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538. Study of Radioisotope Uptake and Efflux in NIS Expressing Cells Goes High-Throughput
CANCER-TARGETED GENE & CELL THERAPY II 536. p16-Mediated Inhibition of HypoxiaInduced Cancer Cell Migration
Yi Lu,1 Liyuan Li.1 Pathology and Laboratory Medicine, University of Tennessee Health Science Center, Memphis, TN.
1
One general characteristic of fast-growing solid tumors including breast cancer is the development of intratumoral hypoxia. The activation of hypoxia-inducible factor-1α (HIF-1α) by intratumoral hypoxia stimulates a group of downstream genes including vascular endothelial growth factor (VEGF) that are responsible for tumor malignant progression. Loss of p16 expression occurs in many cancers including breast cancer. Our studies showed that, by ectopic expression via adenoviral-mediated gene transfer, p16 downregulates VEGF gene expression by neutralizing the transactivation of HIF1α, and suppresses breast cancer angiogenesis and metastasis. To investigate whether p16 directly affects one or more aspects of the metastatic cascade, MDA-MB-231 cells infected with lentivirus expressing Tet-on inducible p16 were used to study the p16 effects on growth, adhesion and migration of the breast cancer cells. We found that p16 inhibits MDA-MB-231 cell proliferation (45.6% inhibition) and hypoxia–induced cell migration (36% inhibition), but has no apparent effect on cell adhesion. The p16-mediated inhibition of hypoxia-induced cell migration parallels with p16’s inhibition of HIF1α transactivation activity. These results indicate that p16’s ability to suppress breast tumor metastasis may result from a combined p16mediated actions including inhibition of cell proliferation, migration, and angiogenesis. This study suggests that p16 gene therapy may have clinical potentials to treat breast cancer due to p16’s multiplelevel anti-cancer capabilities: inhibiting proliferation, inducing apoptosis, blocking tumor angiogenesis, inhibiting cell migration and suppressing metastasis.
537. Lentivirus-Mediated DR5 Agonistic Chimeric Antibody Expression for Cancer Gene Therapy
Dexian Zheng,1 Juan Shi,1 Yanxin Liu,1 Shilian Liu.1 Biochem Mol Biol, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China.
1
TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces cancer cell death via death receptors on the target tumor cells without side effect on most normal cells. Up to date, five TRAIL receptors have been found in human, i.e., TRAIL-R1 (DR4) and TRAIL-R2 (DR5), which contain a conserved death domain motif so that named as death receptor (DR), TRAIL-R3 (DcR1), TRAIL-R4 (DcR2), and osteoprotegerin (OPG), which are without an intact death domain (named as decoy receptor, DcR), so that can not transfer any cell death signals. The agonists of death receptors (DR4/DR5) including soluble TRAIL and agonistic antibodies against DR4 or DR5 have been emerging as the anticancer therapeutics in clinical trails in recent years. We previously reported an adeno-associated virus-mediated TRAIL expression for cancer gene therapy, which demonstrated that rAAV2/5-sTRAIL may provide an effective form of therapy for lung and liver cancer. The rAAV2/5-sTRAIL transduction of the tumors resulted in a statistically significant reduction in lung cancer growth and prolonged survival of the tumor-bearing animals. Intraportal vein injection of rAAV-TRAIL into the liver of an orthotopic transplanted mouse model mimicking liver cancer metastasis also resulted in a significant suppression of tumor growth and prolonged survival, while toxicity to normal hepatocyte was not detected. These results strongly suggest that rAAVTRAIL- mediated gene delivery could be a promising approach for the treatment of lung and liver cancers. Since high cost of engineering antibody limits the application of antibody therapy, an alternative approach is to produce antibody in vivo, which can provide high S206
concentration and full pure antibody for a long period of time. To this end, we have developed AAV-scFv of a mAb, AD5-10, to DR5 for lung and liver cancer therapy and now we report a lentivirus vector-mediated expression of the mouse-human chimeric antibody (Zaptuzumab), by linking the heavy chain and the light chain with 2A/Furin self-processing peptide in an unique open reading frame, with a srong tumoricidal activity to human colon cancer in nude mice. Lentiviral expression of Zaptuzumab showed specific binding activity to its antigen DR5 and possessed a significant apoptosis-inducing activity in various tumor cell lines. The lentivirus-mediated chimeric antibody expression showed a strong apoptosis-inducing activity in vitro and inoculated s.c. human colon HCT116 tumor in nude mice was suppressed significantly in vivo. Moreover, the tumoricidal efficacy of the viral expressed mouse-human chimeric antibody was synergisted by mitomycin C and prolonged the life span of human lung A549 tumor-bearing nude mice. These data suggest that the lentivirus-mediated and 2A peptide-based anti-DR5 chimeric antibody expression may have clinical utility against cancer and may represent rational adjuvant therapies in combination with chemotherapy.
538. Study of Radioisotope Uptake and Efflux in NIS Expressing Cells Goes High-Throughput Patrycja J. Lech,1 David Dingli,1 Stephen J. Russell.1 1 Department of Molecular Medicine, Mayo Clinic, Rochester, MN.
NIS is a Na+/I- symporter that mediates active I- transport into thyroid follicular cells and salivary, gastric, intestinal and mammary tissue. NIS mediated I- uptake in thyroid follicular cells has been successfully exploited for the diagnosis and treatment of thyroid cancer and its metastases using radioiodide for over 50 yrs. The profound clinical impact of NIS as an imaging and therapeutic gene has inspired its utility for the delivery of radiation doses selectively to cancer cells both in vitro and in vivo, where the NIS gene is either endogenously expressed (e.g. breast cancer) or transduced. This strategy is highly translational as it can use both diagnostic (99mTc, 125 124 I, I)) and therapeutic (131I, 186Re, 188Re, 211At) radioisotopes and can enhance current and developing treatment modalities such as radiotherapy, chemoradiotherapy and oncolytic virotherapy. Here we describe a high-throughput platform for NIS mediated radioisotope uptake by NIS expressing cells in a 96 well format. This platform is highly robust and reproducible and can be exploited to study and/ or manipulate the kinetics and electrophysiological properties of NIS. Our current studies are focusing on the mechanism behind 125I efflux following 125I uptake, as a means of increasing radioiodide concentrations retained in NIS expressing cells in order to increase the therapeutic and diagnostic index of this tool. The cell line used in this platform is Mel624 stably transduced with VSV-G pseudotyped lentiviral vector encoding NIS and neomycin (Mel624-NIS). This cell line was chosen for its robustness and ability to remain adhered to the wells following three washes required to remove residual 125I following uptake. The accumulation of 125I in Mel624-NIS cells is 30-50 times higher than control Mel624-NIS cells treated with perchlorate (ClO4-), a known competitive inhibitor of I- accumulation via NIS. Currently, an individual can assay six 96 well plates per day (360–576 wells) taking into account the time it may take to prepare the buffers, dilutions, perform the 125I uptake and quantify the 125I in cells or media using a gamma counter.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
Yi Lu,1 Liyuan Li.1 Pathology and Laboratory Medicine, University of Tennessee Health Science Center, Memphis, TN.
1
One general characteristic of fast-growing solid tumors including breast cancer is the development of intratumoral hypoxia. The activation of hypoxia-inducible factor-1α (HIF-1α) by intratumoral hypoxia stimulates a group of downstream genes including vascular endothelial growth factor (VEGF) that are responsible for tumor malignant progression. Loss of p16 expression occurs in many cancers including breast cancer. Our studies showed that, by ectopic expression via adenoviral-mediated gene transfer, p16 downregulates VEGF gene expression by neutralizing the transactivation of HIF1α, and suppresses breast cancer angiogenesis and metastasis. To investigate whether p16 directly affects one or more aspects of the metastatic cascade, MDA-MB-231 cells infected with lentivirus expressing Tet-on inducible p16 were used to study the p16 effects on growth, adhesion and migration of the breast cancer cells. We found that p16 inhibits MDA-MB-231 cell proliferation (45.6% inhibition) and hypoxia–induced cell migration (36% inhibition), but has no apparent effect on cell adhesion. The p16-mediated inhibition of hypoxia-induced cell migration parallels with p16’s inhibition of HIF1α transactivation activity. These results indicate that p16’s ability to suppress breast tumor metastasis may result from a combined p16mediated actions including inhibition of cell proliferation, migration, and angiogenesis. This study suggests that p16 gene therapy may have clinical potentials to treat breast cancer due to p16’s multiplelevel anti-cancer capabilities: inhibiting proliferation, inducing apoptosis, blocking tumor angiogenesis, inhibiting cell migration and suppressing metastasis.
537. Lentivirus-Mediated DR5 Agonistic Chimeric Antibody Expression for Cancer Gene Therapy
Dexian Zheng,1 Juan Shi,1 Yanxin Liu,1 Shilian Liu.1 Biochem Mol Biol, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China.
1
TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces cancer cell death via death receptors on the target tumor cells without side effect on most normal cells. Up to date, five TRAIL receptors have been found in human, i.e., TRAIL-R1 (DR4) and TRAIL-R2 (DR5), which contain a conserved death domain motif so that named as death receptor (DR), TRAIL-R3 (DcR1), TRAIL-R4 (DcR2), and osteoprotegerin (OPG), which are without an intact death domain (named as decoy receptor, DcR), so that can not transfer any cell death signals. The agonists of death receptors (DR4/DR5) including soluble TRAIL and agonistic antibodies against DR4 or DR5 have been emerging as the anticancer therapeutics in clinical trails in recent years. We previously reported an adeno-associated virus-mediated TRAIL expression for cancer gene therapy, which demonstrated that rAAV2/5-sTRAIL may provide an effective form of therapy for lung and liver cancer. The rAAV2/5-sTRAIL transduction of the tumors resulted in a statistically significant reduction in lung cancer growth and prolonged survival of the tumor-bearing animals. Intraportal vein injection of rAAV-TRAIL into the liver of an orthotopic transplanted mouse model mimicking liver cancer metastasis also resulted in a significant suppression of tumor growth and prolonged survival, while toxicity to normal hepatocyte was not detected. These results strongly suggest that rAAVTRAIL- mediated gene delivery could be a promising approach for the treatment of lung and liver cancers. Since high cost of engineering antibody limits the application of antibody therapy, an alternative approach is to produce antibody in vivo, which can provide high S206
concentration and full pure antibody for a long period of time. To this end, we have developed AAV-scFv of a mAb, AD5-10, to DR5 for lung and liver cancer therapy and now we report a lentivirus vector-mediated expression of the mouse-human chimeric antibody (Zaptuzumab), by linking the heavy chain and the light chain with 2A/Furin self-processing peptide in an unique open reading frame, with a srong tumoricidal activity to human colon cancer in nude mice. Lentiviral expression of Zaptuzumab showed specific binding activity to its antigen DR5 and possessed a significant apoptosis-inducing activity in various tumor cell lines. The lentivirus-mediated chimeric antibody expression showed a strong apoptosis-inducing activity in vitro and inoculated s.c. human colon HCT116 tumor in nude mice was suppressed significantly in vivo. Moreover, the tumoricidal efficacy of the viral expressed mouse-human chimeric antibody was synergisted by mitomycin C and prolonged the life span of human lung A549 tumor-bearing nude mice. These data suggest that the lentivirus-mediated and 2A peptide-based anti-DR5 chimeric antibody expression may have clinical utility against cancer and may represent rational adjuvant therapies in combination with chemotherapy.
538. Study of Radioisotope Uptake and Efflux in NIS Expressing Cells Goes High-Throughput Patrycja J. Lech,1 David Dingli,1 Stephen J. Russell.1 1 Department of Molecular Medicine, Mayo Clinic, Rochester, MN.
NIS is a Na+/I- symporter that mediates active I- transport into thyroid follicular cells and salivary, gastric, intestinal and mammary tissue. NIS mediated I- uptake in thyroid follicular cells has been successfully exploited for the diagnosis and treatment of thyroid cancer and its metastases using radioiodide for over 50 yrs. The profound clinical impact of NIS as an imaging and therapeutic gene has inspired its utility for the delivery of radiation doses selectively to cancer cells both in vitro and in vivo, where the NIS gene is either endogenously expressed (e.g. breast cancer) or transduced. This strategy is highly translational as it can use both diagnostic (99mTc, 125 124 I, I)) and therapeutic (131I, 186Re, 188Re, 211At) radioisotopes and can enhance current and developing treatment modalities such as radiotherapy, chemoradiotherapy and oncolytic virotherapy. Here we describe a high-throughput platform for NIS mediated radioisotope uptake by NIS expressing cells in a 96 well format. This platform is highly robust and reproducible and can be exploited to study and/ or manipulate the kinetics and electrophysiological properties of NIS. Our current studies are focusing on the mechanism behind 125I efflux following 125I uptake, as a means of increasing radioiodide concentrations retained in NIS expressing cells in order to increase the therapeutic and diagnostic index of this tool. The cell line used in this platform is Mel624 stably transduced with VSV-G pseudotyped lentiviral vector encoding NIS and neomycin (Mel624-NIS). This cell line was chosen for its robustness and ability to remain adhered to the wells following three washes required to remove residual 125I following uptake. The accumulation of 125I in Mel624-NIS cells is 30-50 times higher than control Mel624-NIS cells treated with perchlorate (ClO4-), a known competitive inhibitor of I- accumulation via NIS. Currently, an individual can assay six 96 well plates per day (360–576 wells) taking into account the time it may take to prepare the buffers, dilutions, perform the 125I uptake and quantify the 125I in cells or media using a gamma counter.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy