catalytic role in tissue remodeling in vivo, but it is secreted by cells as a stable, inactive zymogen, pro-MMP-9, and requires activation for catalytic function. A number of proteolytic enzymes activate pro-MMP-9 in vitro, but the natural activator(s) of MMP-9 is unknown. To examine MMP-9 activation in a cellular setting we employed cultures of human tumor cells (MDA-MB-231 breast carcinoma cells) that were induced to produce MMP-9 over a 200-fold concentration range (0.03-8.1 nM). The levels of tissue inhibitors of metalloproteinase (TIMPs) in the induced cultures remain relatively constant at 1-4 nM. Quantitation of the zymogen/active enzyme status of MMP-9 in the MDA-MB-231 cultures indicates that even in the presence of potential activators, the molar ratio of endogenous MMP-9 to TIMP dictates whether pro-MMP-9 activation can progress. When the MMP-9/TIMP ratio exceeds 1.0, MMP-9 activation progresses, but through an interacting protease cascade involving plasmin and stromelysin 1 (MMP-3). Plasmin, generated by the endogenous urokinase-type plasminogen activator, is not an efficient activator of pro-MMP-9, neither the secreted pro-MMP-9 nor the very low levels of pro-MMP-9 associated with intact cells. Although plasmin can proteolytically process pro-MMP-9, this limited action does not yield an enzymatically active MMP-9, nor does it cause the MMP-9 to be more susceptible to activation. Plasmin, however, is very efficient at generating active MMP-3 (stromelysin-1) from exogenously added pro-MMP-3. The activated MMP-3 becomes a potent activator of the 92-kDa pro-MMP-9, yielding an 82-kDa species that is enzymatically active in solution and represents up to 50-75% conversion of the zymogen. The activated MMP-9 enhances the invasive phenotype of the cultured cells as their ability to both degrade extracellular matrix and transverse basement membrane is significantly increased following zymogen activation. That this enhanced tissue remodelling capability is due to the activation of MMP-9 is demonstrated through the use of a specific anti-MMP-9 blocking monoclonal antibody.

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To examine MMP-9 activation in a cellular setting we employed cultures of human tumor cells that were induced to produce MMP-9 over a 200-fold concentration range (0.03 to 8.1 nM). The secreted levels of TIMPs in all the induced cultures remain relatively constant at 1-4 nM. Quantitation of the zymogen/active enzyme status of MMP-9 in the cultures indicates that even in the presence of potential activators, the molar ratio of endogenous MMP-9 to TIMP dictates whether proMMP-9 activation can progress. When the MMP-9/TIMP ratio exceeds 1.0, MMP-9 activation progresses, but only via an interacting protease cascade involving plasmin and stromelysin 1 (MMP-3). Plasmin, generated by the endogenous plasminogen activator (uPA), is not an efficient activator of proMMP-9. Plasmin, however, is very efficient at generating active MMP-3 from exogenously added proMMP-3. The activated MMP-3, when its concentration exceeds that to TIMP, becomes a potent activator of proMMP-9. Addition to the cultures of already-activated MMP-3 relinquishes the requirement for plasminogen and proMMP-3 additions and results in direct activation of the endogenous proMMP-9. The activated MMP-9 enhances the invasive phenotype of the cultured cells as their ability to transverse basement membrane is significantly increased following zymogen activation. That this enhanced tissue remodeling capability is due to the activation of MMP-9 is demonstrated through the use of a specific anti-MMP-9-blocking monoclonal antibody.

Two members of the matrix metalloproteinase (MMP)1 family of enzymes are expressed at elevated levels in highly aggressive human tumor cells and have been implicated in the catalytic functions of extracellular proteolysis. The zymogen forms of these enzymes are designated proMMP-2 and proMMP-9, also known as 72kDa and 92kDa type IV collagenases/gelatinases, respectively. The MMP family of enzymes can be activated in vitro by a number of compounds including the organomercurial 4-aminophenylmercuric acetate (APMA). The natural or in vivo activators of MMP-2 and MMP-9 are at present unknown. A partially purified preparation of MMP-9 was used to immunize mice for the isolation of monoclonal antibodies (mAbs). Three IgG1 mAbs were identified by immunoreactivity with purified MMP-9 and are designated 6-6B, 7-11C, and 8-3H. These mAbs react specifically with MMP-9 by ELISA and Western blot. Additionally, these mAbs react with N-glycanase treated 92kDa protein. These mAbs were tested for their ability to inhibit enzyme activation in a radio-labeled gelatin assay. The 6-6B mAb inhibited the activation of MMP-9, but had no effect on MMP-2. These mAbs are highly specific to human MMP-9 and the 6-6B mAb will be extremely useful for examining the autolytic and catalytic activity of MMP-9 in normal and abnormal biological processes.

