Laminin-5 promotes adhesion and migration of epithelial cells: identification of a migration-related element in the gamma2 chain gene (LAMC2) with activity in transgenic mice.

The effects of laminin-5 and its subunit gamma2 chain on cell adhesion and migration were studied, and a migration-related cis-acting element was identified in the gamma2 chain gene (LAMC2) using promoter-reporter gene constructs in transgenic mice. Intact laminin-5 molecules, but not recombinant gamma2 chain promoted cell adhesion of human keratinocytes and mouse squamous carcinoma cells, indicating that the gamma2 chain does not contain a cellular binding site. However, the gamma2 chain as such is probably involved in the process of cell locomotion, as antibodies against the short arm of the chain inhibited migration of carcinoma cells in an in vitro assay. Further evidence for the involvement of the gamma2 chain in cell migration was obtained by the identification of a cis-acting element in a promoter-lacZ reporter gene construct that was active in migratory epithelial cells of healing wounds in mice made transgenic by microinjection of the construct into fertilized oozytes. The migration active element was located in the sequence between -613 and +55. The same construct, and another one containing 5900 base pairs of the 5' flanking region, yielded very limited expression in cells of normal tissues. The limited expression was, however, only observed in epithelial cells of different tissues, i.e. cell types that normally express laminin-5 in vivo. The results show that the sequence between -613 and +55 contains elements that can drive expression during epithelial cell migration and that also partially confers more general epithelium expression. However, elements outside -5900 and +55 are needed for normal epithelium expression of the LAMC2 gene.

Fibroblasts stimulate human ovarian cancer cell invasion and expression of 72-kDa gelatinase A (MMP-2).

OBJECTIVE: The host-tumor interactions and tumor stroma may participate in the regulation of invasive behavior of tumor cells. In order to better understand the human ovarian cancer invasion we explored the possibility that normal fibroblasts could participate in the control of the spread of human ovarian cancer. RESULTS: A 3.5-fold increase (from 2.83 +/- 0.97 to 10.2 +/- 3.43%) in human ovarian cancer cell (Ovcar-3) invasion through a reconstituted basement membrane was noted when normal fibroblasts (CRL 1295) were added to the invasion chambers in conjunction with tumor cells. Conditioned medium from either fibroblasts or Ovcar-3 also enhanced the in vitro invasion of Ovcar-3 by 2- to 2.5-fold (from 2.83 +/- 0.97 to 5.71 +/- 3.5 and to 7.15 +/- 1.2%, respectively) compared to nonstimulated control cells. Zymographic analysis and assays of mRNA for the 72-kDa matrix metalloproteinase (MMP-2) showed that Ovcar-3 cells alone produced very low levels of MMP-2; the expression of this gelatinase was detectable in zymography only with stimulation by incubation of these cells with conditioned media from either fibroblasts or ovarian cancer cells themselves. Interestingly, MMP-2 activity was increased also in fibroblasts when using either ovarian cancer cell-conditioned (to 178 +/- 67%) or fibroblast-conditioned medium (to 215 +/- 61%) and the gene expression for MMP-2 was similarly increased in both fibroblasts and Ovcar-3 cells when using either fibroblast-conditioned medium or ovarian cancer cell-conditioned medium from 1.00 +/- 0.25 to 2.20 +/- 0.50 and 1.86 +/- 0.10 in fibroblasts and from 1.00 +/- 0.26 to 1.60 +/- 0.34 and 2.15 +/- 0.30 in Ovcar-3 cells, respectively. CONCLUSIONS: These results show that interplay between tumor cells and normal cells in the control of invasion and secretion of proteolytic enzymes may involve not only paracrine but also autocrine elements. Thus, such interactions are possible and may play an important role in the spread of cancer.

Ovarian cancer cell invasion is inhibited by paclitaxel.

Overproduction of matrix metalloproteinases (MMPs) and alterations in adhesive and migratory behavior are common characteristics of metastatic cancer cells. Ovarian cancer is a highly invasive type of malignancy. The effect of the antineoplastic drug paclitaxel on human ovarian cancer cell (Ovcar-3) invasion was studied using an in vitro invasion assay with reconstituted basement membrane. The effect of treatment with paclitaxel was also determined separately on certain invasion-associated events, such as the secretion of 72 kDa type IV collagenase (gelatinase A/MMP-2), the expression of the tissue inhibitor of metalloproteinase-2 (TIMP-2), cell attachment and migration. Ovcar-3 cell attachment, migration and in vitro invasion were significantly decreased after paclitaxel treatment (P = 0.02, P < 0.01 and P = 0.001, respectively) whereas no alteration in the secretion of latent MMP-2 was noted. However, the intracellular localization of the immunoreactive protein for MMP-2 was altered in response to paclitaxel treatment. Interestingly, paclitaxel increased the appearance of TIMP-2 protein in culture medium (P = 0.002) but did not change the expression of mRNA for TIMP-2 in Ovcar-3 cells. These data show that paclitaxel is an effective suppressor of Ovcar-3 cell invasion. It inhibits attachment and migratory activities of the cells but also causes a release of TIMP-2 protein into the tissue culture medium.

Polymorphonuclear leukocyte chemotaxis induced by zinc, copper and nickel in vitro.

Metallic dental restorations and prosthetic constructions are susceptible to corrosion in oral environment, resulting in the release of various heavy metal ions. Chloride salts of zinc, copper, nickel, chromium, iron and gold were tested for their ability to promote the migration of polymorphonuclear leukocytes (PMNs). Using a modified Boyden chamber assay for chemotaxis zinc, copper and nickel enhanced the migration of PMN cells in concentration range of 0.5-1.0 mM, whereas no augmentation in migratory activity was noted using chromium or iron. In contrast, an inhibition in migratory activity was observed in cells directed toward gold ions. Exposure of cells to zinc, copper or nickel ions induced an orientation reaction in leukocytes in a similar fashion as the polarization reaction induced by a potent peptide chemoattractant, N-formylmethionylleucylphenylalanine (fMLP), in these cells. Exposure of PMN cells to zinc or nickel in chemotactic concentrations stimulated the chemotaxis of these cells to fMLP 2-fold, whereas pretreatment of the cells with zinc prior to assay markedly decreased the subsequent chemotactic migration of the cells to this metal or to fMLP. The enhanced locomotion of PMN cells induced by zinc, copper or nickel ions was found to be in greater extent due to an increase in directed migration (chemotaxis) rather than an augmentation in random movement (chemokinesis) as assessed by Zigmond-Hirsch checkerboard analysis. These results suggest that zinc, copper and nickel ions attract leukocytes by inducing and promoting the chemotactic response in these cells, which may modulate the inflammatory response of host tissue around such metals.

Modulation of M(r) 72,000 and M(r) 92,000 type-IV collagenase (gelatinase A and B) gene expression by interferons alpha and gamma in human melanoma.

The purpose of this study was to determine how interferons alpha and gamma influence the expression of M(r) 72,000 type-IV collagenase (gelatinase A) and M(r) 92,000 type-VI collagenase (gelatinase B) genes and whether there are differences in their gene expression. Special emphasis was focused on the treatment time. Total cellular RNA from A2058 human melanoma cells treated for various time periods with IFN-alpha or gamma was analyzed by Northern- and slot-blot hybridization. Both M(r) 72,000 and M(r) 92,000 type-IV collagenase mRNAs were detectable in A2058 cells and mRNA levels for both gelatinases were significantly up-regulated in the cells treated for a short time period with either IFN-alpha or gamma. In contrast, a long-term treatment (7 days) with these drugs markedly down-regulated the genes for both gelatinase A and B. Zymographic analysis showed that human melanoma primarily secretes the gelatinase-A activity, which showed changes similar to those seen in the corresponding mRNA after the treatments with interferons. The expression of gelatinase-B activity was, however, detectable only transiently during the stimulating phase with IFN-alpha. Western immunoblot analysis showed that alterations in the levels of immunoreactive protein of gelatinase A in the cells correlated with the mRNA levels after the treatments. These findings suggest that IFN-alpha and IFN-gamma are potent regulators of both M(r) 72,000 and M(r) 92,000 type-IV collagenase/gelatinase A and B genes in human melanoma showing biphasic and parallel effects on mRNA levels of both enzymes, depending on the treatment time, and that the M(r) 72,000 metalloproteinase/gelatinase A is the predominant basement-membrane-degrading type-IV collagenase in human melanoma.

