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Increased Common Fragile Site Expression, Cell Proliferation Defects, and Apoptosis following Conditional Inactivation of Mouse Hus1 in Primary Cultured Cells

Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853, USA.
Molecular Biology of the Cell (Impact Factor: 4.55). 04/2007; 18(3):1044-55. DOI: 10.1091/mbc.E06-10-0957
Source: PubMed

ABSTRACT Targeted disruption of the mouse Hus1 cell cycle checkpoint gene results in embryonic lethality and proliferative arrest in cultured cells. To investigate the essential functions of Hus1, we developed a system for the regulated inactivation of mouse Hus1 in primary fibroblasts. Inactivation of a loxP site-flanked conditional Hus1 allele by using a cre-expressing adenovirus resulted in reduced cell doubling, cell cycle alterations, and increased apoptosis. These phenotypes were associated with a significantly increased frequency of gross chromosomal abnormalities and an S-phase-specific accumulation of phosphorylated histone H2AX, an indicator of double-stranded DNA breaks. To determine whether these chromosomal abnormalities occurred randomly or at specific genomic regions, we assessed the stability of common fragile sites, chromosomal loci that are prone to breakage in cells undergoing replication stress. Hus1 was found to be essential for fragile site stability, because spontaneous chromosomal abnormalities occurred preferentially at common fragile sites upon conditional Hus1 inactivation. Although p53 levels increased after Hus1 loss, deletion of p53 failed to rescue the cell-doubling defect or increased apoptosis in conditional Hus1 knockout cells. In summary, we propose that Hus1 loss leads to chromosomal instability during DNA replication, triggering increased apoptosis and impaired proliferation through p53-independent mechanisms.

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    • "Several studies reported that multiple components of the ATR pathway such as CHK1 [44], HUS1 [45], and Claspin [46], or other ATR substrates, profoundly affected CFS integrity when defective. But how can replication checkpoint promote CFS stability, and most importantly, are the genetic determinants controlling replication checkpoint functions after genome-wide replication arrest conserved even when replication is perturbed specifically at CFS? "
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    ABSTRACT: Common fragile sites (CFS) are heritable nonrandomly distributed loci on human chromosomes that exhibit an increased frequency of chromosomal breakage under conditions of replication stress. They are considered the preferential targets for high genomic instability from the earliest stages of human cancer development, and increased chromosome instability at these loci has been observed following replication stress in a subset of human genetic diseases. Despite their biological and medical relevance, the molecular basis of CFS fragility in vivo has not been fully elucidated. At present, different models have been proposed to explain how instability at CFS arises and multiple factors seem to contribute to their instability. However, all these models involve DNA replication and suggest that replication fork stalling along CFS during DNA synthesis is a very frequent event. Consistent with this, the maintenance of CFS stability relies on the ATR-dependent checkpoint, together with a number of proteins promoting the recovery of stalled replication forks. In this review, we discuss mainly the possible causes that threaten the integrity of CFS in the light of new findings, paying particular attention to the role of the S-phase checkpoint.
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    • "This indicates that apoptosis could occur in a manner independent of p53, which suggests that there must be another mechanism involved. Zhu and Weiss25 have demonstrated that inactivation of mouse Hus1 protein in primary cultured cells leads to chromosomal instability during DNA replication, triggering apoptosis and impairing proliferation through p53-independent mechanisms. In the present case, it is possible that Hus1 protein was blocked, causing apoptosis without activation of p53. "
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    • "Loss of CHK1, but not CHK2, induces breaks at CFSs [120]. Inactivation of HUS1, a member of PCNA-related 9-1-1complex that promotes CHK1 phosphorylation by ATR and is involved in DNA repair, causes increased chromosomal instability at CFSs [121]. SMC1 is necessary for sister chromatid cohesion and DNA repair. "
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    ABSTRACT: Genomic instability, a hallmark of cancer, occurs preferentially at specific genomic regions known as common fragile sites (CFSs). CFSs are evolutionarily conserved and late replicating regions with AT-rich sequences, and CFS instability is correlated with cancer. In the last decade, much progress has been made toward understanding the mechanisms of chromosomal instability at CFSs. However, despite tremendous efforts, identifying a cancer-associated CFS gene (CACG) remains a challenge and little is known about the function of CACGs at most CFS loci. Recent studies of FATS (for Fragile-site Associated Tumor Suppressor), a new CACG at FRA10F, reveal an active role of this CACG in regulating DNA damage checkpoints and suppressing tumorigenesis. The identification of FATS may inspire more discoveries of other uncharacterized CACGs. Further elucidation of the biological functions and clinical significance of CACGs may be exploited for cancer biomarkers and therapeutic benefits.
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