- Email: [email protected]
263. An Efficient Targeted Gene Therapy Using Brain Tumor-Specific Promoter
CANCER - TARGETED GENE THERAPY I with Ad-∆E1B19/55 plus radiation showed large areas of necrosis and abundant apoptosis with induction of p53. In relative comparison, combination of Ad-∆E1B19/55 and radiation was superior over that of Ad-∆E1B55 and radiation combination treatment. Overall, this study presents strong therapeutic rationale for combination of radiation therapy and E1B 19kDa-deleted oncolytic Ad.
261. Cells
Adenovirus Infection of Epithelial Cancer
Robert Strauss,1 Pavel Sova,1 Ying Liu,1 ZongYi Li,1 Sari Pesonen,2 Akseli Hemminki,2 Pascal Fender,3 Andre Lieber.1 1 Medicine, University of Washington, Seattle, WA; 2Cancer Gene Therapy Group, University of Helsinki, Helsinki, Finland; 3Institut de Biologie Structural, Grenoble, France. The majority of solid cancers are of epithelial origin. A hallmark of epithelial cells are tight and adherens junctions. Tight and adherens junctions seal intercellular spaces and significantly limit the perfusion of anti-tumor agents such as drugs, antibodies, and immune cells within the tumor, thus severely diminishing the efficacy of such therapeutic modalities. The epithelial phenotype of cancer cells also represents a barrier to infection with commonly used adenoviruses that target CD46 or the coxsackie-adenovirus receptor (CAR), due to trapping of these receptors in tight and adherens junctions which explains, in part, why these serotypes were inefficient in cancer gene therapy. We found however, that specific human species B adenoviruses, namely Ad3, Ad7, Ad11, and Ad14 (AdB-group 2) that use a yet unknown receptor (receptor X), which is different from CAR and CD46, efficiently infect epithelial cancer cells. This makes these serotypes potential tools for virotherapy of cancer as well as for gene transfer into normal epithelial tissue. In addition, these serotypes are important pathogens, exemplified by recent outbreaks of a highly pathogenic new Ad14 stain. So far, our studies on mechanisms of AdB-group 2 infection of epithelial cells have shown: i) These Ads use at least two binding moieties on the cell membrane. The C-terminal part of the Ad fiber (the fiber knob) binds to sulfated carbohydrate chains of heparin-sulfate proteoglycans (HSPG). This Ad3knob – interaction with heparin sulfate glucosaminoglycans (HSGAGs) allows for subsequent high affinity attachment and/or access to receptor X, whereby receptor X is either the protein part of the HSPG that interacts with the Ad3 knob or an independent non-HSPG protein. ii) Ad binding to epithelial cells involves both the Ad3 fiber and the Ad3 penton or a composite fiber-penton moeity formed in Ad3 virions. iii) During virus replication, Ad pentons and fibers selfassemble in dodecahedra (PtDd) formed through interaction of 12 penton bases with protruding fibers. These PtDd are thought to disturb tight junctions, thus favoring lateral virus spreading. iv) Recombinant PtDd (produced in insect cells) can enter epithelial cancer cells and can faciliate uptake of Ads (and potentially other tumor agents) for which tight and adherens junctions represent a barrier.
