Rapamycin decreases DNA damage accumulation and enhances cell growth of WRN-deficient human fibroblasts.
Ontology highlight
ABSTRACT:
SUBMITTER: Saha B
PROVIDER: S-EPMC4032596 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Elastin deficiency because of heterozygous loss of an ELN allele in Williams syndrome causes obstructive aortopathy characterized by medial thickening and fibrosis and consequent aortic stiffening. Previous work in Eln-null mice with a severe arterial phenotype showed that inhibition of mTOR (mechanistic target of rapamycin), a key regulator of cell growth, lessened the aortic obstruction but did not prevent early postnatal death. We investigated the effects of mTOR inhibition in Eln-null mice partially rescued by human ELN that manifest a less severe arterial phenotype and survive long term.Thoracic aortas of neonatal and juvenile mice with graded elastin deficiency exhibited increased signaling through both mTOR complex 1 and 2. Despite lower predicted wall stress, there was increased phosphorylation of focal adhesion kinase, suggestive of greater integrin activation, and increased transforming growth factor-?-signaling mediators, associated with increased collagen expression. Pharmacological blockade of mTOR by rapalogs did not improve luminal stenosis but reduced mechanosignaling (in delayed fashion after mTOR complex 1 inhibition), medial collagen accumulation, and stiffening of the aorta. Rapalog administration also retarded somatic growth, however, and precipitated neonatal deaths. Complementary, less-toxic strategies to inhibit mTOR via altered growth factor and nutrient responses were not effective.In addition to previously demonstrated therapeutic benefits of rapalogs decreasing smooth muscle cell proliferation in the absence of elastin, we find that rapalogs also prevent aortic fibrosis and stiffening attributable to partial elastin deficiency. Our findings suggest that mTOR-sensitive perturbation of smooth muscle cell mechanosensing contributes to elastin aortopathy.
| S-EPMC5574180 | biostudies-literature
Project description:Telomeres cap the ends of eukaryotic chromosomes and prevent them from being recognized as DNA breaks. We have shown that certain DNA damage responses induced during senescence and, at times of telomere uncapping, also can be induced by treatment of cells with small DNA oligonucleotides homologous to the telomere 3' single-strand overhang (T-oligos), implicating this overhang in generation of these telomere-based damage responses. Here, we show that T-oligo-treated fibroblasts contain gammaH2AX foci and that these foci colocalize with telomeres. T-oligos with nuclease-resistant 3' ends are inactive, suggesting that a nuclease initiates T-oligo responses. We therefore examined WRN, a 3'-->5' exonuclease and helicase mutated in Werner syndrome, a disorder characterized by aberrant telomere maintenance, premature aging, chromosomal rearrangements, and predisposition to malignancy. Normal fibroblasts and U20S osteosarcoma cells rendered deficient in WRN showed reduced phosphorylation of p53 and histone H2AX in response to T-oligo treatment. Together, these data demonstrate a role for WRN in processing of telomeric DNA and subsequent activation of DNA damage responses. The T-oligo model helps define the role of WRN in telomere maintenance and initiation of DNA damage responses after telomere disruption.
| S-EPMC1586178 | biostudies-literature
Project description:Plasmodium parasites are exposed to endogenous and exogenous oxidative stress during their complex life cycle. To minimize oxidative damage, the parasites use glutathione (GSH) and thioredoxin (Trx) as primary antioxidants. We previously showed that disruption of the Plasmodium berghei gamma-glutamylcysteine synthetase (pbggcs-ko) or the glutathione reductase (pbgr-ko) genes resulted in a significant reduction of GSH in intraerythrocytic stages, and a defect in growth in the pbggcs-ko parasites. In this report, time course experiments of parasite intraerythrocytic development and morphological studies showed a growth delay during the ring to schizont progression. Morphological analysis shows a significant reduction in size (diameter) of trophozoites and schizonts with increased number of cytoplasmic vacuoles in the pbggcs-ko parasites in comparison to the wild type (WT). Furthermore, the pbggcs-ko mutants exhibited an impaired response to oxidative stress and increased levels of nuclear DNA (nDNA) damage. Reduced GSH levels did not result in mitochondrial DNA (mtDNA) damage or protein carbonylations in neither pbggcs-ko nor pbgr-ko parasites. In addition, the pbggcs-ko mutant parasites showed an increase in mRNA expression of genes involved in oxidative stress detoxification and DNA synthesis, suggesting a potential compensatory mechanism to allow for parasite proliferation. These results reveal that low GSH levels affect parasite development through the impairment of oxidative stress reduction systems and damage to the nDNA. Our studies provide new insights into the role of the GSH antioxidant system in the intraerythrocytic development of Plasmodium parasites, with potential translation into novel pharmacological interventions.
| S-EPMC4934901 | biostudies-literature
Project description:Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal premature ageing disease in children. HGPS is one of several progeroid syndromes caused by mutations in the LMNA gene encoding the nuclear structural proteins lamins A and C. In classic HGPS the mutation G608G leads to the formation of a toxic lamin A protein called progerin. During post-translational processing progerin remains farnesylated owing to the mutation interfering with a step whereby the farnesyl moiety is removed by the enzyme ZMPSTE24. Permanent farnesylation of progerin is thought to be responsible for the proteins toxicity. Farnesyl is generated through the mevalonate pathway and three drugs that interfere with this pathway and hence the farnesylation of proteins have been administered to HGPS children in clinical trials. These are a farnesyltransferase inhibitor (FTI), statin and a bisphosphonate. Further experimental studies have revealed that other drugs such as N-acetyl cysteine, rapamycin and IGF-1 may be of use in treating HGPS through other pathways. We have shown previously that FTIs restore chromosome positioning in interphase HGPS nuclei. Mis-localisation of chromosomes could affect the cells ability to regulate proper genome function. Using nine different drug treatments representing drug regimes in the clinic we have shown that combinatorial treatments containing FTIs are most effective in restoring specific chromosome positioning towards the nuclear periphery and in tethering telomeres to the nucleoskeleton. On the other hand, rapamycin was found to be detrimental to telomere tethering, it was, nonetheless, the most effective at inducing DNA damage repair, as revealed by COMET analyses.
| S-EPMC6223735 | biostudies-literature
Project description:Loss of Werner syndrome protein function causes Werner syndrome, characterized by increased genomic instability, elevated cancer susceptibility and premature aging. Although WRN is subject to acetylation, phosphorylation and sumoylation, the impact of these modifications on WRN's DNA metabolic function remains unclear. Here, we examined in further depth the relationship between WRN acetylation and its role in DNA metabolism, particularly in response to induced DNA damage. Our results demonstrate that endogenous WRN is acetylated somewhat under unperturbed conditions. However, levels of acetylated WRN significantly increase after treatment with certain DNA damaging agents or the replication inhibitor HU. Use of DNA repair-deficient cells or repair pathway inhibitors further increase levels of acetylated WRN, indicating that induced DNA lesions and their persistence are at least partly responsible for increased acetylation. Notably, acetylation of WRN correlates with inhibition of DNA synthesis, suggesting that replication blockage might underlie this effect. Moreover, WRN acetylation modulates its affinity for and activity on certain DNA structures, in a manner that may enhance its relative specificity for physiological substrates. Our results also show that acetylation and deacetylation of endogenous WRN is a dynamic process, with sirtuins and other histone deacetylases contributing to WRN deacetylation. These findings advance our understanding of the dynamics of WRN acetylation under unperturbed conditions and following DNA damage induction, linking this modification not only to DNA damage persistence but also potentially to replication stalling caused by specific DNA lesions. Our results are consistent with proposed metabolic roles for WRN and genomic instability phenotypes associated with WRN deficiency.
| S-EPMC4122284 | biostudies-literature
Project description:Inhibition of mTOR signaling using rapamycin has been shown to increase lifespan and healthspan in multiple model organisms; however, the precise mechanisms for the beneficial effects of rapamycin remain uncertain. We have previously reported that rapamycin delays senescence in human cells and that enhanced mitochondrial biogenesis and protection from mitochondrial stress is one component of the benefit provided by rapamycin treatment. Here, using two models of senescence, replicative senescence and senescence induced by the presence of the Hutchinson-Gilford progeria lamin A mutation, we report that senescence is accompanied by elevated glycolysis and increased oxidative phosphorylation, which are both reduced by rapamycin. Measurements of mitochondrial function indicate that direct mitochondria targets of rapamycin are succinate dehydrogenase and matrix alanine aminotransferase. Elevated activity of these enzymes could be part of complex mechanisms that enable mitochondria to resume their optimal oxidative phosphorylation and resist senescence. This interpretation is supported by the fact that rapamycin-treated cultures do not undergo a premature senescence in response to the replacement of glucose with galactose in the culture medium, which forces a greater reliance on oxidative phosphorylation. Additionally, long-term treatment with rapamycin increases expression of the mitochondrial carrier protein UCP2, which facilitates the movement of metabolic intermediates across the mitochondrial membrane. The results suggest that rapamycin impacts mitochondrial function both through direct interaction with the mitochondria and through altered gene expression of mitochondrial carrier proteins.
| S-EPMC6060207 | biostudies-literature
Project description:Mouse embryo fibroblasts deficient for the c-Jun proto-oncogene (c-Jun-/- MEF) undergo p53-dependent premature senescence in conventional culture. This phenotype becomes evident only after several cell divisions, suggesting that senescence may result from exposure to unknown environmental factors. Here, we show that c-Jun-/- MEF can proliferate successfully in low oxygen (3% O2), indicating that premature senescence under conventional culture conditions is a consequence of hyperoxic stress. c-Jun-/- MEF exhibit higher basal levels of DNA damage compared to normal fibroblasts in high but not low oxygen, implying that senescence results from chronic accumulation of spontaneous DNA damage. This accumulation may be attributable, at least in part, to inefficient repair, since DNA damage induced by gamma ionizing radiation and H2O2 persists for longer in c-Jun-/- MEF than in wild-type MEF. Unexpectedly, p53 expression, phosphorylation, and transcriptional activity are largely unaffected by oxygen exposure, indicating that the accumulation of spontaneous DNA damage does not result in chronic activation of p53 as judged by conventional criteria. Finally, we find that c-Jun associates with nuclear foci containing gammaH2AX and ATM following irradiation, suggesting a potential role for c-Jun in DNA repair processes per se.
