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972. Inhibition of TGF-β by RNA Interference To Modulate Breast Cancer Metastasis
CANCER-TARGETED GENE THERAPY: OTHER VIRUSES AND NEW APPROACHES Results: Viability and proliferation assays, don’t’ show significative changes than the control at 24 hours. But, testosterone has very strong effect on the estrogen and progesterone effect at 36 hours on transcriptional effects. We are exploring the effect on proliferation for large time and cross over the expression pattern of Krox-1, on this cell lines and comparatively with myoblast cells Supported by PAPIIT IN220606-2 of the National University of México, FGRA-México, and the Ministry of Health México.
971. Novel Ribozyme-Based Strategy for Cancer Chemoprevention Suparna Mukhopadhyay,1 Wolfgang Zacharias,1 William G. McGregor.1 1 Pharmacology, University of Louisville, Louisville, KY. Recent advances in understanding the molecular mechanisms of carcinogen-induced mutagenesis indicate that most mutations are dependent on the activity of newly-described translesion DNA polymerases, which have reduced base-pairing requirements and are able to replicate past DNA damage with potentially mutagenic consequences. In human cells in culture, reducing the level of one such polymerase, REV1, with a gene-specific ribozyme greatly reduces carcinogen-induced mutagenesis. To examine the hypothesis that reducing the mutagenic load will reduce the incidence of cancer, we are developing strategies to reduce REV1 in vivo in mouse models of carcinogenesis. We constructed vectors that express a ribozyme targeting mouse REV1 in a region homologous to the human mRNA used in our published studies. In order to improve the efficiency of gene-specific knockdown, DNA coding for the ribozyme was cloned into expression cassettes designed to target the transcripts either to the nuclear or to the cytoplasmic compartment. Using quantitative real-time PCR for REV1 mRNA with GAPDH as an internal control, we consistently found that the greatest suppression occurred when both compartments were targeted. When both compartments were targeted, REV1 expression was consistently suppressed by 8090%. UV-induced mutant frequency at the HPRT locus in these cells was also reduced up to 80%, but UV-induced cytotoxicity was not affected. We plan to deliver these plasmids to the skin of excision repair deficient hairless xpa knockout mice, which develop squamous cell skin cancer with 100% penetrance after low doses of UV. Delivery of these expression cassettes to carcinogen-exposed tissue presents a novel chemopreventive strategy to reduce the frequency of mutations and the incidence of cancer.
β by RNA Interference To 972. Inhibition of TGF-β Modulate Breast Cancer Metastasis Lakisha D. Moore,1 Selvarangan Pponnazhagan.1 1 Pathology, The University of Alabama at Birmingham, Birmingham, AL. Transforming Growth Factor-β (TGF-β) has been shown to perform a dual function as both a tumor suppressor in early stage carcinogenesis and a pro-metastatic factor in late-stage tumor progression. Early studies have shown that inhibition of TGF-β signaling pathway can alter various aspects of tumor metastasis such as migration, invasion, angiogenesis and immune suppression. While these methods have shown modest success, several limitations prevent the wide spread use of these inhibitors as an anti-metastatic therapy. RNA interference (RNAi) has emerged as an effective tool for gene specific silencing in vitro and in vivo. The central hypothesis
S374
of the proposed studies is that adeno-associated virus (AAV) mediated deliver of siRNA sequences targeting TGF-β1 in MDAMB 435 breast cancer cells can alter the metastatic phenotype at defined time-points of tumor progression and/or act as an adjuvant to primary chemotherapies. Several siRNA target sequences have been screened to determine silencing capacity and two specific targets, siRNA4 and siRNA7 showed ∼90% inhibition of TGFβ1 transcripts by Northern blot and at least a 5-6 fold reduction by RTPCR analysis. The two siRNA sequences, along with a scrambled sequence, were cloned into a tet-inducible expression vector and used to generate a stable cell line that constitutively expresses this construct. This cell line is being using in various in vitro assays such as migration, invasion and gene expression, to determine the effects of TGF-β silencing. This cell line will later be used in an orthotopic breast cancer model to determine the exact time-point at which TGF-β silencing is most effective. A second set of studies are proceeding in parallel to determine the transduction of AAV serotypes 1-6 both in vitro and in vivo. Initial in vitro data has shown that AAV2 and AAV6 show transduction efficiencies of 40% - 60% in MDA-MB 435 and MDA-MB 231 cells. In vivo transduction studies, of the primary tumor mass, are ongoing using AAV-βgal, Ad-GFP and AAV-GFP. Ongoing studies will determine the effects of inhibition on SMAD-independent signaling pathways that can play a role in detachment, migration, immune-suppression and angiogenesis. This inducible siRNA will allow better insight into the TGF-β “switch” from tumor suppressor to pro-metastatic factor along disease progression and possible development of a TGF-β targeted anti-cancer therapy. If promising, this therapy may also be used in combination with current chemotherapies to provide a synergistic effect against breast cancer metastasis.