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
262. Serotype Chimeric and Fiber Mutated Adenovirus Ad5/19p-HIT for Targeting Renal Cancer and Untargeting the Liver
Iulia Diaconu,1,2 Laura Denby,3 Sari Pesonen,1,2 Vincenzo Cerullo,1,2 Gerd J. Bauerschmitz,4 Kilian Guse,1,2 Maria Rajecki,1,2 João D. Dias,1,2 Kimmo Taari,5 Anna Kanerva,1,2,6 Andrew H. Baker,3 Akseli Hemminki.1,2 1 Cancer Gene Therapy Group, Molecular Cancer Biology Program & Transplantation Laboratory & Haartman Institute & Finnish Institute for Molecular Medicine, University of Helsinki, Helsinki, Finland; 2HUSLAB, Helsinki University Central Hospital (HUCH), Helsinki, Finland; 3British Heart Foundation Glasgow Cardiovascular Research Center, University of Glasgow, Glasgow, United Kingdom; 4Department of Obstetrics and Gynecology, Heinrich-Heine University, Düsseldorf, Germany; 5Department of Urology, Helsinki University Central Hospital (HUCH), Helsinki, Finland; 6Department of Obstetrics and Gynecology, Helsinki University Central Hospital (HUCH), Helsinki, Finland. Despite some recent advances, patients with advanced renal cell carcinoma (RCC) cannot usually be cured. Alteration of the natural tropism of adenoviruses may permit more specific gene transfer to target tissues. The aim of this study was to utilize novel targeting moieties for adenoviral gene therapy of RCC. Previous work in rats suggested that utilization of Ad5/19p (Ad5 capsid with Ad19p fiber) with kidney vascular targeting moieties HTTHREP (HTT), HITSLLS (HIT) and APASLYN (APA) placed into the fiber knob might be useful for targeting kidney vasculature. Therefore, we sought to investigate the utility of Ad5/19p variants for gene delivery to human RCC cell lines, clinical samples and orthotopic murine models of metastatic RCC. Six different human RCC cell lines were infected with Ad5/19p variants but only Ad5/19p-HIT showed increased transduction, and only in one cell line. Thus, we analyzed human normal and cancerous kidney specimens fresh from patients, which might better mimic the three dimensional architecture of clinical tumors and found that Ad5/19p-HIT showed transduction levels similar to Ad5. In mice, we found that intraperitoneal and intravenous Ad5/19p-HIT transduced tumors at levels comparable to Ad5, while intratumoral Ad5/19p-HIT was even superior to Ad5. Liver tropism was significantly reduced in comparison to Ad5. Improvements in tumor to liver transduction ratios suggested that Ad5/19p-HIT may be promising for systemic gene delivery to kidney tumors.
263. An Efficient Targeted Gene Therapy Using Brain Tumor-Specific Promoter Toshio Yawata,1 Eri Ishida,1 Yu Kawanishi,1 Masakazu Tamura,1 Keiji Shimizu.1 1 Neurosurgery, Kochi Medical School, Nankoku, Kochi, Japan.
Despite many efforts to develop effective therapy, the outcome of malignant glioma remains poor. Gene therapy for this disease using retroviral vector is attractive, because the virus can infect only mitotic cells. Previously, we reported the eradication of mouse glioma by retroviral-mediated gene therapy. In this study, a tumor-specific targeting system was studied to develop the effective and safe gene therapy. We searched for genes expressing at high frequency in brain tumors but not in normal human astrocyte (NHA) among cancer testis antigen (CTA) genes. MAGEA3 and SSX4 were identified as tumor-specific genes. The promoter of both genes was cloned into luciferase reporter vector and the activity was measured in glioma, telomerase-immortalized fibroblast and normal human astrocyte cells. The SSX4 promoter but not MAGEA3 showed the tumorspecific activity. The minimal promoter of SSX4 was defined as a 256 bp fragment upstream of transcriptional start site. In order to define a useful tumor-specific promoter for targeting in context of retroviral-mediated gene therapy, the SSX4 promoter were used to S103
CANCER - TARGETED GENE THERAPY I restrict the expression of suicide gene HSVtk in retroviral vector. The glioma cell lines transduced with the retroviral vector were killed efficiently by addition of ganciclovir (GCV), but telomeraseimmortalized fibroblast BJ-5ta cells were not. Mouse glioma RSV-M cells transduced with the retroviral vector were transplanted into S.C. of syngeneic mouse. The administration of GCV suppressed the tumor growth completely. These results support identification of a tumor-specific promoter capable of providing safe and effective retroviral-mediated gene therapy for malignant glioma.
