Brk/PTK6 and Involucrin Expression May Predict Breast Cancer Cell Responses to Vitamin D3.

The process of human embryonic mammary development gives rise to the structures in which mammary cells share a developmental lineage with skin epithelial cells such as keratinocytes. As some breast carcinomas have previously been shown to express high levels of involucrin, a marker of keratinocyte differentiation, we hypothesised that some breast tumours may de-differentiate to a keratinocyte-derived 'evolutionary history'. To confirm our hypothesis, we investigated the frequency of involucrin expression along with that of Brk, a tyrosine kinase expressed in up to 86% of breast carcinomas whose normal expression patterns are restricted to differentiating epithelial cells, most notably those in the skin (keratinocytes) and the gastrointestinal tract. We found that involucrin, a keratinocyte differentiation marker, was expressed in a high proportion (78%) of breast carcinoma samples and cell lines. Interestingly, tumour samples found to express high levels of involucrin were also shown to express Brk. 1,25-dihydroxyvitamin D3, a known differentiation agent and potential anti-cancer agent, decreased proliferation in the breast cancer cell lines that expressed both involucrin and Brk, whereas the Brk/involucrin negative cell lines tested were less susceptible. In addition, responses to 1,25-dihydroxyvitamin D3 were not correlated with vitamin D receptor expression. These data contribute to the growing body of evidence suggesting that cellular responses to 1,25-dihydroxyvitamin D3 are potentially independent of vitamin D receptor status and provide an insight into potential markers, such as Brk and/or involucrin that could predict therapeutic responses to 1,25-dihydroxyvitamin D3.

Matrix metalloproteinase 8 (collagenase 2) induces the expression of interleukins 6 and 8 in breast cancer cells.

Matrix metalloproteinase 8 (MMP-8) is a tumor-suppressive protease that cleaves numerous substrates, including matrix proteins and chemokines. In particular, MMP-8 proteolytically activates IL-8 and, thereby, regulates neutrophil chemotaxis in vivo. We explored the effects of expression of either a WT or catalytically inactive (E198A) mutant version of MMP-8 in human breast cancer cell lines. Analysis of serum-free conditioned media from three breast cancer cell lines (MCF-7, SK-BR-3, and MDA-MB-231) expressing WT MMP-8 revealed elevated levels of IL-6 and IL-8. This increase was mirrored at the mRNA level and was dependent on MMP-8 catalytic activity. However, sustained expression of WT MMP-8 by breast cancer cells was non-permissive for long-term growth, as shown by reduced colony formation compared with cells expressing either control vector or E198A mutant MMP-8. In long-term culture of transfected MDA-MB-231 cells, expression of WT but not E198A mutant MMP-8 was lost, with IL-6 and IL-8 levels returning to base line. Rare clonal isolates of MDA-MB-231 cells expressing WT MMP-8 were generated, and these showed constitutively high levels of IL-6 and IL-8, although production of the interleukins was no longer dependent upon MMP-8 activity. These studies support a causal connection between MMP-8 activity and the IL-6/IL-8 network, with an acute response to MMP-8 involving induction of the proinflammatory mediators, which may in part serve to compensate for the deleterious effects of MMP-8 on breast cancer cell growth. This axis may be relevant to the recognized ability of MMP-8 to orchestrate the innate immune system in inflammation in vivo.

Reversible transdifferentiation of blood vascular endothelial cells to a lymphatic-like phenotype in vitro.

Blood vascular cells and lymphatic endothelial cells (BECs and LECs, respectively) form two separate vascular systems and are functionally distinct cell types or lineages with characteristic gene expression profiles. Interconversion between these cell types has not been reported. Here, we show that in conventional in vitro angiogenesis assays, human BECs of fetal or adult origin show altered gene expression that is indicative of transition to a lymphatic-like phenotype. This change occurs in BECs undergoing tubulogenesis in fibrin, collagen or Matrigel assays, but is independent of tube formation per se, because it is not inhibited by a metalloproteinase inhibitor that blocks tubulogenesis. It is also reversible, since cells removed from 3D tubules revert to a BEC expression profile upon monolayer culture. Induction of the lymphatic-like phenotype is partially inhibited by co-culture of HUVECs with perivascular cells. These data reveal an unexpected plasticity in endothelial phenotype, which is regulated by contact with the ECM environment and/or cues from supporting cells.

