Expression of cyclins A, E and topoisomerase II alpha correlates with centrosome amplification and genomic instability and influences the reliability of cytometric S-phase determination.
(9/2256)
BACKGROUND: The progression of normal cells through the cell cycle is meticulously regulated by checkpoints guaranteeing the exact replication of the genome during S-phase and its equal division at mitosis. A prerequisite for this achievement is synchronized DNA-replication and centrosome duplication. In this context the expression of cyclins A and E has been shown to play a principal role. RESULTS: Our results demonstrated a correlation between centrosome amplification, cell cycle fidelity and the level of mRNA and protein expression of cyclins A and E during the part of the cell cycle defined as G1-phase by means of DNA content based histogram analysis. It is shown that the normal diploid breast cell line HTB-125, the genomically relatively stable aneuploid breast cancer cell line MCF-7, and the genomically unstable aneuploid breast cancer cell line MDA-231 differ remarkably concerning both mRNA and protein expression of the two cyclins during G1-phase. In MDA-231 cells the expression of e.g. cyclin A mRNA was found to be ten times higher than in MCF-7 cells and about 500 times higher than in HTB-125 cells. Topoisomerase II alpha showed high mRNA expression in MDA compared to MCF-7 cells, but the difference in protein expression was small. Furthermore, we measured centrosome aberrations in 8.4% of the MDA-231 cells, and in only 1.3% of the more stable aneuploid cell line MCF-7. MDA cells showed 27% more incorporation of BrdU than reflected by S-phase determination with flow cytometric DNA content analysis, whereas these values were found to be of the same size in both HTB-125 and MCF-7 cells. CONCLUSIONS: Our data indicate that the breast cancer cell lines MCF-7 and MDA-231, although both DNA-aneuploid, differ significantly regarding the degree of cell cycle disturbance and centrosome aberrations, which partly could explain the different genomic stability of the two cell lines. The results also question the reliability of cytometric DNA content based S-phase determination in genomically unstable tumor cell populations. (+info)
A genome-wide screen identifies 27 genes involved in transposon silencing in C. elegans.
(10/2256)
Transposon jumps are a major cause of genome instability. In the C. elegans strain Bristol N2, transposons are active in somatic cells, but they are silenced in the germline, presumably to protect the germline from mutations. Interestingly, the transposon-silencing mechanism shares factors with the RNAi machinery. To better understand the mechanism of transposon silencing, we performed a genome-wide RNAi screen for genes that, when silenced, cause transposition of Tc1 in the C. elegans germline. We identified 27 such genes, among which are mut-16, a mutator that was previously found but not identified at the molecular level, ppw-2, a member of the argonaute family, and several factors that indicate a role for chromatin structure in the regulation of transposition. Some of the newly identified genes are also required for cosuppression and therefore represent the shared components of the two pathways. Since most of the newly identified genes have clear homologs in other species, and since transposons are found from protozoa to human, it seems likely that they also protect other genomes against transposon activity in the germline. (+info)
Regional differences of somatic CAG repeat instability do not account for selective neuronal vulnerability in a knock-in mouse model of SCA1.
(11/2256)
Expression of unstable translated CAG repeats is the mutational mechanism in nine different neurodegenerative disorders. Although the products of genes harboring these repeats are widely expressed, a subset of neurons is vulnerable in each disease accounting for the different phenotypes. Somatic instability of the expanded CAG repeat has been implicated as a factor mediating the selective striatal neurodegeneration in Huntington disease. It remains unknown, however, whether such a mechanism contributes to the selective neurodegeneration in other polyglutamine diseases or not. To address this question, we investigated the pattern of CAG repeat instability in a knock-in mouse model of spinocerebellar ataxia type 1 (SCA1). Small pool PCR analysis on DNA from various neuronal and non-neuronal tissues revealed that somatic repeat instability was most remarkable in the striatum. In the two vulnerable tissues, cerebellum and spinal cord, there were substantial differences in the profiles of mosaicism. These results suggest that in SCA1 there is no clear causal relationship between the degree of somatic instability and selective neuronal vulnerability. The finding that somatic instability is most pronounced in the striatum of various knock-in models of polyglutamine diseases highlights the role of trans-acting tissue- or cell-specific factors in mediating the instability. (+info)
Reduction of stability of arabidopsis genomic and transgenic DNA-repeat sequences (microsatellites) by inactivation of AtMSH2 mismatch-repair function.
