Role of the SIKVAV site of laminin in promotion of angiogenesis and tumor growth: an in vivo Matrigel model.

BACKGROUND: Angiogenesis (vascularization) has a critical role in tumor growth and metastasis, and peptides containing the SIKVAV amino acid sequence (Ser-Ile-Lys-Val-Ala-Val) have been shown to stimulate many angiogenic activities in vitro. The use of model systems to identify agents that stimulate or inhibit angiogenesis may lead to the development of new antitumor strategies. PURPOSE: Our purpose was to use an in vivo murine model system to study the angiogenic activity of a synthetic peptide derived from the laminin A protein chain and containing the SIKVAV amino acid sequence. We also examined the ability of the peptide to enhance tumor growth in vivo. METHODS: The SIKVAV-containing peptide was mixed with Matrigel, a reconstituted basement membrane extract used to assay stimulation of angiogenesis. The mixture was subcutaneously injected into C57BL/6 mice. At various times after injection, the Matrigel plug was excised, and angiogenic activity was assessed by histologic examination and immunohistochemical staining with an antibody to the von Willebrand factor (vWF), an endothelium-specific antigen. In other experiments, the mixture of peptide and Matrigel was co-injected with B16F10 murine melanoma cells into C57BL/6 mice, and the resultant tumors were assessed for size and vascularization. RESULTS: When co-injected with Matrigel at doses as low as 10 micrograms, the SIKVAV-containing peptide stimulated angiogenesis fourfold greater than that seen in controls, and maximum angiogenic activity was observed 2 weeks after injection. This peptide was angiogenic in a dose-dependent manner up to a 100-micrograms dose. When co-injected with Matrigel and B16F10 melanoma cells, the peptide enhanced tumor growth by approximately 2.5-fold, and tumor vascularization was significantly increased (P = .027) over that observed after injection with melanoma cells and Matrigel alone. CONCLUSIONS: These data demonstrate that the laminin-derived SIKVAV-containing peptide is angiogenic in a new in vivo model system and can enhance tumor vascularization and growth.

Enhanced tumor growth of both primary and established human and murine tumor cells in athymic mice after coinjection with Matrigel.

Previously we found that the reconstituted basement membrane matrix Matrigel, when premixed with human small-cell lung carcinoma cells and injected subcutaneously into athymic mice, permitted tumor growth, whereas cells injected in the absence of Matrigel did not form tumors. In the present study, we examined additional cell types and determined some of the underlying mechanisms involved in the promotion of tumor formation by Matrigel. The tumor cell lines that we studied included transformed mouse Englebreth-Holm-Swarm tumor cells (T-EHS), human submandibular carcinoma A253 cells, mouse melanoma B16F10 cells, human epidermoid carcinoma KB cells, and human primary renal cell carcinoma cells. When coinjected subcutaneously with Matrigel, these cell lines formed rapidly proliferating tumors. Primary biopsy specimens of human colon carcinoma, when dispersed and coinjected with Matrigel, also formed tumors. Only A253, KB, and B16F10 cells formed small tumors in the absence of Martrigel, but a fivefold to tenfold increase in tumor size was observed in the presence of Matrigel. These data demonstrate a useful method for improving the growth of human tumors in athymic mice.

Melanoma cells selected for adhesion to laminin peptides have different malignant properties.

Laminin is an important promoter of cell-matrix interactions. A number of active laminin domains have been defined by use of synthetic peptides. The Tyr-Ile-Gly-Ser-Arg (YIGSR) sequence on the B1 chain in laminin can decrease tumor growth and metastasis, whereas another sequence containing Ser-Ile-Lys-Val-Ala-Val (SIKVAV) on the A chain can increase tumor growth and metastasis. Here, we selected B16-F10 melanoma cells by adherence or nonadherence to either YIGSR- or SIKVAV-coated dishes and established 3 B16-F10 variants: YIGSR-adherent cells (Y+), YIGSR-nonadherent cells (Y-), and SIKVAV-nonadherent cells (S-). SIKVAV-adherent cells were not selected because most of F10 cells attached to the SIKVAV-coated dish. These cell lines proliferated at the same rate as the parent F10 cells and attached equally to laminin, collagen IV, and fibronectin. Y+ cells produced rapidly growing tumors after s.c. injection and twice as many lung colonies as the parental F10 cells after i.v. injection. In contrast, Y- cells produced more slowly growing tumors after s.c. injection and produced one-third of the lung colonies relative to the parent cells after i.v. injection. S- cells produced slowly growing tumors after s.c. injection and yielded similar numbers but smaller colonies in the lung than the parental B16-F10 cells after i.v. injection. These data suggest that interactions of melanoma cells with the YIGSR site on laminin are probably important for both colony formation in a target organ (lung) and subsequent tumor growth, while the SIKVAV-containing site on laminin may be more important for tumor growth.

LZIP-1 and LZIP-2: two novel members of the bZIP family.

A large family of bZIP proteins, containing a basic DNA-binding domain and a leucine zipper, have been described that recognize the CRE and AP-1 elements. Here, we have identified two new members, designated LZIP-1 and LZIP-2. The murine cDNA for LZIP-1 coded for a 379-amino-acid (aa) residue protein containing several distinct domains, including a Ser-rich region, a basic DNA-binding region, and an unusually long leucine zipper. A second form, LZIP-2, contained an additional 25 aa in the N-terminal region. Western immunoblotting revealed that antibody raised against part of recombinant LZIP-1 detected both forms in a variety of tissues. Gel mobility shift assays demonstrated that the recombinant protein possessed specific DNA-binding activity for both the CRE AP-1 sites. The present identification of two more ubiquitous members of the bZIP family emphasizes the complex nature of transcription factor interactions at the CRE and AP-1 sites.

Localization of a laminin-binding protein in brain.

A 110,000 mol.wt laminin-binding protein from newborn mouse brain recognizes a neurite promoting laminin A chain site and is related to the beta-amyloid precursor protein. In the present study, we examined the expression of 110,000 mol.wt laminin-binding protein in brains of adult mice, rats, and non-human primates. Essentially identical immunoreactivities were observed across species with distinct staining of cortical pyramidal neurons with apical dendrites, cerebellar basket cell axons, hippocampal mossy fibers, and fine labeling of processes throughout the brain. Colocalization of immunoreactivities to 110,000 mol.wt laminin-binding protein and to laminin in neurons of the adult rat brain was observed. Electron microscopy demonstrated that 110,000 mol.wt laminin-binding protein-like immunoreactivity is intracellular and is possibly associated with the neuronal cytoskeleton. Western blot analysis revealed that anti-110,000 mol.wt laminin-binding protein also recognizes a 140,000 mol.wt protein in the pellet, in addition to the 110,000 mol.wt protein in the Triton soluble extract. Antibody fractions specific to the two reactive protein species (110,000 mol.wt and 140,000 mol.wt) exhibited cross-reactivity on immunoblots and revealed similar immunohistochemical staining in adult brain. Results suggest a significant interaction between laminin-like molecules and 110,000 mol.wt laminin-binding protein-like molecules in normal brain function, in response to CNS injury and possibly in the pathogenesis of Alzheimer's disease.

Angiogenesis as a component of epithelial-mesenchymal interactions.

Here we review the role of angiogenesis as it pertains to the interactions between the epithelium and the mesenchyme, especially during tumor growth and metastasis. We illustrate and discuss several models of angiogenesis including endothelial tube formation on Matrigel. Finally, we examine angiogenic factors using the Matrigel model and investigate several other matrix molecules for their importance in angiogenesis and epithelial/stromal interactions.

The role of basement membrane in angiogenesis and tumor growth.