Mycobacterium tuberculosis upregulates microglial matrix metalloproteinase-1 and -3 expression and secretion via NF-kappaB- and Activator Protein-1-dependent monocyte networks.

Inflammatory tissue destruction is central to pathology in CNS tuberculosis (TB). We hypothesized that microglial-derived matrix metalloproteinases (MMPs) have a key role in driving such damage. Analysis of all of the MMPs demonstrated that conditioned medium from Mycobacterium tuberculosis-infected human monocytes (CoMTb) stimulated greater MMP-1, -3, and -9 gene expression in human microglial cells than direct infection. In patients with CNS TB, MMP-1/-3 immunoreactivity was demonstrated in the center of brain granulomas. Concurrently, CoMTb decreased expression of the inhibitors, tissue inhibitor of metalloproteinase-2, -3, and -4. MMP-1/-3 secretion was significantly inhibited by dexamethasone, which reduces mortality in CNS TB. Surface-enhanced laser desorption ionization time-of-flight analysis of CoMTb showed that TNF-alpha and IL-1beta are necessary but not sufficient for upregulating MMP-1 secretion and act synergistically to drive MMP-3 secretion. Chemical inhibition and promoter-reporter analyses showed that NF-kappaB and AP-1 c-Jun/FosB heterodimers regulate CoMTb-induced MMP-1/-3 secretion. Furthermore, NF-kappaB p65 and AP-1 c-Jun subunits were upregulated in biopsy granulomas from patients with cerebral TB. In summary, functionally unopposed, network-dependent microglial MMP-1/-3 gene expression and secretion regulated by NF-kappaB and AP-1 subunits were demonstrated in vitro and, for the first time, in CNS TB patients. Dexamethasone suppression of MMP-1/-3 gene expression provides a novel mechanism explaining the benefit of steroid therapy in these patients.

Cutting edge: the metalloproteinase ADAM17/TNF-alpha-converting enzyme regulates proteolytic shedding of the MHC class I-related chain B protein.

MHC class I-related chain (MIC) A/B are transmembrane proteins expressed in pathological conditions that are ligands for the activating receptor NKG2D found on cytotoxic lymphocytes. Soluble NKG2D ligands are detected in sera of patients suffering from multiple types of cancer where they are associated with reduced levels of receptor expression and compromised function of NK and CTLs. In this study, we report the identification of a metalloproteinase involved in the cleavage process of MIC; inhibition and knockdown of ADAM17/TACE blocks the shedding of these proteins. Strikingly, the recruitment of both enzyme and substrate to detergent-resistant membrane microdomains is crucial for efficient proteolysis. These findings provide a novel insight into the molecular mechanisms of MIC shedding.

Brk protects breast cancer cells from autophagic cell death induced by loss of anchorage.

Brk, a tyrosine kinase expressed in a majority of breast tumors, but not normal mammary tissue, promotes breast carcinoma cell proliferation. Normal epithelial cells are dependent on cell-cell or cell-matrix interactions for survival and undergo apoptosis after disruption of these interactions. Tumor cells are less sensitive to the induction of apoptosis and are predicted to have the potential to disseminate. We investigated whether Brk has further roles in breast tumor progression by relating its expression to tumor grade and demonstrating its role in the regulation of carcinoma cell survival under non-adherent conditions. Brk expression was determined by reverse transcription PCR on RNA extracted from surgical samples of human breast cancers. Breast carcinoma cell survival in suspension culture was examined when Brk protein levels were suppressed by RNA interference. Additionally, the effect of experimentally overexpressing Brk in otherwise Brk-negative breast carcinoma cells was assessed. Brk mRNA expression was notably higher in grade 3 breast tumors, as compared with lower tumor grades. In suspension culture, Brk suppression increased the rate of cell death, as compared with controls, and this cell death program exhibited characteristics of autophagy but not of apoptosis. Conversely, experimental expression of Brk in Brk-negative cells increased cell survival whereas kinase-inactive Brk did not. Therefore, Brk enhances breast carcinoma cell survival in suspension, suggesting a role for Brk in supporting breast cancer cell dissemination.

