[Show abstract][Hide abstract] ABSTRACT: A quantitative in vivo angiogenesis model employing collagen onplants placed on the chick embryo chorioallantoic membrane (CAM) has been used in this study to assess the spatial and temporal associations between neutrophil-like inflammatory cells, namely chicken heterophils, and the development of new blood vessels. Previously we have demonstrated that monocytes/macrophages infiltrating the onplants were associated with extracellular matrix remodeling and angiogenesis, in particular by delivering MMP-13 collagenase. By introducing chicken gelatinase B (chMMP-9) as a specific marker for heterophils, we now show that the onset and extent of angiogenesis induced by purified growth factors or by human HT-1080 fibrosarcoma cells correlated with the initial influx of chMMP-9-positive heterophils. This early heterophil arrival was followed by the infiltration of monocytes/macrophages and appeared to sustain further blood vessel formation. The disruption of inflammatory cell influx by 2 mechanistically distinct anti-inflammatory drugs, cortisone and ibuprofen, significantly inhibited angiogenesis, indicating a functional involvement of these inflammatory cells in new blood vessel development. A direct addition of isolated heterophils or purified chMMP-9 into the HT-1080 onplants engrafted into cortisone- or ibuprofen-treated embryos reversed the antiangiogenic effects of the drugs. The exogenously added heterophils induced in vivo a further infiltration of endogenous heterophils and monocytes and dramatically rescued the impaired angiogenesis, highlighting the importance of early inflammatory leukocytes in tumor-induced angiogenesis. Moreover, purified heterophils incorporated into onplants lacking growth factors or tumor cells induced angiogenesis in nontreated embryos, further indicating a direct proangiogenic role for neutrophil-like leukocytes.
[Show abstract][Hide abstract] ABSTRACT: Entry of malignant cells into the vasculature, i.e. intravasation, requires proteolytic remodeling of the extracellular matrix so that tumor cells may pass through the local stroma and penetrate the vessel wall. The circulatory system then provides a means of transporting tumor cells to distant sites where they establish metastatic lesions. We utilized activity based protein profiling (ABPP) to compare the active serine hydrolase repertoire of high-intravasating (HT-hi/diss) and low-intravasating (HT-lo/diss) variants of the human fibrosarcoma HT-1080 cell line, recently isolated in our laboratory, to determine which enzyme(s) play a role in intravasation. ABPP is a chemical proteomic technique that uses active site directed probes linked to a reporter group to measure the activity levels of individual enzymes of a given class in a complex mixture of proteins. ABPP combined with mass spectroscopy can identify enzymes that have altered activity levels between experimental groups, such as metastatic and non-metastatic tumor cells. Since proteins are detected based on activity rather than abundance, ABPP can distinguish between active enzymes and their inactive zymogen or their inhibitor bound forms, thereby providing an advantage compared to other methods of profiling. ABPP analysis of HT-hi/diss and HT-lo/diss cells revealed multiple serine hydrolases with altered activity between the two variants, with the largest difference being the activity of urokinase plasminogen activator (uPA). While the level of uPA expression has been implicated in the metastatic process, we show that activation of uPA is a key step due to the fact that uPA protein levels in the two variants are the same. The failure to activate uPA may play a large role in the inability of HT-lo/diss cells to intravasate and metastasize, while the active uPA in HT-hi/diss cells likely contributes to the intravasation ability of these cells. To determine the biological significance of this difference in uPA activity, we used the HT-lo/diss and HT-hi/diss variants in a chick-embryo chorioallantoic membrane (CAM) assay to quantitatively measure intravasation and metastasis in vivo. We also took advantage of the activity based nature of the ABPP system to screen small molecule inhibitors of uPA for their specificity and efficacy, to determine the optimal uPA inhibitor for use in the CAM assay. Inhibition of uPA activity with natural (PAI-1) or synthetic (amiloride) inhibitors significantly diminished HT-hi/diss intravasation and metastasis in vivo. Additionally, treatment of HT-lo/diss tumors with exogenous active uPA increased the number of intravasated cells in vivo 2-3 fold. These results indicate that active uPA promotes tumor cell intravasation and that uPA activation appears to be a key step in tumor progression. These experiments highlight the efficiency of ABPP analysis in the identification of specific proteases that regulate tumor progression. The combination of ABPP analysis with the chick embryo CAM assay has proven to be a powerful and rapid technique for identifying potential regulators of tumor dissemination and for modulating their activities in vivo to confirm their effect on the metastatic process.