(12/2256)
Highly conserved mismatch repair (MMR) systems promote genomic stability by correcting DNA replication errors, antagonizing homeologous recombination, and responding to various DNA lesions. Arabidopsis and other plants encode a suite of MMR protein orthologs, including MSH2, the constant component of various specialized eukaryotic mismatch recognition heterodimers. To study MMR roles in plant genomic stability, we used Arabidopsis AtMSH2::TDNA mutant SALK_002708 and AtMSH2 RNA-interference (RNAi) lines. AtMSH2::TDNA and RNAi lines show normal growth, development, and fertility. To analyze AtMSH2 effects on germ line DNA fidelity, we measured insertion-deletion mutation of dinucleotide-repeat sequences (microsatellite instability) at nine loci in 16 or more progeny of two to four different wild-type or AtMSH2-deficient plants. Scoring 992 total alleles revealed 23 (2.3%) unique and 51 (5.1%) total repeat length shifts ([+2], [-2], [+4], or [-4] bp). For the six longest repeat loci, the corresponding frequencies were 22/608 and 50/608. Two of four AtMSH2-RNAi plants showed similar microsatellite instability. In wild-type progeny, only one unique repeat length allele was found in 576 alleles tested. This endogenous microsatellite instability, shown for the first time in MMR-defective plants, is similar to that seen in MMR-defective yeast and mice, indicating that plants also use MMR to promote germ line fidelity. We used a frameshifted reporter transgene, (G)(7)GUS, to measure insertion-deletion reversion as blue-staining beta-glucuronidase-positive leaf spots. Reversion rates increased only 5-fold in AtMSH2::TDNA plants, considerably less than increases in MSH2-deficient yeast or mammalian cells for similar mononucleotide repeats. Thus, MMR-dependent error correction may be less stringent in differentiated leaf cells than in plant equivalents of germ line tissue. (+info)
Elg1 forms an alternative PCNA-interacting RFC complex required to maintain genome stability.
(13/2256)
BACKGROUND: Genome instability is a hallmark of cancer and plays a critical role in generating the myriad of phenotypes selected for during tumor progression. However, the mechanisms that prevent genome rearrangements remain poorly understood. RESULTS: To elucidate the mechanisms that ensure genome stability, we screened a collection of candidate genes for suppressors of gross chromosomal rearrangements (GCRs) in budding yeast. One potent suppressor gene encodes Elg1, a conserved but uncharacterized homolog of the large RFC subunit Rfc1 and the alternative RFC subunits Ctf18/Chl12 and Rad24. Our results are consistent with the hypothesis that Elg1 forms a novel and distinct RFC-like complex in both yeast and human cells. We find that Elg1 is required for efficient S phase progression and telomere homeostasis in yeast. Elg1 interacts physically with the PCNA homolog Pol30 and the FEN-1 homolog Rad27. The physical and genetic interactions suggest a role for Elg1 in Okazaki fragment maturation. Furthermore, Elg1 acts in concert with the alternative Rfc1-like proteins Rad24 and Ctf18 to enable Rad53 checkpoint kinase activation in response to replication stress. CONCLUSIONS: Collectively, these results reveal that Elg1 forms a novel and conserved alternative RFC complex. Furthermore, we propose that genome instability arises at high frequency in elg1 mutants due to a defect in Okazaki fragment maturation. (+info)
Large-scale genomic instability predicts long-term outcome for women with invasive stage I ovarian cancer.
(14/2256)
BACKGROUND: The objective was to evaluate the value of DNA ploidy using high-resolution image cytometry in predicting long-term survival of patients with early ovarian cancer. PATIENTS AND METHODS: A retrospective analysis of 284 cases with FIGO stage I ovarian carcinoma treated during the period 1982-1989 was performed. Clinical follow-up information was available for all patients. RESULTS: Patients with diploid and tetraploid tumors had a 10-year relapse-free survival of 95% and 89%, respectively, compared with 70% and 29% for polyploid and aneuploid tumors, respectively. DNA ploidy analysis was the strongest predictor of survival in multivariate analysis (diploid/tetraploid versus polyploid/aneuploid; relative hazard 9.0) followed by histological grade, including clear cell tumors in the group of poorly differentiated tumors (grade 1-2 versus grade 3 or clear cell; relative hazard 2.7), and FIGO stage (Ib/Ic versus Ia; relative hazard 2.0). In a stratified Kaplan-Meier analysis, patients with grade 1-2, diploid or tetraploid tumors had a 10-year relapse-free survival of 95%, forming a low-risk group. Patients with grade 3 or clear cell, diploid or tetraploid tumors had 10-year relapse-free survival of 86%, forming an intermediate-risk group, while all patients with aneuploid/polyploid tumors formed a high-risk group, with 10-year relapse-free survival of 34%. CONCLUSIONS: This study points to the importance of including DNA ploidy analysis by image cytometry when selecting patients with early ovarian cancer for adjuvant treatment after surgery. (+info)
Genomic instability induced by mutations in Saccharomyces cerevisiae POL1.
(15/2256)
Mutations of chromosome replication genes can be one of the early events that promote genomic instability. Among genes that are involved in chromosomal replication, DNA polymerase alpha is essential for initiation of replication and lagging-strand synthesis. Here we examined the effect of two mutations in S. cerevisiae POL1, pol1-1 and pol1-17, on a microsatellite (GT)(16) tract. The pol1-17 mutation elevated the mutation rate 13-fold by altering sequences both inside and downstream of the (GT)(16) tract, whereas the pol1-1 mutation increased the mutation rate 54-fold by predominantly altering sequences downstream of the (GT)(16) tract in a RAD52-dependent manner. In a rad52 null mutant background pol1-1 and pol1-17 also exhibited different plasmid and chromosome loss phenotypes. Deletions of mismatch repair (MMR) genes induce a differential synergistic increase in the mutation rates of pol1-1 and pol1-17. These findings suggest that perturbations of DNA replication in these two pol1 mutants are caused by different mechanisms, resulting in various types of mutations. Thus, mutations of POL1 can induce a variety of mutator phenotypes and can be a source of genomic instability in cells. (+info)
High-throughput molecular analysis of urine sediment for the detection of bladder cancer by high-density single-nucleotide polymorphism array.
(16/2256)
The detection of urothelial malignancies remains challenging. The majority of patients diagnosed with bladder cancer require life-long surveillance for disease recurrence. Monitoring strategies rely predominantly on invasive endoscopic techniques, which are inconvenient and uncomfortable. Multiple in vitro diagnostic technologies have been developed to supplant the contemporary standard of care. The U.S. Food and Drug Administration has approved several assays, but [because of inferior performance characteristics (low sensitivity and specificity)] these tests have not made a significant impact on practice, to date. We sought to develop a test for bladder cancer with better performance characterization detection based on a novel molecular approach. Matched urine and peripheral blood lymphocyte samples were obtained before surgery from 31 patients with bladder cancer (10 pTa, 4 pT1, and 17 pT2>or). DNA from these samples was subjected to allelic imbalance analysis using HuSNP chips and was validated in parallel with microsatellite analysis for loss of heterozygosity and microsatellite instability. Peripheral blood lymphocyte and urine DNA obtained from 14 individuals without clinical evidence of genitourinary malignancy served as controls. Thirty-one of 31 (100%) urine DNA samples from patients with bladder tumors were found to have 24 or more single-nucleotide polymorphism (SNP) DNA alterations. In general, SNP alterations were more common in urine samples from pT2>or tumors than pTa or pT1 tumors. SNP alterations were not identified in nine normal control subjects and in four of five patients with hematuria. These data support the noninvasive HuSNP chip assay in urine DNA as a valuable tool for the detection of bladder cancer (on a high-throughput-automated platform). (+info)