Maura C. Kibbey

The laminins: a family of basement membrane glycoproteins important in cell differentiation and tumor metastases

Laminins are a family of basement membrane-derived glycoproteins that are very biologically active with a number of diverse cell types. The response of the cells is dependent on the cell type and various cell-specific intracellular events are activated. Multiple active sites on laminin and cellular receptors have been described. Both laminin and the synthetic peptides that define the active sites may have important clinical uses. For example, the neurite-promoting peptides may be useful in vivo in regeneration studies because of their potent activity with neural cells and their lack of antigenicity. Also, peptides, such as YIGSR, that inhibit angiogenesis are potentially useful for treating the vascularization of the eye that occurs in conditions such as diabetes mellitus. Likewise, the angiogenic peptide SIKVAV, because of its role in endothelial cell block vessel formation, may be useful for treating ischemia. The recent progress that has been made in characterizing basic mechanisms of action of laminin has laid the groundwork for more direct studies of its clinical relevance.

The ?1 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.

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

SummaryLaminin, 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 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.

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.

Maintenance of the EHS sarcoma and Matrigel preparation

Matrigel is a reconstituted basement membrane extract which is rich in laminin, growth factors, entactin/nidogen, type IV collagen, and heparan sulfate proteoglycan (perlecan). This basement membrane extract can be used for promotion and maintenance of a differentiated phenotype in cell cultures, as well as for a variety of in vitro and in vivo uses. The Engelbreth-Holm-Swarm (EHS) sarcoma is a rich source of both individual basement membrane components and Matrigel. Maintenance of the EHS tumor in mice and its subsequent harvesting for the preparation of Matrigel are described.

Enhancement of tumor growth by basement membrane: Modulation of growth and angiogenesis by laminin-derived synthetic peptides

The study of human cancers has been impaired by a lack of good animal models. Human cancers are difficult to grow in culture, and the use of immunologically deficient mice has had limited success in propagating human tumors [1]. If a tumor is able to grow in a mouse, its ability to subsequently survive in culture is enhanced. We and others have found much greater take and growth of both murine and human tumor cells and primary isolates in mice when the cells are premixed with a basement membrane extract (Matrigel) and injected subcutaneously [2, 3, 4, 5]. In addition, we have been able to further increase the growth of solid tumors by the inclusion of a synthetic angiogenic peptide derived from the laminin A chain [6]. In contrast, anti-angiogenic peptides reduce the tumor growth even when intra-peritoneal injections of the peptide are initiated several days after tumor growth has begun [3]. Although these subcutaneous tumors do not frequently metastasize, the growth of such tumors is an important first step in developing animal models to study the metastatic and phenotypic properties of human cancer.

Basement Membrane Laminin-Derived Peptide Sikvav Promotes Angiogenesis and Tumor Growth

The basement membrane is a thin extracellular matrix which underlies endothelial cells in vessels and forms a barrier to the passage of macromolecules and cells (Martin et al, 1988). Basement membranes also provide structural support and are very biologically active (Kleinman et al, 1987; Beck et al, 1990). The major and constant components of basement membranes include laminin, collagen IV, entactin, heparan sulfate proteoglycan and various growth factors (Martin et al, 1988; Vukicevic et al, 1992). These components interact with each other to form a highly elastic and organized structure.