Journal of Thrombosis and Haemostasis 01/2006; 4:194-194. · 6.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Proteolytic modifications of the extracellular matrix proteins and cell surface receptors by matrix metalloproteinases (MMPs) are critically important for tumor cell dissemination and metastasis. Functional activity of MMPs is tightly regulated through gene and protein expression as well as the degree of proenzyme activation and inhibition of the activated enzyme by natural tissue inhibitors of metalloproteinases (TIMPs). A great deal of information has accumulated regarding the role of MMPs and TIMPs in tumor cell invasion and tumor-induced angiogenesis. However, only limited data are available on the role of the MMP/TIMP system in tumor cell intravasation, although it is one of the rate-limiting steps in the metastatic cascade. To analyze intravasation of tumor cells, we have generated two variants of human HT-1080 fibrosarcoma, which differ 100-fold in their ability to enter the vasculature of the chorioallantoic membrane (CAM) and metastasize to the secondary organs of the chick embryo. The availability of these high (HT-hi/diss) and low (HT-lo/diss) disseminating cell variants allowed us to investigate the role of MMPs and TIMPs specifically in tumor cell intravasation. By quantitative PCR and Western blot analyses, HT-hi/diss and HT-lo/diss cells were initially profiled for in vitro mRNA and protein expression levels of nine MMPs (MMP-1, -2, -3, -7, -8, -9, -10, -13, and -14) and three TIMPs (TIMP-1, -2, and -3), known to be expressed in the parental HT-1080 cells. To delineate tumor and host MMPs and TIMPs, which are actually involved in tumor cell intravasation, in vivo mRNA and protein profiling analyses were performed on CAM primary tumors derived from the two HT-1080 cell variants. Human MMP-1 and MMP-9 were more abundant in HT-hi/diss variant than in HT-lo/diss variant both in cell cultures in vitro and in primary tumors in vivo. In contrast, human MMP-2 and TIMP-2 were consistently expressed at higher levels in the HT-lo/diss cells and tumors. To further assess the role of MMP/TIMP system in tumor cell intravasation, activity based protein profiling (ABPP) of the two HT-1080 cell variants was performed with a metalloprotease-specific probe. Surprisingly, ABPP indicated only minor differences in MMP activity between the two HT-1080 cell variants. This discrepancy between results of the different profiling methods could indicate that many of the tumor MMPs are functionally inactive due to complex formation with TIMPs. Functional roles of human TIMP-1, -2 and -3 in intravasation of HT-1080 cell variants were evaluated by downregulation of protein expression with the respective siRNA. Our findings on downregulation of individual TIMPs and their combinations indicate that a fine balance between tumor MMPs and their natural inhibitors TIMPs might determine success of an intravasation event during tumor cell dissemination.
Journal of Thrombosis and Haemostasis 01/2006; 4. · 6.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The human tumor/chick embryo model involving grafting of human HT-1080 fibrosarcoma cells on the chorioallantoic membrane was used in conjunction with quantitative real-time Alu PCR to select in vivo a pair of isogenic cell lines (HT-hi/diss and HT-lo/diss), dramatically differing in their ability to disseminate from the primary tumor (i.e., intravasate into the chorioallantoic membrane vasculature and metastasize to the lungs). During an immunohistochemical time course study, HT-hi/diss cells were sequentially visualized having escaped from the primary tumors, engaged with the blood vessels, and eventually observed inside the chorioallantoic membrane capillaries, thus reflecting early intravasating events. In contrast, HT-lo/diss cells seemed restricted to their primary tumor. Importantly, after i.v. inoculation, both variants arrested, extravasated, and proliferated in host tissues with similar efficiencies, highlighting that the observed earlier events at the periphery of the primary tumor could account for their differential dissemination. In a mechanistic probing of these events, we determined that HT-hi/diss intravasation was sensitive to a broad-range matrix metalloproteinase (MMP) inhibitor. To analyze the possible role of individual MMPs, membrane-bound MMP-14 and secreted MMP-9 were individually down-regulated in HT-hi/diss cells with their corresponding small interfering RNAs. Despite efficient down-regulation of MMP-14, neither intravasation nor metastasis of HT-hi/diss cells was affected significantly. However, a substantial down-regulation of MMP-9 was accompanied by a surprising 3-fold increase in intravasation and metastasis. The results emphasize a rising awareness that targeting certain MMPs might result in an enhanced malignancy, exemplified herein at the intravasation level as this step of the metastatic cascade is dissected and quantified.