Expansion of the tumor-cell mass is dependent on both the degree of tumor vascularization and the rate of angiogenesis. Blood vessel growth is controlled, in part, by the matrix surrounding it, in particular, the basement membrane underlying the endothelium. Here we illustrate that laminin, a major component of basement membrane, has several biologically active sites that can bind to endothelial and tumor cells, and have the ability to regulate angiogenesis and tumor growth. We show that synthetic peptides at two sites in the laminin B1 chain (the RGD and YIGSR sequences) inhibit angiogenesis, whereas a third site in the A chain, designated SIK-VAV, stimulates vessel and tumor cell growth. By developing strategies that promote or inhibit the activities of these sites in laminin, we may obtain methods to inhibit angiogenesis and subsequent tumor growth.

Transfection of murine P19S18 embryonal carcinoma cells with the oncogene neu induces an epithelioid phenotype.

An embryonal carcinoma cell line, P19S18, was transfected with the rat oncogene neu to investigate the function of its protein product, p185*, in a multipotential cellular environment. Levels of message for p185* were determined by in situ hybridization analysis and two highly expressing clones, PnnA and PnnB, were isolated. As demonstrated by indirect immunofluorescence and immunoprecipitation, these neu-transfected cells synthesized a full length rat p185*. The transfectants do not resemble typical embryonal carcinoma cells either before or after differentiation is induced by retinoic acid treatment. They are much larger, flatter, "epithelioid" cells. These cells have lost the expression of stage specific embryonic antigen-1 (SSEA-1), but do synthesize and assemble the basement membrane components laminin and fibronectin. These results suggest that expression of the neu oncogene in a multipotential cell line may induce the synthesis of proteins indicative of an epithelioid phenotype due to the presence of p185*.

Effect of Matrigel and laminin peptide YIGSR on tumor growth and metastasis.

Basement membrane has a variety of effects on tumor cells and promotes malignant behavior. Tumor cell growth is enhanced both in vitro and in vivo in mice in the presence of basement membrane. This has led to the ability to grow various tumors including human biopsy specimens in nude mice. Furthermore, low cell numbers can be used when coinjected with Matrigel, a basement membrane extract. The basement membrane glycoprotein laminin is important in promoting invasive behavior and the level of a 32/67 kDa laminin receptor has been shown to correlate with malignancy. A sequence of five amino acids, tyrosine-isoleucine-glycine-serine-arginine (YIGSR) has been shown to recognize this receptor and to reduce experimental metastases (tail vein injection resulting in colonization of the lung) and subcutaneous tumor growth. This peptide is active in both models either when coinjected or when daily intraperitoneal injections are given after tumor growth has initiated. YIGSR does not effect cell arrest but does inhibit angiogenesis which is necessary for tumor growth. YIGSR also appears to have an additional antitumor effect via its interaction with a specific receptor. YIGSR-adherent cells established after 30 successive selections on YIGSR-coated dishes in vitro formed more lung colonies after intravenous injection and larger tumors after subcutaneous injection than the parent B16F10 melanoma cells. The YIGSR-non-adherent cells formed fewer lung colonies and smaller subcutaneous tumors. These data demonstrate the importance of laminin-tumor cell interactions in malignancies and suggest that a short sequence from laminin has multiple effects in reducing tumor growth and spread.(ABSTRACT TRUNCATED AT 250 WORDS)

A 110-kD nuclear shuttling protein, nucleolin, binds to the neurite-promoting IKVAV site of laminin-1.

