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Anti-cancer gene therapy using genetically modified MSCs expressing HSV-TK with controlled gene expression by pro-drug GCV
ABSTRACTS / Bone 43 (2008) S38–S75
PP2, or two independent Src kinase-dead mutants prevented Src activation and CTGF induction by TGF-beta1. The inhibition of Src prevented TGF-beta1 induced Smad 2 & 3 activation and Smad nuclear translocation in osteoblasts. In addition, inhibiting Src prevented the Erk activation. When we treated cells with the Erk inhibitor, PD98059, it inhibited TGF-beta1-induced CTGF promoter activity and protein expression but had no effect on Smad activation or Smad nuclear translocation. Using electro-mobility shift assays we found that treatment with PD98059 impaired transcriptional complex formation on the Smad binding element (SBE) of the CTGF promoter, demonstrating that Erk activation was required for SBE transactivation. Conclusion: Src is an essential upstream signaling partner of both Erk and Smads for TGF-beta1 induction of CTGF in osteoblasts, and that Src and Erk have independent effects on Smad signaling required for the formation of a transcriptionally active complex on the CTGF promoter. doi:10.1016/j.bone.20 08.08.011
A10 Anti-cancer gene therapy using genetically modified MSCs expressing HSV-TK with controlled gene expression by pro-drug GCV Chao Song, Zhen Wang, Juanjuan Xiang, Jingqun Tang, David Hirst, Gang Li Department of Orthopedics, Queens University Medical College, Belfast, UK Department of Orthopedics, Hong Kong Chinese University, China Introduction: Mesenchymal stem cells (MSCs) are capable of homing to many tissues after injuries and participate in the healing processes. Cancer/tumour development shares many common characteristics with the wound healing process, and cancer/tumour development has been long regarded as wounds that never heal. In this study we investigated the distribution of systemically delivered MSCs in tumor bearing animals and evaluated the anti-tumour effects of HSV-TK (herpes simplex virus thymidine kinase) gene modified MSCs in vitro and in vivo. Methods: MSCs cells were obtained from the bone marrows of GFP transgenic rats, cultured and characterized as previously described. MSCs were transfected with luciferase or HSV-TK gene by lentiviral vector. The cytotoxic effects of HSV-TK-MSCs on the tumor cells were confirmed by in vitro co-culture experiments with tumor cell lines. PC3 (human prostate cancer) or RIF-1 (mouse myofibroma cell line) cells were used to establish tumor bearing animal models in nude (intra-dermal tumor model) or C3H-HEN (lung metastasis model) mice. 1 × 106 luciferase-MSCs were injected through the tail vein into the tumor bearing animals. The distributions of luciferase-MSCs in both tumor models were confirmed using in vivo image system (IVIS 200, Xenogen, USA) and immunostaining methods. To test if HSV-TK-MSCs can inhibit tumor growth following their homing into tumours, Luciferase-PC3 intra-dermal model and luciferase-RIF-1 lung metastasis model were injected intravenously with 1 × 106 HSV-TK-MSCs twice (day 3 and day 11) and the pro-drug GCV was injected to activate the suicide gene HSV-TK between days 4-9 and days 11-16. Tumor size (intra-dermal tumor model) or density of luciferase in the lung were measured and compared. Results: 72 hours after the luciferase-MSCs injection, MSCs were found in the tumor sites and they were present till 30 days after, demonstrated by both in vivo imaging and immunohistochemistry. The rate of tumor growth in the HSV-TK experimental group is
S41
much lower than the other groups, significant inhibition of tumor growth up to 41.27% was observed in PC3 subcutaneous tumor model by day 25, and up to 98.44% in RIF-1 lung metastasis model by day 27. No obvious side effect was observed in the animals that received MSCs-TK/GCV treatment. Conclusion: Bone marrow MSCs have the capability to home to the tumor microenvironments. MSCs transduced by suicide gene system (HSV-TK), which the expression is controlled, may be used for site and time specific anti-cancer gene therapy. doi:10.1016/j.bone.20 08.08.012
A11 Effects of silencing connective tissue growth factor (CTGF) by RNA interference on expression of matrix metalloproteinase-1 and -2 in human osteoblasts Yiqun Peng, Guoliang Sui, Eryuan Liao Endocrinology Institute, Xiaya Second Hospital, Zhongnan University, Department of Endocrinology, Yantaishan Hospital, Yantai, Shandong, China Objective: Our previous study found that estrogen could inhibit the connective tissue growth factor (CTGF) expression in human osteoblasts, but so little is known about its biological effects. To observe the effects of down-regulating CTGF on the expression of alkaline phosphatase, matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-2 (MMP-2) in human osteoblasts, as well as the effects of inhibiting endogenous CTGF expression on the activity of human osteoblasts, we knockdown CTGF expression selectively by small interference RNA. Methods: Three siRNAs (siRNA1, siRNA2, siRNA3) targeting 3 locus of human CTGF mRNA (440, 875, 910) specifically were designed, and were transfected into the human osteoblasts mediated by cationic liposome, using the blank and non-specific siRNA as controls. The cells were collected after transfection with siRNAs for 48 h. The expression level of CTGF mRNA was detected by Northern blotting, while ALP, MMP-1, MMP-2 were examined by real time-PCR. The protein expression of CTGF, MMP-1, MMP-2 were determined by Western blotting and the activity of human osteoblasts was demonstrated by MTT. Results: After being synthesized by transcription in vitro, siRNA1 (corresponding to 440 site) and siRNA3 (corresponding to 910 site) can inhibit mRNA and protein expression of CTGF when transferred into human osteoblasts, and siRNA1 downregulated these expression considerably more, with 70% of depressant effect on protein expression of CTGF, whereas siRNA2 (corresponding to 875 site) had no significant role in CTGF expression. With down-regulation of CTGF expression, mRNA expression of ALP and MMP-1, and MMP-1 protein expression were reduced significantly, but without significant change in MMP-2 mRNA and protein expression. Meanwhile, we found that the survival rates of osteoblasts in siRNA1 group and siRNA3 group were decreased significantly after CTGF expression was inhibited. Conclusion: The downregulated expression of CTGF can inhibit mRNA expression of ALP and MMP-1, reduce MMP-1 protein expression and cell activity, rather than MMP-2. It suggests that CTGF be a kind of necessary cytokines maintaining survival and differentiation of human osteoblasts, and play an important role in the maintening balance in bone metabolism by regulating MMPs expression. doi:10.1016/j.bone.20 08.08.013
PP2, or two independent Src kinase-dead mutants prevented Src activation and CTGF induction by TGF-beta1. The inhibition of Src prevented TGF-beta1 induced Smad 2 & 3 activation and Smad nuclear translocation in osteoblasts. In addition, inhibiting Src prevented the Erk activation. When we treated cells with the Erk inhibitor, PD98059, it inhibited TGF-beta1-induced CTGF promoter activity and protein expression but had no effect on Smad activation or Smad nuclear translocation. Using electro-mobility shift assays we found that treatment with PD98059 impaired transcriptional complex formation on the Smad binding element (SBE) of the CTGF promoter, demonstrating that Erk activation was required for SBE transactivation. Conclusion: Src is an essential upstream signaling partner of both Erk and Smads for TGF-beta1 induction of CTGF in osteoblasts, and that Src and Erk have independent effects on Smad signaling required for the formation of a transcriptionally active complex on the CTGF promoter. doi:10.1016/j.bone.20 08.08.011
A10 Anti-cancer gene therapy using genetically modified MSCs expressing HSV-TK with controlled gene expression by pro-drug GCV Chao Song, Zhen Wang, Juanjuan Xiang, Jingqun Tang, David Hirst, Gang Li Department of Orthopedics, Queens University Medical College, Belfast, UK Department of Orthopedics, Hong Kong Chinese University, China Introduction: Mesenchymal stem cells (MSCs) are capable of homing to many tissues after injuries and participate in the healing processes. Cancer/tumour development shares many common characteristics with the wound healing process, and cancer/tumour development has been long regarded as wounds that never heal. In this study we investigated the distribution of systemically delivered MSCs in tumor bearing animals and evaluated the anti-tumour effects of HSV-TK (herpes simplex virus thymidine kinase) gene modified MSCs in vitro and in vivo. Methods: MSCs cells were obtained from the bone marrows of GFP transgenic rats, cultured and characterized as previously described. MSCs were transfected with luciferase or HSV-TK gene by lentiviral vector. The cytotoxic effects of HSV-TK-MSCs on the tumor cells were confirmed by in vitro co-culture experiments with tumor cell lines. PC3 (human prostate cancer) or RIF-1 (mouse myofibroma cell line) cells were used to establish tumor bearing animal models in nude (intra-dermal tumor model) or C3H-HEN (lung metastasis model) mice. 1 × 106 luciferase-MSCs were injected through the tail vein into the tumor bearing animals. The distributions of luciferase-MSCs in both tumor models were confirmed using in vivo image system (IVIS 200, Xenogen, USA) and immunostaining methods. To test if HSV-TK-MSCs can inhibit tumor growth following their homing into tumours, Luciferase-PC3 intra-dermal model and luciferase-RIF-1 lung metastasis model were injected intravenously with 1 × 106 HSV-TK-MSCs twice (day 3 and day 11) and the pro-drug GCV was injected to activate the suicide gene HSV-TK between days 4-9 and days 11-16. Tumor size (intra-dermal tumor model) or density of luciferase in the lung were measured and compared. Results: 72 hours after the luciferase-MSCs injection, MSCs were found in the tumor sites and they were present till 30 days after, demonstrated by both in vivo imaging and immunohistochemistry. The rate of tumor growth in the HSV-TK experimental group is
S41
much lower than the other groups, significant inhibition of tumor growth up to 41.27% was observed in PC3 subcutaneous tumor model by day 25, and up to 98.44% in RIF-1 lung metastasis model by day 27. No obvious side effect was observed in the animals that received MSCs-TK/GCV treatment. Conclusion: Bone marrow MSCs have the capability to home to the tumor microenvironments. MSCs transduced by suicide gene system (HSV-TK), which the expression is controlled, may be used for site and time specific anti-cancer gene therapy. doi:10.1016/j.bone.20 08.08.012
A11 Effects of silencing connective tissue growth factor (CTGF) by RNA interference on expression of matrix metalloproteinase-1 and -2 in human osteoblasts Yiqun Peng, Guoliang Sui, Eryuan Liao Endocrinology Institute, Xiaya Second Hospital, Zhongnan University, Department of Endocrinology, Yantaishan Hospital, Yantai, Shandong, China Objective: Our previous study found that estrogen could inhibit the connective tissue growth factor (CTGF) expression in human osteoblasts, but so little is known about its biological effects. To observe the effects of down-regulating CTGF on the expression of alkaline phosphatase, matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-2 (MMP-2) in human osteoblasts, as well as the effects of inhibiting endogenous CTGF expression on the activity of human osteoblasts, we knockdown CTGF expression selectively by small interference RNA. Methods: Three siRNAs (siRNA1, siRNA2, siRNA3) targeting 3 locus of human CTGF mRNA (440, 875, 910) specifically were designed, and were transfected into the human osteoblasts mediated by cationic liposome, using the blank and non-specific siRNA as controls. The cells were collected after transfection with siRNAs for 48 h. The expression level of CTGF mRNA was detected by Northern blotting, while ALP, MMP-1, MMP-2 were examined by real time-PCR. The protein expression of CTGF, MMP-1, MMP-2 were determined by Western blotting and the activity of human osteoblasts was demonstrated by MTT. Results: After being synthesized by transcription in vitro, siRNA1 (corresponding to 440 site) and siRNA3 (corresponding to 910 site) can inhibit mRNA and protein expression of CTGF when transferred into human osteoblasts, and siRNA1 downregulated these expression considerably more, with 70% of depressant effect on protein expression of CTGF, whereas siRNA2 (corresponding to 875 site) had no significant role in CTGF expression. With down-regulation of CTGF expression, mRNA expression of ALP and MMP-1, and MMP-1 protein expression were reduced significantly, but without significant change in MMP-2 mRNA and protein expression. Meanwhile, we found that the survival rates of osteoblasts in siRNA1 group and siRNA3 group were decreased significantly after CTGF expression was inhibited. Conclusion: The downregulated expression of CTGF can inhibit mRNA expression of ALP and MMP-1, reduce MMP-1 protein expression and cell activity, rather than MMP-2. It suggests that CTGF be a kind of necessary cytokines maintaining survival and differentiation of human osteoblasts, and play an important role in the maintening balance in bone metabolism by regulating MMPs expression. doi:10.1016/j.bone.20 08.08.013