Degradation of the extracellular matrix (ECM) is a critical feature of cancer cell invasion. Accumulating evidence suggests that proteolytic enzymes, including the matrix metalloproteinase (MMP) family, contribute to these multifactorial processes. Important features of these enzymes that exist in normal and neoplastic invasive processes are the induction of proteinase expression and the regulation of proteolytic activity. A common feature of the enzymes cited above is that they are produced as zymogens and require an activation step to become catalytically active. In addition, the expression of these enzymes requires stimuli that induce at the level of transcription whereas others are transcriptionally overexpressed. A correlation between proteolysis and malignant progression has been established. Specific inhibitors of serine and metalloproteinases have been used in in vitro and in vivo modelsI4 to demonstrate inhibition of tumor cell invasion and metastasis. The exact role of these specific proteinases in the mechanisms involved in the formation of metastatic lesions remains unclear.The interaction of tumor cells with the ECM and basement membrane occurs at multiple stages in the metastatic cascade. Benign adenoma and in situ carcinomas are characterized by a continuous basement membrane that separates the epithelium from the underlying stroma, whereas invasive carcinomas demonstrate zones of basement membrane loss around the invading tumor cells. Attention has been focused on proteolytic enzymes whose substrate specificities include ECM and basement membrane components. Some enzymes that have been implicated in the invasive processes of cancer cells include the MMPsS and urokinase-type plasminogen activator (uPA).~ Natural inhibitors of these enzymes have been identified and include the tissue inhibitors of metalloproteinases-1 and -2 (TIMP-1 and TIMP-2)7-9 and plasminogen activator inhibitor types-1 and -2 (PAI-1 and PAI-2).1°Positive correlations between the expression of MMPs and uPA and the malignant phenotype have been demonstrated in vitro using numerous tumor and transformed cell liness4 These correlative studies have been supported by analyses using specific inhibitors. Inhibitors of MMPs or uPA have been shown to block tumor cell invasion across native or reconstituted basement membranes in v i t r~.~J *~'~ The ability of TIMP to prevent matrix degradation has been demonstrated in cell model systems. Inhibition of tumor cell invasion and metastasis in an in vivo animal model has been demonstrated using anti-uPA antibodiesZ or in vivo injections of TIMP.I3 A causal role for MMPs in cancer cell invasion was demonstrated using antisense TIMP RNA.14 These studies demonstrated that a reduction in TIMP levels resulted in a change of the cellular phenotype to one that was invasive in an in vitro model and metastatic in an in vivo model. These analyses implicate the MMPs in cancer cell invasion.The MMPs represent a family of structurally related enzymes that are grouped 324

The degradation of extracellular matrix (ECM) components in normal and pathologic processes has been linked to the presence of a family of hydrolytic enzymes known as matrix metalloproteinases (MMPs).' Two members of this family, the 72-kDa (MMP-2) and the 92-kDa (MMP-9) enzyme, are potent gelatinases. MMP-2 and MMP-9 are highly expressed in invasive tumors, suggesting a role in the degradation of ECM and basement membrane during tumor cell invasion and metastasis.' Studies with natural inhibitors, such as the tissue inhibitor of metalloproteinases (TIMP-1 and TIMP-2), have demonstrated the importance of MMPs in cancer in that they inhibit tumor cell invasion in vitro and reduce metastasis in in vivo animal models.Monoclonal antibodies (mAbs) are useful reagents in assessing the role(s) of specific MMPs in tumor cell invasion and metastasis. Antihuman MMP-1 and MMP-2 mAbs neutralized enzymatic activity in solution using radiolabeled collagens type I and IV, respectively.' The physiologic importance of matrix degradation by MMP-2 during tumor cell invasion in vitro was demonstrated using anti-MMP-2 mAbs.2 Similar studies with anti-MMP-9 mAbs have not been reported. Monoclonal antibodies generated against amino-and carboy-terminal peptides of MMP-9 have been described, but their ability to inhibit enzyme activity has not been tested?To study the role of MMP-9 in tumor cell invasion we generated mAbs against partially purified preparations of MMP-2 and MMP-9 from human fibrosarcoma cell line HT1080.4 Three IgGl mAbs designated 6-6B, 7-llC, and 8-3H were identified and an IgGzb-producing switch variant of 6-6B was isolated. The mAbs are specific for MMP-9 and do not react with MMP-2 by ELISA! All four mAbs are reactive by immunoblot to MMP-9 and not to MMP-2, as shown by French et al. (this volume). In addition to the immunoreactivity analyses, the 6-6B mAb neutralizes the in vitro activation of During in vitro activation of proMMP-9, products with apparent molecular weights of 82-83,000 and 67-75,000 are generated.3,5,6 The formation of the lowest molecular weight species has been correlated with an increase in catalytic activity3z6 and may be generated following cleavage of a carboxyterminal ~e p t i d e .~ To determine if 6-6B and 8-3H mAbs react with this low molecular form of MMP-9, immunoprecipitation analyses were performed. With a highly purified preparation of MMP-9, conversion of the enzyme to the lower molecular species after activation with APMA was assessed by silver-stained SDS-PAGE and gelatin zymography (FIG. 1). The starting material contained predominantly the 92-kDa enzyme and a small amount of the 83-kDa form of the enzyme.Over a 6-h period with APMA, most of the enzyme converted to the 83-kDa form and a small amount of the 67-kDa form was visible by silver staining. With gelatin zymography, the lowest molecular species was apparent by 3 h and had increased by 6-h. Immunoreactivity of mAbs with various molecular weight species of MMP-9 was assessed by immunoprecipitation followed by gelatin zymog...

We have investigated whether variability in the apoptotic pathway may account for the differential susceptibility to apoptosis-induction by 9-nitrocamptothecin (9-NC) in cell subpopulations derived from the human ovarian cancer cell line, SKOV-3. Quantitative differences in the apoptotic fractions of cells were assessed by flow cytometry, whereas major regulatory and executing components of the apoptotic machinery were investigated by Western blot analysis using specific antibodies. The results indicate that indeed the apoptotic pathway was activated by 9-NC in some, but not all, cells of the SKOV-3 cell line, suggesting that 9-NC alone may partially be effective for treatment of patients with ovarian cancer.