TGF-[beta]1 limits plaque growth, stabilizes plaque structure, and prevents aortic dilation in apolipoprotein E-null mice.

OBJECTIVE: Impairment of transforming growth factor (TGF)-beta1 signaling accelerates atherosclerosis in experimental mice. However, it is uncertain whether increased TGF-beta1 expression would retard atherosclerosis. The role of TGF-beta1 in aneurysm formation is also controversial. We tested whether overexpression of active TGF-beta1 in hyperlipidemic mice affects atherogenesis and aortic dilation. METHODS AND RESULTS: We generated apolipoprotein E-null mice with transgenes that allow regulated overexpression of active TGF-beta1 in their hearts. Compared to littermate controls, these mice had elevated cardiac and plasma TGF-beta1, less aortic root atherosclerosis (P< or =0.002), fewer lesions in the thoracic and abdominal aortae (P< or =0.01), less aortic root dilation (P<0.001), and fewer pseudoaneurysms (P=0.02). Mechanistic studies revealed no effect of TGF-beta1 overexpression on plasma lipids or cytokines, or on peripheral lymphoid organ cells. However, aortae of TGF-beta1-overexpressing mice had fewer T-lymphocytes, more collagen, less lipid, lower expression of inflammatory cytokines and matrix metalloproteinase-13, and higher expression of tissue inhibitor of metalloproteinase-2. CONCLUSIONS: When overexpressed in the heart and plasma, TGF-beta1 is an antiatherogenic, vasculoprotective cytokine that limits atherosclerosis and prevents aortic dilation. These actions are associated with significant changes in cellularity, collagen and lipid accumulation, and gene expression in the artery wall.

Tumour-associated tenascin-C isoforms promote breast cancer cell invasion and growth by matrix metalloproteinase-dependent and independent mechanisms.

INTRODUCTION: The stromal microenvironment has a profound influence on tumour cell behaviour. In tumours, the extracellular matrix (ECM) composition differs from normal tissue and allows novel interactions to influence tumour cell function. The ECM protein tenascin-C (TNC) is frequently up-regulated in breast cancer and we have previously identified two novel isoforms - one containing exon 16 (TNC-16) and one containing exons 14 plus 16 (TNC-14/16). METHODS: The present study has analysed the functional significance of this altered TNC isoform profile in breast cancer. TNC-16 and TNC-14/16 splice variants were generated using PCR-ligation and over-expressed in breast cancer cells (MCF-7, T47D, MDA-MD-231, MDA-MB-468, GI101) and human fibroblasts. The effects of these variants on tumour cell invasion and proliferation were measured and compared with the effects of the large (TNC-L) and fully spliced small (TNC-S) isoforms. RESULTS: TNC-16 and TNC-14/16 significantly enhanced tumour cell proliferation (P < 0.05) and invasion, both directly (P < 0.01) and as a response to transfected fibroblast expression (P < 0.05) with this effect being dependent on tumour cell interaction with TNC, because TNC-blocking antibodies abrogated these responses. An analysis of 19 matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases 1 to 4 (TIMP 1 to 4) revealed that TNC up-regulated expression of MMP-13 and TIMP-3 two to four fold relative to vector, and invasion was reduced in the presence of MMP inhibitor GM6001. However, this effect was not isoform-specific but was elicited equally by all TNC isoforms. CONCLUSIONS: These results demonstrate a dual requirement for TNC and MMP in enhancing breast cancer cell invasion, and identify a significant role for the tumour-associated TNC-16 and TNC-14/16 in promoting tumour invasion, although these isoform-specific effects appear to be mediated through MMP-independent mechanisms.

Distinct functions of natural ADAM-15 cytoplasmic domain variants in human mammary carcinoma.

Adamalysins [a disintegrin and metalloproteinase (ADAM)] are a family of cell surface transmembrane proteins that have broad biological functions encompassing proteolysis, adhesion, and cell signal regulation. We previously showed that the cytoplasmic domain of ADAM-15 interacts with Src family protein tyrosine kinases and the adaptor protein growth factor receptor binding protein 2 (Grb2). In the present study, we have cloned and characterized four alternatively spliced forms of ADAM-15, which differ only in their cytoplasmic domains. We show that the four ADAM-15 variants were differentially expressed in human mammary carcinoma tissues compared with normal breast. The expression of the individual isoforms did not correlate with age, menopausal status, tumor size or grade, nodal status, Nottingham Prognostic Index, or steroid hormone receptor status. However, higher levels of two isoforms (ADAM-15A and ADAM-5B) were associated with poorer relapse-free survival in node-negative patients, whereas elevated ADAM-15C correlated with better relapse-free survival in node-positive, but not in node-negative, patients. The expression of ADAM-15A and ADAM-15B variants in MDA-MB-435 cells had differential effects on cell morphology, with adhesion, migration, and invasion enhanced by expression of ADAM-15A, whereas ADAM-15B led to reduced adhesion. Using glutathione S-transferase pull-down assays, we showed that the cytoplasmic domains of ADAM-15A, ADAM-15B, and ADAM-15C show equivalent abilities to interact with extracellular signal-regulated kinase and the adaptor molecules Grb2 and Tks5/Fish, but associate in an isoform-specific fashion with Nck and the Src and Brk tyrosine kinases. These data indicate that selective expression of ADAM-15 variants in breast cancers could play an important role in determining tumor aggressiveness by interplay with intracellular signaling pathways.

Spontaneous metastasis in matrix metalloproteinase 3-deficient mice.

Matrix metalloproteinases (MMPs) have been linked to the metastatic potential of tumor cells due to their ability to degrade the extracellular matrix. MMP-3 (stromelysin-1) is upregulated in a wide variety of human tumors. We used the MMTV-PyMT breast cancer model to determine if MMP-3 is involved in tumorigenesis and metastatic growth. In this model the stromal expression of MMP-3 mRNA resembles the predominant MMP-3 expression pattern observed in human ductal breast carcinomas. We studied a cohort of 63 PyMT transgenic mice, either deficient for MMP-3 or wild-type controls. The degree of metastasis did not differ significantly between the two groups of mice, although the median lung metastasis volume was more than threefold increased in MMTV-PyMT mice deficient in MMP-3. Likewise, primary tumor growth rate and lymph node metastasis were not significantly affected by MMP-3-deficiency. By comparing mRNA levels in MMP-3-deficient PyMT tumors with PyMT wild-type tumors we excluded compensatory transcriptional changes of other MMPs or their specific inhibitors. Thus, we conclude that genetic ablation of MMP-3 does not significantly affect tumor growth and metastasis in the MMTV-PyMT model.

Tissue inhibitor of metalloproteinases-1 promotes liver metastasis by induction of hepatocyte growth factor signaling.

Balanced expression of proteases and their inhibitors is one prerequisite of tissue homeostasis. Metastatic spread of tumor cells through the organism depends on proteolytic activity and is the death determinant for cancer patients. Paradoxically, increased expression of tissue inhibitor of metalloproteinases-1 (TIMP-1), a natural inhibitor of several endometalloproteinases, including matrix metalloproteinases and a disintegrin and metalloproteinase-10 (ADAM-10), in cancer patients is negatively correlated with their survival, although TIMP-1 itself inhibits invasion of some tumor cells. Here, we show that elevated stromal expression of TIMP-1 promotes liver metastasis in two independent tumor models by inducing the hepatocyte growth factor (HGF) signaling pathway and expression of several metastasis-associated genes, including HGF and HGF-activating proteases, in the liver. We also found in an in vitro assay that suppression of ADAM-10 is in principle able to prevent shedding of cMet, which may be one explanation for the increase of cell-associated HGF receptor cMet in livers with elevated TIMP-1. Similar TIMP-1-associated changes in gene expression were detected in livers of patients with metastatic colorectal cancer. The newly identified role of TIMP-1 to create a prometastatic niche may also explain the TIMP-1 paradoxon.

The ADAM metalloproteinases.

The ADAMs (a disintegrin and metalloproteinase) are a fascinating family of transmembrane and secreted proteins with important roles in regulating cell phenotype via their effects on cell adhesion, migration, proteolysis and signalling. Though all ADAMs contain metalloproteinase domains, in humans only 13 of the 21 genes in the family encode functional proteases, indicating that at least for the other eight members, protein-protein interactions are critical aspects of their biological functions. The functional ADAM metalloproteinases are involved in "ectodomain shedding" of diverse growth factors, cytokines, receptors and adhesion molecules. The archetypal activity is shown by ADAM-17 (tumour necrosis factor-alpha convertase, TACE), which is the principal protease involved in the activation of pro-TNF-alpha, but whose sheddase functions cover a broad range of cell surface molecules. In particular, ADAM-17 is required for generation of the active forms of Epidermal Growth Factor Receptor (EGFR) ligands, and its function is essential for the development of epithelial tissues. Several other ADAMs have important sheddase functions in particular tissue contexts. Another major family member, ADAM-10, is a principal player in signalling via the Notch and Eph/ephrin pathways. For a growing number of substrates, foremost among them being Notch, cleavage by ADAM sheddases is essential for their subsequent "regulated intramembrane proteolysis" (RIP), which generates cleaved intracellular domains that translocate to the nucleus and regulate gene transcription. Several ADAMs play roles in spermatogenesis and sperm function, potentially by effecting maturation of sperm and their adhesion and migration in the uterus. Other non-catalytic ADAMs function in the CNS via effects on guidance mechanisms. The ADAM family are thus fundamental to many control processes in development and homeostasis, and unsurprisingly they are also linked to pathological states when their functions are dysregulated, including cancer, cardiovascular disease, asthma, Alzheimer's disease. This review will provide an overview of current knowledge of the human ADAMs, discussing their structure, function, regulation and disease involvement.

Laser-capture microdissection in prostate cancer research: establishment and validation of a powerful tool for the assessment of tumour-stroma interactions.

OBJECTIVES: To describe our experience with the optimization and validation of laser-capture microdissection (LCM) for biomarker analysis in prostate tissues. As LCM allows the separation of benign and malignant epithelial structures and stromal elements, it not only allows identification of the source of the biomarker, but might also accentuate gene or protein expression changes by reducing contamination by other cellular elements. MATERIALS AND METHODS: In all, 19 fresh-frozen prostate tissue samples were subjected to LCM, with the cDNA being analysed using quantitative polymerase chain reaction for several genes, to identify the optimum number of cells for capture, as well as gene markers assessing for the purity of the captured cells. The localization was further confirmed by in situ hybridization. RESULTS: Prostate-specific antigen (PSA) and cytokeratin 8, were expressed solely by epithelial cells, whereas hepatocyte growth factor (HGF) and tissue inhibitor of metalloproteinases-3 (TIMP3) were expressed only by stromal cells, and the levels of transcripts of these genes were unaltered between benign and malignant tissues. CONCLUSIONS: These data suggest that PSA, cytokeratin 8, HGF and TIMP3 are reliable gene markers of purity of epithelial and stromal compartments for LCM of prostate tumours. Although this technique is not new and is increasingly used in laboratories, it needs optimization and stringent validation criteria before data analysis. This applies to all tissue types subjected to LCM.

Analysis of hypoxia-associated gene expression in prostate cancer: lysyl oxidase and glucose transporter-1 expression correlate with Gleason score.