| S-EPMC517871 | biostudies-other
Project description:ObjectivesR-loop is a three-stranded nucleic acid structure of RNA/DNA hybrid, which occurs naturally during transcription, and more R-loop accumulation can trigger serious DNA damage. There has been increasing attention to the issue of R-loop accumulation acted as a target for cancer therapy. However, the regulation of R-loop-associated proteins is poorly explored.Material and methodQuantitative real-time PCR and Western blot were used to measure the expression of C1orf109 in cell lines. In addition, C1orf109L (C1orf109 longest isoform) protein binding partner was identified and validated using immunoprecipitation-mass spectrometric (IP-MS) and immunoprecipitation assays. DNA-RNA immunoprecipitation (DR-IP) and immunofluorescence determined the C1orf109L location on R-loop. R-loop accumulation was determined by immunofluorescence. Cell cycle was determined by flow cytometry. Finally, time-lapse assay and cell counting were conducted to determined cell survival in response to camptothecin (CPT).ResultsWe found that C1orf109L could mediate cell cycle arrest in the G2/M phase and DNA damage depended on R-loop accumulation. Meanwhile, C1orf109L could bind with DHX9 to trigger R-loop accumulation. And C1orf109L was competitive with PARP1 binding to DHX9, which would block the function of DHX9-PARP1 to prevent the R-loop accumulation. Furthermore, C1orf109L could enhance the chemosensitivity of CPT, a chemotherapeutic drug capable of promoting R-loop formation.ConclusionsOur data demonstrate that C1orf109L triggers R-loop accumulation and DNA damage to arrest cell cycle.
| S-EPMC7507383 | biostudies-literature
Project description:The Hedgehog pathway is activated in various types of malignancies. We previously reported that inhibition of SMO or GLI prevents osteosarcoma growth in vitro and in vivo. Recently, it has been reported that arsenic trioxide (ATO) inhibits cancer growth by blocking GLI transcription. In this study, we analyzed the function of ATO in the pathogenesis of osteosarcoma. Real-time PCR showed that ATO decreased the expression of Hedgehog target genes, including PTCH1, GLI1, and GLI2, in human osteosarcoma cell lines. WST-1 assay and colony formation assay revealed that ATO prevented osteosarcoma growth. These findings show that ATO prevents GLI transcription and osteosarcoma growth in vitro. Flow cytometric analysis showed that ATO promoted apoptotic cell death. Comet assay showed that ATO treatment increased accumulation of DNA damage. Western blot analysis showed that ATO treatment increased the expression of ?H2AX, cleaved PARP, and cleaved caspase-3. In addition, ATO treatment decreased the expression of Bcl-2 and Bcl-xL. These findings suggest that ATO treatment promoted apoptotic cell death caused by accumulation of DNA damage. In contrast, Sonic Hedgehog treatment decreased the expression of ?H2AX induced by cisplatin treatment. ATO re-induced the accumulation of DNA damage attenuated by Sonic Hedgehog treatment. These findings suggest that ATO inhibits the activation of Hedgehog signaling and promotes apoptotic cell death in osteosarcoma cells by accumulation of DNA damage. Finally, examination of mouse xenograft models showed that ATO administration prevented the growth of osteosarcoma in nude mice. Because ATO is an FDA-approved drug for treatment of leukemia, our findings suggest that ATO is a new therapeutic option for treatment of patients with osteosarcoma.
| S-EPMC3704531 | biostudies-literature
Project description:Human BRCA1 mutation carriers and BRCA1-deficient mouse mammary glands contain an abnormal population of mammary luminal progenitors that can form 3D colonies in a hormone-independent manner. The intrinsic cellular regulatory defect in these presumptive breast cancer precursors is not known. We have discovered that nuclear factor kappaB (NF-?B) (p52/RelB) is persistently activated in a subset of BRCA1-deficient mammary luminal progenitors. Hormone-independent luminal progenitor colony formation required NF-?B, ataxia telangiectasia-mutated (ATM), and the inhibitor of kappaB kinase, IKK?. Progesterone (P4)-stimulated proliferation resulted in a marked enhancement of DNA damage foci in Brca1(-/-) mouse mammary. In vivo, NF-?B inhibition prevented recovery of Brca1(-/-) hormone-independent colony-forming cells. The majority of human BRCA1(mut/+) mammary glands showed marked lobular expression of nuclear NF-?B. We conclude that the aberrant proliferative capacity of Brca1(-/-) luminal progenitor cells is linked to the replication-associated DNA damage response, where proliferation of mammary progenitors is perpetuated by damage-induced, autologous NF-?B signaling.
| S-EPMC6850408 | biostudies-literature