264. Selective Retargeting of Adenoviral Vector to Metastatic Breast Cancer
Shilpa Bhatia,1 Ana Nedeljkovic-Kurepa,1 Yoshi Odaka,1 Angel A. Rivera,2 Alexander Pereboev,2 David T. Curiel,2 J. Michael Mathis.1 1 Gene Therapy Program, Department of Cellular Biology and Anatomy, LSU Health Sciences Center, Shreveport, LA; 2Division of Human Gene Therapy, Departments of Medicine, Surgery, and Pathology, University of Alabama at Birmingham, Birmingham, AL. The success of gene therapy relies on efficient and targeted delivery systems. Adenovirus vectors have a number of advantages for gene therapy. However, because of their lack of tumor tropism and their tendenacy to induce liver infection following systemic administration, they cannot be used for systemic attack on metastatic disease. The present study addresses this issue by retargeting adenovirus to breast cancer cells over expressing the chemokine receptor CXCR4. Many solid tumors (e.g., colon, lung, and breast) and hematopoietic tumors over express CXCR4. The CXCR4 receptor belongs to the large superfamily of G protein-coupled receptors, and is known to participate in a number of biological processes including organogenesis, hematopoiesis, and immune response. Recent evidence has highlighted the role of CXCR4 in cancer, particularly in metastasis due to dysregulation of the receptor leading to enhanced signaling. We used sCAR-CXCL12, a bispecific adapter molecule with the ectodomain of CAR fused to the human CXCR4 ligand CXCL12 (also known as SDF-1). We hypothesized sCAR-CXCL12 would retarget adenovirus vectors to CXCR4-positive breast cancer metastases. Infectivity assays in the absence and presence of ligand were performed in human breast cancer cells and normal liver tissue. Cells were infected with increasing titers of Ad-CMV-GFPLuc with and without ligand. Forty-eight hours post-infection, cells were harvested and analysed for GFP expression by fluorescence microscopy and flow cytometry. Time-dependence of infectivity was also determined by incubating cells with optimum titer of AdCMV-GFP-Luc vector in the presence of retargeting ligand. The binding specificity of the adapter protein was determined by indirect ELISA assay. In vitro, ELISA assay demonstrated strong binding of the sCAR-CXCL12 bispecific adapter molecule both to Ad5 fiber protein and CXCR4 receptor. In human breast cancer cells, a marked enhancement of infectivity was observed using an sCAR-CXCL12 retargeted adenovirus compared to an untargeted vector both in a dose- and time-dependent manner. In contrast, an sCAR-CXCL12 retargeted adenovirus showed a decreased infectivity in normal liver tissue compared to an untargeted vector. In this study, we report that sCAR-CXCL12 can dramatically redirect an adenoviral gene therapy vector to CXCR4-positive breast cancer cells. This bispecific adapter protein should, therefore, be a powerful agent to retarget adenovirus vectors to tumor metastases. Our future goal is to investigate the capacity of this agent to re-direct adenoviral vectors in vivo using breast cancer metastasis models.
265. Impact of Acid Ceramidase Expression on Head and Neck Cancer Proliferation, a Novel Target for Head and Neck Gene Therapy
Xiang Liu,1 Sarah Tucker Marrison,1 William D. Meacham,1 Alex McPherson,1 Joe Cheng,1 Lorianne S. Turner,1 Terry Day,3 Yusuf A. Hannun,2 James S. Norris.1 1 Microbiology and Immunology, Medical University of South Carolina, Charleston, SC; 2Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC; 3Otolaryngology, Medical University of South Carolina, Charleston, SC. Within the complex network of intracellular signals that control cell properties and fate, ceramide has been recognized as a crucial lipid second messenger with anti-proliferative and pro-apoptotic activities. Our preliminary data indicated that 70% of head and neck (H&N) tumors have up-regulated acid ceramidase (AC) expression compared with normal tissue, suggesting unscheduled ceramide metabolism is operative during tumor progression. AC is a lysosomal enzyme that deacylates ceramide to generate sphingosine. This lipid is the substrate for sphingosine kinase-1 (SK1), that forms the potent mitogen S1P which has anti-apoptotic and pro-proliferative functions. AC is the central regulator of the balance of the cellular levels of ceramide, sphingosine, and S1P, and is integral in determining cell growth or death. In this study, we examined the role of AC and its impact on cell proliferation in H&N cancers. SCC1, SCC14A, and SCC22, three independent H&N cancer cell lines with differing levels of AC, were examined and their proliferative capacity were measured. As demonstrated by MTS assay, cell lines characterized with higher AC expression by Western blotting exhibited greater