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CCL3–CCR5 Axis regulates intratumoral accumulation of leukocytes and fibroblasts, and promotes angiogenesis in murine lung metastasis process
Abstracts / Cytokine 48 (2009) 46–90 transcription of CCL13. Knock-down of STAT5 abrogates CCL13 expression and strongly reduces migration of monocytic cells. Furthermore, the use of pharmacological inhibitors as well as siRNA technology identifies the active p38 MAPK as important stabilizer of CCL13 mRNA through a mechanism involving phosphorylation of tristetraprolin, an important destabilizer of short-lived mRNAs. Despite potent OSM-induced signal transduction in all types of fibroblasts analyzed, only synovial fibroblasts secrete CCL13 which might be indicative of tissue-specific imprinting of different fibroblasts during development. doi:10.1016/j.cyto.2009.07.136
PP1-014 CCL3–CCR5 Axis regulates intratumoral accumulation of leukocytes and fibroblasts, and promotes angiogenesis in murine lung metastasis process Yu Wu, Ying-Yi Li, Tomohisa Baba, Naofumi Mukaida, Poster Presentation I CCL3–CCR5 Axis regulates intratumoral accumulation of leukocytes and fibroblasts, and promotes angiogenesis in murine lung metastasis process Yu Wu, Ying-Yi Li, Tomohisa Baba, Naofumi Mukaida, Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa 920-0934, Japan Metastasis proceeds through interaction between cancer cells and residenT cells such as leukocytes and fibroblasts. An intravenous injection of a mouse renal cell carcinoma, Renca, into wild-type (WT) mice, resulted in multiple metastasis foci in lungs and was associated with intratumoral accumulation of macrophages, granulocytes, and fibroblasts. A chemokine, CCL3, was detected in infiltrating cells and to a lesser degree, tumor cells, together with an infiltration of leukocytes expressing CCR5, a specific receptor for CCL3. A deficiency of CCL3 or CCR5 gene markedly reduced the number of metastasis foci in the lung, and the analysis using bone marrow chimeric mice revealed that both bone marrow- and non-bone marrow-derived cells contributed to metastasis formation. CCL3- and CCR5-deficient mice exhibited a reduction in intratumoral accumulation of macrophages, granulocytes, and fibroblasts. Moreover, intratumroal neovascularization, an indispensable process for metastasis, was attenuated in these gene deficient mice. Intrapulmonary expression of matrix metalloproteinase (MMP)-9 and hepatocyte growth factor (HGF) was enhanced in WT mice and the increases were markedly diminished in CCL3- and CCR5-deficient mice. Furthermore, MMP-9 protein was detected in macrophages and granulocytes, the cells which also express CCR5 and in vitro stimulation by CCL3 induced macrophages to express MMP-9. Intratumoral fibroblasts expressed CCR5 and HGF protein. In vitro CCL3 stimulated fibroblasts to express HGF. Collectively, the CCL3-CCR5 axis appears to regulate intratumoral trafficking of leukocytes and fibroblasts, and MMP-9 and HGF expression, and as a consequence to accelerate neovascularization and subsequent metastasis formation.
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C1q may induce apoptosis of prostate cancer cells by activating WOX1 and destabilizing cell adhesion. Downregulation of C1q enhances prostate hyperplasia and cancerous formation due to failure of WOX1 activation. [supported in part by NSC Taiwan (96-2320-B-006-014, NSC 96-2628-B-006-045-MY3, NSC 96-2628-B-006-041-MY3), NHRI Taiwan (EX97-9705BI), and DoD, USA (BC075692)]. doi:10.1016/j.cyto.2009.07.138
PP1-016 Specific genetic alterations in the gene for IL-15 can enhance its translation efficiency in cells that show translational down-regulation T.G. Wu, C.F. Grewe, D.W. Idossa, M.R. DeWall, W.R. Fleischmann Jr., Poster Presentation I Specific genetic alterations in the gene for IL-15 can enhance its translation efficiency in cells that show translational down-regulation T.G. Wu, C.F. Grewe, D.W. Idossa, M.R. DeWall, W.R. Fleischmann Jr., The University of Minnesota Medical School, Minneapolis, MN 55455, USA B16 melanoma cells transfected with the gene for the short signal peptide isoform of IL-15 accumulate high levels of IL-15 mRNA and cell-associated IL-15 protein (79 pg/million cells). They also serve as an efficacious cancer vaccine. However, while RM-1 prostate cancer cells transfected with the same IL-15 gene accumulate high levels of IL-15 mRNA, they show translational regulation of the IL-15 mRNA and accumulate only very low levels of IL-15 protein (2.4 pg/million cells). Regulatory regions in the signal peptide as well as in the 50 and 30 coding regions of IL-15 mRNA have been suggested to have down-regulatory effects but have not been specifically identified (Bamford RN, et al. J Immunol 1998;160:4418–26). The Vienna RNA program was used to analyze the putative secondary structure of the IL-15 mRNA. First, it was noted that the Kozak sequence was located in a largely double-stranded region. Second, it was noted that the IL-15 mRNA contains six hairpin loops that might be involved in translational regulation. A series of genetically altered constructs of IL15 were created and used to transfect RM-1 cells. A construct designed to release the Kozak sequence from its double-stranded region increased IL-15 translation by 2.9-fold from 2.4 pg/million cells to 7.0 pg/million cells. Constructs designed to disrupt hairpin loops 1–2–4, 1–3–4, and 2–3 implicate hairpin loop 3 but not loops 1, 2 or 4 in down-regulation of IL-15 translation. Constructs with a disruption of hairpin loop 3 increased IL-15 translation by an additional 4.9-fold to 34 pg/million cells. Thus, by altering the environment of the Kozak sequence and by disrupting hairpin loop 3 in the 50 end of the IL-15 mRNA, the translational efficiency of IL-15 mRNA has been increased by 14-fold. Further increases in translational efficiency might be expected by genetically altering hairpin loop 5 or 6 at the 30 end of the IL-15 mRNA. The results support the hypothesis that cancer cells transfected with genetically modified constructs of the short signal peptide isoform of IL-15 can be made to translate sufficient quantities of IL-15 to serve as a cancer vaccine.
doi:10.1016/j.cyto.2009.07.137 doi:10.1016/j.cyto.2009.07.139
PP1-015 Complement C1q signals dancing of tumor suppressor WWOX/WOX1 on cell surface for apoptosis Nan-Shan Chang, Poster Presentation I Complement C1q signals dancing of tumor suppressor WWOX/WOX1 on cell surface for apoptosis Nan-Shan Chang, Institute of Molecular Medicine, National Cheng Kung University Medical College, Tainan 70101, Taiwan, ROC, Department of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY 13210, USA We determined whether specific serum complement components coordinate the activation of tumor suppressors p53 and WOX1 (also named WWOX or FOR) and kinases ERK and JNK1 in human prostate DU145 and neuroblastoma SK-N-SH cells. Cells were cultured overnight in 1% normal human serum, or in human serum depleted of an indicated complement protein. Under complement C1q- or C6-free conditions, WOX1 and ERK were mainly present in the cytoplasm without phosphorylation, whereas phosphorylated JNK1 was greatly accumulated in the nuclei. Exogenous C1q rapidly restored the WOX1 activation (with Tyr33 phosphorylation) in less than 2 hr. Without serum complement C9, p53 became activated spontaneously. Notably, exogenous C1q significantly induced apoptosis of WOX1-overexpressing DU145 or SK-N-SH cells, but not vehicle-expressing cells. A dominant negative and Y33R mutant of WOX1 blocked the apoptotic effect. C1q did not enhance p53-mediated apoptosis. By total internal reflection fluorescence (TIRF) and time-lapse microscopy, we determined that C1q induced dynamic formation (or dancing) of WOX1-rich microvilli, which were assembled as clusters in between cells, thus destabilizing cell adherence. These cells then underwent shrinkage, membrane blebbing and death. Remarkably, as determined by immunostaining, benign prostatic hyperplasia and prostate cancer were shown to have a significantly reduced expression of tissue C1q, compared to age-matched normal prostate tissues. In conclusion, complement
PP1-017 IFN-beta pro-apoptotic and antiproliferative activity is superior to IFNalpha in adult T-cell leukemia: Ex vivo response predicts survival Johan Van Weyenbergh, Ricardo Khouri, Daniele Decanine, Kristof Theys, Koen Deforche, Aline Clara Silva, Lourdes Farre, Achilea Bittencourt, Anne-Mieke Vandamme, Poster Presentation I IFN-beta pro-apoptotic and antiproliferative activity is superior to IFN-alpha in adult T-cell leukemia: Ex vivo response predicts survival Johan Van Weyenbergh 1, Ricardo Khouri 1,2, Daniele Decanine 1, Kristof Theys 2, Koen Deforche 2, Aline Clara Silva 1, Lourdes Farre 1, Achilea Bittencourt 3, Anne-Mieke Vandamme 2, 1 CPqGM-FIOCRUZ, Salvador-BA, Brazil, 2 Rega Institute, KU Leuven, Belgium, 3 HUPES-UFBA, Salvador-BA, Brazil Background: Adult T-cell leukemia (ATL) is an aggressive CD4+ leukemia which usually develops several decades after HTLV-1 infection. IFN-alpha in combination with AZT is currently the major treatment option in ATL, but therapeutic success of this combination therapy is limited. No bona fide surrogate markers for survival and/or therapeutic failure are currently available for clinical use in ATL. Methods: Molecular and clinical data (>2 years follow-up) were obtained from thirty ATL patients. We have quantified immunological markers (flow cytometry, proliferation, cytokines and apoptosis-related markers) ex vivo in plasma and in vitro in peripheral blood mononuclear cells. For multivariate analysis, molecular data were combined with clinical data as parameters for Bayesian network learning and classification modeling. Gene expression profiling was performed in IFN-treated primary ATL cells and ATL-derived cell lines. Results: Using univariate analysis, we found that proliferation of primary cells from ATL patients was significantly decreased by both IFNalpha (p < 0.05) and IFN-beta (p < 0.01), but IFN-alpha-induced antiproliferative activity was only significant in mild clinical forms (chronic and smoldering), whereas IFN-
PP1-014 CCL3–CCR5 Axis regulates intratumoral accumulation of leukocytes and fibroblasts, and promotes angiogenesis in murine lung metastasis process Yu Wu, Ying-Yi Li, Tomohisa Baba, Naofumi Mukaida, Poster Presentation I CCL3–CCR5 Axis regulates intratumoral accumulation of leukocytes and fibroblasts, and promotes angiogenesis in murine lung metastasis process Yu Wu, Ying-Yi Li, Tomohisa Baba, Naofumi Mukaida, Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa 920-0934, Japan Metastasis proceeds through interaction between cancer cells and residenT cells such as leukocytes and fibroblasts. An intravenous injection of a mouse renal cell carcinoma, Renca, into wild-type (WT) mice, resulted in multiple metastasis foci in lungs and was associated with intratumoral accumulation of macrophages, granulocytes, and fibroblasts. A chemokine, CCL3, was detected in infiltrating cells and to a lesser degree, tumor cells, together with an infiltration of leukocytes expressing CCR5, a specific receptor for CCL3. A deficiency of CCL3 or CCR5 gene markedly reduced the number of metastasis foci in the lung, and the analysis using bone marrow chimeric mice revealed that both bone marrow- and non-bone marrow-derived cells contributed to metastasis formation. CCL3- and CCR5-deficient mice exhibited a reduction in intratumoral accumulation of macrophages, granulocytes, and fibroblasts. Moreover, intratumroal neovascularization, an indispensable process for metastasis, was attenuated in these gene deficient mice. Intrapulmonary expression of matrix metalloproteinase (MMP)-9 and hepatocyte growth factor (HGF) was enhanced in WT mice and the increases were markedly diminished in CCL3- and CCR5-deficient mice. Furthermore, MMP-9 protein was detected in macrophages and granulocytes, the cells which also express CCR5 and in vitro stimulation by CCL3 induced macrophages to express MMP-9. Intratumoral fibroblasts expressed CCR5 and HGF protein. In vitro CCL3 stimulated fibroblasts to express HGF. Collectively, the CCL3-CCR5 axis appears to regulate intratumoral trafficking of leukocytes and fibroblasts, and MMP-9 and HGF expression, and as a consequence to accelerate neovascularization and subsequent metastasis formation.
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C1q may induce apoptosis of prostate cancer cells by activating WOX1 and destabilizing cell adhesion. Downregulation of C1q enhances prostate hyperplasia and cancerous formation due to failure of WOX1 activation. [supported in part by NSC Taiwan (96-2320-B-006-014, NSC 96-2628-B-006-045-MY3, NSC 96-2628-B-006-041-MY3), NHRI Taiwan (EX97-9705BI), and DoD, USA (BC075692)]. doi:10.1016/j.cyto.2009.07.138
PP1-016 Specific genetic alterations in the gene for IL-15 can enhance its translation efficiency in cells that show translational down-regulation T.G. Wu, C.F. Grewe, D.W. Idossa, M.R. DeWall, W.R. Fleischmann Jr., Poster Presentation I Specific genetic alterations in the gene for IL-15 can enhance its translation efficiency in cells that show translational down-regulation T.G. Wu, C.F. Grewe, D.W. Idossa, M.R. DeWall, W.R. Fleischmann Jr., The University of Minnesota Medical School, Minneapolis, MN 55455, USA B16 melanoma cells transfected with the gene for the short signal peptide isoform of IL-15 accumulate high levels of IL-15 mRNA and cell-associated IL-15 protein (79 pg/million cells). They also serve as an efficacious cancer vaccine. However, while RM-1 prostate cancer cells transfected with the same IL-15 gene accumulate high levels of IL-15 mRNA, they show translational regulation of the IL-15 mRNA and accumulate only very low levels of IL-15 protein (2.4 pg/million cells). Regulatory regions in the signal peptide as well as in the 50 and 30 coding regions of IL-15 mRNA have been suggested to have down-regulatory effects but have not been specifically identified (Bamford RN, et al. J Immunol 1998;160:4418–26). The Vienna RNA program was used to analyze the putative secondary structure of the IL-15 mRNA. First, it was noted that the Kozak sequence was located in a largely double-stranded region. Second, it was noted that the IL-15 mRNA contains six hairpin loops that might be involved in translational regulation. A series of genetically altered constructs of IL15 were created and used to transfect RM-1 cells. A construct designed to release the Kozak sequence from its double-stranded region increased IL-15 translation by 2.9-fold from 2.4 pg/million cells to 7.0 pg/million cells. Constructs designed to disrupt hairpin loops 1–2–4, 1–3–4, and 2–3 implicate hairpin loop 3 but not loops 1, 2 or 4 in down-regulation of IL-15 translation. Constructs with a disruption of hairpin loop 3 increased IL-15 translation by an additional 4.9-fold to 34 pg/million cells. Thus, by altering the environment of the Kozak sequence and by disrupting hairpin loop 3 in the 50 end of the IL-15 mRNA, the translational efficiency of IL-15 mRNA has been increased by 14-fold. Further increases in translational efficiency might be expected by genetically altering hairpin loop 5 or 6 at the 30 end of the IL-15 mRNA. The results support the hypothesis that cancer cells transfected with genetically modified constructs of the short signal peptide isoform of IL-15 can be made to translate sufficient quantities of IL-15 to serve as a cancer vaccine.
