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366. Prostate-Specific Expression of Human Coxsackie Adenovirus Receptor (hCAR) in Transgenic Mice
GENE REGULATION: IN VIVO APPLICATIONS expression has a therapeutic effect on hyperglycemia in various diabetic rodents resulting increased serum adiponectin levels. And yet an immediate hypoglycemic effect of adiponectin in NOD mice and STZ-induced diabetic rats needs further studies that may provide adiponectin has an alternative action to regulate hyperglycemia.
366. Prostate-Specific Expression of Human Coxsackie Adenovirus Receptor (hCAR) in Transgenic Mice Yunhua Bao,1 Weidan Peng,1 Amy Verbitsky,1 Jipeng Chen,1 Lily Wu,2 Katherine Rauen,3 Janet Sawicki.1 1 Lankenau Institute for Medical Research, Wynnewood, PA; 2 Department of Urology, UCLA School of Medicine, Los Angeles, CA; 3Cancer Research Institute, University of California, San Francisco, San Francisco, CA. Transgenic mice provide useful animal models with which to test the effectiveness of new therapeutic strategies for the treatment of diseases, including prostate cancer. Their use in testing adenoviralbased gene therapies, however, is limited because of the restricted expression of the Coxsackie adenovirus receptor (CAR) on the surface of mouse cells. This receptor for subclass C, serotypes 2 and 5 adenoviruses, is required for the attachment of adenovirus to the cell membrane and for efficient infection of cells. Adenovirus-based gene therapy holds much promise for the treatment of late-stage androgen-independent prostate cancer, a disease for which there is currently no effective cure. To develop a transgenic mouse model that would be more suitable for testing adenoviral-based gene therapy for prostate cancer, we introduced a PSA/hCAR transgene into mice, our aim being to overexpress CAR specifically on the surface of prostate cells. In this transgene, a chimeric enhancer/promoter of the human prostate specific antigen (PSA) gene, PSE-BC, drives expression of a truncated human CAR coding sequence, lacking the cytoplasmic tail. Immunohistochemical analysis of CAR demonstrated elevated amounts of CAR in the prostate of transgenic mice as compared to their non-transgenic littermates. Analysis of several other organs including bladder, liver, lung, and mammary gland suggested that this increase in CAR expression was prostatespecific. Western analysis of tissue extracts prepared from transgenic and non-transgenic sibs, however, showed that while hCAR expression is robust in the prostate, it is also expressed at a very low level in the lung. To functionally assay for an increase in the efficiency of viral infection of the prostate, we are administering adenoviruses containing reporter transgenes by tail vein injection to transgenic and non-transgenic mice. Double transgenic mice derived from breeding PSA/hCAR mice to existing transgenic mouse models for prostate cancer, for example TRAMP, should provide an improved mouse model for testing the efficacy of adenoviral-based therapies for the treatment of prostate cancer.
367. Early Gene Expression Signatures in Human Mesangial Cells Following Adenoviral Transduction U. Y. Bhatt,1 N. S. Shen,1 A. B. Bringardner,1 M. Husa,1 T. J. Sferra,1 N. S. Nahman, Jr.1 1 Internal Medicine, The Ohio State University, Columbus, OH, United States. Recombinant adenoviruses (rAv) are potent gene delivery vehicles. Adenoviral transduction begins with engagement of rAv to the common coxsackie adenovirus receptor (CAR). The complete function of CAR and the signaling pathways activated by viral engagement remain undefined. We theorize that rAv binding to CAR, and subsequent internalization, is associated with a distinct series of cellular events and that assessing the gene expression signature following transduction is a reasonable approach to defining the S144
process. To test this question, human mesangial cells were grown to confluency and exposed to rAv in transduction media or media alone. After 20 minutes, the cells were washed and harvested for RNA. After synthesis of biotinylated cRNA, the samples were hybridized to the U133A GeneChip (Affymetrix) and analyzed using MicroArray Suite v5.0 (Affymetrix). Of the 22,832 genes probed, there was a significant increase in 573 genes (2.57%) and decrease in 490 genes (2.20%). To define the magnitude of change, gene intensities altered by 3-fold or greater were isolated. Using this criteria, 33 genes were noted to be up-regulated. The function of proteins encoded by these genes included cell signaling / signal transduction (13), transcription (9), cellular metabolism (2), and unknown (9). Increased gene expression was noted to occur in genes encoding G-proteins, known to be involved in adenoviral transduction. In contrast, only 7 genes were noted to be downregulated by greater than 3-fold. The majority of these genes were involved in signal transduction. Conclusion: Gene expression patterns within 20 minutes of adenoviral transduction are associated with the modification of genes regulating cell signaling and transcription, indicating rapid cellular responses to viral internalization. Assessing gene expression signatures at later time points may be a useful tool for delineating both additional consequences of rAv infection, as well as the functional characteristics of CAR.
