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Downregulation of ATF2 desensitizes oral squamous cell carcinoma to apoptosis
LIPID MEDIATED GENE TRANSFER LIPID MEDIATED GENE TRANSFER
466. Downregulation of ATF2 Desensitizes Oral Squamous Cell Carcinoma to Apoptosis
κβ Decoy Related 465. Delivery of NF-κβ Oligodeoxynucleotides Reduces ProInflammatory Cytokine Responses Associated with Plasmid DNA/Lipid Mediated Gene Transfer to Murine Lungs
Sleiman N. Razzouk,1 Svetlana M. Dolgilevich,1 Dianne C. Duffey.1 1 Otolaryngology, Mount Sinai School of Medicine, New York, NY.
Anusha Varathalingam,1,3 Ian A. Pringle,1,3 Seng H. Cheng,2 Deborah R. Gill,1,3 Stephen C. Hyde.1,3 1 Gene Medicine Group, University of Oxford, Oxford, Oxfordshire, United Kingdom; 2Genzyme Corporation, Cambridge, MA; 3UK Cystic Fibrosis Gene Therapy Consortium, United Kingdom. Non-viral mediated gene transfer is being investigated as an approach for the treatment of lung diseases such as Cystic Fibrosis (CF). However, an important limiting factor is the inflammatory responses generated by such vectors in patients and animal models. Gene delivery of plasmid DNA complexed with cationic lipid to the murine lung results in increased levels of pro-inflammatory cytokines, which includes tumour necrosis factor alpha (TNF-α) (Scheule et al., 1997, Human Gene Therapy, 8: 689-707). Clinical studies have also indicated an inflammatory response following dosing of plasmid DNA/lipid complexes to the lungs of patients with CF (Alton et al., 1999, Lancet, 353 947). It is believed that the inflammation occurs, at least in part, in response to the unmethylated bacterial CpG sequences delivered to the cytoplasm of the target cells which results in the activation of Nuclear Factor κβ (NF-κβ). NF-κβ is a key regulator of inflammatory and immune responses and is essential for the transcription of multiple pro-inflammatory molecules including TNF-α, interleukin 12 (IL-12) and interferons (IFN) and has become a target for anti-inflammatory treatment. Previous studies have employed an NF-κβ decoy strategy using synthetic oligodeoxynucleotides (ODNs) to block the binding of NF-κβ to the promoter region of its targeted genes following systemic delivery of non-viral vectors to the mouse lung. The addition of the oligodeoxynucleotides inhibited TNF-α in a dose-dependent manner (Tan et al., 2002, Molecular Therapy, 804-812). This study investigates the effect of NF-κβ decoy oligodeoxynucleotides on the inflammatory response following topical delivery of plasmid DNA complexed with Genzyme lipid 67 (GL67) to the mouse lung. Female BALB/c mice were dosed intranasally with 40µg of luciferase expressing plasmid pCIKLux and 40µg of synthetic single stranded phosphorothioate ODN, containing an NF-κβ consensus binding sequence (S+) complexed with cationic lipid GL67 in total volume of 100µl. A scrambled form of the ODN (S11), without a consenus NF-κβ binding site was used as a control. Mouse lungs were harvested 24hrs after dosing and luciferase activity and proinflammatory cytokine levels were measured. With topical delivery of the S+/pCIKLux/GL67 complex, there were 80% and 70% reductions in TNF-α (ANOVA with Fisher’s PLSD, P=0.0001) and IFN-γ (P=0.004) respectively compared to dosing with pCIKLux/ GL67. Furthermore, reporter gene expression was retained at maximum levels after co-delivery of the S+ ODN (ANOVA, P=0.820). This type of approach may allow reduced inflammation in the lung following delivery of GL67 complexes. Reduction in inflammation may also lead to improved duration of transgene expression by avoiding cytokine induced promoter attenuation. Further experiments are in progress to detect cytokine levels induced by intransanal delivery of other gene transfer formulations codelivered with NF-κβ ODNs.
Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright © The American Society of Gene Therapy
Introduction: Oral squamous cell carcinomas (OSCC) are aggressive cancer types because of their invasiveness,ability to metastasize locoregionally, and relative resistance to nonsurgical therapies. Transcription factors are molecular targets with known importance in mechanisms of tumorigenesis of OSCC. For example, the transcription factors NF-kB and AP-1 are upregulated in OSCC in a constitutive fashion. This activation correlates functionally with the production of pro-inflammatory cytokines that enhance angiogenesis, mitogenesis, and invasion. ATF2 has been implicated in the regulation of genes that play roles in apoptosis, survival and tumor progression in other cell systems. It also partners with many transcription factors such as c-Jun to initiate transcriptional activity. Recently, ATF2 has also been identified as an executor of apoptosis in poorly differentiated tumor cells. Here we demonstrate that ATF2 can be downregulated in OSCC using siRNA. We also show that decreasing ATF2 in the human oral tongue carcinoma cell line UMSCC 9 affects resistance of cells to staurosporin-mediated growth inhibition. Methods: UMSCC9 were transfected for 48 hours with different concentrations of siRNA duplex (0-200 nM) corresponding to nucleotides 523-541 of ATF2. SiRNA Cyclophilin B was used as control. Western immunoblotting was performed to confirm effects on baseline levels of ATF2 expressed in treated cells. Cells were cotreated for 2-3 days with staurosporin at different concentrations (0-100 ng/mL). Cell viability was determined using MTT assay. In addition, the transcription activity of ATF2 was measured using Luciferase assay with TRE promoter known to interact with ATF2 and c-Jun. Results: Expression of ATF2 protein was decreased in the presence of siRNA in a dose-dependent manner. A total suppression was observed at 200nM of siRNA showing that siRNA transfection is efficient in affecting gene transcription in OSCC. In addition, the presence of staurosporin induces cell death in a dose-dependent fashion, but siRNA/ATF2-transfected cells showed decreased proliferation by 30% compared to siRNA/CypB-transfected cells. Luciferase measurement showed an increase in TRE-mediated transcription of siRNA/ATF2-transfected cells compared to control cells. These findings indicate that expression of another transcription factor is likely upregulated to compensate for the ATF2 decrease. Conclusion: These findings highlight the role of ATF2 in OSCC baseline transcription and cell survival in the presence of staurosporin. This study also demonstrates that siRNA’s are useful tools in modulating protein expression in OSCC cells.
467. Cationic Liposome-Based Delivery of DNA Modulating Enzymes to Specifically Regulate Gene Expression in Carcinoma Derived Cell Lines Pamela M. J. McLaughlin,1 Monika Trzpis,1 Ieneke T. F. van der Gun,1 Martin C. Harmsen,1 Marcel H. J. Ruiters.1 1 Pathology and Laboratory Medicine; Medical Biology, University Hospital Groningen, Groningen, Netherlands. Gene therapy so far has not fulfilled its promise, mainly because efficiency and specificity of both the gene transferred and the transferring device are not sufficient enough. A new and potentially powerful way to treat diseases is to interfere with the endogenous gene expression of the gene of interest using DNA modifying enzymes, such as restriction enzymes and DNA-methyltransferases. These enzymes are commonly used in a wide range of genomic analysis, and by altering their specificity by coupling of gene-specific S177
466. Downregulation of ATF2 Desensitizes Oral Squamous Cell Carcinoma to Apoptosis
κβ Decoy Related 465. Delivery of NF-κβ Oligodeoxynucleotides Reduces ProInflammatory Cytokine Responses Associated with Plasmid DNA/Lipid Mediated Gene Transfer to Murine Lungs
Sleiman N. Razzouk,1 Svetlana M. Dolgilevich,1 Dianne C. Duffey.1 1 Otolaryngology, Mount Sinai School of Medicine, New York, NY.
Anusha Varathalingam,1,3 Ian A. Pringle,1,3 Seng H. Cheng,2 Deborah R. Gill,1,3 Stephen C. Hyde.1,3 1 Gene Medicine Group, University of Oxford, Oxford, Oxfordshire, United Kingdom; 2Genzyme Corporation, Cambridge, MA; 3UK Cystic Fibrosis Gene Therapy Consortium, United Kingdom. Non-viral mediated gene transfer is being investigated as an approach for the treatment of lung diseases such as Cystic Fibrosis (CF). However, an important limiting factor is the inflammatory responses generated by such vectors in patients and animal models. Gene delivery of plasmid DNA complexed with cationic lipid to the murine lung results in increased levels of pro-inflammatory cytokines, which includes tumour necrosis factor alpha (TNF-α) (Scheule et al., 1997, Human Gene Therapy, 8: 689-707). Clinical studies have also indicated an inflammatory response following dosing of plasmid DNA/lipid complexes to the lungs of patients with CF (Alton et al., 1999, Lancet, 353 947). It is believed that the inflammation occurs, at least in part, in response to the unmethylated bacterial CpG sequences delivered to the cytoplasm of the target cells which results in the activation of Nuclear Factor κβ (NF-κβ). NF-κβ is a key regulator of inflammatory and immune responses and is essential for the transcription of multiple pro-inflammatory molecules including TNF-α, interleukin 12 (IL-12) and interferons (IFN) and has become a target