doi:10.1016/j.cyto.2009.07.137 doi:10.1016/j.cyto.2009.07.139
PP1-015 Complement C1q signals dancing of tumor suppressor WWOX/WOX1 on cell surface for apoptosis Nan-Shan Chang, Poster Presentation I Complement C1q signals dancing of tumor suppressor WWOX/WOX1 on cell surface for apoptosis Nan-Shan Chang, Institute of Molecular Medicine, National Cheng Kung University Medical College, Tainan 70101, Taiwan, ROC, Department of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY 13210, USA We determined whether specific serum complement components coordinate the activation of tumor suppressors p53 and WOX1 (also named WWOX or FOR) and kinases ERK and JNK1 in human prostate DU145 and neuroblastoma SK-N-SH cells. Cells were cultured overnight in 1% normal human serum, or in human serum depleted of an indicated complement protein. Under complement C1q- or C6-free conditions, WOX1 and ERK were mainly present in the cytoplasm without phosphorylation, whereas phosphorylated JNK1 was greatly accumulated in the nuclei. Exogenous C1q rapidly restored the WOX1 activation (with Tyr33 phosphorylation) in less than 2 hr. Without serum complement C9, p53 became activated spontaneously. Notably, exogenous C1q significantly induced apoptosis of WOX1-overexpressing DU145 or SK-N-SH cells, but not vehicle-expressing cells. A dominant negative and Y33R mutant of WOX1 blocked the apoptotic effect. C1q did not enhance p53-mediated apoptosis. By total internal reflection fluorescence (TIRF) and time-lapse microscopy, we determined that C1q induced dynamic formation (or dancing) of WOX1-rich microvilli, which were assembled as clusters in between cells, thus destabilizing cell adherence. These cells then underwent shrinkage, membrane blebbing and death. Remarkably, as determined by immunostaining, benign prostatic hyperplasia and prostate cancer were shown to have a significantly reduced expression of tissue C1q, compared to age-matched normal prostate tissues. In conclusion, complement
PP1-017 IFN-beta pro-apoptotic and antiproliferative activity is superior to IFNalpha in adult T-cell leukemia: Ex vivo response predicts survival Johan Van Weyenbergh, Ricardo Khouri, Daniele Decanine, Kristof Theys, Koen Deforche, Aline Clara Silva, Lourdes Farre, Achilea Bittencourt, Anne-Mieke Vandamme, Poster Presentation I IFN-beta pro-apoptotic and antiproliferative activity is superior to IFN-alpha in adult T-cell leukemia: Ex vivo response predicts survival Johan Van Weyenbergh 1, Ricardo Khouri 1,2, Daniele Decanine 1, Kristof Theys 2, Koen Deforche 2, Aline Clara Silva 1, Lourdes Farre 1, Achilea Bittencourt 3, Anne-Mieke Vandamme 2, 1 CPqGM-FIOCRUZ, Salvador-BA, Brazil, 2 Rega Institute, KU Leuven, Belgium, 3 HUPES-UFBA, Salvador-BA, Brazil Background: Adult T-cell leukemia (ATL) is an aggressive CD4+ leukemia which usually develops several decades after HTLV-1 infection. IFN-alpha in combination with AZT is currently the major treatment option in ATL, but therapeutic success of this combination therapy is limited. No bona fide surrogate markers for survival and/or therapeutic failure are currently available for clinical use in ATL. Methods: Molecular and clinical data (>2 years follow-up) were obtained from thirty ATL patients. We have quantified immunological markers (flow cytometry, proliferation, cytokines and apoptosis-related markers) ex vivo in plasma and in vitro in peripheral blood mononuclear cells. For multivariate analysis, molecular data were combined with clinical data as parameters for Bayesian network learning and classification modeling. Gene expression profiling was performed in IFN-treated primary ATL cells and ATL-derived cell lines. Results: Using univariate analysis, we found that proliferation of primary cells from ATL patients was significantly decreased by both IFNalpha (p < 0.05) and IFN-beta (p < 0.01), but IFN-alpha-induced antiproliferative activity was only significant in mild clinical forms (chronic and smoldering), whereas IFN-