Cancer Research 01/2006; 65(23):10959-69. · 8.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cancer arises by the accumulation of genetic alterations in DNA leading to aberrant gene transcription. Expression-profiling studies have correlated genomewide expression signatures with malignancy. However, functional analysis elucidating the contribution and synergy of genes in specific cancer cell phenotypes remains a formidable obstacle. Herein, we describe an alternative genetic approach for identification of genes involved in tumor progression by using a library of zinc finger artificial transcription factors (ATFs) and functional screening of tumor cells as a source of genetic plasticity and clonal selection. We isolated a six-zinc finger transcriptional activator (TF 20-VP, TF 20 containing the VP64 activator domain) that acts to reprogram a drug-sensitive, poorly invasive, and nonmetastatic cell line into a cell line with a drug-resistant, highly invasive, and metastatic phenotype. Differential expression profiles of cells expressing TF 20-VP followed by functional studies, both in vitro and in animal models, revealed that invasion and metastasis requires co-regulation of multiple target genes. Significantly, the E48 antigen, associated with poor metastasis-free survival in head and neck cancer, was identified as one specific target of TF 20-VP. We have shown phenotypic modulation of tumor cell behavior by E48 expression, including enhanced cell migration in vitro and tumor cell dissemination in vivo. This study demonstrates the use of ATFs to identify the group of genes that cooperate during tumor progression. By co-regulating multiple targets, ATFs can be used as master genetic switches to reprogram and modulate complex neoplastic phenotypes.
Proceedings of the National Academy of Sciences 09/2005; 102(33):11716-21. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: TMPRSS2 is a type II transmembrane-bound serine protease that has gained interest owing to its highly localized expression in the prostate and its overexpression in neoplastic prostate epithelium. Once activated, the serine protease domain of TMPRSS2 is released from the cell surface into the extracellular space. PAR (protease-activated receptor)-2 belongs to a family of G-protein-coupled receptors (PAR-1-4) that are activated by specific serine proteases, which are expressed in many normal and malignant cell types. Previous in vitro studies on prostate cancer cells suggest a role for PAR-2 in prostate cancer metastasis. A polyclonal anti-human TMPRSS2 antibody was generated against the TMPRSS2 serine protease domain. The antibody showed specific reactivity with recombinant expressed TMPRSS2, and so was used to extract and purify the cleaved active TMPRSS2 protease from prostate cancer cells. Reverse transcriptase PCR and Western blot analysis were used to show the expression of both TMPRSS2 and PAR-2 in the androgen-dependent LNCaP prostate cancer cell line. Treatment of LNCaP cells with the cellular immunopurified TMPRSS2 protease induced a transient increase in intracellular calcium, which is indicative of G-protein-coupled-receptor activation. This calcium mobilization was inhibited by cellular pre-treatment with a specific PAR-2 antagonist, but not with a PAR-1 antagonist; inhibition of the protease activity also failed to mobilize calcium, suggesting that TMPRSS2 is capable of cleaving and thereby activating the PAR-2 receptor. The calcium mobilization was also inhibited by cellular pre-treatment with suramin or 2-APB (2-aminoethoxydiphenyl borate), indicating that a G-protein pathway is involved and that subsequent calcium release is mainly from intracellular stores. The present study describes how TMPRSS2 may contribute to prostate tumour metastasis via the activation of PAR-2.