for anti-inflammatory treatment. Previous studies have employed an NF-κβ decoy strategy using synthetic oligodeoxynucleotides (ODNs) to block the binding of NF-κβ to the promoter region of its targeted genes following systemic delivery of non-viral vectors to the mouse lung. The addition of the oligodeoxynucleotides inhibited TNF-α in a dose-dependent manner (Tan et al., 2002, Molecular Therapy, 804-812). This study investigates the effect of NF-κβ decoy oligodeoxynucleotides on the inflammatory response following topical delivery of plasmid DNA complexed with Genzyme lipid 67 (GL67) to the mouse lung. Female BALB/c mice were dosed intranasally with 40µg of luciferase expressing plasmid pCIKLux and 40µg of synthetic single stranded phosphorothioate ODN, containing an NF-κβ consensus binding sequence (S+) complexed with cationic lipid GL67 in total volume of 100µl. A scrambled form of the ODN (S11), without a consenus NF-κβ binding site was used as a control. Mouse lungs were harvested 24hrs after dosing and luciferase activity and proinflammatory cytokine levels were measured. With topical delivery of the S+/pCIKLux/GL67 complex, there were 80% and 70% reductions in TNF-α (ANOVA with Fisher’s PLSD, P=0.0001) and IFN-γ (P=0.004) respectively compared to dosing with pCIKLux/ GL67. Furthermore, reporter gene expression was retained at maximum levels after co-delivery of the S+ ODN (ANOVA, P=0.820). This type of approach may allow reduced inflammation in the lung following delivery of GL67 complexes. Reduction in inflammation may also lead to improved duration of transgene expression by avoiding cytokine induced promoter attenuation. Further experiments are in progress to detect cytokine levels induced by intransanal delivery of other gene transfer formulations codelivered with NF-κβ ODNs.
Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright © The American Society of Gene Therapy
Introduction: Oral squamous cell carcinomas (OSCC) are aggressive cancer types because of their invasiveness,ability to metastasize locoregionally, and relative resistance to nonsurgical therapies. Transcription factors are molecular targets with known importance in mechanisms of tumorigenesis of OSCC. For example, the transcription factors NF-kB and AP-1 are upregulated in OSCC in a constitutive fashion. This activation correlates functionally with the production of pro-inflammatory cytokines that enhance angiogenesis, mitogenesis, and invasion. ATF2 has been implicated in the regulation of genes that play roles in apoptosis, survival and tumor progression in other cell systems. It also partners with many transcription factors such as c-Jun to initiate transcriptional activity. Recently, ATF2 has also been identified as an executor of apoptosis in poorly differentiated tumor cells. Here we demonstrate that ATF2 can be downregulated in OSCC using siRNA. We also show that decreasing ATF2 in the human oral tongue carcinoma cell line UMSCC 9 affects resistance of cells to staurosporin-mediated growth inhibition. Methods: UMSCC9 were transfected for 48 hours with different concentrations of siRNA duplex (0-200 nM) corresponding to nucleotides 523-541 of ATF2. SiRNA Cyclophilin B was used as control. Western immunoblotting was performed to confirm effects on baseline levels of ATF2 expressed in treated cells. Cells were cotreated for 2-3 days with staurosporin at different concentrations (0-100 ng/mL). Cell viability was determined using MTT assay. In addition, the transcription activity of ATF2 was measured using Luciferase assay with TRE promoter known to interact with ATF2 and c-Jun. Results: Expression of ATF2 protein was decreased in the presence of siRNA in a dose-dependent manner. A total suppression was observed at 200nM of siRNA showing that siRNA transfection is efficient in affecting gene transcription in OSCC. In addition, the presence of staurosporin induces cell death in a dose-dependent fashion, but siRNA/ATF2-transfected cells showed decreased proliferation by 30% compared to siRNA/CypB-transfected cells. Luciferase measurement showed an increase in TRE-mediated transcription of siRNA/ATF2-transfected cells compared to control cells. These findings indicate that expression of another transcription factor is likely upregulated to compensate for the ATF2 decrease. Conclusion: These findings highlight the role of ATF2 in OSCC baseline transcription and cell survival in the presence of staurosporin. This study also demonstrates that siRNA’s are useful tools in modulating protein expression in OSCC cells.
467. Cationic Liposome-Based Delivery of DNA Modulating Enzymes to Specifically Regulate Gene Expression in Carcinoma Derived Cell Lines Pamela M. J. McLaughlin,1 Monika Trzpis,1 Ieneke T. F. van der Gun,1 Martin C. Harmsen,1 Marcel H. J. Ruiters.1 1 Pathology and Laboratory Medicine; Medical Biology, University Hospital Groningen, Groningen, Netherlands. Gene therapy so far has not fulfilled its promise, mainly because efficiency and specificity of both the gene transferred and the transferring device are not sufficient enough. A new and potentially powerful way to treat diseases is to interfere with the endogenous gene expression of the gene of interest using DNA modifying enzymes, such as restriction enzymes and DNA-methyltransferases. These enzymes are commonly used in a wide range of genomic analysis, and by altering their specificity by coupling of gene-specific S177