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Title: ATM-deficient human fibroblast cells are resistant to low levels of DNA double-strand break induced apoptosis and subsequently undergo drug-induced premature senescence

Abstract

Highlights: Black-Right-Pointing-Pointer A-T cells were not hypersensitive to low levels of DNA DSBs. Black-Right-Pointing-Pointer A-T cells have enhanced Akt but defect in activation of p53 and apoptotic proteins. Black-Right-Pointing-Pointer A-T cells underwent premature senescence after DNA damage accumulated. Black-Right-Pointing-Pointer Chemotherapeutic effect in cancer therapy may be associated with premature senescence. -- Abstract: DNA DSBs are induced by IR or radiomimetic drugs such as doxorubicin. It has been indicated that cells from ataxia-telangiectasia patients are highly sensitive to radiation due to defects in DNA repair, but whether they have impairment in apoptosis has not been fully elucidated. A-T cells showed increased sensitivity to high levels of DNA damage, however, they were more resistant to low doses. Normal cells treated with combination of KU55933, a specific ATM kinase inhibitor, and doxorubicin showed increased resistance as they do in a similar manner to A-T cells. A-T cells have higher viability but more DNA breaks, in addition, the activations of p53 and apoptotic proteins (Bax and caspase-3) were deficient, but Akt expression was enhanced. A-T cells subsequently underwent premature senescence after treatment with a low dose of doxorubicin, which was confirmed by G2 accumulation, senescent morphology, and SA-{beta}-gal positive until 15 days repair incubation.more » Finally, A-T cells are radio-resistant at low doses due to its defectiveness in detecting DNA damage and apoptosis, but the accumulation of DNA damage leads cells to premature senescence.« less

Authors:
; ; ; ; ;  [1];  [1]
  1. Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, 26 Kyunghee-daero, Dongdaemun-gu, Seoul 130-701 (Korea, Republic of)
Publication Date:
OSTI Identifier:
22210377
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 430; Journal Issue: 1; Other Information: Copyright (c) 2012 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; APOPTOSIS; DNA; DNA REPAIR; DOXORUBICIN; FIBROBLASTS; INCUBATION; MORPHOLOGY; NEOPLASMS; PROTEINS; RADIOMIMETIC DRUGS; STRAND BREAKS; THERAPY