proliferative capability. Decreased ceramide levels were seen in SCC14A consistent with its higher endogenous AC level and greater cell growth. Moreover, cell lines generated from metastatic H&N tumor tissues had higher AC levels compared with cell lines generated from primary H&N tumors isolated from the same patient. They grew faster especially under stress conditions such as when cultured in low-glucose media. Down-regulation of AC by siRNA in these cells resulted in reduced cell growth confirming the function of AC in H&N cancer proliferation. In order to study the association of AC expression with cell proliferation, we over-expressed AC in the H&N cancer cell line SCC1. Cells over-expressing AC exhibited increased cell proliferation compared with control cells and have altered ceramide levels. While examining gene expression profiles in AC over-expressing cells our microarray data indicated that expression of AC resulted in significant specific up-regulation of a small group of genes involved in oncogenesis and tumor cell proliferation including TGFA, IL1, IL8, FOSL1, HB-EGF, DUSP6, FGF2 and LIF. This gives additional support on our observation that AC over-expression promotes cell proliferation activities. Taken together, our data indicate that H&N cancer cells over-expressing AC exhibit aberrant ceramide metabolism and gene regulation that ultimately facilitates enhanced tumor progression. As the increased capacity of cells to proliferate impacts tumor malignancy and survival, understanding the role of AC in cell proliferation and tumorigenesis could greatly impact head and neck cancer diagnosis and treatment. Supported by NIH/NCI PO1 CA97132.
266. Design and Characterization of Catalytic Oligonucleotides That Can Cleave Lukemogenic Chimeric RNAs
Bo-Ra Choi,1 Woo-Hyung Choi,1 Se-Ho Kim,1 Dong-Eun Kim.1 Bioscience and Biotechnology, Konkuk University, Seoul, Korea, Republic of.
1
RNA-cleaving oligonucleotides such as hammerhead ribozymes and deoxyoligoribozyme were designed to abolish an expression S104
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
261. Cells
Adenovirus Infection of Epithelial Cancer
Robert Strauss,1 Pavel Sova,1 Ying Liu,1 ZongYi Li,1 Sari Pesonen,2 Akseli Hemminki,2 Pascal Fender,3 Andre Lieber.1 1 Medicine, University of Washington, Seattle, WA; 2Cancer Gene Therapy Group, University of Helsinki, Helsinki, Finland; 3Institut de Biologie Structural, Grenoble, France. The majority of solid cancers are of epithelial origin. A hallmark of epithelial cells are tight and adherens junctions. Tight and adherens junctions seal intercellular spaces and significantly limit the perfusion of anti-tumor agents such as drugs, antibodies, and immune cells within the tumor, thus severely diminishing the efficacy of such therapeutic modalities. The epithelial phenotype of cancer cells also represents a barrier to infection with commonly used adenoviruses that target CD46 or the coxsackie-adenovirus receptor (CAR), due to trapping of these receptors in tight and adherens junctions which explains, in part, why these serotypes were inefficient in cancer gene therapy. We found however, that specific human species B adenoviruses, namely Ad3, Ad7, Ad11, and Ad14 (AdB-group 2) that use a yet unknown receptor (receptor X), which is different from CAR and CD46, efficiently infect epithelial cancer cells. This makes these serotypes potential tools for virotherapy of cancer as well as for gene transfer into normal epithelial tissue. In addition, these serotypes are important pathogens, exemplified by recent outbreaks of a highly pathogenic new Ad14 stain. So far, our studies on mechanisms of AdB-group 2 infection of epithelial cells have shown: i) These Ads use at least two binding moieties on the cell membrane. The C-terminal part of the Ad fiber (the fiber knob) binds to sulfated carbohydrate chains of heparin-sulfate proteoglycans (HSPG). This Ad3knob – interaction with heparin sulfate glucosaminoglycans (HSGAGs) allows for subsequent high affinity attachment and/or access to receptor X, whereby receptor X is either the protein part of the HSPG that interacts with the Ad3 knob or an independent non-HSPG protein. ii) Ad binding to epithelial cells involves both the Ad3 fiber and the Ad3 penton or a composite fiber-penton moeity formed in Ad3 virions. iii) During virus replication, Ad pentons and fibers selfassemble in dodecahedra (PtDd) formed through interaction of 12 penton bases with protruding fibers. These PtDd are thought to disturb tight junctions, thus favoring lateral virus spreading. iv) Recombinant PtDd (produced in insect cells) can enter epithelial cancer cells and can faciliate uptake of Ads (and potentially other tumor agents) for which tight and adherens junctions represent a barrier.
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
262. Serotype Chimeric and Fiber Mutated Adenovirus Ad5/19p-HIT for Targeting Renal Cancer and Untargeting the Liver
Iulia Diaconu,1,2 Laura Denby,3 Sari Pesonen,1,2 Vincenzo Cerullo,1,2 Gerd J. Bauerschmitz,4 Kilian Guse,1,2 Maria Rajecki,1,2 João D. Dias,1,2 Kimmo Taari,5 Anna Kanerva,1,2,6 Andrew H. Baker,3 Akseli Hemminki.1,2 1 Cancer Gene Therapy Group, Molecular Cancer Biology Program & Transplantation Laboratory & Haartman Institute & Finnish Institute for Molecular Medicine, University of Helsinki, Helsinki, Finland; 2HUSLAB, Helsinki University Central Hospital (HUCH), Helsinki, Finland; 3British Heart Foundation Glasgow Cardiovascular Research Center, University of Glasgow, Glasgow, United Kingdom; 4Department of Obstetrics and Gynecology, Heinrich-Heine University, Düsseldorf, Germany; 5Department of Urology, Helsinki University Central Hospital (HUCH), Helsinki, Finland; 6Department of Obstetrics and Gynecology, Helsinki University Central Hospital (HUCH), Helsinki, Finland. Despite some recent advances, patients with advanced renal cell carcinoma (RCC) cannot usually be cured. Alteration of the natural tropism of adenoviruses may permit more specific gene transfer to target tissues. The aim of this study was to utilize novel targeting moieties for adenoviral gene therapy of RCC. Previous work in rats suggested that utilization of Ad5/19p (Ad5 capsid with Ad19p fiber) with kidney vascular targeting moieties HTTHREP (HTT), HITSLLS (HIT) and APASLYN (APA) placed into the fiber knob might be useful for targeting kidney vasculature. Therefore, we sought to investigate the utility of Ad5/19p variants for gene delivery to human RCC cell lines, clinical samples and orthotopic murine models of metastatic RCC. Six different human RCC cell lines were infected with Ad5/19p variants but only Ad5/19p-HIT showed increased transduction, and only in one cell line. Thus, we analyzed human normal and cancerous kidney specimens fresh from patients, which might better mimic the three dimensional architecture of clinical tumors and found that Ad5/19p-HIT showed transduction levels similar to Ad5. In mice, we found that intraperitoneal and intravenous Ad5/19p-HIT transduced tumors at levels comparable to Ad5, while intratumoral Ad5/19p-HIT was even superior to Ad5. Liver tropism was significantly reduced in comparison to Ad5. Improvements in tumor to liver transduction ratios suggested that Ad5/19p-HIT may be promising for systemic gene delivery to kidney tumors.
263. An Efficient Targeted Gene Therapy Using Brain Tumor-Specific Promoter Toshio Yawata,1 Eri Ishida,1 Yu Kawanishi,1 Masakazu Tamura,1 Keiji Shimizu.1 1 Neurosurgery, Kochi Medical School, Nankoku, Kochi, Japan.
