Lower phosphorylation of p38 MAPK blocks the oxidative stress-induced senescence in myeloid leukemic CD34⁺CD38⁻ cells

Abstract

Leukemia seems to depend on a small population of “leukemia stem cells (LSCs)” for its growth and metastasis. However, the precise surviving mechanisms of LSCs remain obscure. Cellular senescence is an important obstacle for production and surviving of tumor cells. In this study we investigated the activated state of a pathway, in which reactive oxygen species (ROS) induces cellular senescence through DNA damage and phophorylation of p38 MAPK (p38), in myeloid leukemic CD34⁺CD38⁻ cells. Bone marrow samples were obtained from patients with acute myeloid leukemia (AML, n=11) and chronic myeloid leukemia (CML, n=9). CD34⁺CD38⁻ cells were isolated from mononuclear cells from these bone marrow samples, and K562 and KG1a cells (two kinds of myeloid leukemia cell lines) by mini-magnetic activated cell sorting. Hematopoietic stem cells (HSCs) from human cord blood served as controls. Intracellular ROS level was detected by flow cytometry. DNA damage defined as the γH2AX level was measured by immunofluorescence staining. Real-time RT-PCR was used to detect the expression of p21, a senescence-associated gene. Western blotting and immunofluorescence staining were employed to determine the p38 expression and activation. The proliferation and apoptosis of CD34⁺CD38⁻ cells were detected by MTT assay and flow cytometry. Our results showed that ROS and DNA damage were substantially accumulated and p38 was less phosphorated in myeloid leukemic CD34⁺CD38⁻ cells as compared with HSCs and H₂O₂-induced senescent HSCs. Furthermore, over-phosphorylation of p38 by anisomycin, a selective activator of p38, induced both the senescence-like growth arrest and apoptosis of CD34⁺CD38⁻ cells from K562 and KG1a cell lines. These findings suggested that, although excessive accumulation of oxidative DNA damage was present in LSCs, the relatively decreased phosphorylation of p38 might help leukemic cells escape senescence and apoptosis.

Keywords

reactive oxygen species / DNA damage / p38 MAPK / cellular senescence / leukemia stem cells / myeloid

References

[1] ChanW.I., HuntlyB.J.. Leukemia stem cells in acute myeloid leukemia. Semin Oncol, 2008, 35(4): 326-335

[2] EppertK., TakenakaK., LechmanE.R., et al.. Stem cell gene expression programs influence clinical outcome in human leukemia. Nat Med, 2011, 17(9): 1086-1093

[3] ViscontiR., GriecoD.. New insights on oxidative stress in cancer. Curr Opin Drug Discov Devel, 2009, 12(2): 240-245

[4] NakaK., MuraguchiT., HoshiiT., et al.. Regulation of reactive oxygen species and genomic stability in hematopoietic stem cells. Antioxid Redox Signal, 2008, 10(11): 1883-1894

[5] WangY., SchulteB.A., LaRueA.C., et al.. Total body irradiation selectively induces murine hematopoietic stem cell senescence. Blood, 2006, 107(1): 358-366

[6] ColladoM., SerranoM.. Senescence in tumours: evidence from mice and humans. Nat Rev Cancer, 2010, 10(1): 51-57

[7] ColladoM., GilJ., EfeyanA., et al.. Tumour biology: senescence in premalignant tumours. Nature, 2005, 436(7051): 642

[8] SallmyrA., FanJ., DattaK., et al.. Internal tandem duplication of FLT3 (FLT3/ITD) induces increased ROS production, DNA damage, and misrepair: implications for poor prognosis in AML. Blood, 2008, 111(6): 3173-3182

[9] SattlerM., VermaS., ShrikhandeG., et al.. The BCR/ABL tyrosine kinase induces production of reactive oxygen species in hematopoietic cells. J Biol Chem, 2000, 275(32): 24273-24278

[10] KoptyraM., FalinskiR., NowickiM.O., et al.. BCR/ABL kinase induces self-mutagenesis via reactive oxygen species to encode imatinib resistance. Blood, 2006, 108(1): 319-327

[11] RassoolF.V., GaymesT.J., OmidvarN., et al.. Reactive oxygen species, DNA damage, and error-prone repair: a model for genomic instability with progression in myeloid leukemia?. Cancer Res, 2007, 67(18): 8762-8771

[12] ItoK., HiraoA., AraiF., et al.. Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells. Nat Med, 2006, 12(4): 446-451

[13] WangY., LiuL., PazhanisamyS.K., et al.. Total body irradiation causes residual bone marrow injury by induction of persistent oxidative stress in murine hematopoietic stem cells. Free Radic Biol Med, 2010, 48(2): 348-356

[14] WangY., KellnerJ., LiuL., et al.. Inhibition of p38 mitogen-activated protein kinase promotes ex vivo hematopoietic stem cell expansion. Stem Cells Dev, 2011, 20(7): 1143-1152

[15] ZouJ., ZouP., LouY., et al.. The cross-talk between ROS and p38MAPKalpha in the ex vivo expanded human umbilical cord blood CD133(+) cells. J Huazhong Univ Sci Technolog Med Sci, 2011, 31(5): 591-595

[16] DoladoI., SwatA., AjenjoN., et al.. p38alpha MAP kinase as a sensor of reactive oxygen species in tumorigenesis. Cancer Cell, 2007, 11(2): 191-205

[17] AmbrosinoC., NebredaA.R.. Cell cycle regulation by p38 MAP kinases. Biol Cell, 2001, 93(1–2): 47-51

[18] ZhouL., OpalinskaJ., VermaA.. p38 MAP kinase regulates stem cell apoptosis in human hematopoietic failure. Cell Cycle, 2007, 6(5): 534-537

[19] HaqR., BrentonJ.D., TakahashiM., et al.. Constitutive p38HOG mitogen-activated protein kinase activation induces permanent cell cycle arrest and senescence. Cancer Res, 2002, 62(17): 5076-5082

[20] BulavinD.V., FornaceA.J.. p38 MAP kinase’s emerging role as a tumor suppressor. Adv Cancer Res, 2004, 92: 95-118