[Show abstract][Hide abstract] ABSTRACT: The American horseshoe crab Limulus polyphemus contains α2-macroglobulin (α2M) in the hemolymph plasma and hemocytes. α2M from Limnulus shows many of the typical characteristics of mammalian α2M, including the presence of an internal thiol-ester, reactivity with a diversity of endopeptidases, a unique proteinase-trapping mechanism, and reactivity with the mammalian α2M receptor. Additionally, Limulusα2M has the unique property that it regulates the limulin-based hemolytic system of the plasma. A cDNA encoding Limulusα2M has been obtained from a hemocyte cDNA library. The open reading frame encodes an N-terminal signal sequence of 25 amino acid residues and a mature protein of 1482 residues. The entire amino acid sequence is similar to those of the mammalian α2Ms (28–29% identity) and contains common features found in mammalian α2Ms, a bait region, an internal thiol-ester site, and a receptor-binding domain. However, the N-terminal portion (positions 24–105) has no sequence similarity with those of mammalian α2Ms, and it is structurally related to that of the human complement factor C8γ chain, consistent with a role for Limulus α2M in host defense. The component sugar analysis of Limulus α2M showed the existence of a complex type of oligosaccharide chain similar to those of mammalian α2M. However, unlike mammalian α2M, no sialic acid was detected in Limulusα2M and it contained approximately 3 mol/mol N-acetylgalactosamine, suggesting the presence of O-linked sugar chains, which have not been found in mammalian α2M.Expression of α2M was detected in hemocyies, but not in hepatopancreas, heart, stomach, intestine, coxal gland, brain and skeletal muscle. Furthermore, immunoblotting of large and small granules of the hemocytes with antiserum against α2M indicated the presence of the α2M in large granules. Trypsin-treated Limulusα2M, but not the native α2M, displaced methylamine-treated human 125I-α2M from the human α2M receptor with a Kd of 30 nM, suggesting conservation of the proteinase-clearance mechanisms between mammalian and arthropod evolutionary lineages.
European Journal of Biochemistry. 07/2004; 242(3):822 - 831.
[Show abstract][Hide abstract] ABSTRACT: We have demonstrated previously that new blood vessel formation induced by angiogenic growth factors in onplants placed on the chorioallantoic membrane (CAM) of the chick embryos is critically dependent on the cleavage of fibrillar collagen by a previously unidentified interstitial collagenase. In the present study we have used a quantitative CAM angiogenesis system to search for and functionally characterize host avian collagenases responsible for the collagen remodeling associated with angiogenesis. Among the matrix metalloproteinases (MMPs) identified in the CAM onplant tissue, the chicken MMP-13 (chMMP-13) was the only enzyme whose induction and expression coincided with the onset of angiogenesis and blood vessel formation. The chMMP-13 cDNA has been cloned and recombinantly expressed. The chMMP-13 protein has been purified, characterized in vitro, and examined in situ in the CAM. MMP-13-positive cells appear in the CAM shortly after angiogenic stimulation and then accumulate in the collagen onplant tissue. Morphologically, the chMMP-13-containing cells appear as hematopoietic cells of monocyte/macrophage lineage. In vitro, the chMMP-13 proenzyme is rapidly and efficiently activated through the urokinase plasminogen activator/plasminogen/plasmin cascade into a collagenase capable of cleaving native but not the (r/r) mutant collagenase-resistant collagen. Surprisingly, nanogram levels of purified chMMP-13 elicit an angiogenic response in the CAM onplants comparable with that induced by the angiogenic growth factors. The chMMP-13-mediated response was efficiently blocked by select protease inhibitors indicating that plasmin-activated chMMP-13 can function as an angiogenic factor in vivo. Altogether, the results of this study extend the physiological role of MMP-13, previously associated with cartilage/bone resorption, to the collagen remodeling involved in the angiogenic cascade.
Journal of Biological Chemistry 07/2004; 279(26):27633-45. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report the identification and characterization of mouse matriptase-2 (m-matriptase-2), an 811-amino-acid protein composed of an N-terminal cytoplasmic domain, a membrane-spanning domain, two CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domains, three LDLR (low-density-lipoprotein receptor class A) domains and a C-terminal serine-protease domain. All m-matriptase-2 protein domain boundaries corresponded with intron/exon junctions of the encoding gene, which spans approx. 29 kb and comprises 18 exons. Matriptase-2 is highly conserved in human, mouse and rat, with the rat matriptase-2 gene ( r-maltriptase-2 ) predicted to encode transmembrane and soluble isoforms. Western-blot analysis indicated that m-matriptase-2 migrates close to its theoretical molecular mass of 91 kDa, and immunofluorescence analysis was consistent with the proposed surface membrane localization of this protein. Reverse-transcription PCR and in-situ -hybridization analysis indicated that m-matriptase-2 expression overlaps with the distribution of mouse hepsin (m-hepsin, a cell-surface serine protease identified in hepatoma cells) in adult tissues and during embryonic development. In adult tissues both are expressed at highest levels in liver, kidney and uterus. During embryogenesis m-matriptase-2 expression peaked between days 12.5 and 15.5. m-hepsin expression was biphasic, with peaks at day 7.5 to 8.5 and again between days 12.5 and 15.5. In situ hybridization of embryonic tissues indicated abundant expression of both m-matriptase-2 and m-hepsin in the developing liver and at lower levels in developing pharyngo-tympanic tubes. While m-hepsin was detected in the residual embryonic yolk sac and with lower intensity in lung, heart, gastrointestinal tract, developing kidney tubules and epithelium of the oral cavity, m-matriptase-2 was absent in these tissues, but strongly expressed within the nasal cavity by olfactory epithelial cells. Mechanistic insight into the potential role of this new transmembrane serine protease is provided by its novel expression profile in embryonic and adult mouse.
[Show abstract][Hide abstract] ABSTRACT: Monoclonal antibody technology has generated invaluable tools for both the analytical and clinical sciences. However, standard immunization approaches frequently fail to provide monoclonal antibodies with the desired specificity. Subtractive immunization provides a powerful alternative to standard immunization and allows for the production of truly unique antibodies. With the intent of targeting specific epitopes within the proteome, subtractive immunization has been broadly and successfully implemented for the production of monoclonal antibodies otherwise unobtainable by standard immunization. Subtractive immunization utilizes a distinct immune tolerization approach that can substantially enhance the generation of monoclonal antibodies to desired antigens. The approach is based on tolerizing the host animal to immunodominant or otherwise undesired antigen(s) (tolerogen) that may be structurally or functionally related to the antigen of interest. Tolerization of the host animal can be achieved through one of three methods: High Zone, Neonatal, or Drug-induced tolerization. The tolerized animal is then inoculated with the desired antigen (immunogen) and antibodies generated by the subsequent immune response are screened for the desired antigenic reactivity. Over the past 15 years a large number of investigators have used the subtractive approach with cleverly chosen tolerogen-immunogen combinations and successfully generated uniquely reactive antibodies which are often neutralizing or function-blocking. This review will focus on the implementation of subtractive immunization for the production of antibodies otherwise unobtainable by standard immunization.
Biochemical and Biophysical Research Communications 05/2003; 303(3):733-44. · 2.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have previously used a subtractive immunization (SI) approach to generate monoclonal antibodies (mAbs) against proteins preferentially expressed by the highly metastatic human epidermoid carcinoma cell line, M(+)HEp3. Here we report the immunopurification, identification and characterization of SIMA135/CDCP1 (subtractive immunization M(+)HEp3 associated 135 kDa protein/CUB domain containing protein 1) using one of these mAbs designated 41-2. Protein expression levels of SIMA135/CDCP1 correlated with the metastatic ability of variant HEp3 cell lines. Protein sequence analysis predicted a cell surface location and type I orientation of SIMA135/CDCP1, which was confirmed directly by immunocytochemistry. Analysis of deglycosylated cell lysates indicated that up to 40 kDa of the apparent molecular weight of SIMA135/CDCP1 is because of N-glycosylation. Western blot analysis using a antiphosphotyrosine antibody demonstrated that SIMA135/CDCP1 from HEp3 cells is tyrosine phosphorylated. Selective inhibitor studies indicated that an Src kinase family member is involved in the tyrosine phosphorylation of the protein. In addition to high expression in M(+)HEp3 cells, the SIMA135/CDCP1 protein is expressed to varying levels in 13 other human tumor cell lines, manifesting only a weak correlation with the reported metastatic ability of these tumor cell lines. The protein is not detected in normal human fibroblasts and endothelial cells. Northern blot analysis indicated that SIMA135/CDCP1 mRNA has a restricted expression pattern in normal human tissues with highest levels of expression in skeletal muscle and colon. Immunohistochemical analysis indicated apical and basal plasma membrane expression of SIMA135/CDCP1 in epithelial cells in normal colon. In colon tumor, SIMA135/CDCP1 expression appeared dysregulated showing extensive cell surface as well as cytoplasmic expression. Consistent with in vitro shedding experiments on HEp3 cells, SIMA135/CDCP1 was also detected within the lumen of normal and cancerous colon crypts, suggesting that protein shedding may occur in vivo. Thus, specific immunodetection followed by proteomic analysis allows for the identification and partial characterization of a heretofore uncharacterized human cell surface antigen.
[Show abstract][Hide abstract] ABSTRACT: Many serine proteases play important regulatory roles in complex biological systems, but only a few have been linked directly with capillary morphogenesis and angiogenesis. Here we provide evidence that serine protease activities, independent of the plasminogen activation cascade, are required for microvascular endothelial cell reorganization and capillary morphogenesis in vitro. A homology cloning approach targeting conserved motifs present in all serine proteases, was used to identify candidate serine proteases involved in these processes, and revealed 5 genes (acrosin, testisin, neurosin, PSP and neurotrypsin), none of which had been associated previously with expression in endothelial cells. A subsequent gene-specific RT-PCR screen for 22 serine proteases confirmed expression of these 5 genes and identified 7 additional serine protease genes expressed by human endothelial cells, urokinase-type plasminogen activator, protein C, TMPRSS2, hepsin, matriptase/MT-SP1, dipeptidylpeptidase IV, and seprase. Differences in serine protease gene expression between microvascular and human umbilical vein endothelial cells (HUVECs) were identified and several serine protease genes were found to be regulated by the nature of the substratum, ie. artificial basement membrane or fibrillar type I collagen. mRNA transcripts of several serine protease genes were associated with blood vessels in vivo by in situ hybridization of human tissue specimens. These data suggest a potential role for serine proteases, not previously associated with endothelium, in vascular function and angiogenesis.
Thrombosis and Haemostasis 04/2003; 89(3):561-72. · 6.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mammalian urokinase-type plasminogen activator (uPA) is produced as a stable single polypeptide chain zymogen and requires a distinct proteolytic cleavage to become an active, two-chain enzyme. In contrast, chicken uPA, both native and recombinant, is found predominantly as a two-chain, active enzyme even in the absence of plasmin, a physiological activator. Here we show that the proclivity to autoactivate is not a unique property of the chicken uPA catalytic domain but requires sequences distinct from and independent of the serine protease domain. Human/chicken chimeric uPA molecules and point mutants were used to determine the structural requirements for uPA autoactivation versus zymogen stability. The amino terminal fragment of chicken uPA engineered onto the human uPA molecule can induce the autoactivation of the human uPA. In fact, the first twenty residues of the chicken uPA are necessary and sufficient to induce the autoactivation of chicken and human uPA. These results indicate that sequence motifs, distal to the active site, control the substrate specificity and catalytic efficiency of uPA activity in autolytic activation.
Thrombosis and Haemostasis 03/2003; 89(2):382-92. · 6.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A quantitative assessment of rate-limiting steps in metastasis has always been challenging because of the difficulty of detecting small tumor cell populations. We have developed a highly sensitive assay for monitoring the metastatic dissemination of human tumor cells in the chick embryo and used this assay to investigate the relative efficacy of sequential stages in the metastatic cascade for two malignant human tumor cells lines, HEp3 and HT1080. This assay is based on the real-time PCR amplification of human alu sequences and exhibits a high sensitivity (25 cells/lung) with a large linear range (50-100,000 cell/lung). The assay is optimized for a high number of replicate in vivo assays (50-100 animals/assay) and can be applied in both experimental and spontaneous metastasis models. Using quantitative alu PCR, we determined that HEp3 spontaneously metastasizes very efficiently and rapidly, generating secondary growth in the lung exceeding 1-2 x 10(4) cells/lung in 7 days. In contrast, spontaneous HT1080 metastasis is 50-100-fold less efficient, resulting in only 200-400 cells/lung in 7 days. By taking advantage of the sensitivity and specificity of the real-time alu PCR assay we were also able to quantitatively assess multiple steps in metastasis including intravasation, arrest of tumor cells in secondary organs of the embryo, and the initial growth and expansion of the arrested tumor cells. A comparative analysis of HEp3 and HT1080 metastasis demonstrates that the relatively low-to-moderate metastatic rate of HT1080 is caused by two distinct deficiencies, an 8-10-fold lower rate of intravasation and a delayed onset of HT1080 growth expansion in the secondary organ. Thus, a very facile metastasis model system coupled with the sensitive, real-time PCR-based assay allows for the identification and quantification of rate-limiting steps in the metastatic cascade for select human tumor cell lines.
Cancer Research 01/2003; 62(23):7083-92. · 8.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although tumors frequently show elevated protease activities, the concept of anti-proteolytic cancer therapy has lost momentum after failure of clinical trials with broad-spectrum matrix metalloproteinase inhibitors. Thus we need to adapt our design strategies for protease inhibitors. Here, we employed a series of seven structurally fine-modulated and pharmacokinetically closely related synthetic 4-amidinobenzylamine-based inhibitors with distinct selectivity for prototypical serine proteases in a murine T cell lymphoma liver metastasis model. This in vivo screening revealed efficacy of urokinase inhibitors but no correlation between urokinase selectivity or affinity and anti-metastatic effect. In contrast, factor Xa-selective inhibitors were more potent, demonstrating factor Xa or a factor Xa-like serine protease likely to be more determinant in this model. Factor Xa selectivity, but not affinity, significantly improved anti-metastatic efficacy. For example, factor Xa inhibitors CJ-504 and CJ-510 exert similar affinity for factor Xa (K(i)=14 nM versus 8.8 nM) but CJ-504 was 70-fold more selective for factor Xa. This correlated with higher anti-metastatic efficacy (58.8% with CJ-504; 28.2% with CJ-510). Our results show that among the protease inhibitors employed that have affinities in the nanomolar range, the strategy of selectivity-optimization is superior to further improvement of affinity to significantly enhance anti-metastatic efficacy. This appreciation may be important for the future rational design of new anti-proteolytic agents for cancer therapy.
[Show abstract][Hide abstract] ABSTRACT: Dysregulated proteolysis is a hallmark of cancer. Malignant cells require a range of proteolytic activities to enable growth, survival, and expansion. Serine proteases of the S1 or trypsin-like family have well recognized roles in the maintenance of normal homeostasis as well as in the pathology of diseases such as cancer. Recently a rapidly expanding subgroup of S1 proteases has been recognized that are directly anchored to plasma membranes. These membrane anchored serine proteases are anchored either via a carboxy-terminal transmembrane domain (Type I), a carboxy terminal hydrophobic region that functions as a signal for membrane attachment via a glycosyl-phosphatidylinositol linkage (GPI-anchored), or via an amino terminal proximal transmembrane domain (Type II or TTSP). The TTSPs also encode multiple domains in their stem regions that may function in regulatory interactions. The serine protease catalytic domains of these enzymes show high homology but also possess features indicating unique substrate specificities. It is likely that the membrane anchored serine proteases have evolved to perform complex functions in the regulation of cellular signaling events at the plasma membrane and within the extracellular matrix. Disruption or mutation of several of the genes encoding these proteases are associated with disease. Many of the membrane anchored serine proteases show restricted tissue distribution in normal cells, but their expression is widely dysregulated during tumor growth and progression. Diagnostic or therapeutic targeting of the membrane anchored serine proteases has potential as promising new approaches for the treatment of cancer and other diseases.
Cancer and metastasis reviews 01/2003; 22(2-3):237-58. · 7.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The primary structure determination of the dimeric invertebrate alpha(2)-macroglobulin (alpha(2)M) from Limulus polyphemus has been completed by determining its sites of glycosylation and disulfide bridge pattern. Of seven potential glycosylation sites for N-linked glycosylation, six (Asn(275), Asn(307), Asn(866), Asn(896), Asn(1089), and Asn(1145)) carry common glucosamine-based carbohydrates groups, whereas one (Asn(80)) carries a carbohydrate chain containing both glucosamine and galactosamine. Nine disulfide bridges, which are homologues with bridges in human alpha(2)M, have been identified (Cys(228)-Cys(269), Cys(456)-Cys(580), Cys(612)-Cys(799), Cys(657)-Cys(707), Cys(849)-Cys(876), Cys(874)-Cys(910), Cys(946)-Cys(1328), Cys(1104)-Cys(1155), and Cys(1362)-Cys(1475)). In addition to these bridges, Limulus alpha(2)M contains three unique bridges that connect Cys(361) and Cys(382), Cys(1370) and Cys(1374), respectively, and Cys(719) in one subunit with the same residue in the other subunit of the dimer. The latter bridge forms the only interchain disulfide bridge in Limulus alpha(2)M. The location of this bridge within the bait region is discussed and compared with other alpha-macroglobulins. Several peptides identified in the course of determining the disulfide bridge pattern provided evidence for the existence of two forms of Limulus alpha(2)M. The two forms have a high degree of sequence identity, but they differ extensively in large parts of their bait regions suggesting that they have different inhibitory spectra. The two forms (Limulus alpha(2)M-1 and -2) are most likely present in an approximately 2:1 ratio in the hemolymph of each animal, and they can be partially separated on a Mono Q column at pH 7.4 by applying a shallow gradient of NaCl.
Journal of Biological Chemistry 12/2002; 277(46):43698-706. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The primary structure determination of the dimeric invertebrate α2-macroglobulin (α2M) from Limulus polyphemus has been completed by determining its sites of glycosylation and disulfide bridge pattern. Of seven potential glycosylation
sites forN-linked glycosylation, six (Asn275, Asn307, Asn866, Asn896, Asn1089, and Asn1145) carry common glucosamine-based carbohydrates groups, whereas one (Asn80) carries a carbohydrate chain containing both glucosamine and galactosamine. Nine disulfide bridges, which are homologues
with bridges in human α2M, have been identified (Cys228–Cys269, Cys456–Cys580, Cys612–Cys799, Cys657–Cys707, Cys849–Cys876, Cys874–Cys910, Cys946–Cys1328, Cys1104–Cys1155, and Cys1362–Cys1475). In addition to these bridges, Limulus α2M contains three unique bridges that connect Cys361 and Cys382, Cys1370 and Cys1374, respectively, and Cys719 in one subunit with the same residue in the other subunit of the dimer. The latter bridge forms the only interchain disulfide
bridge in Limulus α2M. The location of this bridge within the bait region is discussed and compared with other α-macroglobulins. Several peptides
identified in the course of determining the disulfide bridge pattern provided evidence for the existence of two forms of Limulusα2M. The two forms have a high degree of sequence identity, but they differ extensively in large parts of their bait regions
suggesting that they have different inhibitory spectra. The two forms (Limulus α2M-1 and -2) are most likely present in an ∼2:1 ratio in the hemolymph of each animal, and they can be partially separated
on a Mono Q column at pH 7.4 by applying a shallow gradient of NaCl.
Journal of Biological Chemistry 11/2002; 277(46):43698-43706. · 4.65 Impact Factor