A laminin graft replaces neurorrhaphy in the restorative surgery of the rat sciatic nerve.

We investigated the role of laminin in functional recovery of a peripheral nerve injury using electrophysiological and behavioral approaches on the rat sciatic nerve in vivo. These studies were complemented by neurofilament protein immunocytochemistry on the sciatic nerve 20 days after an operation, in which an 8-mm piece of the nerve was removed and replaced by a graft of laminin, its neurite outgrowth-promoting peptide, a control peptide, collagen, or by resuturing of the removed piece of the nerve. Electrophysiological measurements of muscle strength 4 months after the sciatic nerve transection showed that a laminin graft was as effective as neurorrhaphy in supporting functional recovery of an injured peripheral nerve. A laminin graft also significantly reduced autotomy in the operated animals. Immunocytochemistry confirmed that both a laminin graft and resuturing supported growth of the 200-kDa neurofilament-positive axons into the distal stump of the nerve within 20 days of operation. A graft with a neurite outgrowth-promoting peptide of the B2 chain of laminin supported similar axon growth, whereas another peptide graft also derived from laminin or a collagen graft did not support axon growth. All grafts allowed Schwann cell growth into the distal stumps of the nerves, but neurites accompanied them only in the regeneration-supporting grafts and in the resutured nerves. The Schwann cells of the regenerating nerves expressed high levels of the neurite outgrowth-promoting domain of the B2 chain of laminin, whereas the Schwann cells of the degenerating nerves failed to express this domain in the distal stumps of the degenerating nerves. These results provide the first in vivo evidence for the functional role of laminin in peripheral nerve regeneration. As the neurite outgrowth-promoting domain of the B2 chain of laminin is as efficient as laminin or resuturing in supporting a short-term recovery of an injured sciatic nerve, this area may be a regeneration-promoting domain of this glycoprotein. More importantly, as grafting significantly reduces post-traumatic pain behavior in the operated animals, the laminin graft surgery may provide a useful method for clinical restoration of the injured peripheral nerves.

70 K type IV collagenase (gelatinase).

Type IV collagenase (gelatinase) is a 70,000 dalton neutral metalloproteinase that specifically cleaves type IV collagen in addition to degrading denatured collagen (gelatin). It is secreted in a latent proenzyme form that is converted proteolytically in the extracellular space to a 62,000 dalton active enzyme. The primary structure, enzymatic properties as well as gene structure, demonstrate that type IV collagenase is closely related with the other well characterized metalloproteinases, interstitial collagenase and stromelysin. However, the structure of type IV collagenase differs from the others in that it is larger and contains three internal repeats that resemble the type II domains of fibronectin. Also, initial characterization of the promoter region of the gene indicates that its regulation differs from the other proteinase genes. Type IV collagenase is presumably required for the normal turnover of basement membranes. Augmented activity is linked with the invasive potential of tumor cells and the enzyme is believed to play a major role in the penetration of basement membranes by metastatic cells. Measurements of enzyme activity and mRNA levels as well as immunostaining of a variety of tumor cells and tissues suggest that assays for the enzyme may have value in the follow-up of malignant growth.

Recombinant interferon alpha and gamma modulate the invasive potential of human melanoma in vitro.

We have studied the effects of interferons (IFNs) on the attachment, collagenase IV activity, chemotactic migration and in vitro invasion of human melanoma (A2058) cells treated for various time periods with human recombinant interferon alpha (hrIFN-alpha) or gamma (hrIFN-gamma). The cells treated with hrIFN alpha for a short time period attached more readily to purified basement membrane components, type IV collagen and laminin, than control cells. The stimulating effect of hrIFN gamma on the attachment was seen, however, when the cells were treated for a longer period of time (3 days) with this drug. The short-term treatment with hrIFN alpha also enhanced the in vitro invasion of cells through a reconstituted basement membrane compared to findings with untreated control cells. Pre-treatment of 3 days or more was, however, needed for hrIFN gamma to promote the invasion of A2058 cells. Both IFNs increased the secretion of basement membrane (type IV) collagen degrading metalloproteinase (collagenase IV) activity from human melanoma cells. Further, chemotaxis, i.e., directed migration of A2058 cells to laminin, was enhanced by both IFNs. In contrast, the attachment, collagenase IV activity, chemotaxis, and in vitro invasion were markedly inhibited when the cells were treated for an extended time period (7 days) with the IFNs. Interferons also inhibited cell proliferation after 4 days of exposure. These results suggest that time of treatment with interferons modulates the invasive capacity of human melanoma cells in vitro, causing initially a transient enhancement of invasion followed by an inhibition of invasive propensity after extended exposure to these drugs, and that different biochemical steps required for successful invasion are regulated in parallel by interferons alpha and gamma.

Modulation of type-IV collagenase activity and invasive behavior of metastatic human melanoma (A2058) cells in vitro by monoclonal antibodies to type-IV collagenase.

Monoclonal antibodies (MAbs) against human type-IV collagenase were developed and used for studies on enzyme activity and tumor-cell invasion in vitro. Fifteen MAb clones were generated against the enzyme purified form serum-free culture medium of human melanoma cells (A2058). Five clones affecting the activity of type-IV collagenase were selected for further characterization. All the selected clones could be used for a single-step purification of type-IV collagenase using IgG-Sepharose affinity columns. One of the antibodies activated the enzyme when 3H-proline-labelled type-IV collagen was used as substrate. The activation was dependent on the enzyme antibody ratio. Four clones caused more than 30% inhibition of the activity, maximal inhibition being 50%. Interestingly, the same antibody which activated the enzyme also increased the invasion of A2058 cells through a reconstituted basement membrane in an in vitro invasion assay. The 4 inhibitory antibodies decreased the penetration of A2058 cells through the reconstituted basement membrane. The results strongly support previous findings about the importance of type-IV collagenase in tumor-cell invasion.

A new animal model for human colon cancer metastasis.

An animal model for human colon cancer metastasis is described in which spontaneously metastasizing colonic tumors are formed after injection of human colon cancer cells into the cecal wall of young athymic nude mice. Lymphatic and vascular invasion were demonstrated histologically after injection of both well- and poorly-differentiated cell lines, and metastases were found in a pattern similar to that of naturally occurring human colonic cancer. In contrast, little or no visceral organ involvement could be demonstrated after s.c. injection. Cells with increased liver-metastasizing potential were obtained by serial selection in this system. These cells had an enhanced ability to penetrate a reconstituted basement membrane in the presence of partially purified liver extract when compared to lung or brain extracts in a modified Boyden chamber assay. These results demonstrate the ability of human epithelial tumor cells to metastasize reproducibly in an animal model system, which may be useful for studying many aspects of the pathogenesis of cancer metastasis. In addition, it is suggested that local invasion by colon cancer cells may be influenced in part by tissue-specific factors.

An animal model for colon cancer metastasis: establishment and characterization of murine cell lines with enhanced liver-metastasizing ability.

A detailed understanding of the pathogenesis of colon cancer metastasis has been hindered by the lack of appropriate animal models which accurately reflect events in this complex process. An animal model for colon cancer metastasis is described in which spontaneously metastasizing colonic tumors are formed after injection of murine colon cancer cells into the cecal wall of BALB/c mice. Using this model, tumor cells with different liver-metastasizing potential were selected and shown to possess several properties known to be associated with other metastatic cell lines. The ability of tumor cells to invade a reconstituted basement membrane and to secrete type IV collagenase was directly proportional to their metastatic ability. In addition, liver-metastasizing cells preferentially migrated toward liver extracts in a Boyden chamber assay, as compared to extracts of brain or lung, and adhered rapidly to highly purified hepatic sinusoidal endothelial cells versus hepatic parenchymal cells in vitro. This model may thus be useful for studying many aspects of the pathogenesis of colon cancer metastasis.

Basement membrane and the invasive activity of metastatic tumor cells.

Many, but not all, metastatic tumor cells have a similar phenotype. These are transformed cells with a high affinity for basement membranes and the ability to produce basement membrane degrading enzymes. Such characteristics help the cells traverse this critical barrier. Traversal may be a rather rare event unless the cells respond to local factors that amplify the numbers of cells recruited to the site and induce the cells to invade. These factors may include tissue-specific attractants and matrix molecules such as laminin. Understanding the specific steps involved in the invasion process should allow development of antimetastatic regimens directed at those activities specific to metastatic tumor cells. Due to the heterogeneity of tumor cells, other mechanisms for metastasis undoubtably exist.

Laminin promotes rabbit neutrophil motility and attachment.

Polymorphonuclear neutrophils (PMN) traverse basement membrane to reach sites of infection. We have studied the role of laminin, a specific basement membrane component, in this process using three assay systems. In the Boyden chamber, laminin was found to stimulate chemotaxis of neutrophils while fibronectin did not. Co-incubation of cells with antibody to laminin blocked this chemotaxis, while antibody to fibronectin was without effect. In the human amnion system, neutrophils were shown to penetrate through the tissue when the peptide chemoattractant f-Met-Leu-Phe was placed on the opposing side. Antibody to laminin, but not to fibronectin, blocked this penetration. In an attachment assay system, laminin, but not fibronectin, was found to increase dispase-treated neutrophil attachment to type IV (basement membrane) collagen-coated plastic and to a plastic substrate itself. Electrophoretic analysis of PMN extract indicated the presence of laminin, and indirect immunofluorescence suggested that laminin is localized on the surface of the neutrophils. These data suggest that PMN can bind laminin on their cell surfaces, use laminin to attach to basement (type IV) membrane collagen, and migrate toward a gradient of laminin. These properties may be important for the passage of neutrophils from the circulation to sites of infection.

Use of a reconstituted basement membrane to measure cell invasiveness and select for highly invasive tumor cells.

Malignant cells must traverse basement membranes during their migration to sites distant from the primary tumor. Since basement membranes are thought to be a critical barrier to the passage of tumor cells, we have constructed a model basement membrane-stromal matrix consisting of laminin and type IV collagen reconstituted onto a disk of type I collagen for use in an in vitro assay of invasiveness. Metastatic tumor cells and leukocytes are able to cross this barrier, whereas nonmetastatic tumor cells, fibroblasts, and epidermal cells cannot penetrate it. Those tumor cells that penetrate the barriers were found, when isolated and subcultured, to be more invasive and to produce more metastases than the parental population. This assay system should be useful for studying the invasiveness of tumor cells and for isolating highly invasive variants.

Migration of tumor cells to organ-derived chemoattractants.

Certain tissues contain unique factors which are chemotactic for metastatic tumor cell lines. Extracts of bone, brain, liver, and lung were tested for their ability to promote either the migration or the chemoinvasion, i.e., their penetration through a reconstituted basement membrane barrier, of various metastatic tumor cells. Using a modified Boyden chamber assay for chemotaxis, B16-Br2 melanoma cells, which metastasize to brain, migrated most actively to brain extract. Lung-directed T241-PM2 fibrosarcoma cells migrated selectively to lung extract. Further, murine M50-76 reticulum cell sarcoma cells, which metastasize to liver and ovaries, were preferentially attracted to liver extract, and MCF-7 breast adenocarcinoma cells with high bone and brain colonization potential were found to migrate most actively to bone and brain extracts. Partial purification of tissue extracts showed that the factors in brain and liver are of different molecular weights. These data suggest that tissue-specific factors in different target tissues attract tumor cells which home to those sites.