ABSTRACT
Common fragile sites have been involved in neoplastic transformation, although their molecular basis is still poorly understood. Here, we demonstrate that inhibition of the SMC1 by RNAi is sufficient to induce fragile site expression. By investigating normal, ATM- and ATR-deficient cell lines, we provide evidence that the contribution of SMC1 in preventing the collapse of stalled replication fork is an Atr-dependent pathway. Using a fluorescent antibody specific for gamma-H2AX, we show that very rare discrete nuclear foci appear 1 and 2 h after exposure to aphidicolin and/or RNAi-SMC1, but became more numerous and distinct after longer treatment times. In this context, fragile sites might be viewed as an in vitro phenomenon originating from double-strand breaks formed because of a stalled DNA replication that lasted too long to be managed by physiological rescue acting through the Atr/Smc1 axis. We propose that in vivo, following an extreme replication block, rare cells could escape checkpoint mechanisms and enter mitosis with a defect in genome assembly, eventually leading to neoplastic transformation.
Subject(s)
Cell Cycle Proteins/physiology , Chromosomal Proteins, Non-Histone/physiology , Chromosome Fragile Sites , DNA-Binding Proteins/physiology , Protein Serine-Threonine Kinases/physiology , Tumor Suppressor Proteins/physiology , Aphidicolin/pharmacology , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/antagonists & inhibitors , Cell Nucleus , Chromosomal Proteins, Non-Histone/antagonists & inhibitors , Chromosomes/drug effects , DNA Replication/drug effects , Enzyme Inhibitors/pharmacology , Fibroblasts/metabolism , HeLa Cells , Histones/immunology , Histones/metabolism , Humans , Oligonucleotides, Antisense/pharmacology , RNA InterferenceABSTRACT
The relative contribution of aneuploidy and gene mutations to human tumorigenesis is not yet known. Studies in mice have demonstrated that even single point mutations in oncogenes and tumor suppressor genes can dramatically increase tumor frequency. However, models to evaluate the definitive role of aneuploidy and genomic instability are not yet available. Human fibroblast cells have long been used as a tool for investigating proliferation, senescence, immortalization, and tumorigenesis, all processes that are strongly interrelated. We have now used antisense and ribozyme-mediated temporary inhibition of BUB1 to study the consequences of mitotic checkpoint failure on the development of aneuploidy. The analysis of cell colonies selected by soft agar growth showed evidence of chromosome instability and delayed senescence, without being tumorigenic in nude mice. Our data suggest that chromosomal instability and aneuploidy are early changes that precede tumorigenicity in the multistep process leading to neoplastic transformation.
