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.

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.

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.