The basement membrane protein laminin and the IKVAV-containing sequence from the laminin alpha 1 chain have been found to promote the differentiation of primary neurons and a variety of neural cell lines. We previously reported that a 110-kd IKVAV-binding protein (LBP110) isolated from brain appears to be a member of the beta-amyloid precursor protein (APP) family by immunologic and functional studies, which showed that LBP110/APP is also important in neurite outgrowth (Kibbey et al.: Proc Natl Acad Sci USA 90:10150-10153, 1993). In the preparation of this binding protein, a contaminating IKVAV-binding protein of identical molecular weight, nucleolin, was also identified. Here we have studied the relationship between these binding proteins. We find that nucleolin binds specifically to the IKVAV sequence independently of LBP110/ApP. We have also demonstrated significant levels of nucleolin in mature brain and in differentiating neural cells, suggesting that nucleolin functions not only in cell proliferation and in ribosome biogenesis as was previously reported, but also in the differentiation and maintenance of neural tissue. Our identification of cytoplasmic and cell-surface nucleolin, an IKVAV-binding protein, suggests that this protein may function in signalling by extra-cellular matrix.

Laminin SIKVAV peptide induction of monocyte/macrophage prostaglandin E2 and matrix metalloproteinases.

The laminin-derived synthetic peptide containing the SIKVAV (Ser-Ile-Lys-Val-Ala-Val) amino acid sequence has been previously shown to regulate tumor invasion, metastasis, and angiogenesis. Here, we demonstrate that this peptide also modulates human monocyte responses. Moreover, the monocytic responses elicited by this peptide are influenced by the culture conditions. When elutriated monocytes were cultured on SIKVAV substrate or in suspension with this peptide, the synthesis of prostaglandin E2, interstitial collagenase, and gelatinase B was induced and was further enhanced in the presence of concanavalin A (ConA). However, when monocytes were adhered before adding soluble SIKVAV, the peptide alone failed to induce the production of prostaglandin E2 or matrix metalloproteinases. If adherent monocytes were exposed to SIKVAV in the presence of ConA, this peptide enhanced the ConA induced production of these mediators. In contrast to SIKVAV, the intact laminin molecule failed to influence these monocyte responses. This is the first demonstration that a laminin derived peptide is capable of inducing or enhancing monocyte inflammatory responses that may influence a number of biological activities such as wound healing or excessive connective tissue destruction associated with chronic inflammation.

Laminin SIKVAV peptide-induced angiogenesis in vivo is potentiated by neutrophils.

Angiogenesis has been investigated in vivo using subcutaneously injected reconstituted basement membrane (Matrigel) supplemented with angiogenic factors. Previously we found that the laminin-derived synthetic peptide containing SIKVAV (ser-ile-lys-val-ala-val) promoted angiogenesis in vivo. In parallel studies, it was observed that new vessel formation in response to this peptide occurred several days after basic fibroblast growth factor-induced angiogenesis. Since this delay suggested that SIKVAV-induced angiogenesis may be secondary to other events, we investigated here earlier time points to determine if both indirect and direct mechanisms of angiogenesis are involved. We found that neutrophils are continuously recruited to the SIKVAV-containing plugs between 4 hours to 3 days following the initial injection. By day 7, columns of endothelial cells begin to migrate into the plug and form small blood vessels. In contrast, neutropenic mice had a 62% reduction in SIKVAV-induced angiogenesis when compared to control mice. Freshly isolated neutrophils also degraded laminin, the major component of the basement membrane Matrigel. These cells also produced factors in response to SIKVAV peptide which induced proliferation of human umbilical vein endothelial cells relative to a control peptide. In vitro experiments utilizing human neutrophils demonstrated that these cells migrate to the SIKVAV peptide and possess a specific cell surface SIKVAV binding protein of approximately 56 kD. These data suggest that neutrophils are induced to migrate to the Matrigel plugs, at least in part, by SIKVAV peptide, where they may release their own angiogenic factors and degrade the matrix, thus physically facilitating cell migration and liberating additional angiogenic matrix fragments and/or cytokines.

110/140 laminin-binding protein immunoreactivity in spinal dorsal root ganglia: a capsaicin-insensitive reduction induced by constriction injury of the sciatic nerve in rats.