973. SEquence-Enabled Reassembly of βLactamase (SEER-LAC): A Sensitive Method for the Detection of Double-Stranded DNA Aik T. Ooi,1,3 Cliff I. Stains,2 Indraneel Ghosh,2 David J. Segal.1,3 Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ; 2Department of Chemistry, University of Arizona, Tucson, AZ; 3Department of Pharmacology/Genome Center, University of California, Davis, Davis, CA. 1
This work describes the development of a new methodology for the detection of specific double-stranded DNA sequences. We previously showed that two inactive fragments of green fluorescent protein, each coupled to engineered zinc finger DNA-binding proteins, were able to reassemble an active reporter complex in the presence of a predefined DNA sequence [Stains et al., (2005) J. Am Chem. Soc. 127:10782]. This system, designated SEquence-Enabled Reassembly (SEER), was demonstrated in vitro to produce a DNAconcentration-dependent signal. Here we endow the SEER system with catalytic capability using the reporter enzyme TEM1 βlactamase. This system could distinguish target from non-target DNA in less than 5 minutes, representing a more than 1,000-fold improvement over our previous SEER design. A single base pair substitution in the DNA binding sequence reduced the signal to nearly background levels. Substitution of a different custom zinc finger DNA-binding domain produced a signal only on the new cognate target. These results present SEER as a rapid and sensitive method for the detection of double-stranded DNA sequences. The results also suggest the use of SEER to activate prodrugs in cells as a strategy to achieve DNA sequence-dependant cell death.
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
971. Novel Ribozyme-Based Strategy for Cancer Chemoprevention Suparna Mukhopadhyay,1 Wolfgang Zacharias,1 William G. McGregor.1 1 Pharmacology, University of Louisville, Louisville, KY. Recent advances in understanding the molecular mechanisms of carcinogen-induced mutagenesis indicate that most mutations are dependent on the activity of newly-described translesion DNA polymerases, which have reduced base-pairing requirements and are able to replicate past DNA damage with potentially mutagenic consequences. In human cells in culture, reducing the level of one such polymerase, REV1, with a gene-specific ribozyme greatly reduces carcinogen-induced mutagenesis. To examine the hypothesis that reducing the mutagenic load will reduce the incidence of cancer, we are developing strategies to reduce REV1 in vivo in mouse models of carcinogenesis. We constructed vectors that express a ribozyme targeting mouse REV1 in a region homologous to the human mRNA used in our published studies. In order to improve the efficiency of gene-specific knockdown, DNA coding for the ribozyme was cloned into expression cassettes designed to target the transcripts either to the nuclear or to the cytoplasmic compartment. Using quantitative real-time PCR for REV1 mRNA with GAPDH as an internal control, we consistently found that the greatest suppression occurred when both compartments were targeted. When both compartments were targeted, REV1 expression was consistently suppressed by 8090%. UV-induced mutant frequency at the HPRT locus in these cells was also reduced up to 80%, but UV-induced cytotoxicity was not affected. We plan to deliver these plasmids to the skin of excision repair deficient hairless xpa knockout mice, which develop squamous cell skin cancer with 100% penetrance after low doses of UV. Delivery of these expression cassettes to carcinogen-exposed tissue presents a novel chemopreventive strategy to reduce the frequency of mutations and the incidence of cancer.
