Thrombin induces neoangiogenesis in the chick chorioallantoic membrane.

Most tumors have constitutively active tissue factor on their surface, capable of generating thrombin in the surrounding environment, and thrombosis is associated with cancer. Thrombin is known to induce a malignant phenotype by enhancing tissue adhesion and cell growth in vitro and in vivo in mice. Because tumors require angiogenesis for growth, we examined whether thrombin induces neoangiogenesis in a physiologically intact in vivo model. Thrombin (0.1 U mL-1) induced neoangiogenesis in the chick chorioallantoic membrane over a 24-72-h period by approximately 2-3-fold. This was inhibited by the potent thrombin inhibitor, hirudin and shown to have its mode of action by ligation of the thrombin protease-activated receptor, PAR-1. The thrombin receptor activation peptide, SFLLRNPNDKYEPF (200 microm) also enhanced neoangiogenesis c. 2-3-fold. Thrombin-induced neoangiogenesis was accompanied by the induction of vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) mRNA at 24-48 h (approximately 2-fold) as determined by semi-quantitative reverse transcriptase-polymerase chain reaction. Thrombin-induced neoangiogenesis was inhibited to baseline level by the specific angiogenesis receptor inhibitors KDR-Fc (vs. VEGF) and Tie-2-Fc (vs. Ang-1 and Ang-2), as well as the non-specific angiogenesis inhibitor thrombospondin-1. Thrombin-induced neoangiogenesis was also inhibited to baseline level by agents known to inhibit thrombin receptor signaling in other cells: G-coupled protein receptor inhibitor, pertussis toxin (40 pg per egg), protein kinase C inhibitor, bisindolylmaleimide (1 microm per egg), MAP kinase inhibitor, PD980598 (10 microm per egg) and PI3 kinase inhibitor, LY294002 (0.25 microm per egg). Thus angiogenesis is stimulated by thrombosis, which could help explain the enhancement of experimental tumorigenesis by thrombin.

Slow induction of RecA by DNA damage in Mycobacterium tuberculosis.

In mycobacteria, as in most bacterial species, the expression of RecA is induced by DNA damage. However, the authors show here that the kinetics of recA induction in Mycobacterium smegmatis and in Mycobacterium tuberculosis are quite different: whilst maximum expression in M. smegmatis occurred 3-6 h after addition of a DNA-damaging agent, incubation for 18-36 h was required to reach peak levels in M. tuberculosis. This is despite the fact that the M. tuberculosis promoter can be activated more rapidly when transferred to M. smegmatis. In addition, it is demonstrated that in both species the DNA is sufficiently damaged to give maximum induction within the first hour of incubation with mitomycin C. The difference in the induction kinetics of recA between the two species was mirrored by a difference in the levels of DNA-binding-competent LexA following DNA damage. A decrease in the ability of LexA to bind to the SOS box was readily detected by 2 h in M. smegmatis, whilst a decrease was not apparent until 18-24 h in M. tuberculosis and then only a very small decrease was observed.

Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo.

Evidence is provided that proteolytic cleavage of collagen type IV results in the exposure of a functionally important cryptic site hidden within its triple helical structure. Exposure of this cryptic site was associated with angiogenic, but not quiescent, blood vessels and was required for angiogenesis in vivo. Exposure of the HUIV26 epitope was associated with a loss of alpha1beta1 integrin binding and the gain of alphavbeta3 binding. A monoclonal antibody (HUIV26) directed to this site disrupts integrin-dependent endothelial cell interactions and potently inhibits angiogenesis and tumor growth. Together, these studies suggest a novel mechanism by which proteolysis contributes to angiogenesis by exposing hidden regulatory elements within matrix-immobilized collagen type IV.

Plasminogen activator inhibitor-1 regulates tumor growth and angiogenesis.

