Cellular and Molecular Life Sciences

, Volume 67, Issue 10, pp 1713–1722 | Cite as

Role of Chk1 in the differentiation program of hematopoietic stem cells

  • Laura Carrassa
  • Elisa Montelatici
  • Lorenza Lazzari
  • Stefano Zangrossi
  • Matteo Simone
  • Massimo Broggini
  • Giovanna Damia
Research Article

Abstract

Hematopoietic stem cells (HSC) isolated from umbilical cord blood (UCB) were treated with ionizing radiation (IR) and sensitivity and IR induced checkpoints activation were investigated. No difference in the sensitivity and in the activation of DNA damage pathways was observed between CD133+ HSC and cells derived from them after ex vivo expansion. Chk1 protein was very low in freshly isolated CD133+ cells, and undetectable in ex vivo expanded UCB CD133+ cells. Chk1 was expressed only on day 3 of the ex vivo expansion. This pattern of Chk1 expression was corroborated in CD133+ cells isolated from peripheral blood apheresis collected from an healthy donor. Treatment with a specific Chk1 inhibitor resulted in a strong reduction in the percentage of myeloid precursors (CD33+) and an increase in the percentage of lymphoid precursors (CD38+) compared to untreated cells, suggesting a possible role for Chk1 in the differentiation program of UCB CD133+ HSC.

Keywords

Stem and progenitor cells Umbilical cord blood Chk1 DNA damage Differentiation 

Notes

Acknowledgments

We thank Dr Patrick Casara and Dr John Hickman who respectively synthesized and kindly provided the Chk1 inhibitor compound. A special thank to JD Baggott that kindly edited the paper. This study was supported by grants from “Fondazione I1 Sangue”, Ministero della Salute, Istituto Superiore di Sanità, Sixth FP “Thercord”, Foundation Novussanguis and Jérome Lejeune. The generous contribution of the Italian Association for Cancer Research and of FIRB is also gratefully acknowledged.

Supplementary material

18_2010_274_MOESM1_ESM.tif (174 kb)
UCB CD133+ cell growth curve from isolation up to day 14 of ex vivo expansion with the cytokine cocktail. Mean ± SD of three experiments performed from UCB of three different donors. The bottom panel in the figure gives a larger view of the growth curve from day 0 to day 6 of ex vivo expansion (TIFF 174 kb)
18_2010_274_MOESM2_ESM.tif (146 kb)
Cytotoxic effect of IR in UCB CD133+ cells the day of isolation (♦), in the ex vivo expanded population at day 3 from isolation (■) and in the partly differentiated cells at day 14 from isolation (●). Data are expressed as percentages of the untreated control sample; mean ± SD of 3 different experiments done in triplicate (TIFF 146 kb)
18_2010_274_MOESM3_ESM.tif (567 kb)
Western blot analysis showing Chk1, pS317Chk1, pCdc25C, Cdc25C and Ran protein levels of UCB CD133+ cells, untreated or treated with the Chk1 inhibitor as described in Materials and Methods (TIFF 566 kb)
18_2010_274_MOESM4_ESM.tif (313 kb)
(A) Upper panel: Table representing the relative proportion of cells positive for the differentiation markers selected both in untreated and in Chk1 inhibitor AZD- 7762 treated cells at day 14 of the ex vivo expansion. Lower panel: Western Blot Analysis showing pS317 Chk1 and total Chk1 protein levels of UCB CD 133+ cells untreated or treated with AZD 7762. (B) Upper panel: Table representing the decrease in percentage of CD 33 positive HL 60 cells after treatment with CHIR-124 (120 nM) and AZD 7762 (500 nM). Lower panel: Western Blot Analysis showing pS317 Chk1 and total Chk1 protein levels of HL-60 cells untreated or treated with AZD 7762 and CHIR-124 (TIFF 312 kb)

References

  1. 1.
    Bakkenist CJ, Kastan MB (2004) Initiating cellular stress responses. Cell 118:9–17CrossRefPubMedGoogle Scholar
  2. 2.
    Zhou BB, Elledge SJ (2000) The DNA damage response: putting checkpoints in perspective. Nature 408:433–439CrossRefPubMedGoogle Scholar
  3. 3.
    Hurley PJ, Bunz F (2007) ATM and ATR: components of an integrated circuit. Cell Cycle 6:414–417PubMedGoogle Scholar
  4. 4.
    Syljuasen RG, Sorensen CS, Hansen LT, Fugger K, Lundin C, Johansson F, Helleday T, Sehested M, Lukas J, Bartek J (2005) Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage. Mol Cell Biol 25:3553–3562CrossRefPubMedGoogle Scholar
  5. 5.
    Carrassa L, Sanchez Y, Erba E, Damia G (2009) U2OS cells lacking Chk1 undergo aberrant mitosis and fail to activate the spindle checkpoint. J Cell Mol Med 13:1565–1576PubMedGoogle Scholar
  6. 6.
    Lam MH, Liu Q, Elledge SJ, Rosen JM (2004) Chk1 is haploinsufficient for multiple functions critical to tumor suppression. Cancer Cell 6:45–59CrossRefPubMedGoogle Scholar
  7. 7.
    Shimada M, Niida H, Zineldeen DH, Tagami H, Tanaka M, Saito H, Nakanishi M (2008) Chk1 is a histone H3 threonine 11 kinase that regulates DNA damage-induced transcriptional repression. Cell 132:221–232CrossRefPubMedGoogle Scholar
  8. 8.
    Gluckman E, Broxmeyer HA, Auerbach AD, Friedman HS, Douglas GW, Devergie A, Esperou H, Thierry D, Socie G, Lehn P (1989) Hematopoietic reconstitution in a patient with Fanconi’s anemia by means of umbilical-cord blood from an HLA-identical sibling. N Engl J Med 321:1174–1178PubMedCrossRefGoogle Scholar
  9. 9.
    Brunstein CG, Wagner JE (2006) Cord blood transplantation for adults. Vox Sang 91:195–205CrossRefPubMedGoogle Scholar
  10. 10.
    Rocha V, Labopin M, Sanz G, Arcese W, Schwerdtfeger R, Bosi A, Jacobsen N, Ruutu T, de Lima M, Finke J, Frassoni F, Gluckman E (2004) Transplants of umbilical-cord blood or bone marrow from unrelated donors in adults with acute leukemia. N Engl J Med 351:2276–2285CrossRefPubMedGoogle Scholar
  11. 11.
    Lazzari L, Lucchi S, Rebulla P, Porretti L, Puglisi G, Lecchi L, Sirchia G (2001) Long-term expansion and maintenance of cord blood haematopoietic stem cells using thrombopoietin, Flt3-ligand, interleukin (IL)-6 and IL-11 in a serum-free and stroma-free culture system. Br J Haematol 112:397–404CrossRefPubMedGoogle Scholar
  12. 12.
    De Felice L, Di Pucchio T, Mascolo MG, Agostini F, Breccia M, Guglielmi C, Ricciardi MR, Tafuri A, Screnci M, Mandelli F, Arcese W (1999) Flt3LP3nduces the ex-vivo amplification of umbilical cord blood committed progenitors and early stem cells in short-term cultures. Br J Haematol 106:133–141CrossRefPubMedGoogle Scholar
  13. 13.
    Schoemans H, Theunissen K, Maertens J, Boogaerts M, Verfaillie C, Wagner J (2006) Adult umbilical cord blood transplantation: a comprehensive review. Bone Marrow Transplant 38:83–93CrossRefPubMedGoogle Scholar
  14. 14.
    Bryder D, Rossi DJ, Weissman IL (2006) Hematopoietic stem cells: the paradigmatic tissue-specific stem cell. Am J Pathol 169:338–346CrossRefPubMedGoogle Scholar
  15. 15.
    Yin AH, Miraglia S, Zanjani ED, Almeida-Porada G, Ogawa M, Leary AG, Olweus J, Kearney J, Buck DW (1997) AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood 90:5002–5012PubMedGoogle Scholar
  16. 16.
    Zangiacomi V, Balon N, Maddens S, Lapierre V, Tiberghien P, Schlichter R, Versaux-Botteri C, Deschaseaux F (2008) Cord blood-derived neurons are originated from CD133+/CD34 stem/progenitor cells in a cell-to-cell contact dependent manner. Stem Cells Dev 17:1005–1016CrossRefPubMedGoogle Scholar
  17. 17.
    Ivanova NB, Dimos JT, Schaniel C, Hackney JA, Moore KA, Lemischka IR (2002) A stem cell molecular signature. Science 298:601–604CrossRefPubMedGoogle Scholar
  18. 18.
    Aladjem MI, Spike BT, Rodewald LW, Hope TJ, Klemm M, Jaenisch R, Wahl GM (1998) ES cells do not activate p53-dependent stress responses and undergo p53-independent apoptosis in response to DNA damage. Curr Biol 8:145–155CrossRefPubMedGoogle Scholar
  19. 19.
    Chuykin IA, Lianguzova MS, Pospelova TV, Pospelov VA (2008) Activation of DNA damage response signaling in mouse embryonic stem cells. Cell Cycle 7:2922–2928PubMedGoogle Scholar
  20. 20.
    Tse AN, Rendahl KG, Sheikh T, Cheema H, Aardalen K, Embry M, Ma S, Moler EJ, Ni ZJ, Lopes de Menezes DE, Hibner B, Gesner TG, Schwartz GK (2007) CHIR-124, a novel potent inhibitor of Chk1, potentiates the cytotoxicity of topoisomerase I poisons in vitro and in vivo. Clin Cancer Res 13:591–602CrossRefPubMedGoogle Scholar
  21. 21.
    Zabludoff SD, Deng C, Grondine MR, Sheehy AM, Ashwell S, Caleb BL, Green S, Haye HR, Horn CL, Janetka JW, Liu D, Mouchet E, Ready S, Rosenthal JL, Queva C, Schwartz GK, Taylor KJ, Tse AN, Walker GE, White AM (2008) AZD7762, a novel checkpoint kinase inhibitor, drives checkpoint abrogation and potentiates DNA-targeted therapies. Mol Cancer Ther 7:2955–2966CrossRefPubMedGoogle Scholar
  22. 22.
    Kaneko YS, Watanabe N, Morisaki H, Akita H, Fujimoto A, Tominaga K, Terasawa M, Tachibana A, Ikeda K, Nakanishi M (1999) Cell-cycle-dependent and ATM-independent expression of human Chk1 kinase. Oncogene 18:3673–3681CrossRefPubMedGoogle Scholar
  23. 23.
    Carrassa L, Broggini M, Erba E, Damia G (2004) Chk1, but not Chk2, is involved in the cellular response to DNA damaging agents: differential activity in cells expressing or not p53. Cell Cycle 3:1177–1181PubMedGoogle Scholar
  24. 24.
    Ganzinelli M, Carrassa L, Crippa F, Tavecchio M, Broggini M, Damia G (2008) Checkpoint kinase 1 down-regulation by an inducible small interfering RNA expression system sensitized in vivo tumors to treatment with 5-fluorouracil. Clin Cancer Res 14:5131–5141CrossRefPubMedGoogle Scholar
  25. 25.
    Sanchez Y, Wong C, Thoma RS, Richman R, Wu Z, Piwnica-Worms H, Elledge SJ (1997) Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25. Science 277:1497–1501CrossRefPubMedGoogle Scholar
  26. 26.
    Leung-Pineda V, Ryan CE, Piwnica-Worms H (2006) Phosphorylation of Chk1 by ATR is antagonized by a Chk1-regulated protein phosphatase 2A circuit. Mol Cell Biol 26:7529–7538CrossRefPubMedGoogle Scholar
  27. 27.
    Damia G, D’Incalci M (2007) Targeting DNA repair as a promising approach in cancer therapy. Eur J Cancer 43:1791–1801CrossRefPubMedGoogle Scholar
  28. 28.
    Pierelli L, Scambia G, Fattorossi A, Bonanno G, Battaglia A, Rumi C, Marone M, Mozzetti S, Rutella S, Menichella G, Romeo V, Mancuso S, Leone G (1998) Functional, phenotypic and molecular characterization of cytokine low-responding circulating CD34+ haemopoietic progenitors. Br J Haematol 102:1139–1150CrossRefPubMedGoogle Scholar
  29. 29.
    Buschfort-Papewalis C, Moritz T, Liedert B, Thomale J (2002) Down-regulation of DNA repair in human CD34(+) progenitor cells corresponds to increased drug sensitivity and apoptotic response. Blood 100:845–853CrossRefPubMedGoogle Scholar
  30. 30.
    Bracker TU, Giebel B, Spanholtz J, Sorg UR, Klein-Hitpass L, Moritz T, Thomale J (2006) Stringent regulation of DNA repair during human hematopoietic differentiation: a gene expression and functional analysis. Stem Cells 24:722–730CrossRefPubMedGoogle Scholar
  31. 31.
    Giono LE, Manfredi JJ (2006) The p53 tumor suppressor participates in multiple cell cycle checkpoints. J Cell Physiol 209:13–20CrossRefPubMedGoogle Scholar
  32. 32.
    Bartek J, Lukas J (2003) Chk1 and Chk2 kinases in checkpoint control and cancer. Cancer Cell 3:421–429CrossRefPubMedGoogle Scholar
  33. 33.
    Zachos G, Black EJ, Walker M, Scott MT, Vagnarelli P, Earnshaw WC, Gillespie DA (2007) Chk1 is required for spindle checkpoint function. Dev Cell 12:247–260CrossRefPubMedGoogle Scholar
  34. 34.
    Zachos G, Rainey MD, Gillespie DA (2005) Chk1-dependent S-M checkpoint delay in vertebrate cells is linked to maintenance of viable replication structures. Mol Cell Biol 25:563–574CrossRefPubMedGoogle Scholar
  35. 35.
    Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K, Luo G, Carattini Rivera S, DeMayo F, Bradley A, Donehower LA, Elledge SJ (2000) Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev 14:1448–1459CrossRefPubMedGoogle Scholar
  36. 36.
    Takai H, Tominaga K, Motoyama N, Minamishima YA, Nagahama H, Tsukiyama T, Ikeda K, Nakayama K, Nakanishi M (2000) Aberrant cell cycle checkpoint function and early embryonic death in Chk1(−/−) mice. Genes Dev 14:1439–1447PubMedGoogle Scholar
  37. 37.
    Niida H, Tsuge S, Katsuno Y, Konishi A, Takeda N, Nakanishi M (2005) Depletion of Chk1 leads to premature activation of Cdc2-cyclin B and mitotic catastrophe. J Biol Chem 280:39246–39252CrossRefPubMedGoogle Scholar
  38. 38.
    Zachos G, Rainey MD, Gillespie DA (2003) Chk1-deficient tumour cells are viable but exhibit multiple checkpoint and survival defects. EMBO J 22:713–723CrossRefPubMedGoogle Scholar
  39. 39.
    Zaugg K, Su YW, Reilly PT, Moolani Y, Cheung CC, Hakem R, Hirao A, Liu Q, Elledge SJ, Mak TW (2007) Cross-talk between Chk1 and Chk2 in double-mutant thymocytes. Proc Natl Acad Sci USA 104:3805–3810CrossRefPubMedGoogle Scholar

Copyright information

© Springer Basel AG 2010

Authors and Affiliations

  • Laura Carrassa
    • 1
  • Elisa Montelatici
    • 2
  • Lorenza Lazzari
    • 2
  • Stefano Zangrossi
    • 2
  • Matteo Simone
    • 1
  • Massimo Broggini
    • 1
  • Giovanna Damia
    • 1
  1. 1.Laboratory of Molecular Pharmacology, Department of OncologyIstituto di Ricerche Farmacologiche “Mario Negri”MilanItaly
  2. 2.Cell Factory, Centre for Transfusion Medicine, Cell Therapy and Cryobiology, Department of Regenerative MedicineOspedale Maggiore PoliclinicoMilanItaly

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