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Sensitivity of RECQL4-deficient fibroblasts from Rothmund–Thomson syndrome patients to genotoxic agents

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Abstract

RECQ helicase protein-like 4 (RECQL4) is a member of the human RECQ family of DNA helicases. Two-thirds of patients with Rothmund–Thomson syndrome (RTS) carry biallelic inactivating mutations in the RECQL4 gene. RTS is an autosomal recessive disorder characterized by poikiloderma, sparse hair, small stature, skeletal abnormalities, cataracts, and an increased risk of cancer. Mutations in two other RECQ helicases, BLM and WRN, are responsible for the cancer predisposition conditions Bloom and Werner syndromes, respectively. Previous studies have shown that BLM and WRN-deficient cells demonstrate increased sensitivity to hydroxyurea (HU), camptothecin (CPT), and 4-nitroquinoline 1-oxide (4NQO). Little is known about the sensitivity of RECQL4-deficient cells to these and other genotoxic agents. The purpose of this study was to determine if RTS cells display any distinct cellular phenotypes in response to DNA damaging agents or replication blocks that could provide insight into the molecular function of the RECQL4 protein. Our results show that primary fibroblasts from RTS patients carrying two deleterious RECQL4 mutations, compared to wild type (WT) fibroblasts, have increased sensitivity to HU, CPT, and doxorubicin (DOX), modest sensitivity to other DNA damaging agents including ultraviolet (UV) irradiation, ionizing radiation (IR), and cisplatin (CDDP), and relative resistance to 4NQO. The RECQ family of DNA helicases has been implicated in the regulation of DNA replication, recombination, and repair. Because HU, CPT, and DOX exert their effects primarily during S phase, these results support a greater role for the RECQL4 protein in DNA replication as opposed to repair of exogenous damage.

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References

  • Aubel-Sadron G, Londos-Gagliardi D (1984) Daunorubicin and doxorubicin, anthracycline antibiotics, a physicochemical and biological review. Biochimie 66:333–352

    Article  PubMed  CAS  Google Scholar 

  • Auerbach AD, Verlander PC (1997) Disorders of DNA replication and repair. Curr Opin Pediatr 9:600–616

    Article  PubMed  CAS  Google Scholar 

  • Bachrati CZ, Hickson ID (2008) RecQ helicases: guardian angels of the DNA replication fork. Chromosoma 117:219–233

    Article  PubMed  CAS  Google Scholar 

  • Bagherieh-Najjar MB, de Vries OM, Hille J, Dijkwel PP (2005) Arabidopsis RecQI4A suppresses homologous recombination and modulates DNA damage responses. Plant J 43:789–798

    Article  PubMed  CAS  Google Scholar 

  • Blank A, Bobola MS, Gold B, Varadarajan S, Kolstoe D, Meade EH, Rabinovitch PS, Loeb LA, Silber JR (2004) The Werner syndrome protein confers resistance to the DNA lesions N3-methyladenine and O6-methylguanine: implications for WRN function. DNA Repair (Amst) 3:629–638

    CAS  Google Scholar 

  • Burks LM, Yin J, Plon SE (2007) Nuclear import and retention domains in the amino terminus of RECQL4. Gene 391:26–38

    Article  PubMed  CAS  Google Scholar 

  • Davies SL, North PS, Dart A, Lakin ND, Hickson ID (2004) Phosphorylation of the Bloom’s syndrome helicase and its role in recovery from S-phase arrest. Mol Cell Biol 24:1279–1291

    Article  PubMed  CAS  Google Scholar 

  • Davies SL, North PS, Hickson ID (2007) Role for BLM in replication-fork restart and suppression of origin firing after replicative stress. Nat Struct Mol Biol 14:677–679

    Article  PubMed  CAS  Google Scholar 

  • den Dunnen JT, Antonarakis SE (2001) Nomenclature for the description of human sequence variations. Hum Genet 109:121–124

    Article  CAS  Google Scholar 

  • Dhillon KK, Sidorova J, Saintigny Y, Poot M, Gollahon K, Rabinovitch PS, Monnat RJ Jr (2007) Functional role of the Werner syndrome RecQ helicase in human fibroblasts. Aging Cell 6:53–61

    Article  PubMed  CAS  Google Scholar 

  • Dietschy T, Shevelev I, Stagljar I (2007) The molecular role of the Rothmund–Thomson-, RAPADILINO- and Baller–Gerold-gene product, RECQL4: recent progress. Cell Mol Life Sci 64:796–802

    Article  PubMed  CAS  Google Scholar 

  • Ellis NA, Groden J, Ye TZ, Straughen J, Lennon DJ, Ciocci S, Proytcheva M, German J (1995) The Bloom’s syndrome gene product is homologous to RecQ helicases. Cell 83:655–666

    Article  PubMed  CAS  Google Scholar 

  • Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C (2006) Clonogenic assay of cells in vitro. Nat Protoc 1:2315–2319

    Article  PubMed  CAS  Google Scholar 

  • Grant SG, Wenger SL, Latimer JJ, Thull D, Burke LW (2000) Analysis of genomic instability using multiple assays in a patient with Rothmund–Thomson syndrome. Clin Genet 58:209–215

    Article  PubMed  CAS  Google Scholar 

  • Hanada K, Hickson ID (2007) Molecular genetics of RecQ helicase disorders. Cell Mol Life Sci 64:2306–2322

    Article  PubMed  CAS  Google Scholar 

  • Hicks MJ, Roth JR, Kozinetz CA, Wang LL (2007) Clinicopathologic features of osteosarcoma in patients with Rothmund–Thomson syndrome. J Clin Oncol 25:370–375

    Article  PubMed  Google Scholar 

  • Hisama FM, Chen YH, Meyn MS, Oshima J, Weissman SM (2000) WRN or telomerase constructs reverse 4-nitroquinoline 1-oxide sensitivity in transformed Werner syndrome fibroblasts. Cancer Res 60:2372–2376

    PubMed  CAS  Google Scholar 

  • Hoki Y, Araki R, Fujimori A, Ohhata T, Koseki H, Fukumura R, Nakamura M, Takahashi H, Noda Y, Kito S, Abe M (2003) Growth retardation and skin abnormalities of the Recql4-deficient mouse. Hum Mol Genet 12:2293–2299

    Article  PubMed  CAS  Google Scholar 

  • Honma M, Tadokoro S, Sakamoto H, Tanabe H, Sugimoto M, Furuichi Y, Satoh T, Sofuni T, Goto M, Hayashi M (2002) Chromosomal instability in B-lymphoblasotoid cell lines from Werner and Bloom syndrome patients. Mutat Res 520:15–24

    PubMed  CAS  Google Scholar 

  • Kitao S, Shimamoto A, Goto M, Miller RW, Smithson WA, Lindor NM, Furuichi Y (1999) Mutations in RECQL4 cause a subset of cases of Rothmund–Thomson syndrome. Nat Genet 22:82–84

    Article  PubMed  CAS  Google Scholar 

  • Kumata Y, Tada S, Yamanada Y, Tsuyama T, Kobayashi T, Dong YP, Ikegami K, Murofushi H, Seki M, Enomoto T (2007) Possible involvement of RecQL4 in the repair of double-strand DNA breaks in Xenopus egg extracts. Biochim Biophys Acta 1773:556–564

    Article  PubMed  CAS  Google Scholar 

  • Larizza L, Magnani I, Roversi G (2006) Rothmund–Thomson syndrome and RECQL4 defect: splitting and lumping. Cancer Lett 232:107–120

    Article  PubMed  CAS  Google Scholar 

  • Lebel M, Leder P (1998) A deletion within the murine Werner syndrome helicase induces sensitivity to inhibitors of topoisomerase and loss of cellular proliferative capacity. Proc Natl Acad Sci USA 95:13097–13102

    Article  PubMed  CAS  Google Scholar 

  • Lindor NM, Furuichi Y, Kitao S, Shimamoto A, Arndt C, Jalal S (2000) Rothmund–Thomson syndrome due to RECQ4 helicase mutations: report and clinical and molecular comparisons with Bloom syndrome and Werner syndrome. Am J Med Genet 90:223–228

    Article  PubMed  CAS  Google Scholar 

  • Macris MA, Krejci L, Bussen W, Shimamoto A, Sung P (2006) Biochemical characterization of the RECQ4 protein, mutated in Rothmund–Thomson syndrome. DNA Repair (Amst) 5:172–180

    Article  CAS  Google Scholar 

  • Martin LP, Hamilton TC, Schilder RJ (2008) Platinum resistance: the role of DNA repair pathways. Clin Cancer Res 14:1291–1295

    Article  PubMed  CAS  Google Scholar 

  • Miao ZH, Rao VA, Agama K, Antony S, Kohn KW, Pommier Y (2006) 4-nitroquinoline-1-oxide induces the formation of cellular topoisomerase I-DNA cleavage complexes. Cancer Res 66:6540–6545

    Article  PubMed  CAS  Google Scholar 

  • Miozzo M, Castorina P, Riva P, Dalpra L, Fuhrman Conti AM, Volpi L, Hoe TS, Khoo A, Wiegant J, Rosenberg C, Larizza L (1998) Chromosomal instability in fibroblasts and mesenchymal tumors from 2 sibs with Rothmund–Thomson syndrome. Int J Cancer 77:504–510

    Article  PubMed  CAS  Google Scholar 

  • Nunoshiba T, Demple B (1993) Potent intracellular oxidative stress exerted by the carcinogen 4-nitroquinoline-N-oxide. Cancer Res 53:3250–3252

    PubMed  CAS  Google Scholar 

  • Park SJ, Lee YJ, Beck BD, Lee SH (2006) A positive involvement of RecQL4 in UV-induced S-phase arrest. DNA Cell Biol 25:696–703

    Article  PubMed  CAS  Google Scholar 

  • Petkovic M, Dietschy T, Freire R, Jiao R, Stagljar I (2005) The human Rothmund–Thomson syndrome gene product, RECQL4, localizes to distinct nuclear foci that coincide with proteins involved in the maintenance of genome stability. J Cell Sci 118:4261–4269

    Article  PubMed  CAS  Google Scholar 

  • Poot M, Gollahon KA, Emond MJ, Silber JR, Rabinovitch PS (2002a) Werner syndrome diploid fibroblasts are sensitive to 4-nitroquinoline-N-oxide and 8-methoxypsoralen: implications for the disease phenotype. FASEB J 16:757–758

    PubMed  CAS  Google Scholar 

  • Poot M, Silber JR, Rabinovitch PS (2002b) A novel flow cytometric technique for drug cytotoxicity gives results comparable to colony-forming assays. Cytometry 48:1–5

    Article  PubMed  CAS  Google Scholar 

  • Prince PR, Ogburn CE, Moser MJ, Emond MJ, Martin GM, Monnat RJ Jr (1999) Cell fusion corrects the 4-nitroquinoline 1-oxide sensitivity of Werner syndrome fibroblast cell lines. Hum Genet 105:132–138

    Article  PubMed  CAS  Google Scholar 

  • Rodriguez-Lopez AM, Whitby MC, Borer CM, Bachler MA, Cox LS (2007) Correction of proliferation and drug sensitivity defects in the progeroid Werner’s Syndrome by Holliday junction resolution. Rejuvenation Res 10:27–40

    Article  PubMed  CAS  Google Scholar 

  • Sangrithi MN, Bernal JA, Madine M, Philpott A, Lee J, Dunphy WG, Venkitaraman AR (2005) Initiation of DNA replication requires the RECQL4 protein mutated in Rothmund–Thomson syndrome. Cell 121:887–898

    Article  PubMed  CAS  Google Scholar 

  • Shinya A, Nishigori C, Moriwaki S, Takebe H, Kubota M, Ogino A, Imamura S (1993) A case of Rothmund–Thomson syndrome with reduced DNA repair capacity. Arch Dermatol 129:332–336

    Article  PubMed  CAS  Google Scholar 

  • Sidorova JM, Li N, Folch A, Monnat RJ Jr (2008) The RecQ helicase WRN is required for normal replication fork progression after DNA damage or replication fork arrest. Cell Cycle 7:796–807

    PubMed  CAS  Google Scholar 

  • Siitonen HA, Kopra O, Kaariainen H, Haravuori H, Winter RM, Saamanen AM, Peltonen L, Kestila M (2003) Molecular defect of RAPADILINO syndrome expands the phenotype spectrum of RECQL diseases. Hum Mol Genet 12:2837–2844

    Article  PubMed  CAS  Google Scholar 

  • Smith PJ, Paterson MC (1981) Abnormal responses to mid-ultraviolet light of cultured fibroblasts from patients with disorders featuring sunlight sensitivity. Cancer Res 41:511–518

    PubMed  CAS  Google Scholar 

  • Smith PJ, Paterson MC (1982) Enhanced radiosensitivity and defective DNA repair in cultured fibroblasts derived from Rothmund Thomson syndrome patients. Mutat Res 94:213–228

    PubMed  CAS  Google Scholar 

  • Tamminga RY, Dolsma WV, Leeuw JA, Kampinga HH (2002) Chemo- and radiosensitivity testing in a patient with ataxia telangiectasia and Hodgkin disease. Pediatr Hematol Oncol 19:163–171

    Article  PubMed  CAS  Google Scholar 

  • Van Maldergem L, Siitonen HA, Jalkh N, Chouery E, De RM, Delague V, Muenke M, Jabs EW, Cai J, Wang LL, Plon SE, Fourneau C, Kestila M, Gillerot Y, Megarbane A, Verloes A (2006) Revisiting the craniosynostosis-radial ray hypoplasia association: Baller–Gerold syndrome caused by mutations in the RECQL4 gene. J Med Genet 43:148–152

    Article  PubMed  CAS  Google Scholar 

  • Vasseur F, Delaporte E, Zabot MT, Sturque MN, Barrut D, Savary JB, Thomas L, Thomas P (1999) Excision repair defect in Rothmund Thomson syndrome. Acta Derm Venereol 79:150–152

    Article  PubMed  CAS  Google Scholar 

  • Wang LL, Levy ML, Lewis RA, Chintagumpala MM, Lev D, Rogers M, Plon SE (2001) Clinical manifestations in a cohort of 41 Rothmund–Thomson syndrome patients. Am J Med Genet 102:11–17

    Article  PubMed  CAS  Google Scholar 

  • Wang LL, Gannavarapu A, Kozinetz CA, Levy ML, Lewis RA, Chintagumpala MM, Ruiz-Maldanado R, Contreras-Ruiz J, Cunniff C, Erickson RP, Lev D, Rogers M, Zackai EH, Plon SE (2003) Association between osteosarcoma and deleterious mutations in the RECQL4 gene in Rothmund–Thomson syndrome. J Natl Cancer Inst 95:669–674

    Article  PubMed  CAS  Google Scholar 

  • Werner SR, Prahalad AK, Yang J, Hock JM (2006) RECQL4-deficient cells are hypersensitive to oxidative stress/damage: insights for osteosarcoma prevalence and heterogeneity in Rothmund–Thomson syndrome. Biochem Biophys Res Commun 345:403–409

    Article  PubMed  CAS  Google Scholar 

  • Woo LL, Futami K, Shimamoto A, Furuichi Y, Frank KM (2006) The Rothmund–Thomson gene product RECQL4 localizes to the nucleolus in response to oxidative stress. Exp Cell Res 312:3443–3457

    Article  PubMed  CAS  Google Scholar 

  • Yu CE, Oshima J, Fu YH, Wijsman EM, Hisama F, Alisch R, Matthews S, Nakura J, Miki T, Ouais S, Martin GM, Mulligan J, Schellenberg GD (1996) Positional cloning of the Werner’s syndrome gene. Science 272:258–262

    Article  PubMed  CAS  Google Scholar 

  • Zelle B, Bootsma D (1980) Repair of DNA damage after exposure to 4-nitroquinoline-1-oxide in heterokaryons derived from xeroderma pigmentosum cells. Mutat Res 70:373–381

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors thank the patients and families for their participation in this research. We thank the following ongoing collaborators in our research study: Moise Levy, MD and Richard Lewis, MD. We gratefully acknowledge the Baylor College of Medicine Mental Retardation Developmental Disabilities Research Center, Tissue Culture Core for technical assistance, Stephen M. Gottschalk, MD for scientific advice, and Alison Bertuch, MD, Ph.D. for critical reading of the manuscript and helpful discussions. National Institutes of Health (K08 HD42136-05 to L.W., K12 HD41648-01, HD24064); Doris Duke Charitable Foundation; V-Foundation for Cancer Research.

Conflicts of interest statement

The authors indicated no potential conflicts of interest.

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Authors and Affiliations

  1. Department of Pediatrics, Section of Hematology/Oncology, Texas Children’s Cancer Center, Baylor College of Medicine, 6621 Fannin MC 3-3320, Houston, TX, 77030, USA

    Weidong Jin, Subhendu K. Otta & Lisa L. Wang

  2. Division of Biostatistics, Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX, 77030, USA

    Hao Liu & Yiqun Zhang

  3. Departments of Human and Molecular Genetics and Pediatrics, Section of Hematology/Oncology, Texas Children’s Cancer Center, Baylor College of Medicine, Houston, TX, 77030, USA

    Sharon E. Plon

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  1. Weidong Jin
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  2. Hao Liu
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Correspondence to Lisa L. Wang.

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439_2008_518_MOESM1_ESM.doc

Sensitivity of individual RTS fibroblast samples to CPT compared to WT, BS, and WS controls a Log percentage survival curves and standard deviations for three independent WT controls and one sample each for RTS, WS, and BS fibroblasts b Log percentage survival curves and standard deviations for three independent RTS samples and one WT control and one BS control c Same as b except using three other independent RTS samples (MOESM1 DOC 97kb).

439_2008_518_MOESM2_ESM.doc

Plots of survival fraction curves as a function of various doses of genotoxic agent for indicated fibroblasts. Curves were estimated by the generalized linear regression models fitted for the probability of surviving colonies. LD10 levels for each agent are indicated by the horizontal lines. Fibroblasts were treated with the following agents: a HU; b CPT; c DOX; d UV; e IR; f CDDP; g 4NQO (MOESM2 198kb).

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Jin, W., Liu, H., Zhang, Y. et al. Sensitivity of RECQL4-deficient fibroblasts from Rothmund–Thomson syndrome patients to genotoxic agents. Hum Genet 123, 643–653 (2008). https://doi.org/10.1007/s00439-008-0518-4

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  • DOI: https://doi.org/10.1007/s00439-008-0518-4

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