Oxidative stress and prostate cancer progression are elicited by membrane-type 1 matrix metalloproteinase.

Oxidative stress caused by high levels of reactive oxygen species (ROS) has been correlated with prostate cancer aggressiveness. Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP), which has been implicated in cancer invasion and metastasis, is associated with advanced prostate cancer. We show here that MT1-MMP plays a key role in eliciting oxidative stress in prostate cancer cells. Stable MT1-MMP expression in less invasive LNCaP prostate cancer cells with low endogenous MT1-MMP increased activity of ROS, whereas MT1-MMP knockdown in DU145 cells with high endogenous MT1-MMP decreased activity of ROS. Expression of MT1-MMP increased oxidative DNA damage in LNCaP and in DU145 cells, indicating that MT1-MMP-mediated induction of ROS caused oxidative stress. MT1-MMP expression promoted a more aggressive phenotype in LNCaP cells that was dependent on elaboration of ROS. Blocking ROS activity using the ROS scavenger N-acetylcysteine abrogated MT1-MMP-mediated increase in cell migration and invasion. MT1-MMP-expressing LNCaP cells displayed an enhanced ability to grow in soft agar that required increased ROS. Using cells expressing MT1-MMP mutant cDNAs, we showed that ROS activation entails cell surface MT1-MMP proteolytic activity. Induction of ROS in prostate cancer cells expressing MT1-MMP required adhesion to extracellular matrix proteins and was impeded by anti-ß1 integrin antibodies. These results highlight a novel mechanism of malignant progression in prostate cancer cells that involves ß1 integrin-mediated adhesion, in concert with MT1-MMP proteolytic activity, to elicit oxidative stress and induction of a more invasive phenotype.

ProMMP-2: TIMP-1 complexes identified in plasma of healthy individuals.

Activation of MMPs in tissues is an important component of tissue injury. Based on earlier reports that (latent) proMMP-2 is incapable of forming a complex with TIMP-1, we reasoned that the identification of MMP-2:TIMP-1 complexes in blood might serve as a surrogate marker ("smoking gun") of MMP-2 activation in tissues. Using specific antibodies, we developed a sensitive and specific assay to detect MMP-2:TIMP-1 complexes. We were perplexed to find that approximate 40% of plasma specimens from healthy individuals had detectable levels of the MMP-2:TIMP-1 complexes. Employing recombinant TIMP-1 bound Sepharose beads and Western blots, we demonstrated binding between recombinant proMMP-2 and TIMP-1 proteins. Recombinant MMP-2 lacking the catalytic domain also bound to TIMP-1 coated beads. These data are consistent with TIMP-1 binding to the hemopexin or hinge domain of proMMP-2. The explanation for the presence of plasma proMMP-2:TIMP-1 complexes in selected healthy individuals remains to be determined. In contrast to our immunoassay and bead-binding experiments, proMMP-2 failed to bind to immobilized TIMP-1 employing surface plasmon resonance technology. Additional studies are needed to clarify these contrasting results.

Neutrophil activator of matrix metalloproteinase-2 (NAM).

We have isolated a novel soluble factor(s), neutrophil activator of matrix metalloproteinases (NAM), secreted by unstimulated normal human peripheral blood neutrophils that causes the activation of cell secreted promatrix metalloproteinase-2 (proMMP-2). Partially purified preparations of NAM have been isolated from the conditioned media of neutrophils employing gelatin-Sepharose chromatography and differential membrane filter centrifugation. NAM activity, as assessed by exposing primary human umbilical vein endothelial cells (HUVEC) or HT1080 cells to NAM followed by gelatin zymography, was seen within one hour. Tissue inhibitor of metalloproteinase-2 (TIMP-2) and hydroxamic acid derived inhibitors of MMPs (CT1746 and BB94) abrogated the activation of proMMP-2 by NAM, while inhibitors of serine and cysteine proteases showed no effect. NAM also produced an increase in TIMP-2 binding to HUVEC and HT1080 cell surfaces that was inhibited by TIMP-2, CT1746, and BB94. Time-dependent increases in MT1-MMP protein and mRNA were seen following the addition of NAM to cells. These data support a role for NAM in cancer dissemination.

Community capacity building in CDC's Community Coalition Partnership Programs for the Prevention of Teen Pregnancy.

PURPOSE: To describe lessons learned from the Centers for Disease Control and Prevention's Community Coalition Partnership Program (CCPP) about building a community's capacity to prevent teen pregnancy through strengthening of partnerships, mobilization of community resources, and changes in the number and quality of community programs. METHODS: A multi-component post-test-only evaluation. In-person interviews (n = 364) were conducted with a sample of CCPP project staff, evaluators, and community and agency members from each of the 13 CCPP communities. RESULTS: All partnerships reported that new groups worked together to address teen pregnancy prevention; however, more time, effort, and resources than anticipated were spent engaging these groups and strengthening their partnerships. Respondents reported increases in community awareness of the problem of teen pregnancy and the willingness to discuss the issue. As a result of partnerships' activities, knowledge and skills related to addressing teen pregnancy improved among partnership members, but respondents were concerned that the broader community did not share these gains. To a lesser extent, respondents reported that partners worked together to reduce duplication and fill gaps in services either through increased collaboration and/or differentiation of activities. Respondents from most of the partnerships also reported new programs were developed as a result of the project; however, in several partnerships, only a few programs were developed in their community. Many respondents doubted whether the limited mobilization of resources during the program would translate into increased agency and community capacity. CONCLUSIONS: Overall, increased partner skills, program improvements, and new programs did not appear to be sufficient to affect community capacity. Research is needed to identify the pathways between changes in community capacity and in individual-level behavior that might result in the avoidance or reduction of teen pregnancy.

Coalition models: lessons learned from the CDC's Community Coalition Partnership Programs for the Prevention of Teen Pregnancy.

PURPOSE: To describe the models created by the 13 communities in the Centers for Disease Control and Prevention's Community Coalition Partnership Program (CCPP), and the relationship between key organizational features of the coalitions and the perception by coalition members of interim and community-wide outcomes. METHODS: This study relied on three sources of data: interviews conducted on site with a sample of coalition staff, evaluators, and members (n = 364); a written survey administered after the site visit to those interviewed (n = 216) asking about perceived outcomes and changes between the beginning and end of the project; and a coalition member survey mailed to all coalition members at all sites (n = 341) focusing on perceptions of coalition functioning, outcomes, and satisfaction. RESULTS: A variety of coalition models were developed. Respondents were positive in their assessments of how their coalitions operated even though few were sustained. The coalitions for which members perceived more positive outcomes were better established at the outset of the grant, led by paid staff, and had an area-wide focus, a steering committee, and a hub that was not a community-based organization. Coalitions composed primarily of neighborhood members were difficult to maintain. CONCLUSIONS: Despite members' high ratings, by the end of the funding period most coalitions were no longer functioning. It may be that coalitions are useful but not as permanent structures in communities. Grassroots and individual members not affiliated with an agency may require meaningful incentives to sustain participation. Because maturity of the coalition at the start of the project was a good predictor of sustainability, time should be spent verifying the stage of coalition development before funding.