Cell Biology and Toxicology

, Volume 28, Issue 4, pp 201–212 | Cite as

Ectopic expression of Flt3 kinase inhibits proliferation and promotes cell death in different human cancer cell lines

  • Eystein Oveland
  • Line Wergeland
  • Randi Hovland
  • James B. Lorens
  • Bjørn Tore Gjertsen
  • Kari E. Fladmark
Original Research


Stable ectopic expression of Flt3 receptor tyrosine kinase is usually performed in interleukin 3 (IL-3)-dependent murine cell lines like Ba/F3, resulting in loss of IL-3 dependence. Such high-level Flt3 expression has to date not been reported in human acute myeloid leukemia (AML) cell lines, despite the fact that oncogenic Flt3 aberrancies are frequent in AML patients. We show here that ectopic Flt3 expression in different human cancer cell lines might reduce proliferation and induce apoptotic cell death, involving Bax/Bcl2 modulation. Selective depletion of Flt3-expressing cells occurred in human AML cell lines transduced with retroviral Flt3 constructs, shown here using the HL-60 leukemic cell line. Flt3 expression was investigated in two cellular model systems, the SAOS-2 osteosarcoma cell line and the human embryonic kidney HEK293 cell line, and proliferation was reduced in both systems. HEK293 cells underwent apoptosis upon ectopic Flt3 expression and cell death could be rescued by overexpression of Bcl-2. Furthermore, we observed that the Flt3-induced inhibition of proliferation in HL-60 cells appeared to be Bax-dependent. Our results thus suggest that excessive Flt3 expression has growth-suppressive properties in several human cancer cell lines.


Acute myeloid leukemia Cell death Flt3 Receptor tyrosine kinase 



Acute myeloid leukemia


Flt3 with internal tandem duplication


Interleukin 3


Receptor tyrosine kinase


Wild type



We are grateful to Linn Hodneland, Lena Fjellstad Hansen, Atle Bredhaug, Ann Kristin Frøyset, Lise Fismen, and Hege Avsnes Dale for valuable technical assistance and Ian F. Pryme for proofreading the manuscript. Confocal imaging and electron microscopy were performed at the Molecular Imaging Center (FUGE, Norwegian Research Council), University of Bergen. This work was supported by grants from the Helse Vest Research Fund and The Norwegian Cancer Society (B.T.G.).


  1. Alcalay M, Tiacci E, Bergomas R, Bigerna B, Venturini E, Minardi SP, Meani N, Diverio D, Bernard L, Tizzoni L, Volorio S, Luzi L, Colombo E, Lo Coco F, Mecucci C, Falini B, Pelicci PG. Acute myeloid leukemia bearing cytoplasmic nucleophosmin (NPMc+ AML) shows a distinct gene expression profile characterized by up-regulation of genes involved in stem-cell maintenance. Blood. 2005;106:899–902.PubMedCrossRefGoogle Scholar
  2. Beham A, Marin MC, Fernandez A, Herrmann J, Brisbay S, Tari AM, Lopez-Berestein G, Lozano G, Sarkiss M, McDonnell TJ. Bcl-2 inhibits p53 nuclear import following DNA damage. Oncogene. 1997;15:2767–72.PubMedCrossRefGoogle Scholar
  3. Blume-Jensen P, Hunter T. Oncogenic kinase signalling. Nature. 2001;411:355–65.PubMedCrossRefGoogle Scholar
  4. Bredholt T, Dimba EA, Hagland HR, Wergeland L, Skavland J, Fossan KO, Tronstad KJ, Johannessen AC, Vintermyr OK, Gjertsen BT. Camptothecin and khat (Catha edulis Forsk.) induced distinct cell death phenotypes involving modulation of c-FLIPL, Mcl-1, procaspase-8 and mitochondrial function in acute myeloid leukemia cell lines. Mol Cancer. 2009;8:101.PubMedCrossRefGoogle Scholar
  5. Bruserud O, Hovland R, Wergeland L, Huang TS, Gjertsen BT. Flt3-mediated signaling in human acute myelogenous leukemia (AML) blasts: a functional characterization of Flt3-ligand effects in AML cell populations with and without genetic Flt3 abnormalities. Haematologica. 2003;88:416–28.PubMedGoogle Scholar
  6. Brustugun OT, Fladmark KE, Doskeland SO, Orrenius S, Zhivotovsky B. Apoptosis induced by microinjection of cytochrome c is caspase-dependent and is inhibited by Bcl-2. Cell Death Differ. 1998;5:660–8.PubMedCrossRefGoogle Scholar
  7. Desplat V, Belloc F, Lagarde V, Boyer C, Melo JV, Reiffers J, Praloran V, Mahon FX. Overproduction of BCR-ABL induces apoptosis in imatinib mesylate-resistant cell lines. Cancer. 2005;103:102–10.PubMedCrossRefGoogle Scholar
  8. Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci L, La Starza R, Diverio D, Colombo E, Santucci A, Bigerna B, Pacini R, Pucciarini A, Liso A, Vignetti M, Fazi P, Meani N, Pettirossi V, Saglio G, Mandelli F, Lo-Coco F, Pelicci PG, Martelli MF. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med. 2005;352:254–66.PubMedCrossRefGoogle Scholar
  9. Fearnhead HO, McCurrach ME, O'Neill J, Zhang K, Lowe SW, Lazebnik YA. Oncogene-dependent apoptosis in extracts from drug-resistant cells. Genes Dev. 1997;11:1266–76.PubMedCrossRefGoogle Scholar
  10. Fenski R, Flesch K, Serve S, Mizuki M, Oelmann E, Kratz-Albers K, Kienast J, Leo R, Schwartz S, Berdel WE, Serve H. Constitutive activation of FLT3 in acute myeloid leukaemia and its consequences for growth of 32D cells. Br J Haematol. 2000;108:322–30.PubMedCrossRefGoogle Scholar
  11. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–74.PubMedCrossRefGoogle Scholar
  12. Hayakawa F, Towatari M, Kiyoi H, Tanimoto M, Kitamura T, Saito H, Naoe T. Tandem-duplicated Flt3 constitutively activates STAT5 and MAP kinase and introduces autonomous cell growth in IL-3-dependent cell lines. Oncogene. 2000;19:624–31.PubMedCrossRefGoogle Scholar
  13. Heiss E, Masson K, Sundberg C, Pedersen M, Sun J, Bengtsson S, Ronnstrand L. Identification of Y589 and Y599 in the juxtamembrane domain of Flt3 as ligand-induced autophosphorylation sites involved in binding of Src family kinases and the protein tyrosine phosphatase SHP2. Blood. 2006;108:1542–50.PubMedCrossRefGoogle Scholar
  14. Hovland R, Gjertsen BT, Bruserud O. Acute myelogenous leukemia with internal tandem duplication of the Flt3 gene appearing or altering at the time of relapse: a report of two cases. Leuk Lymphoma. 2002;43:2027–9.PubMedCrossRefGoogle Scholar
  15. Huang GC, Hobbs S, Walton M, Epstein RJ. Dominant negative knockout of p53 abolishes ErbB2-dependent apoptosis and permits growth acceleration in human breast cancer cells. Br J Cancer. 2002;86:1104–9.PubMedCrossRefGoogle Scholar
  16. Irish JM, Anensen N, Hovland R, Skavland J, Borresen-Dale AL, Bruserud O, Nolan GP, Gjertsen BT. Flt3 Y591 duplication and Bcl-2 overexpression are detected in acute myeloid leukemia cells with high levels of phosphorylated wild-type p53. Blood. 2007;109:2589–96.PubMedCrossRefGoogle Scholar
  17. Irish JM, Hovland R, Krutzik PO, Perez OD, Bruserud O, Gjertsen BT, Nolan GP. Single cell profiling of potentiated phospho-protein networks in cancer cells. Cell. 2004;118:217–28.PubMedCrossRefGoogle Scholar
  18. Kerr LE, Birse-Archbold JL, Short DM, McGregor AL, Heron I, Macdonald DC, Thompson J, Carlson GJ, Kelly JS, McCulloch J, Sharkey J. Nucleophosmin is a novel Bax chaperone that regulates apoptotic cell death. Oncogene. 2007;26:2554–62.PubMedCrossRefGoogle Scholar
  19. Koch S, Jacobi A, Ryser M, Ehninger G, Thiede C. Abnormal localization and accumulation of FLT3-ITD, a mutant receptor tyrosine kinase involved in leukemogenesis. Cells Tissues Organs. 2008;188:225–35.PubMedCrossRefGoogle Scholar
  20. Koonin EV, Altschul SF, Bork P. BRCA1 protein products … functional motifs. Nat Genet. 1996;13:266–8.PubMedCrossRefGoogle Scholar
  21. Lee BH, Williams IR, Anastasiadou E, Boulton CL, Joseph SW, Amaral SM, Curley DP, Duclos N, Huntly BJ, Fabbro D, Griffin JD, Gilliland DG. FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model. Oncogene. 2005;24:7882–92.PubMedCrossRefGoogle Scholar
  22. Lorens JB, Bennett MK, Pearsall DM, Throndset WR, Rossi AB, Armstrong RJ, Fox BP, Chan EH, Luo Y, Masuda E, Ferrick DA, Anderson DC, Payan DG, Nolan GP. Retroviral delivery of peptide modulators of cellular functions. Mol Ther. 2000;1:438–47.PubMedCrossRefGoogle Scholar
  23. Lowenberg B, van Putten W, Theobald M, Gmur J, Verdonck L, Sonneveld P, Fey M, Schouten H, de Greef G, Ferrant A, Kovacsovics T, Gratwohl A, Daenen S, Huijgens P, Boogaerts M. Effect of priming with granulocyte colony-stimulating factor on the outcome of chemotherapy for acute myeloid leukemia. N Engl J Med. 2003;349:743–52.PubMedCrossRefGoogle Scholar
  24. Lyman SD, James L, Zappone J, Sleath PR, Beckmann MP, Bird T. Characterization of the protein encoded by the flt3 (flk2) receptor-like tyrosine kinase gene. Oncogene. 1993;8:815–22.PubMedGoogle Scholar
  25. Nakao M, Yokota S, Iwai T, Kaneko H, Horiike S, Kashima K, Sonoda Y, Fujimoto T, Misawa S. Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia. 1996;10:1911–8.PubMedGoogle Scholar
  26. Okura T, Igase M, Kitami Y, Fukuoka T, Maguchi M, Kohara K, Hiwada K. Platelet-derived growth factor induces apoptosis in vascular smooth muscle cells: roles of the Bcl-2 family. Biochim Biophys Acta. 1998;1403:245–53.PubMedCrossRefGoogle Scholar
  27. Oveland E, Fladmark KE, Wergeland L, Gjertsen BT, Hovland R. Proteomics approaches to elucidate oncogenic tyrosine kinase signaling in myeloid malignancies. Curr Pharm Biotechnol. 2006;7:185–98.PubMedCrossRefGoogle Scholar
  28. Oveland E, Gjertsen BT, Wergeland L, Selheim F, Fladmark KE, Hovland R. Ligand-induced Flt3-downregulation modulates cell death associated proteins and enhances chemosensitivity to idarubicin in THP-1 acute myeloid leukemia cells. Leuk Res. 2009;33:276–87.PubMedCrossRefGoogle Scholar
  29. Ozeki K, Kiyoi H, Hirose Y, Iwai M, Ninomiya M, Kodera Y, Miyawaki S, Kuriyama K, Shimazaki C, Akiyama H, Nishimura M, Motoji T, Shinagawa K, Takeshita A, Ueda R, Ohno R, Emi N, Naoe T. Biologic and clinical significance of the FLT3 transcript level in acute myeloid leukemia. Blood. 2004;103:1901–8.PubMedCrossRefGoogle Scholar
  30. Pratz KW, Cortes J, Roboz GJ, Rao N, Arowojolu O, Stine A, Shiotsu Y, Shudo A, Akinaga S, Small D, Karp JE, Levis M. A pharmacodynamic study of the FLT3 inhibitor KW-2449 yields insight into the basis for clinical response. Blood. 2009;113:3938–46.PubMedCrossRefGoogle Scholar
  31. Sargin B, Choudhary C, Crosetto N, Schmidt MH, Grundler R, Rensinghoff M, Thiessen C, Tickenbrock L, Schwable J, Brandts C, August B, Koschmieder S, Bandi SR, Duyster J, Berdel WE, Muller-Tidow C, Dikic I, Serve H. Flt3-dependent transformation by inactivating c-Cbl mutations in AML. Blood. 2007;110:1004–12.PubMedCrossRefGoogle Scholar
  32. Shieh SY, Ikeda M, Taya Y, Prives C. DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell. 1997;91:325–34.PubMedCrossRefGoogle Scholar
  33. Small D, Levenstein M, Kim E, Carow C, Amin S, Rockwell P, Witte L, Burrow C, Ratajczak MZ, Gewirtz AM, et al. STK-1, the human homolog of Flk-2/Flt-3, is selectively expressed in CD34+ human bone marrow cells and is involved in the proliferation of early progenitor/stem cells. Proc Natl Acad Sci U S A. 1994;91:459–63.PubMedCrossRefGoogle Scholar
  34. Stommel JM, Kimmelman AC, Ying H, Nabioullin R, Ponugoti AH, Wiedemeyer R, Stegh AH, Bradner JE, Ligon KL, Brennan C, Chin L, DePinho RA. Coactivation of receptor tyrosine kinases affects the response of tumor cells to targeted therapies. Science. 2007;318:287–90.PubMedCrossRefGoogle Scholar
  35. Swift S, Lorens J, Achacoso P, Nolan GP. Current protocols in immunology. In: Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W, editors. Current protocols in immunology, vol Unit 10.17C, Suppl. 31. New York: Wiley; 1999. p. 14–29.Google Scholar
  36. Thompson J, Finlayson K, Salvo-Chirnside E, Macdonald D, McCulloch J, Kerr L, Sharkey J. Characterisation of the Bax-nucleophosmin interaction: the importance of the Bax C-terminus. Apoptosis. 2008;13:394–403.PubMedCrossRefGoogle Scholar
  37. Tikhomirov O, Carpenter G. Ligand-induced, p38-dependent apoptosis in cells expressing high levels of epidermal growth factor receptor and ErbB-2. J Biol Chem. 2004;279:12988–96.PubMedCrossRefGoogle Scholar
  38. Yamamoto Y, Kiyoi H, Nakano Y, Suzuki R, Kodera Y, Miyawaki S, Asou N, Kuriyama K, Yagasaki F, Shimazaki C, Akiyama H, Saito K, Nishimura M, Motoji T, Shinagawa K, Takeshita A, Saito H, Ueda R, Ohno R, Naoe T. Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood. 2001;97:2434–9.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Eystein Oveland
    • 1
    • 2
  • Line Wergeland
    • 3
    • 5
  • Randi Hovland
    • 4
    • 6
  • James B. Lorens
    • 2
  • Bjørn Tore Gjertsen
    • 5
    • 3
  • Kari E. Fladmark
    • 6
  1. 1.Proteomics Unit at University of Bergen (PROBE)University of BergenBergenNorway
  2. 2.Department of BiomedicineUniversity of BergenBergenNorway
  3. 3.Department of Medicine, Hematology SectionHaukeland University HospitalBergenNorway
  4. 4.Center for Medical Genetics and Molecular MedicineHaukeland University HospitalBergenNorway
  5. 5.Institute of Medicine, Hematology SectionUniversity of BergenBergenNorway
  6. 6.Department of Molecular BiologyUniversity of BergenBergenNorway

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