IP-10 is a C-X-C chemokine that is produced by activated monocytes, fibroblasts, endothelial cells, and keritinocytes in response to stimulation with interferon-gamma and is primarily a chemotactic factor for T-lymphocytes. It has in vivo anti-tumor effects in some experimental models with local tumor injection which have been proposed to be mediated via a T-cell dependent mechanism. We evaluated the effects of retroviral IP-10 gene transduction and protein over-expression in human A375 melanoma xenografts in nude mice using a pCLNCX retroviral vector containing a CMV promoter to drive the IP-10 transgene with Null transfected cells (Null) and Wild Type (WT) tumor lines as controls. Two transfectants with high IP-10 protein expression were identified and used in subsequent experiments. There was no difference on in vitro proliferation rates of the various clones compared to Null transfectants or WT cell lines. After subcutaneous injection of WT A-375 cells, Null transduced or IP-10 transduced clones 2 and 11, both clones showed significant inhibition of tumor growth compared to control or parental tumors. Mixing experiments demonstrated a paracrine bystander effect from IP-10 produced in transduced cells; tumors consisting of 50% IP-10 transduced cells were significantly smaller than tumors consisting of 50% Null transduced cells. These data suggested that systemic administration of the recombinant protein may have similar anti-tumor properties. Microvessel counts in IP-10 transduced clones were markedly less than those in control tumors based on vonWillebrand factor immunostaining. Additional studies were undertaken to characterize the alterations in gene expression profiles in IP-10 transduced melanoma xenografts compared to Null transfectants and WT xenografts using cDNA microarray analysis. Genes that were differentially expressed by at least 3 fold between IP-10 transduced cells and both WT and Null transfectants with a less than 1.5 fold difference between WT type and Null transfectants were considered significant. A cluster analysis was performed to identify genes with similar expression patterns and quantitative (q) RT-PCR was used to confirm expression of genes identified as significantly up or down regulated. There were 49 genes on the 10 K cDNA array that fulfilled the filtering criteria including 3 angiogenic genes that were significantly down regulated in IP-10 tumors as assessed by qRT-PCR: IL-8 by 12.3 fold, TGF by 9.5 fold and tissue plasminogen activator (tPA) by 4.1 fold. Taken together these data demonstrate that potent inhibition of tumor growth by IP-10 may be mediated by inhibition of angiogenesis via down-regulation of pro-angiogenic factors within the tumor microenvironment. These findings have been extended using recombinant IP-10 protein; the effects of recombinant IP-10 on proliferation rates of human umbilical vein endothelial cells (ECs), fibroblasts, and 2 human cell lines, A-375 melanoma and WIDR colon adenocarcinoma were evaluated. IP-10 had a dose dependent and selective inhibition of the proliferation of ECs. In addition, IP-10 inhibited the proliferative effects of VEGF on ECs such that proliferation rates were equivalent to untreated cells. These selective effects on ECs were also evident with respect to induction of apoptosis evaluated using the TUNEL assay. IP-10 selectively caused apoptosis in ECs in vitro but did not affect the percentage of apoptotic fibroblasts or A-375 human melanoma cells. The percentage of ECs gaited for apoptosis increased from 2.8% to over 90% after 1 and 10 ug/mL of IP-10. In contrast the percent of apoptotic A-375 cells increased minimally from 2.5% 7.5% after 24 hours of treatment with IP-10 at 10 ug /mL, the protein had no effect on induction of apoptosis in fibroblasts.To investigate the mechanism for the selective effects of IP-10 on ECs, we characterized the level of IP-10 receptor expression in the various cell types. The IP-10 receptor, CXCR-3, has 2 distinct splice variants CXCR-3A and CXCR-3B which have paradoxical effects after ligand receptor binding. Over-expression of the CXCR-3A receptor results in increased cell survival whereas over-expression of the CXCR-3B receptor results in dramatically reduced DNA synthesis and induction apoptotic cell death. Therefore, the A variant is considered a mediator of cellular activation and EC angiogenesis while the B receptor acts as a cellular inhibitor and promoter of apoptosis. We used qRT-PCR to evaluate mRNA gene expression of the A and B variant of the receptor in different cell types. T lymphocytes stimulated with IL-2 had a preponderance of the high affinity A variant consistent with the literature. Confluent ECs, A-375, WIDR, and fibroblasts expressed mRNA for both variants of the CXCR-3. However, the ratio of proapoptotic B receptor to A receptor mRNA expression was 41.5 in ECs but less than 10 in fibroblasts, WIDR, and A-375 cell lines. We next investigated whether the degree of EC confluence (as a surrogate of activated versus quiescent endothelium) altered the level of CXCR-3B mRNA expression in ECs. Interestingly, ECs that were actively proliferating had the highest levels of the CXCR-3B receptor expression when compared to confluent non-proliferating cells while the A variant was minimal at all levels of endothelial confluence. Recombinant IP-10, administered daily to animals with subcutaneously implanted A-375 xenografts, resulted in a significant inhibition of tumor growth compared to saline treated animals. Immunostaining of tumors harvested from the IP-10 and saline treated mice with anti-CD 31 by a pathologist blinded to the treatment groups showed that microvessel density was much lower in IP-10 treated tumors. Together these data indicate that IP-10 exerts selective inhibitory and pro-apoptotic effects on endothelial cells and those that are activated in a proliferating state may be most sensitive to these effects based upon a relatively higher level of the B variant of the IP-10 receptor. Because IP-10 is so well tolerated additional studies are being conducted combining IP-10 with other anti-angiogenic agents that have complementary effects or with chemotherapy. These experiments will be conducted in animal models of liver or peritoneal metastases. Our laboratory was one of the first to demonstrate that a recombinant human IL-1 receptor antagonist (IL-1ra) improved survival following lethal endotoxemia in murine models and gram negative septemia in rodent models. This research served as the basis for subsequent clinical trials using the recombinant protein in humans suffering from the sepsis syndrome. IL-1ra is a naturally occurring antagonist to IL-1 and competitively binds the IL-1 receptor. The IL-1ra/ receptor complex does not activate the receptor and inhibits the biological activities of IL-1. There are abundant data indicating that generalized host responses to various acute or chronic inflammatory conditions is mediated by the endogenous production of secondary cytokines which mediate multiple pathophysiological effects. IL-1 has been shown to mediate immune complex colitis, rheumatoid arthritis, acute and chronic infection, and has been shown to promote tumor growth and metastases in various experimental models and some clinical settings. We reasoned that IL-1ra may be a useful strategy in cancer treatment either via direct inhibition of tumor cell proliferation and metastatic potential or by secondary effects such as inhibition of the host inflammatory or angiogenic response that promotes local tumor growth and metastatic spread. We performed qRT-PCR for IL-1 on tumor specimens obtained from patients with metastatic colon adenocarcinoma, non-small cell lung cancer, or melanoma. IL-1 gene over-expression (>1,000 copies/105 copies beta-actin) was detected in greater than 50% of all samples tested in all histologies.
| Feldman, Jack L; Neverova, Natalia V; Saywell, Shane A (2005) Modulation of hypoglossal motoneuron excitability by intracellular signal transduction cascades. Respir Physiol Neurobiol 147:131-43 |
| Tandle, Anita T; Mazzanti, Chiara; Alexander, H Richard et al. (2005) Endothelial monocyte activating polypeptide-II induced gene expression changes in endothelial cells. Cytokine 30:347-58 |
| Weinreich, David M; Elaraj, Dina M; Puhlmann, Markus et al. (2003) Effect of interleukin 1 receptor antagonist gene transduction on human melanoma xenografts in nude mice. Cancer Res 63:5957-61 |
