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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TL;DR: The dietary status of niacin (vitamin B3) has the potential to influence DNA repair, genomic stability, and the immune system, eventually having an impact on cancer risk, as well as the side effects of chemotherapy in the cancer patient.
Abstract: The dietary status of niacin (vitamin B3) has the potential to influence DNA repair, genomic stability, and the immune system, eventually having an impact on cancer risk, as well as the side effects of chemotherapy in the cancer patient. In addition to its well-known redox functions in energy metabolism, niacin, in the form of NAD, participates in a wide variety of ADP-ribosylation reactions.Poly(ADP-ribose) is a negatively charged polymer synthesized, predominantly on nuclear proteins, by at least seven different enzymes. Poly(ADP-ribose) polymerase-1 (PARP-1) is responsible for the majority of polymer synthesis and plays important roles in DNA damage responses, including repair, maintenance of genomic stability, and signaling events for stress responses such as apoptosis. NAD is also used in the synthesis of mono(ADP-ribose), often on G proteins, with poorly understood roles in signal transduction. Last, NAD and NADP are required for the synthesis of cyclic ADP-ribose and nicotinic acid adenine dinucleo...
63 citations
Cites background from "Normal telomere length and chromoso..."
...Reports from the second PARP-null model also found chromosomal instability, but no change in telomere length or end capping (78)....
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TL;DR: The data suggest that murine mTep1 and mVparp, alone or in combination, are dispensable for normal development, telomerase catalysis, telomere length maintenance, and vault structure in vivo.
Abstract: Telomerase is a ribonucleoprotein (RNP) complex that replenishes telomere loss due to incomplete DNA replication in almost all eukaryotes. A loss of telomerase activity leads to an attrition of telomeric DNA which, in turn, is known to trigger end-to-end chromosome fusions, genomic instability, and cell arrest or death (31). Telomerase expression is thus critical to the prolonged viability of several human cell types and in the majority of human cancers (3). Elucidating the molecular machinery that regulates telomerase activity and its ability to maintain telomere length is critical to an understanding of malignant transformation.
Telomerase contains two core components, telomerase reverse transcriptase (TERT) and telomerase RNA, the latter serving as an integral template for the de novo synthesis of telomeric DNA. Both TERT and telomerase RNA are required for the reconstitution of telomerase activity in vitro (2, 75). In mice, the disruption of either component also abolishes telomerase activity, leading to telomere attrition, genetic instability, and eventual infertility (4, 15, 26, 44, 48, 61, 76).
Several other telomerase-associated proteins have been identified in mammals (22); these include telomerase-associated protein 1 (TEP1), which binds telomerase RNA (23, 55) and which was cloned based on its homology to ciliate Tetrahymena thermophila protein p80 (8, 20, 23, 55). T. thermophila p80 was identified as a species copurifying with telomerase, although it subsequently was found unlikely to be a core telomerase component (8, 20, 49). Although deletion of the T. thermophila p80 gene resulted in slight telomere lengthening (51), no change in telomere length was observed upon breeding of mTep1-deficient mice for up to seven generations. Telomerase activity was not affected in the absence of either the p80 gene or mTep1 (47, 51). Disruption of mTep1 did not affect the levels of telomerase RNA or its association with the telomerase RNP (34), and TEP1 appeared to associate with only a fraction of the total telomerase activity in immortalized cell extracts (Y. Liu and L. Harrington, unpublished data). Although these results suggest that TEP1 is nonessential for telomerase function in normal mouse tissues, the possibility of genetic redundancy with other telomerase-associated proteins cannot be excluded. Indeed, other telomerase-associated proteins have been identified and have been shown to interact with telomerase RNA in mammals; these include La, L22, Staufen, DKC, and several heterogeneous nuclear RNPs (11, 16-19, 29, 42, 43, 52).
TEP1 is an integral component of another RNP, the vault particle. Mammalian vaults, the largest known mammalian RNPs (13 MDa), are composed of at least four components, major vault protein (MVP), vault poly(ADP-ribose) polymerase (VPARP), TEP1, and one or more small vault RNAs (vRNAs) (71, 73). Vaults possess a distinct morphology that is highly conserved. Purified vaults display a unique eightfold barrel-like symmetry structure with caps on each end (32, 38). Although the function of the vault particle has remained elusive, its highly conserved structure, its ubiquitous distribution, and its up-regulation in several human drug-resistant cancers have led to the speculation that vaults have an important cellular function and may be carriers involved in intracellular transport (54, 71, 73). The absence of TEP1 completely disrupts the stable association of vRNA with the purified vault particle and results in decreases in the levels and stability of vRNA (34). Therefore, TEP1 is an integral vault protein and is important for the stabilization and recruitment of vRNA to the vault particle (34).
VPARP, the catalytic vault protein component, contains regions with similarity to BRCT, a poly(ADP-ribose) polymerase (PARP) catalytic domain, inter-α-trypsin and putative von Willebrand type A domains, and a C-terminal MVP-interacting domain (35, 71, 73). The putative VPARP catalytic domain shares 28% identity with the catalytic domain of PARP 1 (PARP1). Like that in PARP1, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction, and the substrates for this vault-associated PARP activity are MVP and VPARP itself (35). Thus, VPARP is a unique member of the PARP family. In addition to its association with vaults, VPARP has also been found at other cellular locations, such as the nucleus and mitotic spindle (35), indicating that it may possess multiple roles in vivo. Here, we report that VPARP interacts with TEP1 and associates with telomerase activity in cell extracts, suggesting that VPARP and TEP1 may play roles in both cytoplasmic and nuclear RNP complexes. We generated mice deficient in mVparp or both mTep1 and mVparp and investigated telomerase function and vault structure in their absence.
62 citations
Cites background from "Normal telomere length and chromoso..."
...Although an interaction between PARP1 and TERT was reported in a human breast cancer cell line (5), a yeast twohybrid based assay was unable to detect such an interaction (63)....
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...observed normal telomere length and G-rich overhangs and low frequencies of chromosome end-to-end fusions in Parp1-deficient mice (63)....
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TL;DR: ATR recognizes critically short telomeres as fragile sites and protects them from chromosomal fusions.
Abstract: Telomere shortening caused by incomplete DNA replication is balanced by telomerase-mediated telomere extension, with evidence indicating that the shortest telomeres are preferred substrates in primary cells. Critically short telomeres are detected by the cellular DNA damage response (DDR) system. In budding yeast, the important DDR kinase Tel1 (homologue of ATM [ataxia telangiectasia mutated]) is vital for telomerase recruitment to short telomeres, but mammalian ATM is dispensable for this function. We asked whether closely related ATR (ATM and Rad3 related) kinase, which is important for preventing replicative stress and chromosomal breakage at common fragile sites, might instead fulfill this role. The newly created ATR-deficient Seckel mouse strain was used to examine the function of ATR in telomerase recruitment and telomere function. Telomeres were recently found to resemble fragile sites, and we show in this study that ATR has an important role in the suppression of telomere fragility and recombination. We also find that wild-type ATR levels are important to protect short telomeres from chromosomal fusions but do not appear essential for telomerase recruitment to short telomeres in primary mouse embryonic fibroblasts from the ATR-deficient Seckel mouse model. These results reveal a previously unnoticed role for mammalian ATR in telomere protection and stability.
61 citations
Cites background or methods or result from "Normal telomere length and chromoso..."
...…to have a shorter mean telomere length than other mouse chromosomes in several independent mouse strains (Zijlmans et al., 1997; Hande et al., 1999; Samper et al., 2001a), whereas chromosome 11 was chosen because it is homologous to human chromosome 17, which was also reported to have relatively…...
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...…undetectable telomeres (bottom panel, signal-free ends [SFEs]), which were completely rescued in cells inheriting the wild-type Terc allele (P < 0.0001; compare i with iii), which is in agreement with telomerase preferentially elongating critically short telomeres in MEFs (Samper et al., 2001a)....
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...…has been shown by numerous groups to act preferentially on the shortest telomeres under conditions of telomere shortening similar to those used in the current study (Hemann et al., 2001; Perrem et al., 2001; Samper et al., 2001a; Liu et al., 2002; Teixeira et al., 2004; Zhao et al., 2009)....
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...These results further indicate that increased telomeric those telomeres that fall below a critical threshold are lengthened and not all telomeres in the cell (Hemann et al., 2001; Samper et al., 2001a)....
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...The same metaphases previously captured for Q-FISH were relocated, and SKY images captured and processed according to Samper et al. (2001b)....
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TL;DR: This review summarizes the current knowledge on the mechanisms of telomerase recruitment and activation using insights from studies in mammals and budding and fission yeasts and discusses the differences in telomere homeostasis between normal cells and cancer cells, which may provide a foundation fortelomere/telomerase targeted cancer treatments.
Abstract: Aberrant activation of telomerase occurs in 85-90% of all cancers and underpins the ability of cancer cells to bypass their proliferative limit, rendering them immortal. The activity of telomerase is tightly controlled at multiple levels, from transcriptional regulation of the telomerase components to holoenzyme biogenesis and recruitment to the telomere, and finally activation and processivity. However, studies using cancer cell lines and other model systems have begun to reveal features of telomeres and telomerase that are unique to cancer. This review summarizes our current knowledge on the mechanisms of telomerase recruitment and activation using insights from studies in mammals and budding and fission yeasts. Finally, we discuss the differences in telomere homeostasis between normal cells and cancer cells, which may provide a foundation for telomere/telomerase targeted cancer treatments.
60 citations
TL;DR: The results suggest that PARP-1 abolition impairs the promotion of skin carcinogenesis interfering with the activation of NF-κB and might have an important implication in targeting PARP -1 as a new antineoplastic therapeutic approach.
Abstract: Poly (ADP-ribose) polymerase-1 (PARP-1)-deficient mice are protected against septic shock, type I diabetes, stroke and inflammation. It is now accepted that inflammation and related events, such as activation of NF-kappaB, are key components in the initiation and progression of epithelial cancer and in particular in the neoplastic transformation of keratinocytes and skin carcinogenesis. Here, we report that PARP-1-deficient mice display a strikingly reduced susceptibility to skin carcinogenesis. In parp-1(-/-) mice, development of papilloma-like premalignant lesions induced with DMBA and TPA, is strongly delayed and the final number of tumor-bearing mice and total tumor number were significantly reduced. In addition, epidermis of parp-1(-/-) mice did not show increased proliferation rates after treatment with carcinogen. Deregulated NF-kappaB is a hallmark for tumorigenesis together with the concomitant release of early inflammatory mediators. In the absence of PARP-1, NF-kappaB activation and induction kappaB-target genes did not take place during the promotion of tumor development. These results suggest that PARP-1 abolition impairs the promotion of skin carcinogenesis interfering with the activation of NF-kappaB and might have an important implication in targeting PARP-1 as a new antineoplastic therapeutic approach.
59 citations
Cites background from "Normal telomere length and chromoso..."
...Treatment of PARP-1 knockout mice with either alkylating agents or girradiation reveals an extreme sensitivity and a high genomic instability to both agents (de Murcia et al., 1997; Samper et al., 2001)....
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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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