β by RNA Interference To 972. Inhibition of TGF-β Modulate Breast Cancer Metastasis Lakisha D. Moore,1 Selvarangan Pponnazhagan.1 1 Pathology, The University of Alabama at Birmingham, Birmingham, AL. Transforming Growth Factor-β (TGF-β) has been shown to perform a dual function as both a tumor suppressor in early stage carcinogenesis and a pro-metastatic factor in late-stage tumor progression. Early studies have shown that inhibition of TGF-β signaling pathway can alter various aspects of tumor metastasis such as migration, invasion, angiogenesis and immune suppression. While these methods have shown modest success, several limitations prevent the wide spread use of these inhibitors as an anti-metastatic therapy. RNA interference (RNAi) has emerged as an effective tool for gene specific silencing in vitro and in vivo. The central hypothesis
S374
of the proposed studies is that adeno-associated virus (AAV) mediated deliver of siRNA sequences targeting TGF-β1 in MDAMB 435 breast cancer cells can alter the metastatic phenotype at defined time-points of tumor progression and/or act as an adjuvant to primary chemotherapies. Several siRNA target sequences have been screened to determine silencing capacity and two specific targets, siRNA4 and siRNA7 showed ∼90% inhibition of TGFβ1 transcripts by Northern blot and at least a 5-6 fold reduction by RTPCR analysis. The two siRNA sequences, along with a scrambled sequence, were cloned into a tet-inducible expression vector and used to generate a stable cell line that constitutively expresses this construct. This cell line is being using in various in vitro assays such as migration, invasion and gene expression, to determine the effects of TGF-β silencing. This cell line will later be used in an orthotopic breast cancer model to determine the exact time-point at which TGF-β silencing is most effective. A second set of studies are proceeding in parallel to determine the transduction of AAV serotypes 1-6 both in vitro and in vivo. Initial in vitro data has shown that AAV2 and AAV6 show transduction efficiencies of 40% - 60% in MDA-MB 435 and MDA-MB 231 cells. In vivo transduction studies, of the primary tumor mass, are ongoing using AAV-βgal, Ad-GFP and AAV-GFP. Ongoing studies will determine the effects of inhibition on SMAD-independent signaling pathways that can play a role in detachment, migration, immune-suppression and angiogenesis. This inducible siRNA will allow better insight into the TGF-β “switch” from tumor suppressor to pro-metastatic factor along disease progression and possible development of a TGF-β targeted anti-cancer therapy. If promising, this therapy may also be used in combination with current chemotherapies to provide a synergistic effect against breast cancer metastasis.
973. SEquence-Enabled Reassembly of βLactamase (SEER-LAC): A Sensitive Method for the Detection of Double-Stranded DNA Aik T. Ooi,1,3 Cliff I. Stains,2 Indraneel Ghosh,2 David J. Segal.1,3 Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ; 2Department of Chemistry, University of Arizona, Tucson, AZ; 3Department of Pharmacology/Genome Center, University of California, Davis, Davis, CA. 1
This work describes the development of a new methodology for the detection of specific double-stranded DNA sequences. We previously showed that two inactive fragments of green fluorescent protein, each coupled to engineered zinc finger DNA-binding proteins, were able to reassemble an active reporter complex in the presence of a predefined DNA sequence [Stains et al., (2005) J. Am Chem. Soc. 127:10782]. This system, designated SEquence-Enabled Reassembly (SEER), was demonstrated in vitro to produce a DNAconcentration-dependent signal. Here we endow the SEER system with catalytic capability using the reporter enzyme TEM1 βlactamase. This system could distinguish target from non-target DNA in less than 5 minutes, representing a more than 1,000-fold improvement over our previous SEER design. A single base pair substitution in the DNA binding sequence reduced the signal to nearly background levels. Substitution of a different custom zinc finger DNA-binding domain produced a signal only on the new cognate target. These results present SEER as a rapid and sensitive method for the detection of double-stranded DNA sequences. The results also suggest the use of SEER to activate prodrugs in cells as a strategy to achieve DNA sequence-dependant cell death.
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy