ABSTRACT
Retroviral short hairpin RNA (shRNA)-mediated genetic screens in mammalian cells are powerful tools for discovering loss-of-function phenotypes. We describe a highly parallel multiplex methodology for screening large pools of shRNAs using half-hairpin barcodes for microarray deconvolution. We carried out dropout screens for shRNAs that affect cell proliferation and viability in cancer cells and normal cells. We identified many shRNAs to be antiproliferative that target core cellular processes, such as the cell cycle and protein translation, in all cells examined. Moreover, we identified genes that are selectively required for proliferation and survival in different cell lines. Our platform enables rapid and cost-effective genome-wide screens to identify cancer proliferation and survival genes for target discovery. Such efforts are complementary to the Cancer Genome Atlas and provide an alternative functional view of cancer cells.
Subject(s)
Breast Neoplasms/genetics , Cell Proliferation , Colonic Neoplasms/genetics , Genes, Neoplasm , Genomics/methods , Breast Neoplasms/pathology , Cell Line , Cell Line, Tumor , Cell Survival/genetics , Colonic Neoplasms/pathology , Gene Library , Genetic Vectors , Genome, Human , Humans , MicroRNAs , Oligonucleotide Array Sequence Analysis , RNA, Small Interfering , Retroviridae/geneticsABSTRACT
Telomere dysfunction causes genomic instability. However, the mechanism that initiates this instability when telomeres become short is unclear. We measured the mutation rate and loss of heterozygosity along a chromosome arm in diploid yeast that lacked telomerase to distinguish between mechanisms for the initiation of instability. Sequence loss was localized near chromosome ends in the absence of telomerase but not after breakage of a dicentric chromosome. In the absence of telomerase, the increase in mutation rate is dependent on the exonuclease Exo1p. Thus, exonucleolytic end resection, rather than chromosome fusion and breakage, is the primary mechanism that initiates genomic instability when telomeres become short.
Subject(s)
DNA-Binding Proteins , Genomic Instability , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Telomerase/metabolism , Base Sequence , Cell Cycle , DNA Repair Enzymes , DNA, Fungal/genetics , DNA, Fungal/metabolism , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , Diploidy , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Endonucleases/genetics , Endonucleases/metabolism , Exodeoxyribonucleases/genetics , Exodeoxyribonucleases/metabolism , Gene Deletion , Genes, Fungal , Genome, Fungal , Loss of Heterozygosity , Mutation , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Telomerase/genetics , Telomere/genetics , Telomere/metabolismABSTRACT
Tumour growth is an evolutionary process that is characterized by the selection of clonal populations of cells that acquire distinct genetic changes. Many cancer therapies aim to exploit the specific changes that occur in cancer cells, but understanding the underlying mechanisms of genomic instability that cause these mutations could lead to more effective therapies. If common mechanisms exist for initiating genomic instability in tumours, selection could explain the differences in specific gene mutations that accumulate in different tumour types. The cause of genomic instability in human tumours is unclear, although there is evidence to indicate that telomere dysfunction could make an important contribution.
Subject(s)
Mutation , Neoplasms/genetics , Telomere/physiology , Cellular Senescence , Chromosome Aberrations , Genome , Humans , Models, GeneticABSTRACT
Telomere shortening and telomerase activation both occur in human tumors. Telomere shortening has been proposed to have two conflicting roles in tumorigenesis: tumor suppression and initiation of chromosomal instability. Similarly, while telomerase activation is suggested to be necessary for tumor growth, telomerase may help to stabilize genomic instability. Here we review what is known about these conflicting roles and propose a framework to understand the role of telomerase in cancer progression.