Elevated expression of plasminogen activator inhibitor-1 (PAI-1) in tumors is associated with a poor prognosis in many cancers. Reduced tumor growth and angiogenesis have also been reported in mice deficient in PAI-1. These results suggest that PAI-1 may be required for efficient angiogenesis and tumor growth. In the present study, we demonstrate that PAI-1 can both enhance and inhibit the growth of M21 human melanoma tumors in nude mice and that this appears to be due to PAI-1 regulation of angiogenesis. Quantitative analysis of angiogenesis in a Matrigel implant assay indicated that in PAI-1 null mice angiogenesis was reduced approximately 60% compared with wild-type mice, while in mice overexpressing PAI-1, angiogenesis was increased nearly 3-fold. Furthermore, addition of PAI-1 to implants in wild-type mice enhanced angiogenesis up to 3-fold at low concentrations but inhibited angiogenesis nearly completely at high concentrations. Together, these data demonstrate that PAI-1 is a potent regulator of angiogenesis and hence of tumor growth and suggest that understanding the mechanism of this activity may lead to the development of important new therapeutic agents for controlling pathologic angiogenesis.

Identification of some DNA damage-inducible genes of Mycobacterium tuberculosis: apparent lack of correlation with LexA binding.

The repair of DNA damage is expected to be particularly important to intracellular pathogens such as Mycobacterium tuberculosis, and so it is of interest to examine the response of M. tuberculosis to DNA damage. The expression of recA, a key component in DNA repair and recombination, is induced by DNA damage in M. tuberculosis. In this study, we have analyzed the expression following DNA damage in M. tuberculosis of a number of other genes which are DNA damage inducible in Escherichia coli. While many of these genes were also induced by DNA damage in M. tuberculosis, some were not. In addition, one gene (ruvC) which is not induced by DNA damage in E. coli was induced in M. tuberculosis, a result likely linked to its different transcriptional arrangement in M. tuberculosis. We also searched the sequences upstream of the genes being studied for the mycobacterial SOS box (the binding site for LexA) and assessed LexA binding to potential sites identified. LexA is the repressor protein responsible for regulating expression of these SOS genes in E. coli. However, two of the genes which were DNA damage inducible in M. tuberculosis did not have identifiable sites to which LexA bound. The absence of binding sites for LexA upstream of these genes was confirmed by analysis of LexA binding to overlapping DNA fragments covering a region from 500 bp upstream of the coding sequence to 100 bp within it. Therefore, it appears most likely that an alternative mechanism of gene regulation in response to DNA damage exists in M. tuberculosis.

Inhibition of angiogenesis in vivo by plasminogen activator inhibitor-1.

The process of angiogenesis is important in both normal and pathologic physiology. However, the mechanisms whereby factors such as basic fibroblast growth factor promote the formation of new blood vessels are not known. In the present study, we demonstrate that exogenously added plasminogen activator inhibitor-1 (PAI-1) at therapeutic concentrations is a potent inhibitor of basic fibroblast growth factor-induced angiogenesis in the chicken chorioallantoic membrane. By using specific PAI-1 mutants with either their vitronectin binding or proteinase inhibitor activities ablated, we show that the inhibition of angiogenesis appears to occur via two distinct but apparently overlapping pathways. The first is dependent on PAI-1 inhibition of proteinase activity, most likely chicken plasmin, while the second is independent of PAI-1's anti-proteinase activity and instead appears to act through PAI-1 binding to vitronectin. Together, these data suggest that PAI-1 may be an important factor regulating angiogenesis in vivo.

The chick embryo metastasis model.

The growth and dissemination of malignant tumors continues to have a devastating impact on people throughout the United States and the rest of the world. In fact, it is estimated that well over a half a million new cases of cancer will be diagnosed per year (1). The most commonly used clinical approaches to treat cancer include surgical removal of the primary tumor, chemotherapy, and radiation, all of which have varying degrees of success. Importantly, a major obstacle contributing to the failure of treatment in many cases involves the metastatic dissemination of tumor cells from the primary tumor mass to distant sites. While some progress has been achieved in understanding the complex biochemical and molecular mechanisms regulating tumor invasion, much remains to be learned.

Generation of monoclonal antibodies to cryptic collagen sites by using subtractive immunization.

The extracellular matrix (ECM) plays a fundamental role in the regulation of normal and pathological processes. The most abundantly expressed component found in the ECM is collagen. Triple helical collagen is known to be highly resistant to proteolytic cleavage except by members of the matrix metalloproteinase (MMP) family of enzymes. To date little is known concerning the biochemical consequences of collagen metabolism on human diseases. This is due in part to the lack of specific reagents that can distinguish between proteolyzed and triple helical forms of collagen. Here we used the technique of Subtractive Immunization (SI) to generate two unique monoclonal antibodies (MAbs HUIV26 and HUI77) that react with denatured and proteolyzed forms of collagen, but show little if any reaction with triple helical collagen. Importantly, HUIV26 and HUI77 react with cryptic sites within the ECM of human melanoma tumors, demonstrating their utility for immunohistochemical analysis in vivo. Thus, the generation of these novel MAbs not only identify specific cryptic epitopes within triple helical collagen, but also provide important new reagents for studying the roles of collagen remodeling in normal as well as pathological processes.

Contact with fibrillar collagen inhibits melanoma cell proliferation by up-regulating p27KIP1.

It is known that the extracellular matrix regulates normal cell proliferation, and it is assumed that anchorage-independent malignant cells escape this regulatory function. Here we demonstrate that human M24met melanoma cells remain responsive to growth regulatory signals that result from contact with type I collagen and that the effect on proliferation depends on the physical structure of the collagen. On polymerized fibrillar collagen, M24met cells are growth arrested at the G(1)/S checkpoint and maintain high levels of p27(KIP1) mRNA and protein. In contrast, on nonfibrillar (denatured) collagen, the cells enter the cell cycle, and p27(KIP1) is down-regulated. These growth regulatory effects involve contact between type I collagen and the collagen-binding integrin alpha(2)beta(1), which appears restricted in the presence of fibrillar collagen. Thus melanoma cells remain sensitive to negative growth regulatory signals originating from fibrillar collagen, and the proteolytic degradation of fibrils is a mechanism allowing tumor cells to escape these restrictive signals.

New functions for non-collagenous domains of human collagen type IV. Novel integrin ligands inhibiting angiogenesis and tumor growth in vivo.

Collagen type IV is a major component of the basal lamina of blood vessels. Six genetically distinct collagen type IV chains have been identified and are distributed in a tissue-specific manner. Here we define a novel function for soluble non-collagenous (NC1) domains of the alpha2(IV), alpha3(IV), and alpha6(IV) chains of human collagen type IV in the regulation of angiogenesis and tumor growth. These NC1 domains were shown to regulate endothelial cell adhesion and migration by distinct alpha(v) and beta(1) integrin-dependent mechanisms. Systemic administration of recombinant alpha2(IV), alpha3(IV), and alpha6(IV) NC1 domains potently inhibit angiogenesis and tumor growth, whereas alpha1(IV), alpha4(IV), and alpha5(IV) showed little if any effect. These findings suggest that specific NC1 domains of collagen type IV may represent an important new class of angiogenesis inhibitors.

Contortrostatin, a homodimeric disintegrin, binds to integrin alphavbeta5.

Contortrostatin is a homodimeric disintegrin from snake venom. We have shown that contortrostatin binds to integrins alphaIIbbeta3, alpha5beta1, and alphavbeta3. We now use several criteria to demonstrate the binding of contortrostatin to alphavbeta5. First, incubation of T24 cells, which express alphavbeta3 and alphavbeta5, with antibody against alphavbeta3 failed to completely inhibit adhesion of cells to vitronectin. However, pretreatment of the cells with contortrostatin or the combination of antibodies against alphavbeta3 and alphavbeta5 completely blocked adhesion to vitronectin. By contrast, either anti-alphavbeta5 alone or contortrostatin blocked adhesion of an alphavbeta3-negative T24 subline. Second, contortrostatin as well as anti-alphavbeta5 inhibits invasion of OVCAR-5, which express only alphavbeta5. Third, contortrostatin binds to purified alphavbeta5 in a saturable manner. Finally, radioligand binding assays yielded a K(d) value of 24 nM for [(125)I]contortrostatin binding to alphavbeta5. This investigation identifies alphavbeta5 as a binding site for contortrostatin. Blockage of alphavbeta5 by contortrostatin inhibits alphavbeta5-mediated adhesion and invasion.

Eukaryotic expression cloning with an antimetastatic monoclonal antibody identifies a tetraspanin (PETA-3/CD151) as an effector of human tumor cell migration and metastasis.

A monoclonal antibody (mAb), 50-6, generated by subtractive immunization, was found to specifically inhibit in vivo metastasis of a human epidermoid carcinoma cell line, HEp-3. The cDNA of the cognate antigen of mAb 50-6 was isolated by a modified eukaryotic expression cloning protocol from a HEp-3 library. Sequence analysis identified the antigen as PETA-3/CD151, a recently described member of the tetraspanin family of proteins. The cloned antigen was also recognized by a previously described antimetastatic antibody, mAb 1A5. Inhibition of HEp-3 metastasis by the mAbs could not be attributed to any effect of the antibodies on tumor cell growth in vitro or in vivo. Rather, the antibodies appeared to inhibit an early step in the formation of metastatic foci. In a chemotaxis assay, HEp-3 migration was blocked by both antibodies. HeLa cells transfected with and overexpressing PETA-3/CD151 were more migratory than control transfectants expressing little CD151. The increase in HeLa migration was inhibitable by both mAb 50-6 and mAb 1A5. PETA-3 appears not to be involved in cell attachment because adhesion did not correlate with levels of PETA-3 expression and was unaffected by mAb 50-6 or mAb 1A5. The ability of PETA-3 to mediate cell migration suggests a mechanism by which this protein may influence metastasis. These data identify PETA-3/CD151 as the first member of the tetraspanin family to be linked as a positive effector of metastasis.

Integrin alpha(v)beta3 promotes M21 melanoma growth in human skin by regulating tumor cell survival.

Growth and dissemination of malignant melanoma has a profound impact on our population, and little is known concerning the mechanisms controlling this disease in humans. Evidence is provided that integrin alpha(v)beta3 plays a critical role in M21 melanoma tumor survival within human skin by a mechanism independent of its known role in angiogenesis. Antagonists of alpha(v)beta3 blocked melanoma growth by inducing tumor apoptosis. Moreover, M21 melanoma cell interactions with denatured collagen, a known ligand for alpha(v)beta3, caused a 5-fold increase in the relative Bcl-2:Bax ratio, an event thought to promote cell survival. Importantly, denatured collagen colocalized with alpha(v)beta3-expressing melanoma cells in human tumor biopsies, suggesting that alpha(v)beta3 interaction with denatured collagen may play a critical role in melanoma tumor survival in vivo.

Vascular integrin alpha(v)beta3: a new prognostic indicator in breast cancer.

Blood vessel density is a prognostic indicator of multiple tumor types. Recently, it has been established that tumor-associated blood vessels express elevated levels of integrin alpha(v)beta3. In fact, there is evidence that integrin alpha(v)beta3 identifies the most proliferative endothelial cells within human breast carcinomas. Therefore, we evaluated breast cancer tissue in terms of both blood vessel density and alpha(v)beta3 expression. We found that the antibody LM609 to integrin alpha(v)beta3 preferentially stains the blood vessels of small caliber. Furthermore, comparative studies between LM609 and anti-CD31 antibodies on normal breast indicate that very low and weak expression of integrin alpha(v)beta3 was found on vessels within normal tissue, whereas CD31 antigen was expressed in almost all vasculature. Indeed, expression of integrin alpha(v)beta3 was significantly higher in tumors of patients with metastasis than in those without metastasis. In a series of 197 consecutive patients with invasive breast cancer and long follow-up, vascular expression of integrin alpha(v)beta3 in tumor vascular "hot spots" was found to be the most significant prognostic factor predictive of relapse-free survival in both node-negative and node-positive patients. These findings support the contention that angiogenesis plays a critical role in breast cancer progression and suggest that integrin alpha(v)beta3 is an endothelial cell marker with significant prognostic value and potential usefulness as a target for specific antiangiogenic therapy.

CAS/Crk coupling serves as a "molecular switch" for induction of cell migration.

Carcinoma cells selected for their ability to migrate in vitro showed enhanced invasive properties in vivo. Associated with this induction of migration was the anchorage-dependent phosphorylation of p130CAS (Crk-associated substrate), leading to its coupling to the adaptor protein c-CrkII (Crk). In fact, expression of CAS or its adaptor protein partner Crk was sufficient to promote cell migration, and this depended on CAS tyrosine phosphorylation facilitating an SH2-mediated complex with Crk. Cytokine-stimulated cell migration was blocked by CAS lacking the Crk binding site or Crk containing a mutant SH2 domain. This migration response was characterized by CAS/Crk localization to membrane ruffles and blocked by the dominant-negative GTPase, Rac, but not Ras. Thus, CAS/Crk assembly serves as a "molecular switch" for the induction of cell migration and appears to contribute to the invasive property of tumors.

Disruption of angiogenesis by PEX, a noncatalytic metalloproteinase fragment with integrin binding activity.

Angiogenesis depends on both cell adhesion and proteolytic mechanisms. In fact, matrix metalloproteinase 2 (MMP-2) and integrin alphavbeta3 are functionally associated on the surface of angiogenic blood vessels. A fragment of MMP-2, which comprises the C-terminal hemopexin-like domain, termed PEX, prevents this enzyme binding to alphavbeta3 and blocks cell surface collagenolytic activity. PEX blocks MMP-2 activity on the chick chorioallantoic membrane where it disrupts angiogenesis and tumor growth. Importantly, a naturally occurring form of PEX can be detected in vivo in conjunction with alphavbeta3 expression in tumors and during developmental retinal neovascularization. Levels of PEX in these vascularized tissues suggest that it interacts with endothelial cell alphavbeta3 where it serves as a natural inhibitor of MMP-2 activity, thereby regulating the invasive behavior of new blood vessels.

Insulin-like growth factor receptor cooperates with integrin alpha v beta 5 to promote tumor cell dissemination in vivo.

Tumor cell interactions with adhesion proteins and growth factors likely contribute to the metastatic cascade. Evidence is provided that insulin or insulin-like growth factor-mediated signals cooperate with the commonly expressed integrin alpha v beta 5 to promote spontaneous pulmonary metastasis of multiple tumor cell types in both the chick embryo and severe combined immune deficiency mouse/human chimeric models. Expression of alpha v beta 5 in tumor cells promoted their adhesion to vitronectin in vitro. However, cell motility required cytokine stimulation, which caused redistribution of alpha-actinin to membrane-adhesive sites containing alpha v beta 5. Significantly, ligation of alpha v beta 5 and cytokine receptors were both required for spontaneous pulmonary metastasis of multiple tumor types even though it was not necessary for primary tumor growth. Thus, tumor cell metastasis can be regulated by a functional cooperation between cytokine signaling events and the adhesion receptor alpha v beta 5 in a manner independent of tumor cell growth. These findings provide evidence that integrin ligation, in conjunction with cytokine activation, plays an important role in the dissemination of malignant tumor cells.

A single immunoglobulin-like domain of the human neural cell adhesion molecule L1 supports adhesion by multiple vascular and platelet integrins.

The neural cell adhesion molecule L1 has been shown to function as a homophilic ligand in a variety of dynamic neurological processes. Here we demonstrate that the sixth immunoglobulin-like domain of human L1 (L1-Ig6) can function as a heterophilic ligand for multiple members of the integrin superfamily including alphavbeta3, alphavbeta1, alpha5beta1, and alphaIIbbeta3. The interaction between L1-Ig6 and alphaIIbbeta3 was found to support the rapid attachment of activated human platelets, whereas a corresponding interaction with alphavbeta3 and alphavbeta1 supported the adhesion of umbilical vein endothelial cells. Mutation of the single Arg-Gly-Asp (RGD) motif in human L1-Ig6 effectively abrogated binding by the aforementioned integrins. A L1 peptide containing this RGD motif and corresponding flanking amino acids (PSITWRGDGRDLQEL) effectively blocked L1 integrin interactions and, as an immobilized ligand, supported adhesion via alphavbeta3, alphavbeta1, alpha5beta1, and alphaIIbbeta3. Whereas beta3 integrin binding to L1-Ig6 was evident in the presence of either Ca2+, Mg2+, or Mn2+, a corresponding interaction with the beta1 integrins was only observed in the presence of Mn2+. Furthermore, such Mn2+-dependent binding by alpha5beta1 and alphavbeta1 was significantly inhibited by exogenous Ca2+. Our findings suggest that physiological levels of calcium will impose a hierarchy of integrin binding to L1 such that alphavbeta3 or active alphaIIbbeta3 > alphavbeta1 > alpha5beta1. Given that L1 can interact with multiple vascular or platelet integrins it is significant that we also present evidence for de novo L1 expression on blood vessels associated with certain neoplastic or inflammatory diseases. Together these findings suggest an expanded and novel role for L1 in vascular and thrombogenic processes.