The distribution of 110/140 laminin-binding protein (110/140 LBP) in the spinal dorsal root ganglia (DRG) and its regulation by partial constriction of the sciatic nerve was studied in adult rats. The cross-sectional area of neurons with 110/140 LBP-immunoreactivity (-I) showed an approximately normal frequency distribution. The 110/140 LBP-I was observed in neuronal cell bodies exclusive of the nucleus. Following sciatic nerve constriction, the 110/140 LBP-I was downregulated in the ipsilateral L4-5 DRG. DRG neurons with a cross-sectional area > or = 1600 microns 2 were preferentially affected. Neonatal capsaicin-treatment, a procedure that selectively destroys a subpopulation of DRG neurons with fine unmyelinated axons, had no effect on the reduction of 110/140 LBP in the DRG induced by sciatic nerve constriction. Western immunoblot analysis confirmed a reduction of 110/140 LBP on the side ipsilateral to the constriction. These results demonstrate a LBP within primary sensory neurons and its suppression by peripheral nerve injury. The data support a role for LBP in the adult nervous system.

Role of laminin-1 and TGF-beta 3 in acinar differentiation of a human submandibular gland cell line (HSG).

Previous studies show that culturing an immortalized human submandibular gland cell line (HSG) on Matrigel, a basement membrane extract, induces cytodifferentiation. We have further defined this model system and identified factors involved in HSG cell acinar development and cyto-differentiation. Acinar development is marked by cell migration into multi-cellular spherical structures, cell proliferation and apoptosis of the centrally localized cells. In addition, functional differentiation was determined by indirect immunofluorescence and immunoblot analysis for cystatin, a salivary gland acinar cell-specific protein found to be produced by differentiated HSG cells. Matrigel contains multiple extracellular matrix proteins, however, laminin-1 was identified as the major matrix component that induced HSG cell acinar development and cytodifferentiation. Antibodies against specific components of Matrigel and against cell surface adhesion molecules were added to cells in culture to identify components important for HSG cell acinar differentiation. Immunostaining of HSG cell acini identified TGF-beta 2 and beta 3 as the predominant isoforms within the cells. Neutralizing antibodies directed against TGF-beta 3 significantly decreased (P < or = 0.0002) the size of acini formed. These results indicate that multiple components, including laminin-1 and TGF-beta 3, contribute to HSG cell acinar development. This model system will be useful to study acinar differentiation and salivary gland-specific protein expression in vitro.

Basement membrane and the SIKVAV laminin-derived peptide promote tumor growth and metastases.

Laminin, the major glycoprotein component of basement membrane, promotes the malignant phenotype. Cells which are adherent to laminin are more malignant than the non-adherent cells and in certain tumor cells, the number of laminin receptors is positively correlated with malignancy. Laminin also increases collagenase IV activity, an enzyme demonstrated to be critical for tumor spread. A site on laminin, containing the amino acid sequence SIKVAV, has been identified which when injected intravenously with B16F10 melanoma cells, causes an increase in the number of colonies on the surface of the lungs. This peptide does not affect tumor cell arrest in the vasculature or the immune system. It does promote angiogenesis in various in vitro and in vivo models, thereby facilitating tumor cell survival. When a complex mixture of laminin-enriched basement membrane components (Matrigel) is coinjected with tumor cells subcutaneously, tumor incidence and growth increases. Various tumor cell lines and primary isolates, which previously could not form tumors in mice, can be induced to grow rapidly in the presence of Matrigel. Slowly growing tumors or arrested tumors can also be induced to grow more quickly with additional injections of Matrigel. When an SIKVAV-containing synthetic peptide is coinjected with B16F10 tumor cells and Matrigel subcutaneously in mice, larger tumors are formed than that observed with either Matrigel or cells alone. Such studies define the role of laminin in tumor growth and spread and generate new models for studying therapeutic agents. Of particular interest is the ability to grow primary isolates which generally do not grow in mice.

The alpha1 subunit of laminin-1 promotes the development of neurons by interacting with LBP110 expressed by neural crest-derived cells immunoselected from the fetal mouse gut.

A plasmalemmal protein, LBP110, which binds to the alpha1 chain of laminin-1, is acquired by the neural crest-derived precursors of enteric neurons after they colonize the gut. We tested the hypothesis that laminin-1 interacts with LBP110 to promote enteric neuronal development. The effects of laminin-1 on neuronal development were studied in cultures of cells immunoselected from fetal mouse gut (E14-15) with antibodies to LBP110 or p75NTR, a marker for enteric crest-derived cells. No matter which antibody was used, the development of cells expressing neuronal markers was increased three- to fourfold by culturing the cells on a laminin-1-containing substrate. To determine whether this effect of laminin-1 is due to the selective adherence of a neurocompetent subset of precursors, immunoselected cells were permitted to preadhere to poly-D-lysine. Addition of soluble laminin-1 24 h later promoted neuronal but not glial development. The laminin-1-induced increment in neuronal development was abolished both by a peptide containing the sequence of the LBP110-binding domain, IKVAV, and by antibodies to laminin alpha1 that recognize the IKVAV domain. Neither reagent affected the total number of cells. In contrast, the response to laminin-1 was not affected by control peptides, preimmune sera, or antibodies to laminin beta1. Laminin-1 transiently induced the expression of nuclear Fos immunoreactivity; this action was blocked specifically by the IKVAV peptide. These data are consistent with the hypothesis that LBP110 interacts with the IKVAV domain of laminin alpha1 to promote the differentiation of neurons from enteric crest-derived precursors.

Human neoplastic submandibular intercalated duct cells express an acinar phenotype when cultured on a basement membrane matrix.

Culture of the human neoplastic submandibular gland intercalated duct cell line, HSG, on the basement membrane extract Matrigel induces dramatic morphologic changes and cytodifferentiation. Transmission electron microscopy demonstrated an acinar cell phenotype with polarized cells containing a well-developed Golgi apparatus, multiple microvilli-like projections from the apical surfaces into a lumenal-like area, and numerous granule-like organelles. Amylase, an acinar cell marker, was detected by both immunocytochemical and Northern blot analyses. A 50% reduction in [3H]thymidine incorporation by cells cultured on Matrigel, as compared to cells cultured on tissue culture plates, confirmed the differentiated phenotype of the cells. Multiple components of Matrigel appear to contribute to the morphologic differentiation of the HSG cells since antibodies to both laminin and collagen IV, as well as the laminin-derived bioactive peptide containing SIKVAV, have potent inhibitory effects on HSG cell organization on Matrigel. Collectively, these data indicate that culture of HSG cells on Matrigel is a useful model to study salivary gland acinar development.

Glandular-like morphogenesis of the human submandibular tumor cell line A253 on basement membrane components.

We have studied the interaction of a human tumor cell line, A253, derived from a submandibular gland carcinoma with a differentiation promoting reconstituted basement membrane extract, Matrigel. When cultured on plastic, these cells maintain a flat, cobblestone, epithelial morphology. On Matrigel, A253 cells initially form a honeycomb network of cords of cells which subsequently thickens. With time, these cords of cells become discontinuous and blunted, whereupon multilobular clusters of cells develop. These clusters possess a lumen with polarized, PAS(+) cells containing numerous desmosomes and an abundance of glycogen. Culture of the cells on laminin, the most abundant protein found in Matrigel, also induces this morphologic differentiation. Using synthetic laminin-derived peptides, the biologically active IKVAV-containing site of laminin was most active in attachment assays, as well as in inhibiting glandular-like morphogenesis when added to the media of cells cultured on Matrigel. Antibodies to the cell surface 67- and 32-kDa laminin binding proteins partially inhibited the glandular-like morphogenesis, suggesting that multiple interactions with laminin are likely required for the differentiation process. Our data demonstrate that A253 cells can undergo glandular-like morphogenesis on basement membrane and that laminin appears to be the major initiating factor.