Prostate cancer cells exist under hypoxic conditions. Hypoxia has a detrimental effect on the efficacy of treatment and final outcome in patients with prostate cancer. There have been a large number of endogenous markers of hypoxia described previously across a range of cancer types, both in vitro and in vivo. The aim of this study was to evaluate the expression of a range of hypoxia-associated genes within benign prostatic hypertrophy (BPH) and prostate cancer tissue. Messenger RNA was extracted from primary prostate tissue obtained from 67 men with benign prostatic hypertrophy or prostate cancer (Gleason score 5 to 10). Real-time polymerase chain reaction was performed to quantify the expression levels of 12 hypoxia-associated genes in these tissues. Expression of lysyl oxidase (LOX) and glucose transporter-1 (GLUT-1) genes were significantly higher in prostate cancer compared with BPH tissue (P<0.05) and correlated with Gleason score (LOX: R=0.297, P=0.015; GLUT-1: R=0.274, P=0.026). HIF-2alpha had a negative correlation with Gleason score (R= -0.309, P=0.012). The remaining hypoxia-associated genes did not show any specific pattern of expression in prostate tissue. Numerous molecules have been proposed as endogenous markers of hypoxia. The findings of this study illustrate that not all hypoxia-associated molecules are relevant to prostate cancer in vivo. However, LOX and GLUT-1 are candidate markers of hypoxia in prostate cancer and may prove useful in identifying patients with hypoxic prostate cancer. Not all hypoxia-associated molecules are relevant in prostate cancer in vivo.

Membrane-type 4 matrix metalloproteinase promotes breast cancer growth and metastases.

Membrane-type matrix metalloproteinases (MT-MMP) constitute a subfamily of six distinct membrane-associated MMPs. Although the contribution of MT1-MMP during different steps of cancer progression has been well documented, the significance of other MT-MMPs is rather unknown. We have investigated the involvement of MT4-MMP, a glycosylphosphatidylinositol-anchored protease, in breast cancer progression. Interestingly, immunohistochemical analysis shows that MT4-MMP production at protein level is strongly increased in epithelial cancer cells of human breast carcinomas compared with normal epithelial cells. Positive staining for MT4-MMP is also detected in lymph node metastases. In contrast, quantitative reverse transcription-PCR analysis reveals similar MT4-MMP mRNA levels in human breast adenocarcinomas and normal breast tissues. Stable transfection of MT4-MMP cDNA in human breast adenocarcinoma MDA-MB-231 cells does not affect in vitro cell proliferation or invasion but strongly promotes primary tumor growth and associated metastases in RAG-1 immunodeficient mice. We provide for the first time evidence that MT4-MMP overproduction accelerates in vivo tumor growth, induces enlargement of i.t. blood vessels, and is associated with increased lung metastases. These results identify MT4-MMP as a new putative target to design anticancer strategies.

MMP and TIMP expression in quiescent, dividing, and differentiating human lens cells.

PURPOSE: Matrix metalloproteinases (MMPs) and the tissue inhibitors of the MMPs (TIMPs) have been implicated in lens differentiation, growth, remodeling, and cataract. Hence, a gene expression analysis was undertaken in epithelial and fiber cells dissected from clear human donor lenses. METHODS: The human lens was dissected into three regions: anterior epithelial, equatorial, and fiber cells. Primary lens cell cultures were also analyzed. cDNA was generated by reverse transcription of the mRNA portion of the total RNA isolated from each sample. Gene expression data were generated using quantitative real-time reverse transcription PCR. Data were analyzed in terms of cycle threshold number (C(T)) and were normalized to endogenous 18S expression. Western blot analyses were carried out to confirm the presence of two critical MMPs. RESULTS: Anterior and equatorial samples were uncontaminated by fiber cells because they showed high expression of alpha-crystallin genes but low expression of beta- and gamma-crystallins. The fibers had high expression of these genes and of MIP. MMP genes were expressed at uniformly low levels in the native tissues except for MMP-14 and -15 (MT1- and MT2-MMP, respectively). In fact, MT1-MMP declined in expression from the anterior epithelium to fibers, whereas MT2-MMP increased. The presence of MT1 and MT2-MMP proforms and faster migrating bands, indicating processed or activated forms, was confirmed at the protein level. TIMP genes were uniformly highly expressed in native tissues, with TIMP-3 having the highest expression in the epithelial tissues and TIMP-2 in the fibers. MMP expression was generally elevated in both sets of cultured cells, including MMP-2 and -9. TIMP genes were also relatively highly expressed in the cultured cells. CONCLUSIONS: MMP expression is generally well regulated in native tissues, with relatively low expression of MMPs and high expression of TIMPs. Membrane-type MMPs (MT1 and 2-MMPs) were the most highly expressed; this is important in a tissue with relatively high membrane content but low extracellular space. The striking reciprocal patterns of expression of MT1-MMP and MT2-MMP indicate that these enzymes are of particular significance in lens function.

Increase in gelatinase-specificity of matrix metalloproteinase inhibitors correlates with antimetastatic efficacy in a T-cell lymphoma model.

The recognition that matrix metalloproteinases (MMPs) facilitate tumor cell invasion and metastasis has led to the development of synthetic MMP inhibitors (MMPIs) as cancer therapeutic agents. Because several Phase III trials failed recently to show efficacy of broad-spectrum MMPIs in advanced cancer, the feasibility of MMPs as therapeutic targets has been challenged. However, it has not yet been determined whether MMPIs that have increased specificity may have greater benefit. We show that MMP-9 expression closely correlates with the progression of liver metastasis in a T-cell lymphoma model. MMPIs with greater selectivity/specificity for MMP-9 in vitro showed greater efficacy against liver metastasis in vivo. These data demonstrate a link between increased specificity of MMPIs and enhanced anticancer activity.

Elevated membrane-type matrix metalloproteinases in gliomas revealed by profiling proteases and inhibitors in human cancer cells.

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulate proteolysis of the extracellular matrix and other extracellular proteins, including growth factors and their receptors. The aberrant expression of these genes is common in most cancers. We profiled the RNA levels of every human MMP and TIMP in a variety of cell types (fibroblast, endothelial, hematopoietic, carcinoma, melanoma, and glioma) using quantitative PCR, with the aim of identifying novel expression patterns. Almost all members of the membrane-type (MT-) MMP and TIMP families were elevated in glioma lines compared to carcinomas. In clinical glioma specimens, there were positive correlations between glioma grade and RNA levels of MT-1, MT-2, and MT-6 MMP, TIMP-1 and TIMP-2, and for several growth factors and receptors. These findings suggest that advanced malignant gliomas have elevated levels of membrane-associated MMPs and TIMPs, which may potentially regulate vascularization and invasion. Concurrent elevation of signaling molecules suggests potential bidirectional relationships that enhance tumor aggressiveness.

The role of chondrocyte senescence in osteoarthritis.

Replicative senescence occurs when normal somatic cells stop dividing. Senescent cells remain viable, but show alterations in phenotype, e.g. altered expression of matrix metalloproteinases (MMPs); these enzymes are known to be involved in cartilage destruction. It is assumed that cells deplete their replicative potential during aging, and age is a major risk factor for osteoarthritis (OA). Therefore, we hypothesized that chondrocytes in aging or diseased cartilage become senescent with associated phenotypic changes contributing to development or progression of OA. Articular cartilage was obtained from OA patients undergoing arthroplasty, with 'normal' cartilage from trauma surgery for hip fracture. Senescent cells were identified using the senescence-associated beta-galactosidase (SA-beta-gal) marker. Telomere length was assessed using Southern blot. MMP expression was measured at the mRNA level using Taqman RT-PCR. No SA-beta-gal staining was observed in control cartilage regardless of patient age. In contrast, SA-beta-gal staining was observed in damaged OA cartilage adjacent to the lesion. Cultured chondrocytes isolated from sites near a lesion contained a greater percentage of SA-beta-gal positive cells than cultures isolated from distal sites or normal cartilage. Mean telomere length was shorter in cells near the lesion compared to distal sites in the same joint; thus the former population has undergone cell division. The expression of collagenases MMP-1, -8 and -13 and tissue inhibitor of metalloproteinases (TIMP)-1 was altered in OA cartilage, but no difference was detected between lesion and distal sites in the same joint (i.e. no correlation was found between senescent cells and proteinase/ inhibitor expression).