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Functional Genomic Analysis of Autophagic Cell Death using RNA interference

CATGGCGATATTGT CATGGCGCCAATAT CATGGCGCGCATTT CATGGCGTGGGGAT CATGGCTAATAAAT CATGGCTCAAGGAG CATGGCTGGACTCC CATGGCTGTGGCCA CATGGCTTTCGTGT CATGGCTTTTTGGC CATGGGAACCGACA CATGGGACCGCCCC CATGGGACCGCTCA CATGGGATCACAAT CATGGGCAACGATC CATGGGCAGCAAGC CATGGGCAGCAATT.

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Functional Genomic Analysis of Autophagic Cell Death using RNA interference

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  1. CATGGCGATATTGT CATGGCGCCAATAT CATGGCGCGCATTT CATGGCGTGGGGAT CATGGCTAATAAAT CATGGCTCAAGGAG CATGGCTGGACTCC CATGGCTGTGGCCA CATGGCTTTCGTGT CATGGCTTTTTGGC CATGGGAACCGACA CATGGGACCGCCCC CATGGGACCGCTCA CATGGGATCACAAT CATGGGCAACGATC CATGGGCAGCAAGC CATGGGCAGCAATT Functional Genomic Analysis of Autophagic Cell Death using RNA interference S. Gorski, Genome Sciences Centre, BC Cancer Agency

  2. Overview • Background • Research Aims • Experimental Approach • Preliminary Data

  3. Programmed Cell Death (PCD) Dysfunction Function Cancer Deleting damaged cells Autoimmune diseases Culling cell number Neurodegenerative diseases Deletingstructures Developmental abnormalities Sculpting tissues

  4. Types of Programmed Cell Death (PCD) I. Apoptosis II. Autophagic PCD (adapted from Baehrecke, 2002)

  5. Autophagic cell death in disease • human neurodegenerative diseases (Alzheimer and Parkinson) • cardiomyocyte degeneration • spontaneous regression of human neuroblastoma • tamoxifen-treated mammary carcinoma cells (MCF-7) • bcl-2 antisense treatment of human leukemic HL60 cells • beclin-1 (apg6) promotes autophagy and inhibits tumorigenesis; • expressed at decreased levels in human breast carcinoma

  6. Research Aims • Identify the genes involved in autophagic cell death in vivo. • Determine which genes are necessary and sufficient for • autophagic cell death. • Identify the autophagic cell death genes/pathways associated • with human disease and investigate potential as molecular • markers and/or therapeutic agents.

  7. Specific Aim Identify and characterize genes that are necessary for mammalian autophagic cell death. Experimental Approach RNAi analysis of candidate autophagic cell death genes in human breast carcinoma cell line MCF-7.

  8. siRNA analysis of autophagic cell death genes in human breast carcinoma cells Overview of experimental design Test candidate genes for expression in MCF-7 by RT-PCR Preparation and storage of siRNAs corresponding to selected human candidate autophagic cell death genes MCF-7 cells + Reporter +/- Tamoxifen Transfection (siPORT Lipid, Ambion) 3-4 days incubation time to ensure protein depletion Test for RNA depletion by RT-PCR Microscopy Cell Death Assays Autophagic vacuole assays

  9. Small Interfering RNAs (siRNA) 5’ phosphate siRNA siRNAs are incorporated into RISC (RNA Induced Silencing Complex) siRNAs unwind and guide RISC to a substrate mRNA substrate cleavage (From McManus and Sharp, 2002 & Hannon, 2002)

  10. siRNA in mammalian cells • a new tool to systematically decipher the functions of genes • many types of genes in a variety of mammalian cell lines • have been knocked down successfully • (85-99% knock-down; in McManus and Sharp, 2002) • transfectability of cells is the limiting step • Potential Problems: • may have to try multiple siRNAs per gene • effects are transient

  11. Characteristics of the MCF-7 cell line • human breast carcinoma cell line • amenable to siRNA analyses • treatment with tamoxifen (10-6M) induces • autophagic cell death (Bursch et al. 1996, 2000)

  12. Preliminary Data • Identification of candidate autophagic cell death genes. • “A SAGE Approach to Discovery of Genes Involved in Autophagic Cell Death” • Gorski et al., 2003, Current Biology, 13: 358-363. • Used gene expression profiling in the Drosophila model system • to identify several hundred candidate autophagic cell death genes • 2. Bioinformatics analyses to relate gene expression patterns in • Drosophila autophagic cell death and human cancers

  13. Human cancer (CGAP) Drosophila autophagic cell death SCC-S2 downregulated 7-fold in human mammary gland ductal carcinoma vs normal CG4091 upregulated 102-fold in 16 vs 23 hr salivary glands CLSTN3 downregulated >16-fold in neuroblastoma vs normal brain CG11059 upregulated > 12-fold SIGLEC5 upregulated > 26-fold in ductal carcinoma vs normal kirre downregulated 11-fold Cross-species Gene Expression Comparisons

  14. Autophagic Cell Death Candidate Genes

  15. Acknowledgements Genome Sciences Centre Marco Marra Victor Ling Drosophila PCD Suganthi Chittaranjan Doug Freeman Carrie Anderson Shaun Coughlin Claire Hou Bioinformatics Steven Jones Erin Pleasance Richard Varhol Scott Zuyderduyn GSC Sequencing Group www.bcgsc.ca http://sage.bcgsc.ca/tagmapping/ http://www.bcgsc.ca/lab/fg/dsage/ BC Cancer Agency BC Cancer Foundation NCIC NSERC

  16. Autophagic cell death in normal physiology • Dictyostelium sorocarp formation • insect metamorphosis • intersegmental muscle, gut, salivary glands • mammalian embryogenesis • regression of interdigital webs, sexual anlagen • mammalian adulthood • intestine, mammary gland post-weaning, ovarian • atretic follicles

  17. Questions • What is the relationship between Autophagic PCD and Cancer? • common mechanism in breast and other cancers? • gene mutation • What is the therapeutic potential of autophagic PCD in cancer? • solid tumours • apoptotic-resistant tumours

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