Abstract

Aim: Polycythemia vera (PV) and essential thrombocythemia (ET) are Philadelphia–negative myeloproliferative neoplasms with documented apoptosis impairment at the level of hematopoietic stem cell. However, so far no study has evaluated apoptosis of circulating blood neoplastic cells, including the suicidal death of erythrocytes – eryptosis. Material/Methods: Erythrocytes from 61 patients (24 PV and 37 ET) naïve to and treated with hydroxyurea (HU) and 13 healthy individuals were analysed using flow cytometry to quantify phosphatidylserine (PS) externalization from Annexin-V-binding, calpain activity from 7-amino-4-chloromethylcoumarin (CMAC)-fluorescence, cell volume from forward scattered light (FSC) and cell shape from side scattered light (SSC). Results: Significantly increased levels of calpain activity and PS exposure were observed in both ET and PV naïve patients, indicating enhanced eryptosis. Among HU-treated patients, a significant increase in calpain activity in the ET group and a decrease in the PV group were observed compared to patients without cytoreductive therapy. Among PV patients, FSC was substantially higher in the HU-treated group than in the naïve group, whereas no significant differences were found between HU-treated and HU-naïve groups of ET patients. Conclusions: The enhanced eryptosis in ET and PV patients may be a form of systemic compensation of the pathological bone marrow overproduction of erythrocytes. HU, the basic cytoreductive drug used in ET and PV, may affect eryptosis in PV and ET in different ways depending on disease. The JAK2V617F mutation was not observed to have any effect on eryptosis in ET.

References

• 1. Berlin N.I., Lawrence J.H., Lee H.C.: The life span of the red bloodcell in chronic leukemia and polycythemia. Science, 1951; 114:385–387
  Google Scholar
• 2. Berlin N.I., Reynafarcje C., Lawrence J.H.: Red cell life span inthe polycythemia of high altitude. J. Appl. Physiol., 1954; 7: 271–272
  Google Scholar
• 3. Bosman G.J., Willekens F.L., Werre J.M.: Erythrocyte aging: a morethan superficial resemblance to apoptosis? Cell Physiol. Biochem.,2005; 16: 1–8
  Google Scholar
• 4. Bratosin D., Estaquier J., Petit F., Arnoult D., Quatannens B., TissierJ.P., Slomianny C., Sartiaux C., Alonso C., Huart J.J., Montreuil J., AmeisenJ.C.: Programmed cell death in mature erythrocytes: a modelfor investigating death effector pathways operating in the absenceof mitochondria. Cell Death Differ., 2001; 8: 1143–1156
  Google Scholar
• 5. Campbell P.J., Scott L.M., Buck G., Wheatley K., East C.L., MarsdenJ.T., Duffy A., Boyd E.M., Bench A.J., Scott M.A., Vassiliou G.S., MilliganD.W., Smith S.R., Erber W.N., Bareford D., Wilkins B.S., et al.:Definition of subtypes of essential thrombocythaemia and relationto polycythaemiavera based on JAK2 V617F mutation status: a prospectivestudy. Lancet, 2005; 366: 1945–1953
  Google Scholar
• 6. Dameshek W.: Some speculations on the myeloproliferative syndromes.Blood, 1951; 6: 372–375
  Google Scholar
• 7. Daugas E., Cande C., Kroemer G.: Erythrocytes: death of a mummy.Cell Death Differ., 2001; 8: 1131–1133
  Google Scholar
• 8. DeSesso J.M.: Cell death and free radicals: a mechanism for hydroxyureateratogenesis. Med. Hypotheses, 1979; 5: 937–951
  Google Scholar
• 9. Dogru G., Ay O.I., Erdal M.E., Ay M.E., Tombak A., Karakas U.: Therole of certain gene polymorphisms involved in the apoptotic pathwaysin polycythemia vera and essential thrombocytosis. Adv. Clin.Exp. Med., 2017; 26: 761–765
  Google Scholar
• 10. Föller M., Huber S.M., Lang F.: Erythrocyte programmed celldeath. IUBMB Life, 2008; 60: 661–668
  Google Scholar
• 11. Fujita H., Sakuma R., Tomiyama J., Hamaki T., Ohwada A., KurosawaS., Nishimura S.: Increased phosphatidylserine exposure onthe erythrocyte membrane in patients with polycythaemiavera. Br.J. Haematol., 2011; 152: 238–240
  Google Scholar
• 12. Fujita H., Sakuma R., Tomiyama J., Hamaki T., Ohwada A., NishimuraS.: Relationship between clotting activity and phosphatidylserineexpression on erythrocyte membranes in polycythemia verapatients with the JAK2 V617F mutation. Arch. Physiol. Biochem.,2011; 117: 231–235
  Google Scholar
• 13. Gerotziafas G.T., Van Dreden P., Chaari M., Galea V., Khaterchi A.,Lionnet F., Stankovic-Stojanovic K., Blanc-Brude O., Woodhams B., Maier-Redelsperger M., Girot R., Hatmi M., Elalamy I.: The accelerationof the propagation phase of thrombin generation in patients withsteady-state sickle cell disease is associated with circulating erythrocyte-derived microparticles. Thromb. Haemost., 2012; 107: 1044–1052
  Google Scholar
• 14. Giardina B., Messana I., Scatena R., Castagnola M.: The multiplefunctions of hemoglobin. Crit. Rev. Biochem. Mol. Biol., 1995; 30: 165–196
  Google Scholar
• 15. Haynes J. Jr., Obiako B., Hester R.B., Baliga B.S., Stevens T.: Hydroxyureaattenuates activated neutrophil-mediated sickle erythrocytemembrane phosphatidylserine exposure and adhesion topulmonary vascular endothelium. Am. J. Physiol. Heart Circ. Physiol.,2008; 294: H379–H385
  Google Scholar
• 16. Jiang J., Jordan S.J., Barr D.P., Gunther M.R., Maeda H., MasonR.P.: In vivo production of nitric oxide in rats after administrationof hydroxyurea. Mol. Pharmacol., 1997; 52: 1081–1086
  Google Scholar
• 17. Kwaan H.C., Wang J.: Hyperviscosity in polycythemia vera andother red cell abnormalities. Semin. Thromb. Hemost., 2003; 29: 451–458
  Google Scholar
• 18. Lang K.S., Lang P.A., Bauer C., Duranton C., Wieder T., HuberS.M., Lang F.: Mechanisms of suicidal erythrocyte death. Cell Physiol.Biochem., 2005; 15: 195–202
  Google Scholar
• 19. Laubach J.P., Fu P., Jiang X., Salter K.H., Potti A., Arcasoy M.O.:Polycythemia veraerythroid precursors exhibit increased proliferationand apoptosis resistance associated with abnormal RAS and PI3Kpathway activation. Exp. Hematol., 2009; 37: 1411–1422
  Google Scholar
• 20. Malherbe J.A., Fuller K.A., Mirzai B., Kavanagh S., So C.C., Ip H.W.,Guo B.B., Forsyth C., Howman R., Erber W.N.: Dysregulation of theintrinsic apoptotic pathway mediates megakaryocytic hyperplasiain myeloproliferative neoplasms. J. Clin. Pathol., 2016; 69: 1017–1024
  Google Scholar
• 21. Nathan D.G., Berlin N.I.: Studies of the production and life spanof erythrocytes in myeloid metaplasia. Blood, 1959; 14: 668–682
  Google Scholar
• 22. Philips F.S., Sternberg S.S., Schwartz H.S., Cronin A.P., SodergrenJ.E., Vidal P.M.: Hydroxyurea. I. Acute cell death in proliferating tissuesin rats. Cancer Res., 1967; 27: 61–75
  Google Scholar
• 23. Richter K., Bruschke G.: Erythrocyte life span in polycythemia vera;studies with radiochromium. Klin. Wochenschr., 1959; 37: 150–158
  Google Scholar
• 24. Rupon J.W., Domingo S.R., Smith S.V., Gummadi B.K., Shields H.,Ballas S.K., King S.B., Kim-Shapiro D.B.: The reactions of myoglobin,normal adult hemoglobin, sickle cell hemoglobin and hemin withhydroxyurea. Biophys. Chem., 2000; 84: 1–11
  Google Scholar
• 25. Silva M., Richard C., Benito A., Sanz C., Olalla I., Fernández-LunaJ.L.: Expression of Bcl-x in erythroid precursors from patients withpolycythemia vera. N. Engl. J. Med., 1998; 338: 564–571
  Google Scholar
• 26. Tan X., Shi J., Fu Y., Gao C., Yang X., Li J., Wang W., Hou J., Li H.,Zhou J.: Role of erythrocytes and platelets in the hypercoagulablestatus in polycythemia vera through phosphatidylserine exposureand microparticle generation. Thromb. Haemost., 2013; 109:1025–1032
  Google Scholar
• 27. Tefferi A., Vannucchi A.M., Barbui T.: Essential thrombocythemiatreatment algorithm 2018. Blood Cancer J., 2018; 8: 2
  Google Scholar
• 28. Tefferi A., Vannucchi A.M., Barbui T.: Polycythemia vera treatmentalgorithm 2018. Blood Cancer J., 2018; 8: 3
  Google Scholar
• 29. Thiele J., Kvasnicka H.M.: A critical reappraisal of the WHO classificationof the chronic myeloproliferative disorders. Leuk Lymphoma,2006; 47: 381–396
  Google Scholar
• 30. Tong D., Yu M., Guo L., Li T., Li J., Novakovic V.A., Dong Z., TianY., Kou J., Bi Y., Wang J., Zhou J., Shi J.: Phosphatidylserine-exposingblood and endothelial cells contribute to the hypercoagulablestate in essential thrombocythemia patients. Ann. Hematol., 2018;97: 605–616
  Google Scholar
• 31. Treliński J., Chojnowski K., Cebula-Obrzut B., Smolewski P.: Impairedapoptosis of megakaryocytes and bone marrow mononuclearcells in essential thrombocythemia: correlation with JAK2V617Fmutational status and cytoreductive therapy. Med. Oncol., 2012;29: 2388–2395
  Google Scholar
• 32. Vannucchi A.M., Guglielmelli P., Tefferi A.: Advances in understandingand management of myeloproliferative neoplasms. CACancer J. Clin., 2009; 59: 171–191
  Google Scholar
• 33. Vardiman J.W., Thiele J., Arber D.A., Brunning R.D., Borowitz M.J.,Porwit A., Harris N.L., Le Beau M.M., Hellström-Lindberg E., TefferiA., Bloomfield C.D.: The 2008 revision of the World Health Organization(WHO) classification of myeloid neoplasms and acute leukemia:rationale and important changes. Blood, 2009; 114: 937–951
  Google Scholar
• 34. Yarbro J.W.: Mechanism of action of hydroxyurea. Semin. Oncol.,1992; 19 (3 Suppl. 9): 1–10
  Google Scholar

Full text