368. Expression of a Human Codon-Optimized Bacteriophage T4 Endonuclease V Gene Enhances UV Resistance of Xeroderma Pigmentosum Group A Cells Hsi-Chou Liu,1 Charles J. Link.1 1 Stoddard Cancer Research Institute, Iowa Methodist Medical Center, Des Moines, IA, United States. Cancer development is attributed to the accumulation of mutations in critical genes that alter normal programs of cell proliferation, differentiation and death. Cyclobutane pyrimidine photodimers, the most common DNA lesions generated by ultraviolet (UV) light, are both mutagenic and cytotoxic. Human xeroderma pigmentosum group A (XP-A) cells have genetic defects in DNA repair, and therefore are very sensitive to UV exposure. Endonuclease V from bacteriophage T4 (T4NV) initiates the repair of pyrimidine photodimers via a base excision repair pathway. A human codonoptimized T4NV (hT4NV) gene was constructed to overcome codon usage bias encountered when bacterial genes are expressed in human cells. The hT4NV gene was cloned into a retroviral vector for production of recombinant viruses harboring the hT4NV gene. In addition, the nucleotide sequence for nuclear localization signal (NLS) of the large T-antigen of SV40 virus was added to either the 5’ or 3’ end of the hT4NV gene in order to facilitate the translocation of T4NV into nuclei for DNA repair. Enhanced survival rate over UV (254 nm) illumination was observed in the hT4NV transduced XP20S cells. However, the addition of NLS to either N- or C-terminus of T4NV had no improved effects on the survival of transduced XP20S cells irradiated with UV, when compared with hT4NV transduced XP20S cells. The relatively small size of T4NV may have provided for the endonuclase an easy access to the DNA lesions of pyrimidine photodimers, and exerted a more positive effect than the NLS signal. Fusion to the NLS peptide might also lower the activity of T4NV enzyme. Transient expression of the hT4NV gene might be employed to enhance the repair of sunlight-induced DNA damages in human skin cells.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
366. Prostate-Specific Expression of Human Coxsackie Adenovirus Receptor (hCAR) in Transgenic Mice Yunhua Bao,1 Weidan Peng,1 Amy Verbitsky,1 Jipeng Chen,1 Lily Wu,2 Katherine Rauen,3 Janet Sawicki.1 1 Lankenau Institute for Medical Research, Wynnewood, PA; 2 Department of Urology, UCLA School of Medicine, Los Angeles, CA; 3Cancer Research Institute, University of California, San Francisco, San Francisco, CA. Transgenic mice provide useful animal models with which to test the effectiveness of new therapeutic strategies for the treatment of diseases, including prostate cancer. Their use in testing adenoviralbased gene therapies, however, is limited because of the restricted expression of the Coxsackie adenovirus receptor (CAR) on the surface of mouse cells. This receptor for subclass C, serotypes 2 and 5 adenoviruses, is required for the attachment of adenovirus to the cell membrane and for efficient infection of cells. Adenovirus-based gene therapy holds much promise for the treatment of late-stage androgen-independent prostate cancer, a disease for which there is currently no effective cure. To develop a transgenic mouse model that would be more suitable for testing adenoviral-based gene therapy for prostate cancer, we introduced a PSA/hCAR transgene into mice, our aim being to overexpress CAR specifically on the surface of prostate cells. In this transgene, a chimeric enhancer/promoter of the human prostate specific antigen (PSA) gene, PSE-BC, drives expression of a truncated human CAR coding sequence, lacking the cytoplasmic tail. Immunohistochemical analysis of CAR demonstrated elevated amounts of CAR in the prostate of transgenic mice as compared to their non-transgenic littermates. Analysis of several other organs including bladder, liver, lung, and mammary gland suggested that this increase in CAR expression was prostatespecific. Western analysis of tissue extracts prepared from transgenic and non-transgenic sibs, however, showed that while hCAR expression is robust in the prostate, it is also expressed at a very low level in the lung. To functionally assay for an increase in the efficiency of viral infection of the prostate, we are administering adenoviruses containing reporter transgenes by tail vein injection to transgenic and non-transgenic mice. Double transgenic mice derived from breeding PSA/hCAR mice to existing transgenic mouse models for prostate cancer, for example TRAMP, should provide an improved mouse model for testing the efficacy of adenoviral-based therapies for the treatment of prostate cancer.
367. Early Gene Expression Signatures in Human Mesangial Cells Following Adenoviral Transduction U. Y. Bhatt,1 N. S. Shen,1 A. B. Bringardner,1 M. Husa,1 T. J. Sferra,1 N. S. Nahman, Jr.1 1 Internal Medicine, The Ohio State University, Columbus, OH, United States. Recombinant adenoviruses (rAv) are potent gene delivery vehicles. Adenoviral transduction begins with engagement of rAv to the common coxsackie adenovirus receptor (CAR). The complete function of CAR and the signaling pathways activated by viral engagement remain undefined. We theorize that rAv binding to CAR, and subsequent internalization, is associated with a distinct series of cellular events and that assessing the gene expression signature following transduction is a reasonable approach to defining the S144
process. To test this question, human mesangial cells were grown to confluency and exposed to rAv in transduction media or media alone. After 20 minutes, the cells were washed and harvested for RNA. After synthesis of biotinylated cRNA, the samples were hybridized to the U133A GeneChip (Affymetrix) and analyzed using MicroArray Suite v5.0 (Affymetrix). Of the 22,832 genes probed, there was a significant increase in 573 genes (2.57%) and decrease in 490 genes (2.20%). To define the magnitude of change, gene intensities altered by 3-fold or greater were isolated. Using this criteria, 33 genes were noted to be up-regulated. The function of proteins encoded by these genes included cell signaling / signal transduction (13), transcription (9), cellular metabolism (2), and unknown (9). Increased gene expression was noted to occur in genes encoding G-proteins, known to be involved in adenoviral transduction. In contrast, only 7 genes were noted to be downregulated by greater than 3-fold. The majority of these genes were involved in signal transduction. Conclusion: Gene expression patterns within 20 minutes of adenoviral transduction are associated with the modification of genes regulating cell signaling and transcription, indicating rapid cellular responses to viral internalization. Assessing gene expression signatures at later time points may be a useful tool for delineating both additional consequences of rAv infection, as well as the functional characteristics of CAR.
368. Expression of a Human Codon-Optimized Bacteriophage T4 Endonuclease V Gene Enhances UV Resistance of Xeroderma Pigmentosum Group A Cells Hsi-Chou Liu,1 Charles J. Link.1 1 Stoddard Cancer Research Institute, Iowa Methodist Medical Center, Des Moines, IA, United States. Cancer development is attributed to the accumulation of mutations in critical genes that alter normal programs of cell proliferation, differentiation and death. Cyclobutane pyrimidine photodimers, the most common DNA lesions generated by ultraviolet (UV) light, are both mutagenic and cytotoxic. Human xeroderma pigmentosum group A (XP-A) cells have genetic defects in DNA repair, and therefore are very sensitive to UV exposure. Endonuclease V from bacteriophage T4 (T4NV) initiates the repair of pyrimidine photodimers via a base excision repair pathway. A human codonoptimized T4NV (hT4NV) gene was constructed to overcome codon usage bias encountered when bacterial genes are expressed in human cells. The hT4NV gene was cloned into a retroviral vector for production of recombinant viruses harboring the hT4NV gene. In addition, the nucleotide sequence for nuclear localization signal (NLS) of the large T-antigen of SV40 virus was added to either the 5’ or 3’ end of the hT4NV gene in order to facilitate the translocation of T4NV into nuclei for DNA repair. Enhanced survival rate over UV (254 nm) illumination was observed in the hT4NV transduced XP20S cells. However, the addition of NLS to either N- or C-terminus of T4NV had no improved effects on the survival of transduced XP20S cells irradiated with UV, when compared with hT4NV transduced XP20S cells. The relatively small size of T4NV may have provided for the endonuclase an easy access to the DNA lesions of pyrimidine photodimers, and exerted a more positive effect than the NLS signal. Fusion to the NLS peptide might also lower the activity of T4NV enzyme. Transient expression of the hT4NV gene might be employed to enhance the repair of sunlight-induced DNA damages in human skin cells.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy