Abstract
Retroviral integration mutagenesis and treatment with the frameshift mutagen ICR191 were used to transform v-H-ras expressing PB-3c cells to interleukin-3 (IL-3) independence. Six clones displayed viral integrations into the 3′ region of the IL-3 gene thus acting post-transcriptionally by disrupting the AU-rich instability element. Two clones contained reverse orientation integration into the raf-1 gene revealing an enhancer insertion mechanism. Growth by this mechanism was sensitive to the Raf-1 inhibitor BAY 43-9006 and the Mek inhibitor U0126. Following treatment with ICR191, IL-3-independent clones were recovered and studied by cell fusion. With 21/22 clones, IL-3 independence resulted from a recessive mechanism as cellular hybrids with parental cells reverted to IL-3 dependence. Recessive clone D2c displayed increased phospho-Erk1/2 levels and was growth sensitive to U0126, but not to BAY43-9006. The single dominant clone, D5a, showed no signs of mitogen-activated protein kinases pathway activation but displayed constitutive phosphorylation of Stat5. We conclude that PB-3c has several options to acquire IL-3 growth autonomy involving transcriptional or post-transcriptional mechanisms affecting the distal regulators Erk or Stat5. The reported panel of independent dominant and recessive transformants should provide a useful tool for inhibitor profiling.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 50 print issues and online access
$259.00 per year
only $5.18 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Algate PA, McCubrey JA . (1993). Oncogene 8: 1221–1232.
Andrejauskas E, Moroni C . (1989). EMBO J 8: 2575–2581.
Benjamin D, Colombi M, Moroni C . (2004). Nucleic acids research 32: e89.
Blalock WL, Pearce M, Steelman LS, Franklin RA, McCarthy SA, Cherwinski H et al. (2000). Oncogene 19: 526–536.
Blalock WL, Weinstein-Oppenheimer C, Chang F, Hoyle PE, Wang XY, Algate PA et al. (1999). Leukemia 13: 1109–1166.
D'Andrea R, Rayner J, Moretti P, Lopez A, Goodall GJ, Gonda TJ et al. (1994). Blood 83: 2802–2808.
Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S et al. (1996). Nat Med 2: 561–566.
Gough NM . (1988). Analytical biochemistry 173: 93–95.
Hannemann J, Hara T, Kawai M, Miyajima A, Ostertag W, Stocking C . (1995). Mol Cell Biol 15: 2402–2412.
Hayakawa F, Towatari M, Kiyoi H, Tanimoto M, Kitamura T, Saito H et al. (2000). Oncogene 19: 624–631.
Hirsch HH, Nair AP, Moroni C . (1993). J Exp Med 178: 403–411.
Hoyle PE, Moye PW, Steelman LS, Blalock WL, Franklin RA, Pearce M et al. (2000). Leukemia 14: 642–656.
Ihle JN, Lee JC, Rebar L . (1981). J Immunol 127: 2565–2570.
Jiang X, Ng E, Yip C, Eisterer W, Chalandon Y, Stuible M et al. (2002). Blood 100: 3731–3740.
Joung YH, Park JH, Park T, Lee CS, Kim OH, Ye SK et al. (2003). Exp Mol Med 35: 350–357.
Liu CB, Itoh T, Arai K, Watanabe S . (1999). J Biol Chem 274: 6342–6349.
McCormack MP, Gonda TJ . (1997). Blood 90: 1471–1481.
McCubrey JA, Shelton JG, Steelman LS, Franklin RA, Sreevalsan T, McMahon M. . (2004). Oncogene 23: 7810–7820.
McCubrey JA, Steelman LS, Hoyle PE, Blalock WL, Weinstein-Oppenheimer C, Franklin RA et al. (1998). Leukemia 12: 1903–1929.
Morita S, Kojima T, Kitamura T . (2000). Gene Ther 7: 1063–1066.
Mui AL, Wakao H, O'Farrell AM, Harada N, Miyajima A . (1995). EMBO J 14: 1166–1175.
Nagata Y, Todokoro K . (1996). Biochem Biophys Res Commun 221: 785–789.
Nair AP, Diamantis ID, Conscience JF, Kindler V, Hofer P, Moroni C . (1989). Mol Cell Biol 9: 1183–1190.
Nosaka T, Kitamura T . (2002). Exp Hematol 30: 697–702.
Onishi M, Kinoshita S, Morikawa Y, Shibuya A, Phillips J, Lanier LL et al. (1996a). Exp Hematol 24: 324–329.
Onishi M, Mui AL, Morikawa Y, Cho L, Kinoshita S, Nolan GP et al. (1996b). Blood 88: 1399–1406.
Onishi M, Nosaka T, Misawa K, Mui AL, Gorman D, McMahon M et al. (1998). Mol Cell Biol 18: 3871–3879.
Overell RW, Watson JD, Gallis B, Weisser KE, Cosman D, Widmer MB . (1987). Mol Cell Biol 7: 3394–3401.
Perkins GR, Marshall CJ, Collins MK . (1996). Blood 87: 3669–3675.
Piao X, Bernstein A . (1996). Blood 87: 3117–3123.
Shelton JG, Steelman LS, Lee JT, Knapp SL, Blalock WL, Moye PW et al. (2003). Oncogene 22: 2478–2492.
Shounan Y, Miller M, Symonds G . (1995). Exp Hematol 23: 492–499.
Steelman LS, Pohnert SC, Shelton JG, Franklin RA, Bertrand FE, McCubrey JA . (2004). Leukemia 18: 189–218.
Stoecklin G, Colombi M, Raineri I, Leuenberger S, Mallaun M, Schmidlin M et al. (2002). EMBO J 21: 4709–4718.
Stoecklin G, Gross B, Ming XF, Moroni C . (2003). Oncogene 22: 3554–3561.
Stoecklin G, Hahn S, Moroni C . (1994). J Biol Chem 269: 28591–28597.
Testa U, Riccioni R, Diverio D, Rossini A, Lo Coco F, Peschle C . (2004). Leukemia 18: 219–226.
Wodnar-Filipowicz A, Heusser CH, Moroni C . (1989). Nature 339: 150–152.
Acknowledgements
We thank Dr Don Benjamin and Bernd Rattenbacher for discussion and comments on the manuscript.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kiser, K., Colombi, M. & Moroni, C. Isolation and characterization of dominant and recessive IL-3-independent hematopoietic transformants. Oncogene 25, 6595–6603 (2006). https://doi.org/10.1038/sj.onc.1209673
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/sj.onc.1209673
Keywords
This article is cited by
-
Anti-apoptotic function of Xbp1 as an IL-3 signaling molecule in hematopoietic cells
Cell Death & Disease (2011)
-
Genome-wide shRNA screen reveals increased mitochondrial dependence upon mTORC2 addiction
Oncogene (2011)