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Effects of a conditionally active v-ErbB and an EGF-R inhibitor on transformation of NIH-3T3 cells and abrogation of cytokine dependency of hematopoietic cells

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Abstract

Epidermal growth factor (EGF) and its cognate receptor (EGF-R) are often dysregulated in human neoplasia. Moreover, EGF-R-transformed cell lines have constitutive EGF-R activity, which makes elucidation of its effects difficult to determine. In the following studies, the effects of a novel conditionally activated form of EGF-R, v-ErbB:ER, on the morphological transformation of NIH-3T3 cells and the abrogation of hematopoietic cell cytokine dependence were investigated. The v-ErbB ES-4 oncogene was fused to the hormone binding domain of the estrogen receptor (ER). This construct, v-ErbB:ER, requires β-estradiol or 4-OH tamoxifen for activation. v-ErbB:ER conditionally transformed NIH-3T3 cells and abrogated cytokine dependence of hematopoietic cells. Stimulation of v-ErbB:ER activity resulted in the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and Raf/MEK/ERK kinase cascades. To determine the importance of these signal transduction pathways, the conditionally transformed hematopoietic cells were treated with EGF-R, PI3K and MEK inhibitors. The EGF-R inhibitor AG1478 effectively inhibited MEK, ERK and Akt activation, and induced apoptosis when the cells were grown in response to v-ErbB:ER. Apoptosis was observed at 100- to 1000-fold lower concentrations of AG1478 when the cells were grown in response to v-ErbB:ER as opposed to IL-3. Furthermore, the parental, BCR-ABL- and Raf-transformed cells were only susceptible to the apoptosis-inducing effects of AG1478 at the highest concentrations demonstrating the specificity of these inhibitors. MEK or PI3K inhibitors suppressed ERK or Akt activation, respectively, and induced apoptosis in the v-ErbB:ER-responsive cells. However, MEK and PI3K inhibitors only induced apoptosis at 1000-fold higher concentrations than the EGFR inhibitor. This novel v-ErbB:ER construct and these conditionally transformed cell lines will be useful to further elucidate ErbB-mediated signal transduction and to determine the effectiveness of various inhibitors in targeting different aspects of EGF-R-mediated signal transduction and malignant transformation.

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References

  • Ahn NG, Nahreini TS, Tolwinski NS and Resing KA . (2001). Methods Enzymol., 332, 417–431.

  • Blalock WL, Moye PW, Chang F, Pearce M, Steelman LS, McMahon M and McCubrey JA . (2000b). Leukemia, 14, 1080–1096.

  • Blalock WL, Pearce M, Chang F, Lee J, Pohnert S, Burrows C, Steelman LS, Franklin RA, McMahon M and McCubrey J . (2001). Leukemia, 15, 794–807.

  • Blalock WL, Pearce M, Steelman LS, Franklin RA, McCarthy SA, Cherwinski H, McMahon M and McCubrey JA . (2000a). Oncogene, 19, 526–536.

  • Blalock WL, Steelman LS, Shelton JG, Moye PW, Lee JT, Franklin RA, Mirza A, McMahon M, White MK and McCubrey JA . (2003). Leukemia, 17, 1058–1068.

  • Blalock WL, Weinstein-Oppenheimer C, Chang F, Hoyle PE, Wang XY, Algate PA, Franklin RA, Oberhaus SM, Steelman LS and McCubrey JA . (1999). Leukemia, 13, 1109–1166.

  • Bosch E, Cherwinski H, Peterson D and McMahon M . (1997). Oncogene, 11, 1021–1034.

  • Bruskin A, Jackson J, Bishop JM, McCarley DJ and Schatzman RC . (1990). Oncogene, 5, 15–24.

  • Chang F, Lee JT, Navolanic PM, Steelman JG, Blalock WL, Franklin RA and McCubrey JA . (2003a). Leukemia, 17, 590–603.

  • Chang F, Steelman LS, Lee JT, Shelton JG, Navolanic PM, Blalock WL, Franklin RA and McCubrey JA . (2003c). Leukemia, 17, 1263–1294.

  • Chang F, Steelman LS and McCubrey JA . (2002). Cell Cycle, 1, 220–227.

  • Chang F, Steelman LS, Shelton JG, Lee JT, Navolanic PN, Blalock WL, Franklin RA and McCubrey JA . (2003b). Int. J. Oncol., 22, 469–480.

  • Clark SS, McLaughin J, Timmons M, Pendergast AM, Ben-Neriah Y, Dow LW, Crist W, Rovera G, Smith SD and Witte ON . (1988). Science, 239, 775–777.

  • Davies SP, Reddy H, Caivano M and Cohen P . (2000). Biochem. J., 351, 95–105.

  • Dexter TM, Garland J, Scott D, Scolnick E and Metcalf D . (1980). J. Exp. Med., 152, 1036–1047.

  • Eilers M, Picard D, Yamamoto KR and Bishop JM . (1989). Nature, 340, 66–68.

  • Fan QW, Zhang C, Shokat KM and Weiss WA . (2002). Curr. Biol., 12, 1386–1394.

  • Farrar MA, Alberol-Ila and Perlmutter RM . (1996). Nature, 383, 178–181.

  • Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, Van Dyk DE, Pitts WJ, Earl RA, Hobbs F, Copeland RA, Magolda RL, Scherle PA and Trzaskos JM . (1998). J. Biol. Chem., 273, 18623–18632.

  • Hoyle PE, Moye PW, Steelman LS, Blalock WL, Franklin RA, Pearce M, Cherwinski H, Bosch E, McMahon M and McCubrey JA . (2000). Leukemia, 14, 642–656.

  • Ihle JN, Witthuhn BA, Quelle FW, Yamamoto K and Silvennoinen O . (1995). Annu. Rev. Immunol., 13, 369–399.

  • Kitamura T, Sato M, Arai K-I and Miyajima A . (1991). Cell, 66, 1165–1174.

  • Kitamura T, Tange T, Terasawa T, Chiba S, Kuwaki T, Miyagawa K, Piao YF, Miyazono K, Urabe A and Takaku F . (1989). J. Cell. Physiol., 140, 323–334.

  • Kuan CT, Wilstrand CJ and Bigner DD . (2001). Brain Tumor Pathol., 17, 71–78.

  • Kumar V, Green S, Staub A and Chambon P . (1986). EMBO J., 5, 2231–2236.

  • Larson RA, Daley GQ, Schiffer CA, Porcu P, Pui CH, Marie JP, Steelman LS, Bertrand FE and McCubrey JA . (2003). Leukemia, 17, 2358–2382.

  • Lee Jr JT and McCubrey JA . (2002). Leukemia, 16, 486–507.

  • Luo Z, Tzivion G, Belshaw PJ, Vavvas D, Marshall M and Avruch J . (1996). Nature, 383, 181–185.

  • McCubrey JA, Holland G, McKearn J and Risser R . (1989). Oncogene Res., 4, 97–109.

  • McCubrey JA, May WS, Duronio V and Mufson A . (2000). Leukemia, 14, 9–21.

  • McCubrey JA, Smith SR, Algate PA, deVente JE, White MK and Steelman LS . (1993). Oncogene, 8, 2905–2915.

  • McCubrey JA, Steelman LS, Hoyle PA, Blalock WL, Weinstein-Oppenheimer CR, Franklin RA, Cherwinski H, Bosch E and McMahon M . (1998). Leukemia, 12, 1903–1929.

  • McCubrey JA, Steelman LS, Wang XY, Davidian EW, Hoyle PE, White C, Prevost KD, Algate PA, Robbins P, Mylott D and White MK . (1995). Int. J. Oncol., 7, 573–586.

  • McKearn JP, McCubrey JA and Fagg B . (1985). Proc. Natl. Acad. Sci. USA, 82, 7414–7418.

  • McMahon M . (2001). Methods Enzymol., 332, 401–417.

  • McMahon M, Schatzman RC and Bishop JM . (1991). Mol. Cell. Biol., 11, 4760–4770.

