Abstract:Germline mutations affecting telomere maintenance or DNA repair may, respectively, cause dyskeratosis congenita or Fanconi anaemia, two clinically related bone marrow failure syndromes. Mice expressing p53Δ31, a mutant p53 lacking the C terminus, model dyskeratosis congenita. Accordingly, the increased p53 activity in p53Δ31/Δ31 fibroblasts correlated with a decreased expression of 4 genes implicated in telomere syndromes. Here we show that these cells exhibit decreased mRNA levels for additional genes contrib… Show more
“…Nutlin treatment decreased total FancD2 level in LXSN cells but not in E6 expressing cells (Fig 7C). Similar downregulation of FancD2 was observed in lung fibroblast cells upon Nutlin-induced p53 activation, with no significant change in FancD2 level in p53-deficient counterparts (46). Fig 7.E6 mediated increased FancD2 monoubiquitination is p53 independent, but delayed FancD2 de-ubiquitination is dependent on p53 degradation(A) Immunoblot for p53 in HFK cells transduced with LXSN, E6 and E6 mutant (8S/9A/10T).…”
Section: Resultssupporting
confidence: 56%
“…To evaluate the expression of FancD2 mRNA, two sets of primers were designed to amplify the longer FancD2 isoform from transcript sequence NM_033084.4. The first primer set adapted from Jaber et al (46) was Forward 5’-AGACTGTCAAAATCTGAGGATAAAGAGA-3’ and Reverse 5’-TGGTTGCTTCCTGGTTTTGG-3’; and next set was designed as Forward 5’- CATGGCTGTTCGAGACTTC-3’ and Reverse 5’-CACAAAGAGACGCCCATAC-3’.…”
10Persistent expression of high-risk HPV oncogenes is necessary for the development of anogenital and 11 oropharyngeal cancers. Here, we show that E6/E7 expressing cells are hypersensitive to DNA 12 crosslinking agent cisplatin and have defects in repairing DNA interstrand crosslinks (ICL). Importantly, 13 we elucidate how E6/E7 attenuate the Fanconi anemia (FA) DNA crosslink repair pathway. Though 14 E6/E7 activated the pathway by increasing FancD2 monoubiquitination and foci formation, they inhibited 15 the completion of the repair by multiple mechanisms. E6/E7 impaired FancD2 colocalization with double-16 strand breaks (DSB), which subsequently hindered the recruitment of downstream protein Rad51 to DSB 17 in E6 cells. Further, E6 expression caused delayed FancD2 de-ubiquitination, an important process for 18 effective ICL repair. Delayed FancD2 de-ubiquitination was attributed to the increased chromatin 19 retention of FancD2 hindering USP1 de-ubiquitinating activity, and persistently activated ATR/CHK-20 1/pS565 FancI signaling. E6 mediated p53 degradation did not hamper the cell cycle specific process of 21 FancD2 modifications but abrogated repair by disrupting FancD2 de-ubiquitination. Further, E6 reduced 22 the expression and foci formation of Palb2, which is a repair protein downstream of FancD2. These 23 findings uncover unique mechanisms by which HPV oncogenes contribute to genomic instability and the 24 response to cisplatin therapies. 25 42 High-risk human papillomavirus (HR-HPV) E6/E7 oncoproteins are essential for the development of 43 malignancies of the anogenital tract and oropharynx, with HPV16 being the predominant type (1). 44 Cervical and oropharyngeal cancers are the most common HPV-associated malignancies among females 45 and males, respectively (2). Persistent HPV infection destabilizes the cellular genome which can lead to 46 cancer. Genomic instability is likely the result of the numerous interactions of HPV oncoproteins with 47 host tumor suppressors and DNA damage repair (DDR) proteins. Recently, we demonstrated that high-48 risk HPV oncogenes attenuate double-strand break (DSB) repair by impairing the homologous 49 recombination pathway (3). To further elucidate the mechanisms by which HPV oncogenes impair DDR, 50 the present study focuses on the impact of HPV16 oncogenes on the Fanconi anemia-BRCA (FA or FA-51 BRCA) pathway.
52The FA-BRCA pathway is involved in the repair of intra or interstrand crosslinks (ICL), thereby 53 maintaining genomic stability (4, 5) ( Fig S1). ICLs block DNA replication and transcription and, thus, 54 are highly cytotoxic to cells if not repaired. The FA pathway is composed of 22 FA proteins (identified to 55 date) (6). When any one of the FA genes of the FA-BRCA pathway is mutated, individuals have a 56 spectrum of disorders, called Fanconi Anemia, characterized by bone marrow failure, congenital 57 malformations, cancer predisposition, and cellular sensitivity to ICL-inducing agents (7). Upon exposure 58 to DNA crosslinking agents, and during S-phase o...
“…Nutlin treatment decreased total FancD2 level in LXSN cells but not in E6 expressing cells (Fig 7C). Similar downregulation of FancD2 was observed in lung fibroblast cells upon Nutlin-induced p53 activation, with no significant change in FancD2 level in p53-deficient counterparts (46). Fig 7.E6 mediated increased FancD2 monoubiquitination is p53 independent, but delayed FancD2 de-ubiquitination is dependent on p53 degradation(A) Immunoblot for p53 in HFK cells transduced with LXSN, E6 and E6 mutant (8S/9A/10T).…”
Section: Resultssupporting
confidence: 56%
“…To evaluate the expression of FancD2 mRNA, two sets of primers were designed to amplify the longer FancD2 isoform from transcript sequence NM_033084.4. The first primer set adapted from Jaber et al (46) was Forward 5’-AGACTGTCAAAATCTGAGGATAAAGAGA-3’ and Reverse 5’-TGGTTGCTTCCTGGTTTTGG-3’; and next set was designed as Forward 5’- CATGGCTGTTCGAGACTTC-3’ and Reverse 5’-CACAAAGAGACGCCCATAC-3’.…”
10Persistent expression of high-risk HPV oncogenes is necessary for the development of anogenital and 11 oropharyngeal cancers. Here, we show that E6/E7 expressing cells are hypersensitive to DNA 12 crosslinking agent cisplatin and have defects in repairing DNA interstrand crosslinks (ICL). Importantly, 13 we elucidate how E6/E7 attenuate the Fanconi anemia (FA) DNA crosslink repair pathway. Though 14 E6/E7 activated the pathway by increasing FancD2 monoubiquitination and foci formation, they inhibited 15 the completion of the repair by multiple mechanisms. E6/E7 impaired FancD2 colocalization with double-16 strand breaks (DSB), which subsequently hindered the recruitment of downstream protein Rad51 to DSB 17 in E6 cells. Further, E6 expression caused delayed FancD2 de-ubiquitination, an important process for 18 effective ICL repair. Delayed FancD2 de-ubiquitination was attributed to the increased chromatin 19 retention of FancD2 hindering USP1 de-ubiquitinating activity, and persistently activated ATR/CHK-20 1/pS565 FancI signaling. E6 mediated p53 degradation did not hamper the cell cycle specific process of 21 FancD2 modifications but abrogated repair by disrupting FancD2 de-ubiquitination. Further, E6 reduced 22 the expression and foci formation of Palb2, which is a repair protein downstream of FancD2. These 23 findings uncover unique mechanisms by which HPV oncogenes contribute to genomic instability and the 24 response to cisplatin therapies. 25 42 High-risk human papillomavirus (HR-HPV) E6/E7 oncoproteins are essential for the development of 43 malignancies of the anogenital tract and oropharynx, with HPV16 being the predominant type (1). 44 Cervical and oropharyngeal cancers are the most common HPV-associated malignancies among females 45 and males, respectively (2). Persistent HPV infection destabilizes the cellular genome which can lead to 46 cancer. Genomic instability is likely the result of the numerous interactions of HPV oncoproteins with 47 host tumor suppressors and DNA damage repair (DDR) proteins. Recently, we demonstrated that high-48 risk HPV oncogenes attenuate double-strand break (DSB) repair by impairing the homologous 49 recombination pathway (3). To further elucidate the mechanisms by which HPV oncogenes impair DDR, 50 the present study focuses on the impact of HPV16 oncogenes on the Fanconi anemia-BRCA (FA or FA-51 BRCA) pathway.
52The FA-BRCA pathway is involved in the repair of intra or interstrand crosslinks (ICL), thereby 53 maintaining genomic stability (4, 5) ( Fig S1). ICLs block DNA replication and transcription and, thus, 54 are highly cytotoxic to cells if not repaired. The FA pathway is composed of 22 FA proteins (identified to 55 date) (6). When any one of the FA genes of the FA-BRCA pathway is mutated, individuals have a 56 spectrum of disorders, called Fanconi Anemia, characterized by bone marrow failure, congenital 57 malformations, cancer predisposition, and cellular sensitivity to ICL-inducing agents (7). Upon exposure 58 to DNA crosslinking agents, and during S-phase o...
“…Clearly, the functions of the FA proteins are a necessary part of S and G2 arrest within the ATM signaling. In addition, the regulation of FA signaling by p21 (a cycling-dependent kinase inhibitor) [58] and p53 [59] further supports the role of FA signaling in the downstream of the ATM signaling for regulating cell proliferation. We believe that FA signaling may be involved in coordinating all major events in the checkpoint systems.…”
Section: Fa Signaling and Master Regulators Of Cellular Stress Rementioning
confidence: 99%
“…We believe that FA signaling may be involved in coordinating all major events in the checkpoint systems. In particular, ATM not only participates in cell growth arrest & DNA damage repair, but possibly also cell death programs as hinted from the relation with p53 [59]. However, to fully explore the latter, further and more specific studies are needed.…”
Section: Fa Signaling and Master Regulators Of Cellular Stress Rementioning
The extremely high cancer incidence associated with patients suffering from a rare human genetic disease, Fanconi Anemia (FA), demonstrates the importance of FA genes. Over the course of human tumor development, FA genes perform critical tumor-suppression roles. In doing so, FA provides researchers with a unique genetic model system to study cancer etiology. Here, we review how aberrant function of the twenty-two FA genes and their signaling network contributes to malignancy. From this perspective, we will also discuss how the knowledge discovered from FA research serves basic and translational cancer research.
“…The role of p53 in both apoptosis and cell‐cycle arrest have been well established. Recent study suggests that p53 downregulates the FA DNA repair pathway . It has also been shown that BM failure in FA is triggered by an exacerbated p53/p21 DNA damage response that impairs HSPCs .…”
Emerging evidence have shown that resting quiescent hematopoietic stem cells (HSCs) prefer to utilize anaerobic glycolysis rather than mitochondrial respiration for energy production. Compelling evidence has also revealed that altered metabolic energetics in HSC underlies the onset of certain blood diseases. However, the mechanisms responsible for energetic reprogramming remain elusive. We recently found that Fanconi anemia (FA) HSCs are more dependent on mitochondrial respiration relative to glycolysis in their resting state for energy metabolism. Here we have investigated the role of deficient glycolysis in FA HSC maintenance. We observed significantly reduced glucose consumption, lactate production and ATP production in HSCs but not less primitive multipotent progenitors or restricted hematopoietic progenitors of Fanca−/− and Fancc−/− mice compared to those of wild-type mice, which was associated with an over-activated p53-TIGAR metabolic axis. We have utilized Fanca−/− HSCs deficient for p53 to show that the p53-TIGAR axis suppressed glycolysis in FA HSCs, leading to enhanced pentose phosphate pathway and cellular antioxidant function, and consequently reduced DNA damage and attenuated HSC exhaustion. Furthermore, by using Fanca−/− HSCs carrying the separation-of-function mutant p53R172P transgene that selectively impairs the p53 function in apoptosis but not cell-cycle control, we demonstrated that the cell-cycle function of p53 was not required for glycolytic suppression in FA HSCs. Finally, ectopic expression of the glycolytic rate-limiting enzyme PFKFB3 specifically antagonized p53-TIGAR-mediated metabolic reprogramming in FA HSCs. Together, our results suggest that p53-TIGAR metabolic axis-mediated glycolytic suppression may play a compensatory role in attenuating DNA damage and proliferative exhaustion in FA HSCs.
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