Citation Formats

Park, Jun, Jo, Yong Hwa, Cho, Chang Hoon, Choe, Wonchae, Kang, Insug, Baik, Hyung Hwan, and Yoon, Kyung-Sik, E-mail: sky9999@khu.ac.kr. ATM-deficient human fibroblast cells are resistant to low levels of DNA double-strand break induced apoptosis and subsequently undergo drug-induced premature senescence. United States: N. p., 2013. Web. doi:10.1016/J.BBRC.2012.11.040.
Park, Jun, Jo, Yong Hwa, Cho, Chang Hoon, Choe, Wonchae, Kang, Insug, Baik, Hyung Hwan, & Yoon, Kyung-Sik, E-mail: sky9999@khu.ac.kr. ATM-deficient human fibroblast cells are resistant to low levels of DNA double-strand break induced apoptosis and subsequently undergo drug-induced premature senescence. United States. doi:10.1016/J.BBRC.2012.11.040.
Park, Jun, Jo, Yong Hwa, Cho, Chang Hoon, Choe, Wonchae, Kang, Insug, Baik, Hyung Hwan, and Yoon, Kyung-Sik, E-mail: sky9999@khu.ac.kr. 2013. "ATM-deficient human fibroblast cells are resistant to low levels of DNA double-strand break induced apoptosis and subsequently undergo drug-induced premature senescence". United States. doi:10.1016/J.BBRC.2012.11.040.
@article{osti_22210377,
title = {ATM-deficient human fibroblast cells are resistant to low levels of DNA double-strand break induced apoptosis and subsequently undergo drug-induced premature senescence},
author = {Park, Jun and Jo, Yong Hwa and Cho, Chang Hoon and Choe, Wonchae and Kang, Insug and Baik, Hyung Hwan and Yoon, Kyung-Sik, E-mail: sky9999@khu.ac.kr},
abstractNote = {Highlights: Black-Right-Pointing-Pointer A-T cells were not hypersensitive to low levels of DNA DSBs. Black-Right-Pointing-Pointer A-T cells have enhanced Akt but defect in activation of p53 and apoptotic proteins. Black-Right-Pointing-Pointer A-T cells underwent premature senescence after DNA damage accumulated. Black-Right-Pointing-Pointer Chemotherapeutic effect in cancer therapy may be associated with premature senescence. -- Abstract: DNA DSBs are induced by IR or radiomimetic drugs such as doxorubicin. It has been indicated that cells from ataxia-telangiectasia patients are highly sensitive to radiation due to defects in DNA repair, but whether they have impairment in apoptosis has not been fully elucidated. A-T cells showed increased sensitivity to high levels of DNA damage, however, they were more resistant to low doses. Normal cells treated with combination of KU55933, a specific ATM kinase inhibitor, and doxorubicin showed increased resistance as they do in a similar manner to A-T cells. A-T cells have higher viability but more DNA breaks, in addition, the activations of p53 and apoptotic proteins (Bax and caspase-3) were deficient, but Akt expression was enhanced. A-T cells subsequently underwent premature senescence after treatment with a low dose of doxorubicin, which was confirmed by G2 accumulation, senescent morphology, and SA-{beta}-gal positive until 15 days repair incubation. Finally, A-T cells are radio-resistant at low doses due to its defectiveness in detecting DNA damage and apoptosis, but the accumulation of DNA damage leads cells to premature senescence.},
doi = {10.1016/J.BBRC.2012.11.040},
journal = {Biochemical and Biophysical Research Communications},
number = 1,
volume = 430,
place = {United States},
year = 2013,
month = 1
}
  • Purpose: To better understand the impact of defects in the DNA damage-surveillance network on the various cell-based assays used for the prediction of patient radiosensitivity. Methods and Materials: We examined noncancerous human fibroblast strains from individuals with ataxia telangiectasia (ataxia telangiectasia mutated [ATM] deficient) or Li-Fraumeni syndrome (p53 deficient) using the neutral comet, H2AX phosphorylation, and clonogenic survival assays. Results: Using the comet assay, we found that, compared with normal fibroblasts, cells lacking either ATM or p53 function exhibited a reduced rate of double-strand break (DSB) rejoining early ({<=}4 h) after exposure to 8 Gy of {gamma}-radiation and also exhibitedmore » high levels of unrejoined DSBs later after irradiation. ATM-deficient and p53-deficient fibroblasts also exhibited abnormally increased levels of phosphorylated H2AX ({gamma}-H2AX) at later intervals after irradiation. In the clonogenic assay, ATM-deficient cells exhibited marked radiosensitivity and p53-deficient cells had varying degrees of radioresistance compared with normal fibroblasts. Conclusion: Regardless of whether ataxia telangiectasia and Li-Fraumeni syndrome fibroblasts are DSB-repair deficient per se, it is apparent that p53 and ATM defects greatly influence the cellular phenotype as evidenced by the neutral comet and {gamma}-H2AX assays. Our data suggest that the {gamma}-H2AX levels observed at later intervals after irradiation may represent a reliable measure of the overall DSB rejoining capabilities of human fibroblasts. However, it appears that using this parameter as a predictor of radiosensitivity without knowledge of the cells' p53 status could lead to incorrect conclusions.« less
  • Highlights: • Dyskerin depletion triggers cellular senescence in U2OS osteosarcoma cells. • Dyskerin-depleted cells are resistant to apoptosis induced by genotoxic stress. • Chromatin relaxation sensitizes dyskerin-depleted cells to apoptosis. - Abstract: Dyskerin is a conserved, nucleolar RNA-binding protein implicated in an increasing array of fundamental cellular processes. Germline mutation in the dyskerin gene (DKC1) is the cause of X-linked dyskeratosis congenita (DC). Conversely, wild-type dyskerin is overexpressed in sporadic cancers, and high-levels may be associated with poor prognosis. It was previously reported that acute loss of dyskerin function via siRNA-mediated depletion slowed the proliferation of transformed cell lines. However,more » the mechanisms remained unclear. Using human U2OS osteosarcoma cells, we show that siRNA-mediated dyskerin depletion induced cellular senescence as evidenced by proliferative arrest, senescence-associated heterochromatinization and a senescence-associated molecular profile. Senescence can render cells resistant to apoptosis. Conversely, chromatin relaxation can reverse the repressive effects of senescence-associated heterochromatinization on apoptosis. To this end, genotoxic stress-induced apoptosis was suppressed in dyskerin-depleted cells. In contrast, agents that induce chromatin relaxation, including histone deacetylase inhibitors and the DNA intercalator chloroquine, sensitized dyskerin-depleted cells to apoptosis. Dyskerin is a core component of the telomerase complex and plays an important role in telomere homeostasis. Defective telomere maintenance resulting in premature senescence is thought to primarily underlie the pathogenesis of X-linked DC. Since U2OS cells are telomerase-negative, this leads us to conclude that loss of dyskerin function can also induce cellular senescence via mechanisms independent of telomere shortening.« less
  • To study the effect of nucleotide excision repair on the spectrum of mutations induced in diploid human fibroblasts by UV light (wavelength, 254 nm), the authors synchronized repair-proficient cells and irradiated them when the HPRT gene was about to be replicated (early S phase) so that there would be no time for repair in that gene before replication, or in G, phase 6 h prior to S, and determined the kinds and location of mutations in that gene. As a control, they also compared the spectra of mutations induced in synchronized populations of xeroderma pigmentosum cells (XP12BE cells, which aremore » unable to excise UV-induced DNA damage). Among the 84 mutants sequenced, base substitutions predominated. Of the XP mutants from S or G[sub 1] and the repair-proficient mutants from S, [approximately]62% were G [center dot] C[r arrow]A [center dot] T. In the repair-proficient mutants from G[sub 1], 47% were. In mutants from the repair-proficient cells irradiated in S, 71% (10 of 14) of the premutagenic lesions were located in the transcribed strand; with mutants from such cells irradiated in G[sub 1], only 20% (3 of 15) were. The switch in strand bias supports preferential nucleotide excision repair of UV-induced damage in the transcribed strand of the HPRT gene.« less
  • Isolation of a true self-renewing stem cell from the human brain would be of great interest as a reliable source of neural tissue. Here, we report that human fetal cortical cells grown in epidermal growth factor expressed low levels of telomerase and telomeres in these cultures shortened over time leading to growth arrest after 30 weeks. Following leukemia inhibitory factor (LIF) supplementation, growth rates and telomerase expression increased. This was best demonstrated following cell cycle synchronization and staining for telomerase using immunocytochemistry. This increase in activity resulted in the maintenance of telomeres at approximately 7 kb for more than 60more » weeks in vitro. However, all cultures displayed a lack of oligodendrotye production, decreases in neurogenesis over time and underwent replicative senescence associated with increased expression of p21 before 70 weeks in vitro. Thus, under our culture conditions, these cells are not stable, multipotent, telomerase expressing self-renewing stem cells. They may be more accurately described as human neural progenitor cells (hNPC) with limited lifespan and bi-potent potential (neurons/astrocytes). Interestingly, hNPC follow a course of proliferation, neuronal production and growth arrest similar to that seen during expansion and development of the human cortex, thus providing a possible model neural system. Furthermore, due to their high expansion potential and lack of tumorogenicity, these cells remain a unique and safe source of tissue for clinical transplantation.« less