Edouard F. Potworowski

Protein Kinase C-ζ Regulates Transcription of the Matrix Metalloproteinase-9 Gene Induced by IL-1 and TNF-α in Glioma Cells via NF-κB

The regulation of matrix metalloproteinase-9 (MMP-9) expression in glioma cells is one of the key processes in tumor invasion through the brain extracellular matrix. Although some studies have demonstrated the implication of classic protein kinase C (PKC) isoforms in the regulation of MMP-9 production by phorbol esters or lipopolysaccharide, the involvement of specific PKC isoforms in the signaling pathways leading to MMP-9 expression by inflammatory cytokines remains unclear. Here we report that the atypical PKC-ζ isoform participates in the induction of MMP-9 expression by interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) in rat C6 glioma cells. Indeed, zymography and semi-quantitative reverse transcriptase-PCR analysis showed that pretreatment of C6 cells with PKC-ζ pseudosubstrate abolished MMP-9 activity and gene expression induced by IL-1 or TNF-α. Accordingly, IL-1 and TNF-α were able to induce PKC-ζ activity, as demonstrated by in vitro kinase assay using immunoprecipitated PKC-ζ. Furthermore, stable C6 clones overexpressing PKC-ζ, but not PKC-ε, displayed an up-regulation of MMP-9 constitutive expression as well as an increase ofmmp-9 promoter activity. These processes were inhibited by an NF-κB-blocking peptide and completely prevented by NF-κB-binding site mutation in the mmp-9 promoter. Taken together, these results indicate that PKC-ζ plays a key role in the regulation of MMP-9 expression in C6 glioma cells through NF-κB.

Evidence for the role of promoter methylation in the regulation of MMP-9 gene expression.

Several studies have reported that elevated MMP-9 expression in lymphoma tissues correlated with tumor stage, grade, or prognosis. Because the DNA methylation pattern is critical for gene expression, detailed methylation analysis using genomic bisulfite sequencing was performed on a series of lymphoma cell lines. We found an inverse correlation between level of methylation of the MMP-9 promoter and the level of MMP-9 expression. Treating lymphoma cells with a DNA methylation inhibitor decreased MMP-9 promoter methylation and increased MMP-9 messenger RNA and protein secretion. This increased expression was potentiated by PMA, a known stimulus of MMP-9 in lymphoma cells. Finally, experiments using in vitro methylated MMP-9 promoter constructs confirmed the fact that DNA methylation exerts suppression on transcriptional activity. The results thus indicate that methylation may contribute to the transcriptional activity of the MMP-9 promoter.

ICAM-1 isoforms: specific activity and sensitivity to cleavage by leukocyte elastase and cathepsin G

The extracellular moiety of ICAM‐1 consists of five Ig‐like domains, the first and third domains mediating adhesion to integrin ligands. The ICAM‐1 gene, however, gives rise to the expression of five alternative splice variants containing two, three, or four Ig‐like domains. In this work, we have investigated whether the rearrangement of the architecture of ICAM‐1 affects its structural properties and function. We showed that, in contrast to the common form, all alternative isoforms of ICAM‐1 were susceptible to cleavage by leukocyte elastase and cathepsin G. We found that the length of an isoform did not influence the susceptibility to proteolysis. The molecular diversity provided by the skipping of entire Ig domains and the level of expression on the APC, however, significantly influenced their ability to potentiate the proliferation of T cells. Finally, we found that the expression of minor ICAM‐1 isoforms encoding the third Ig‐like domains was sufficient to sustain neutrophil infiltration in the liver and confer exon‐5‐targeted ICAM‐1‐deficient mice susceptibility to LPS‐induced septic shock. These findings not only demonstrate that ICAM‐1 isoforms are fully functional, but support the concept that alternative RNA splicing in the Ig superfamily may fulfill distinct roles during the development of the immune response.

GELATINASE B (MMP-9), BUT NOT ITS INHIBITOR (TIMP-1), DICTATES THE GROWTH RATE OF EXPERIMENTAL THYMIC LYMPHOMA

Dysregulation of metalloproteinase production at tumor sites contributes to the modification of local stromal tissue necessary for tumor development. Gelatinase B (matrix metalloproteinase‐9, MMP‐9) is one of the key enzymes that have been associated with the progression of several tumors. Paradoxically, MMP‐9 expression by tumor cells, most notably by lymphoma cells, is concomitant with the expression of its physiological inhibitor, TIMP‐1. Not only are both genes often co‐expressed in the most aggressive forms of lymphomas but also both are up‐regulated upon contact with stromal cells. Since TIMP‐1 is known to regulate growth in several cell types and some aggressive lymphoma cells express TIMP‐1 constitutively without MMP‐9, it is unclear whether the over‐expression of MMP‐9 is counterbalanced by TIMP‐1 and whether TIMP‐1 expression alone could favor the development of lymphoma. To gain further insight into the respective roles of MMP‐9 and TIMP‐1 in lymphoma, we generated lymphoma cell lines expressing constitutively high levels of MMP‐9 or TIMP‐1 and compared these cells for the ability to form thymic lymphoma in vivo. Moreover, we generated lymphoma cell lines expressing constitutively high levels of both MMP‐9 and TIMP‐1 to reproduce the net physiological balance resulting from the expression of both genes simultaneously and to determine which gene overrides the other. Our results show that mice injected with lymphoma cells expressing MMP‐9 constitutively developed thymic lymphoma more rapidly than those injected with control lymphoma cells. Over‐expression of TIMP‐1 alone did not significantly influence tumor progression of lymphoma nor did it delay the capacity of MMP‐9 to accelerate the development of thymic lymphoma. Int. J. Cancer 82:743–747, 1999.

Upregulation of galectin-7 in murine lymphoma cells is associated with progression toward an aggressive phenotype.

We have previously shown that ICAM-1-deficient mice were resistant to lymphoma dissemination of intravenously injected 164T2 lymphoma cells. Highly aggressive variants of this cell line, however, could overcome this resistance. To discern the complex pattern of gene expression involved in the evolution of aggressiveness in lymphoma cells, we compared the transcriptome of 164T2 cells with that of their aggressive variants using cDNA arrays. We identified several genes that were differentially expressed in nonmetastatic lymphoma cells and their metastatic variants. Galectin-7, associated with the development of chemically induced mammary carcinoma, was one such gene whose expression was significantly upregulated. We showed that it was constitutively expressed in aggressive variants, at both mRNA and protein levels. Galectin-7 expression in aggressive lymphoma cells was induced upon in vivo selection in several organs, including the thymus, the spleen and kidneys. We also showed that treatment of nonaggressive lymphoma cells with 5-aza-2′-deoxycytidine was sufficient to induce galectin-7 gene expression. This report is the first to show that galectin-7 is expressed in aggressive lymphoma.

Stromelysin-2 (matrix metalloproteinase 10) is inducible in lymphoma cells and accelerates the growth of lymphoid tumors in vivo.

Matrix metalloproteinase (MMP) 10 (stromelysin-2) is known to degrade various components of the extracellular matrix; however, the signals that regulate its expression and its role in lymphoma growth remain unknown. In the present work, we report the up-regulated expression of MMP10 in T lymphoma cells following contact with endothelial cells. The induction of MMP10 was found to be dependent on the specific interaction between LFA-1 and ICAM-1, which play a central role in regulating the expression of genes involved in the rate-limiting steps of lymphoma development. MMP10, but not MMP3 (stromelysin-1), was also up-regulated in human B lymphoma cells following exposure to IL-4, IL-6, and IL-13, but not to IL-1. To gain further insight into the role of MMP10 in lymphoma development, we generated lymphoma cell lines constitutively expressing high levels of MMP10 and studied these cells for their ability to form thymic lymphoma in vivo. Mice injected with lymphoma cells constitutively expressing MMP10 developed thymic lymphoma more rapidly than those injected with control lymphoma cells. These results provide the first in vivo evidence that overexpression of MMP10 promotes tumor development, and indicate that MMP10 induction is an important pathway activated not only upon ICAM-1/LFA-1-mediated intercellular contact, but also following activation of tumor cells with inflammatory cytokines.

Induced expression of MMP-9 in C6 glioma cells is inhibited by PDGF via a PI 3-kinase-dependent pathway.

The involvement of phosphatidylinositol 3 (PI 3)-kinase in the signalling pathways leading to MMP-9 expression in glioma cells remains unclear. Here, we report that PI 3-kinase inhibits MMP-9 expression induced by either IL-1 or TNF-alpha in rat C6 glioma cells. Using zymography and semi-quantitative RT-PCR analysis, we showed that treatment of C6 cells with wortmannin, an inhibitor of PI 3-kinase activity, potentiated the expression of MMP-9 induced by both cytokines. In contrast, platelet-derived growth factor (PDGF), an inducer of PI 3-kinase activity in C6 cells, inhibited IL-1- or TNF-alpha-induced MMP-9 secretion. Accordingly, this inhibition by PDGF was prevented by wortmannin. Furthermore, stable C6 clones over-expressing the dominant-negative form the regulatory subunit of PI 3-kinase potentiated the expression of MMP-9 induced by IL-1 or TNF-alpha. Taken together, these results suggest that PI 3-kinase may act as a negative regulator of MMP-9 expression in C6 glioma cells.

Immunosuppression in mice fed on diets containing beluga whale blubber from the St Lawrence Estuary and the Arctic populations

In order to assess the immunotoxic potential of naturally relevant mixtures of PCBs and other organohalogens, C57Bl/6 mice were fed on diets in which lipids were replaced by blubber of beluga whales from the highly contaminated population of the Saint-Lawrence River, and the less contaminated population from the Arctic. Different ratios of blubber from both sources were mixed in order to allow a dose-response study. Mice were fed for a period of 90 days at the end of which their immunological status was monitored. For general parameters such as body weight, weight of the spleen and the thymus no significant effect of diets were observed. The immunological endpoints such as the blastic transformation of splenocytes and the spleen NK cell activity were not significantly affected by any of the diets compared to control diets. While the different cell subpopulations of peripheral blood and thymus were not affected by the diets, a significant decrease was noted in the CD8+ T cell population in the spleen of mice fed with most of the diets containing beluga blubber. Moreover, the ability of splenic cells to elicit humoral response against sheep red blood cells as well as the potential of peritoneal macrophages to perform phagocytosis were suppressed by all diets containing beluga blubbers. In summary, there was no differences between the groups fed with a blubber diet with low and high organochlorine contamination. However, a clear immunosuppression was demonstrated when these groups were compared to the group fed with beef oil. Despite the fact that we cannot exclude a possible contribution of the fatty acid composition of the beluga blubber to the immunosupression, these results suggest the sensitivity of mouse immune system towards organohalogens, and point out the toxic potential of contaminant mixtures as found in the less contaminated Arctic population.

Gelatinase B (MMP‐9) production and expression by stromal cells in the normal and adult thymus and experimental thymic lymphoma

Correlative and functional evidence support a crucial role for metalloproteinase (MMP) activity in tumor progression. Dysregulation of MMP production at local tumor sites is thought to participate in the remodeling of the local stromal tissue necessary for tumor growth. The extent of damages in local tissues is often reflected by the high concentration of MMP released in the bloodstream of cancer patients. The integrity of the thymic architecture plays a crucial role in the development of mature T cells, but it is compromised by extensive remodeling occurring during the development of thymic lymphomas. In the present work, we have used an experimental thymic lymphoma model to investigate the regulation of MMP‐9 (gelatinase B) production in animals bearing large thymic lymphomas. We show a 3‐fold increase in serum gelatinase B (Gel B) levels in animals bearing thymic lymphoma compared with those found in normal animals and a correlation between these levels and the size of the tumor. Although Gel B was found within the thymic tumor, lymphoma cells did not express in in vivo, indicating that Gel B expression was associated with thymic stromal cells rather than lymphoma cells. This was corroborated by evidence that lymphoma cells have the capacity to stimulate Gel B gene expression in stromal cells. Our results suggest that lymphoma cells can exert a significant control over Gel B expression by local stromal cells, thereby inducing the extensive remodeling necessary for tumor growth. Int. J. Cancer, 71:71–78, 1997.

Abrogation of graft-versus-host reaction by dieldrin in mice

Sublethal exposure to the organochlorine pesticide, dieldrin, decreased the T-cell immune response in mice. Indeed, a transient inhibition of the mixed lymphocyte reactivity (MLR) was noted at 7 days after intraperitoneal exposure to 0.6 LD50 dieldrin. The present study was undertaken to further investigate the effects of dieldrin on the T-cell immune response, using the graft-versus-host reaction (GVHR) as a model, in order to assess T-cell subset efficiency. Lymphoid cells of A/J mice injected intraperitoneally 7 days earlier with 36 mg/kg body weight dieldrin were transferred into H-2-incompatible F1 hybrids. With this model, known to induce a marked GVHR, we have observed that dieldrin inhibited the potential of parental cells to induce a GVHR in hybrid mice. This effect could not be attributed to a direct cell cytotoxicity, nor to the modulation of major T-cell subsets as shown by thymic and peripheral T-cell subpopulation analysis. Collaboration processes between these cellular subsets seem to represent a potential site for the dieldrin-induced suppression.