Despite many efforts to develop effective therapy, the outcome of malignant glioma remains poor. Gene therapy for this disease using retroviral vector is attractive, because the virus can infect only mitotic cells. Previously, we reported the eradication of mouse glioma by retroviral-mediated gene therapy. In this study, a tumor-specific targeting system was studied to develop the effective and safe gene therapy. We searched for genes expressing at high frequency in brain tumors but not in normal human astrocyte (NHA) among cancer testis antigen (CTA) genes. MAGEA3 and SSX4 were identified as tumor-specific genes. The promoter of both genes was cloned into luciferase reporter vector and the activity was measured in glioma, telomerase-immortalized fibroblast and normal human astrocyte cells. The SSX4 promoter but not MAGEA3 showed the tumorspecific activity. The minimal promoter of SSX4 was defined as a 256 bp fragment upstream of transcriptional start site. In order to define a useful tumor-specific promoter for targeting in context of retroviral-mediated gene therapy, the SSX4 promoter were used to S103
CANCER - TARGETED GENE THERAPY I restrict the expression of suicide gene HSVtk in retroviral vector. The glioma cell lines transduced with the retroviral vector were killed efficiently by addition of ganciclovir (GCV), but telomeraseimmortalized fibroblast BJ-5ta cells were not. Mouse glioma RSV-M cells transduced with the retroviral vector were transplanted into S.C. of syngeneic mouse. The administration of GCV suppressed the tumor growth completely. These results support identification of a tumor-specific promoter capable of providing safe and effective retroviral-mediated gene therapy for malignant glioma.
264. Selective Retargeting of Adenoviral Vector to Metastatic Breast Cancer
Shilpa Bhatia,1 Ana Nedeljkovic-Kurepa,1 Yoshi Odaka,1 Angel A. Rivera,2 Alexander Pereboev,2 David T. Curiel,2 J. Michael Mathis.1 1 Gene Therapy Program, Department of Cellular Biology and Anatomy, LSU Health Sciences Center, Shreveport, LA; 2Division of Human Gene Therapy, Departments of Medicine, Surgery, and Pathology, University of Alabama at Birmingham, Birmingham, AL. The success of gene therapy relies on efficient and targeted delivery systems. Adenovirus vectors have a number of advantages for gene therapy. However, because of their lack of tumor tropism and their tendenacy to induce liver infection following systemic administration, they cannot be used for systemic attack on metastatic disease. The present study addresses this issue by retargeting adenovirus to breast cancer cells over expressing the chemokine receptor CXCR4. Many solid tumors (e.g., colon, lung, and breast) and hematopoietic tumors over express CXCR4. The CXCR4 receptor belongs to the large superfamily of G protein-coupled receptors, and is known to participate in a number of biological processes including organogenesis, hematopoiesis, and immune response. Recent evidence has highlighted the role of CXCR4 in cancer, particularly in metastasis due to dysregulation of the receptor leading to enhanced signaling. We used sCAR-CXCL12, a bispecific adapter molecule with the ectodomain of CAR fused to the human CXCR4 ligand CXCL12 (also known as SDF-1). We hypothesized sCAR-CXCL12 would retarget adenovirus vectors to CXCR4-positive breast cancer metastases. Infectivity assays in the absence and presence of ligand were performed in human breast cancer cells and normal liver tissue. Cells were infected with increasing titers of Ad-CMV-GFPLuc with and without ligand. Forty-eight hours post-infection, cells were harvested and analysed for GFP expression by fluorescence microscopy and flow cytometry. Time-dependence of infectivity was also determined by incubating cells with optimum titer of AdCMV-GFP-Luc vector in the presence of retargeting ligand. The binding specificity of the adapter protein was determined by indirect ELISA assay. In vitro, ELISA assay demonstrated strong binding of the sCAR-CXCL12 bispecific adapter molecule both to Ad5 fiber protein and CXCR4 receptor. In human breast cancer cells, a marked enhancement of infectivity was observed using an sCAR-CXCL12 retargeted adenovirus compared to an untargeted vector both in a dose- and time-dependent manner. In contrast, an sCAR-CXCL12 retargeted adenovirus showed a decreased infectivity in normal liver tissue compared to an untargeted vector. In this study, we report that sCAR-CXCL12 can dramatically redirect an adenoviral gene therapy vector to CXCR4-positive breast cancer cells. This bispecific adapter protein should, therefore, be a powerful agent to retarget adenovirus vectors to tumor metastases. Our future goal is to investigate the capacity of this agent to re-direct adenoviral vectors in vivo using breast cancer metastasis models.
265. Impact of Acid Ceramidase Expression on Head and Neck Cancer Proliferation, a Novel Target for Head and Neck Gene Therapy
Xiang Liu,1 Sarah Tucker Marrison,1 William D. Meacham,1 Alex McPherson,1 Joe Cheng,1 Lorianne S. Turner,1 Terry Day,3 Yusuf A. Hannun,2 James S. Norris.1 1 Microbiology and Immunology, Medical University of South Carolina, Charleston, SC; 2Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC; 3Otolaryngology, Medical University of South Carolina, Charleston, SC. Within the complex network of intracellular signals that control cell properties and fate, ceramide has been recognized as a crucial lipid second messenger with anti-proliferative and pro-apoptotic activities. Our preliminary data indicated that 70% of head and neck (H&N) tumors have up-regulated acid ceramidase (AC) expression compared with normal tissue, suggesting unscheduled ceramide metabolism is operative during tumor progression. AC is a lysosomal enzyme that deacylates ceramide to generate sphingosine. This lipid is the substrate for sphingosine kinase-1 (SK1), that forms the potent mitogen S1P which has anti-apoptotic and pro-proliferative functions. AC is the central regulator of the balance of the cellular levels of ceramide, sphingosine, and S1P, and is integral in determining cell growth or death. In this study, we examined the role of AC and its impact on cell proliferation in H&N cancers. SCC1, SCC14A, and SCC22, three independent H&N cancer cell lines with differing levels of AC, were examined and their proliferative capacity were measured. As demonstrated by MTS assay, cell lines characterized with higher AC expression by Western blotting exhibited greater proliferative capability. Decreased ceramide levels were seen in SCC14A consistent with its higher endogenous AC level and greater cell growth. Moreover, cell lines generated from metastatic H&N tumor tissues had higher AC levels compared with cell lines generated from primary H&N tumors isolated from the same patient. They grew faster especially under stress conditions such as when cultured in low-glucose media. Down-regulation of AC by siRNA in these cells resulted in reduced cell growth confirming the function of AC in H&N cancer proliferation. In order to study the association of AC expression with cell proliferation, we over-expressed AC in the H&N cancer cell line SCC1. Cells over-expressing AC exhibited increased cell proliferation compared with control cells and have altered ceramide levels. While examining gene expression profiles in AC over-expressing cells our microarray data indicated that expression of AC resulted in significant specific up-regulation of a small group of genes involved in oncogenesis and tumor cell proliferation including TGFA, IL1, IL8, FOSL1, HB-EGF, DUSP6, FGF2 and LIF. This gives additional support on our observation that AC over-expression promotes cell proliferation activities. Taken together, our data indicate that H&N cancer cells over-expressing AC exhibit aberrant ceramide metabolism and gene regulation that ultimately facilitates enhanced tumor progression. As the increased capacity of cells to proliferate impacts tumor malignancy and survival, understanding the role of AC in cell proliferation and tumorigenesis could greatly impact head and neck cancer diagnosis and treatment. Supported by NIH/NCI PO1 CA97132.
266. Design and Characterization of Catalytic Oligonucleotides That Can Cleave Lukemogenic Chimeric RNAs
Bo-Ra Choi,1 Woo-Hyung Choi,1 Se-Ho Kim,1 Dong-Eun Kim.1 Bioscience and Biotechnology, Konkuk University, Seoul, Korea, Republic of.
1
RNA-cleaving oligonucleotides such as hammerhead ribozymes and deoxyoligoribozyme were designed to abolish an expression S104
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy