Effects of topoisomerase II inhibition in lymphoblasts from patients with progeroid and “chromosome instability” syndromes

Elli, R.; Chessa, Luciana; Antonelli, A.; Petrinelli, P.; Ambra, Roberto; Marcucci, L.

doi:10.1016/0165-4608(95)00294-4

Cited by 37 publications

(23 citation statements)

References 18 publications

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“…It is thus reasonable to ask whether the mutant WS protein might alter the sensitivity of WS cells to agents that block the action of one or the other topoisomerase. Indeed, it has been shown that human WS lymphoblasts show an increased sensitivity to both topoisomerase type I and II inhibitors, camptothecin and etoposide (46,47). Consistent with this, we have shown that the murine mutant ES cells are similarly more sensitive to etoposide and even more sensitive to the type I topoisomerase inhibitor, camptothecin.…”

Section: Discussionsupporting

confidence: 75%

A deletion within the murine Werner syndrome helicase induces sensitivity to inhibitors of topoisomerase and loss of cellular proliferative capacity

Lebel

Leder

1998

Proc. Natl. Acad. Sci. U.S.A.

279

194

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Werner syndrome (WS) is an autosomal recessive disorder characterized by genomic instability and the premature onset of a number of age-related diseases. The gene responsible for WS encodes a member of the RecQ-like subfamily of DNA helicases. Here we show that its murine homologue maps to murine chromosome 8 in a region syntenic with the human WRN gene. We have deleted a segment of this gene and created Wrn-deficient embryonic stem (ES) cells and WS mice. While displaying reduced embryonic survival, live-born WS mice otherwise appear normal during their first year of life. Nonetheless, although several DNA repair systems are apparently intact in homozygous WS ES cells, such cells display a higher mutation rate and are significantly more sensitive to topoisomerase inhibitors (especially camptothecin) than are wild-type ES cells. Furthermore, mouse embryo fibroblasts derived from homozygous WS embryos show premature loss of proliferative capacity. At the molecular level, wild-type, but not mutant, WS protein copurifies through a series of centrifugation and chromatography steps with a multiprotein DNA replication complex.

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Section: Discussionsupporting

confidence: 75%

A deletion within the murine Werner syndrome helicase induces sensitivity to inhibitors of topoisomerase and loss of cellular proliferative capacity

Lebel

Leder

1998

Proc. Natl. Acad. Sci. U.S.A.

279

194

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“…We noticed that murine mutant ES cells are more sensitive to both types I and II topoisomerase inhibitors than are wild type ES cells. Similar results were obtained with human WS cell lines (31,32). Because both of these topoisomerases are involved in DNA replication, it is possible that the WS protein acts in concert with these enzymes as part of a replication structure.…”

Section: Discussionsupporting

confidence: 74%

The Werner Syndrome Gene Product Co-purifies with the DNA Replication Complex and Interacts with PCNA and Topoisomerase I

Lebel¹,

Spillare²,

Harris³

et al. 1999

Journal of Biological Chemistry

233

168

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Werner syndrome (WS) is a recessive disorder characterized by genomic instability and by the premature onset of a number of age-related diseases. To understand the molecular basis of this disease, we deleted a segment of the murine Wrn gene and created Wrn-deficient embryonic stem (ES) cells. At the molecular level, wild type-but not mutant-WS protein co-purifies through a series of centrifugation, chromatography, and sucrose gradient steps with the well characterized 17 S multiprotein DNA replication complex. Furthermore, wild type WS protein co-immunoprecipitates with a prominent component of the multiprotein replication complex, proliferating cell nuclear antigen (PCNA). In vitro studies also indicate that PCNA binds to a region in the N terminus portion of the WS protein containing a potential 3-5 exonuclease domain. Finally, human WS protein also co-immunoprecipitates with both PCNA and topoisomerase I. These results suggest that the WS protein interacts with several components of the DNA replication fork.Werner syndrome (WS) 1 is a rare disorder characterized by the premature onset of a number of processes associated with aging (1, 2). In addition, the proliferative life span of WS fibroblasts is reduced compared with age-matched controls (3)(4)(5). WS cells from patients also exhibit genomic abnormalities such as variegated chromosomal translocations and deletions (6, 7). Most recently, WS cells have been shown to have an attenuation of a p53-dependent apoptotic pathway (8).The WS gene (WRN) product contains seven helicase consensus domains that are 34 -38% identical to the Escherichia coli RecQ gene (9,10) and to the putative yeast helicase Sgs1p (11,12). The Sgs1p is known to interact with types I and II topoisomerases and is required for genome stability in Saccharomyces cerevisiae (11)(12)(13). In addition, an interesting model involving the promotion of extrachromosomal rDNA circles has been proposed for the aging phenotype in yeast Sgs1 mutants (14). Several studies suggest that the WS protein may be implicated in some aspect of DNA replication. WS cells show a prolongation of the S phase of the cell cycle and a decreased rate of DNA synthesis (15). Interestingly, the homologue of the human Werner syndrome gene product in Xenopus laevis is required for the formation of replication foci in egg extracts (16).To study the possible interaction of the mouse WS protein with the DNA replication apparatus, we recently created a deletion of part of the helicase domain of the murine homologue of the WS gene in embryonic stem (ES) cells (17). Biochemical studies of our ES cell lines allowed us to conclude that the WS protein co-purifies with the DNA replication complex and binds to certain of its specific components. EXPERIMENTAL PROCEDURESProtein Analysis-Generation and maintenance of the wild type and homozygous mutant embryonic stem cells have been described previously (17). Protein extraction, immunoprecipitations, and Western blotting analyses were performed as described (8,18). A rabbit polyclonal anti...

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“…EBV-transformed WS and normal lymphoblast cell lines were exposed to different concentrations of VP-16, m-AMSA and 50 cGy X-rays. Our results show higher susceptibility of WS cells for the induction of chromatid-type exchanges after G 2 treatment with both VP-16 and m-AMSA, confirming the data reported by Elli et al (1996), but not with X-rays. Further analysis will performed on the localisation and type of induced chromosomal damage (breaks versus exchanges).…”

Section: P220supporting

confidence: 82%

Mutagenesis

Natarajan¹

1998

Cytogenetic and Genome Research

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Effects of topoisomerase II inhibition in lymphoblasts from patients with progeroid and “chromosome instability” syndromes

Cited by 37 publications

References 18 publications

A deletion within the murine Werner syndrome helicase induces sensitivity to inhibitors of topoisomerase and loss of cellular proliferative capacity

A deletion within the murine Werner syndrome helicase induces sensitivity to inhibitors of topoisomerase and loss of cellular proliferative capacity

The Werner Syndrome Gene Product Co-purifies with the DNA Replication Complex and Interacts with PCNA and Topoisomerase I

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