  • Miller M, Kennewell A, Takayama Y, Bruskin A, Bishop JM, Johnson G and Symonds G . (1990). Oncogene, 5, 1125–1131.

  • Muthuswamy SK, Gilman M and Brugge JS . (1999). Mol. Cell. Biol., 19, 6845–6857.

  • Navolanic PM, Steelman LS and McCubrey JA . (2003). Int. J. Oncol., 22, 237–252.

  • Pritchard CA, Samuels ML, Bosch E and McMahon M . (1995). Mol. Cell. Biol., 15, 9430–9442.

  • Samuels ML, Weber MJ, Bishop JM and McMahon M . (1993). Mol. Cell. Biol., 13, 6241–6252.

  • Sato N, Sakamaki K, Terada N, Arai K and Miyajima A . (1993). EMBO J., 12, 4181–4189.

  • Shelton JG, Blalock WL, White ER, Steelman LS and McCubrey JA . (2004a). Cell Cycle, 3, 503–512.

  • Shelton JG, Chang F, Lee JT, Franklin RA, Steelman LS and McCubrey JA . (2004b). Cell Cycle, 3, 189–196.

  • Shelton JG, Steelman LS, Lee JT, Knapp SL, Blalock WL, Moye PM, Franklin RA, Pohnert SC, Mizra AM, McMahon M and McCubrey JA . (2003). Oncogene, 24, 2478–2492.

  • Shelton JG, Steelman LS, White ER and McCubrey JA . (2004c). Cell Cycle, 3, 372–379.

  • Steelman LS, Algate PA, Blalock WL, Wang XY, Prevost KD, Hoyle PE and McCubrey JA . (1996). Leukemia, 10, 528–542.

  • Steelman LS, Pohnert SC, Shelton JG, Franklin RA, Bertrand FE and McCubrey JA . (2004c). Leukemia, 14, 189–218.

  • Von Lindern M, Deiner EM, Dolznig H, Parren-Van Amelsvoort M, Hayman MJ, Mullner EW and Beug H . (2001). Oncogene, 21, 3651–3664.

  • Weiss RA, Teich NM, Varmus HE and Coffin JM . (1984). RNA Tumor Viruses 2nd edn. Cold Spring Harbor Press: Cold Spring Harbor, New York, 1292pp.

    Google Scholar 

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Acknowledgements

This work was supported in part by Grants (R01CA51025 and R01CA98185) from the NCI (National Cancer Institute, National Institutes of Health) to JAM and Grant (9930099N) from the American Heart Association to RAF. MM is especially grateful to J Michael Bishop, who provided unwavering enthusiasm, support and advice both at the initiation of these studies and on an ongoing basis since.

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Author notes

  1. Thazepadath Sreevalsan: Currently retired

Authors and Affiliations

  1. Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, Greenville, 27858, NC, USA

    James A McCubrey, John G Shelton, Linda S Steelman & Richard A Franklin

  2. Leo Jenkins Cancer Center, Brody School of Medicine at East Carolina University, Greenville, 27858, NC, USA

    James A McCubrey & Richard A Franklin

  3. Department of Microbiology and Immunology, Georgetown University School of Medicine, Washington, 20057, DC, USA

    Thazepadath Sreevalsan

  4. UCSF/Mt Zion Cancer Center, S-329 Box 0128, 2340 Sutter Street, San Francisco, 94115, CA, USA

    Martin McMahon

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  1. James A McCubrey
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  2. John G Shelton
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  3. Linda S Steelman
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  4. Richard A Franklin
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  5. Thazepadath Sreevalsan
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  6. Martin McMahon
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Correspondence to James A McCubrey.

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McCubrey, J., Shelton, J., Steelman, L. et al. Effects of a conditionally active v-ErbB and an EGF-R inhibitor on transformation of NIH-3T3 cells and abrogation of cytokine dependency of hematopoietic cells. Oncogene 23, 7810–7820 (2004). https://doi.org/10.1038/sj.onc.1208055

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