Normal telomere length and chromosomal end capping in poly(ADP-ribose) polymerase–deficient mice and primary cells despite increased chromosomal instability
TL;DR: The results presented here indicate that PARp-1 does not play a major role in regulating telomere length or in telomeric end capping, and the chromosomal instability of PARP-1−/− primary cells can be explained by the repair defect associated to PARP -1 deficiency.
Abstract: Poly(ADP-ribose) polymerase (PARP)-1, a detector of single-strand breaks, plays a key role in the cellular response to DNA damage. PARP-1-deficient mice are hypersensitive to genotoxic agents and display genomic instability due to a DNA repair defect in the base excision repair pathway. A previous report suggested that PARP-1-deficient mice also had a severe telomeric dysfunction consisting of telomere shortening and increased end-to-end fusions (d'Adda di Fagagna, F., M.P. Hande, W.-M. Tong, P.M. Lansdorp, Z.-Q. Wang, and S.P. Jackson. 1999. NAT: Genet. 23:76-80). In contrast to that, and using a panoply of techniques, including quantitative telomeric (Q)-FISH, we did not find significant differences in telomere length between wild-type and PARP-1(-/)- littermate mice or PARP-1(-/)- primary cells. Similarly, there were no differences in the length of the G-strand overhang. Q-FISH and spectral karyotyping analyses of primary PARP-1(-/)- cells showed a frequency of 2 end-to-end fusions per 100 metaphases, much lower than that described previously (d'Adda di Fagagna et al., 1999). This low frequency of end-to-end fusions in PARP-1(-/)- primary cells is accordant with the absence of severe proliferative defects in PARP-1(-/)- mice. The results presented here indicate that PARP-1 does not play a major role in regulating telomere length or in telomeric end capping, and the chromosomal instability of PARP-1(-/)- primary cells can be explained by the repair defect associated to PARP-1 deficiency. Finally, no interaction between PARP-1 and the telomerase reverse transcriptase subunit, Tert, was found using the two-hybrid assay.
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Dissertation•
01 Jan 2008
TL;DR: Dans cette perspective, nous avons etabli des clones telomerase (+) exprimant de maniere stable le transgene P53R175H, decrit comme stimulant the RH, se revelant ainsi incapable of stimuler the recombinaison telomerique.
Abstract: Les telomeres sont des structures nucleoproteiques particulieres conferant une stabilite aux extremites de chromosomes. La plupart des mecanismes telomeriques sont conservee chez les eucaryotes superieurs notamment chez l'homme. Parmi les fonctions exercees, les telomeres developpent des strategies visant a limiter la recombinaison impropre aux telomeres afin de preserver l'integrite chromosomique. Au cours de ma these, j'ai etudie les mecanismes de maintenance et de protection des telomeres chez l'homme, et plus particulierement le role de la recombinaison homologue et recombinaison illegitime. Je me suis plus particulierement interesse au role de P53 dans le maintien des telomeres, a travers sa fonction dans la recombinaison. Bien que son role soit etabli dans la maintenance des telomeres des cellules ALT, sa fonction est en revanche moins bien decrite dans les cellules presentant une activite telomerase. Dans cette perspective, nous avons etabli des clones telomerase (+) exprimant de maniere stable le transgene P53R175H, decrit comme stimulant la RH. Cependant, la surexpression de P53R175H ne modifie pas le profil telomerique des ces cellules, se revelant ainsi incapable de stimuler la recombinaison telomerique. L'absence de phenotype pourrait etre en fait la consequence d'une interference de la telomerase dans la mise en place d'un processus de recombinaison telomerique. Le deuxieme volet de notre etude nous amene a preciser le role la recombinaison dans le maintien des telomeres. Pour cela, nous avons construit des lignees isogeniques invalidees pour les deux voies majeures de la recombinaison grâce a une nouvelle famille de vecteurs ARNi basee sur des vecteurs EBV (brevet CEA N° 0501483) developpe par Biard D. Ce systeme permet d'obtenir une reduction importante, specifique et a long terme (plus de 300 jours en culture) de l'expression d'un gene cible. Plusieurs partenaires du NHEJ (DNA-PKcs, XRCC4, LigIV, Ku70), ou de la RH (RAD54, RAD51, RAD52) ou du complexe MRN ont ete inhibes dans la lignee HeLa (adenocarcinome cervical). Des les premiers jours d'invalidation du gene cible, des alterations de la stabilite des telomeres sont constatees. Ces alterations se maintiennent au cours de la selection (de 100 a 300 jours selon les clones). Ainsi, il semble que l'invalidation du NHEJ conduise a une instabilite telomerique associee a un raccourcissement de la taille des telomeres dans le cas de XRCC4 et LigaseIVKD. Malgre une instabilite telomerique prononcee, les cellules RHKD se distinguent pourtant des clones NHEJKD par des alterations telomeriques particulieres. En effet, la deregulation du RH mene a des signaux heterogenes au sein du meme bras chromosomique. En revanche, les anomalies dites de structure (perte associee a des cassures) sont plus abondantes dans les cellules deficientes pour le NHEJ. Ces resultats suggerent que le NHEJ jouerait un role de protection alors que le RH serait plutot implique dans l'elongation et la replication des telomeres. Cette etude s'est poursuit avec l'examen de clones MRE11KD, RAD50KD, NBS1KD permettant de clarifier dans un premier temps le role de du complexe dans les differentes voies de recombinaison puis sur la maintenance des telomeres. A l'image des autres proteines de la recombinaison, RAD50 preserverait l'integrite telomerique. L'invalidation des proteines NBS1 et MRE11 initie une elongation des telomeres, probable manifestation de l‘activation de la recombinaison telomerique, absente des cellules RAD50KD. Ainsi, la presence de cercles telomeriques extrachromosomiques dans le clone NBS1KD vient etayer cettehypothese. Le complexe MRE11/RAD50/NBS1 constituerait alors un represseur de la RH aux telomeres.
2 citations
Dissertation•
13 Apr 2011
TL;DR: El objetivo del presente proyecto de tesis ha sido the determinacion of los efectos de the inhibicion of PARP, una proteina nuclear implicada en procesos de reconocimiento y reparacion of danos en el ADN.
Abstract: La ausencia de control de los mecanismos apoptoticos proporciona ventajas de supervivencia a las celulas tumorales (1). p53 juega un papel central en la regulacion del crecimiento y muerte celulares (2). En cancer de mama, las alteraciones de las vias apoptoticas incluyen la regulacion negativa de la via de receptores de muerte, mutaciones en p53 y sobre-expresion de BCL-2 (3-5). Estas deficiencias en p53 confieren resistencia a tratamientos quimioterapeuticos usados en clinica, como es el caso de Doxorubicina (DOXO), una potente antraciclina ampliamente usada en protocolos clinicos antineoplasicos (6,7). El objetivo del presente proyecto de tesis ha sido la determinacion de los efectos de la inhibicion de PARP, una proteina nuclear implicada en procesos de reconocimiento y reparacion de danos en el ADN (8,9), sobre la citotoxicidad de doxorubicina y el mecanismo por el cual el uso de inhibidores quimicos de PARP (4-amino 1,8-naftalimida, ANI) sensibiliza la muerte inducida por doxo.
2 citations
Dissertation•
01 Jan 2014
2 citations
Cites background from "Normal telomere length and chromoso..."
...16 telomeres in telomerase-deficient murine embryonic stem cells had been reported (d'Adda di Fagagna et al., 1999; Samper et al., 2001; Gomez et al., 2006)....
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...Moreover, a recruitment of PARP-1 to critically short Introduction 16 telomeres in telomerase-deficient murine embryonic stem cells had been reported (d'Adda di Fagagna et al., 1999; Samper et al., 2001; Gomez et al., 2006)....
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DOI•
01 Jan 2011
TL;DR: In this article, the latent biological effect of childhood sexual abuse (CSA) on adults by quantifying acquired cytogenetic changes and cortisol levels in identical twins who were discordant or concordant for a history of CSA was evaluated.
Abstract: ACQUIRED CYTOGENETIC CHANGES IN ADULT TWINS DISCORDANT FOR A HISTORY OF CHILDHOOD SEXUAL ABUSE Jenni Rebecca Brumelle, B.S., B.A. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth University, 2011 Director: Colleen Jackson-Cook, Ph.D. Department of Pathology The primary study aim was to evaluate the latent biological effect of childhood sexual abuse (CSA) on adults by quantifying acquired cytogenetic changes and cortisol levels in identical twins who were discordant (N=22) or concordant (N=2) for a history of CSA. Although the difference scores for cortisol values between discordant identical cotwins were not significantly different from zero, a trend was observed for the twins exposed to intercourse, the most severe form of CSA, to have a blunted cortisol awakening response. Acquired cytogenetic changes were assessed by scoring telomere lengths and somatic cell abnormality frequencies via a cytokinesis-block
1 citations
01 Jan 2013
1 citations
References
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TL;DR: P16 seems to act in a regulatory feedback circuit with CDK4, D-type cyclins and retinoblastoma protein, and inhibits the catalytic activity of theCDK4/cyclin D enzymes.
Abstract: The division cycle of eukaryotic cells is regulated by a family of protein kinases known as the cyclin-dependent kinases (CDKs). The sequential activation of individual members of this family and their consequent phosphorylation of critical substrates promotes orderly progression through the cell cycle. The complexes formed by CDK4 and the D-type cyclins have been strongly implicated in the control of cell proliferation during the G1 phase. CDK4 exists, in part, as a multi-protein complex with a D-type cyclin, proliferating cell nuclear antigen and a protein, p21 (refs 7-9). CDK4 associates separately with a protein of M(r) 16K, particularly in cells lacking a functional retinoblastoma protein. Here we report the isolation of a human p16 complementary DNA and demonstrate that p16 binds to CDK4 and inhibits the catalytic activity of the CDK4/cyclin D enzymes. p16 seems to act in a regulatory feedback circuit with CDK4, D-type cyclins and retinoblastoma protein.
3,716 citations
"Normal telomere length and chromoso..." refers methods in this paper
...As control for the two-hybrid assay we show interaction between the cell cycle proteins CDK4 and p16 as described previously (Table VI; Serrano et al., 1993)....
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TL;DR: The DNA of telomeres—the terminal DNA-protein complexes of chromosomes—differs notably from other DNA sequences in both structure and function, and has been shown to be essential for telomere maintenance and long-term viability.
Abstract: The DNA of telomeres--the terminal DNA-protein complexes of chromosomes--differs notably from other DNA sequences in both structure and function. Recent work has highlighted its remarkable mode of synthesis by the ribonucleoprotein reverse transcriptase, telomerase, as well as its ability to form unusual structures in vitro. Moreover, telomere synthesis by telomerase has been shown to be essential for telomere maintenance and long-term viability.
3,139 citations
TL;DR: Electron microscopy reported here demonstrated that TRF2 can remodel linear telomeric DNA into large duplex loops (t loops) in vitro, which may provide a general mechanism for the protection and replication of telomeres.
2,413 citations
TL;DR: Results indicate that telomerase is essential for telomere length maintenance but is not required for establishment of cell lines, oncogenic transformation, or tumor formation in mice.
2,066 citations
"Normal telomere length and chromoso..." refers background or methods in this paper
...These Robertsonian fusions are different from those found in late generation telomerase-deficient mice that have critically short telomeres (Blasco et al., 1997)....
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...5 embryos derived from heterozygous crosses as described (Blasco et al., 1997)....
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...Telomeres have an essential role in chromosome stability and are proposed to be biological determinants in the processes of tumorigenesis and aging (for reviews see Blackburn, 1991; Autexier and Greider, 1996; Greider, 1996; Blasco et al., 1997; Lee et al., 1998)....
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...Then the plugs were digested with MboI overnight and run in a pulse field gel electrophoresis as described (Blasco et al., 1997)....
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TL;DR: The history and present situation of Spanish language, culture, literature, cuisine, tourism, and more are explored in more detail in this booklet.
Abstract: TELOMERES DEFINED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579 TELOMERE FUNCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580 SEQUENCE AND STRUCTURE OF TELOMERES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581 SOLUTIONS FOR REPLICATION OF DNA TERMINI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 586 STRUCTURE OF SUBTELOMERIC REGIONS.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589 FORMA TION OF TELOMERES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . .. 591 PROTEINS THAT INTERACT WITH TELOMERES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594 ARE TELOMERES REALLY ESSENTIAL? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597 FUTURE PROSPECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598
1,923 citations
"Normal telomere length and chromoso..." refers background in this paper
...Vertebrate telomeres consist of tandem repeats of the sequence TTAGGG (for review see Blackburn, 1991)....
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...Telomeres have an essential role in chromosome stability and are proposed to be biological determinants in the processes of tumorigenesis and aging (for reviews see Blackburn, 1991; Autexier and Greider, 1996; Greider, 1996; Blasco et al., 1997; Lee et al., 1998)....
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