SEARCH

SEARCH BY CITATION

Keywords:

  • AML1-ETO;
  • leukaemogenesis;
  • stem cell line;
  • pluripotent;
  • in vitro screening

Summary

  1. Top of page
  2. Summary
  3. Methods and materials
  4. Cell culture
  5. Retroviral infection
  6. Western blots
  7. Competitive repopulation assay
  8. Analysis of cultured HSCN1cl10, HSCN1cl10-AE cells and transplant recipients
  9. Results and discussion
  10. Expression of AML1-ETO in murine HPCs
  11. In vitro effects of AML1-ETO
  12. In vivo effects of AML1-ETO
  13. Acknowledgments
  14. References

The AML1-ETO fusion has been associated with up to 40% of acute myeloid leukaemia French–American–British classified M2 cases. This chimaeric protein interferes with normal AML1 function and disrupts critical transcriptional regulation of haematopoiesis. Current evidence suggests that AML1-ETO alone is insufficient to induce leukaemia, but rather is a co-operating event in leukaemogenesis. We developed a pluripotent murine haematopoietic stem cell line expressing the AML1-ETO fusion protein that displays in vitro and in vivo properties consistent with a preleukaemic state, including inhibition of terminal granulocytic differentiation in vitro and the development of non-lymphoid leukaemias in vivo. This cell line represents a potential platform for the introduction and in vitro rapid screening of candidate genes thought to co-operate with AML1-ETO in developing frank leukaemia.

The AML1-ETO fusion has been associated with up to 40% of acute myeloid leukaemia (AML) French–American–British classified M2 cases (Nucifora & Rowley, 1995). This translocation results in a chimaeric protein that interferes with normal AML1 function, disrupting critical transcriptional regulation of haematopoiesis. However, AML1-ETO expression is insufficient for inducing full leukaemic transformation in mice (Yergeau et al, 1997; Okuda et al, 1998; Rhoades et al, 2000), suggesting a requirement for co-operating mutations. In transgenic and Cre-Lox-mediated murine models of the t(8;21) translocation, mice developed leukaemia during their lifespan only when secondary mutations were induced by treatment with N-ethyl-N-nitrosourea (Yuan et al, 2001). The murine cell line, HSCN1cl10, which was developed by expressing the constitutively active intracellular domain of Notch1 in a haematopoietic stem cell (HSC), demonstrates multilineage repopulating potential (Varnum-Finney et al, 2000). Here, we show that AML1-ETO expression has similar effects in HSCN1cl10 cells as in primary marrow cells, establishing a preleukaemic stem cell line with both in vitro and in vivo pluripotent growth properties. This cell line offers an advantage over previously established cell lines or primary marrow cells transduced with AML1-ETO by providing a sufficient supply of cells for in vitro molecular study with concurrent ability for in vivo identification of potentially co-operative mutations in leukaemogenesis.

Cell culture

  1. Top of page
  2. Summary
  3. Methods and materials
  4. Cell culture
  5. Retroviral infection
  6. Western blots
  7. Competitive repopulation assay
  8. Analysis of cultured HSCN1cl10, HSCN1cl10-AE cells and transplant recipients
  9. Results and discussion
  10. Expression of AML1-ETO in murine HPCs
  11. In vitro effects of AML1-ETO
  12. In vivo effects of AML1-ETO
  13. Acknowledgments
  14. References

Cell lines were maintained in Iscoves's modified Dulbecco's medium supplemented with 20% fetal bovine serum and four growth factors (4GF): 100 ng/ml of murine stem cell factor (SCF), human Flt-3-ligand, human interleukin (IL)-6 and 10 ng/ml of human IL-11 (PeproTech, Inc., Rocky Hill, NJ, USA). HSCN1cl10-AE cells were also maintained in neomycin as these cells contain the neomycin resistance gene.

Retroviral infection

  1. Top of page
  2. Summary
  3. Methods and materials
  4. Cell culture
  5. Retroviral infection
  6. Western blots
  7. Competitive repopulation assay
  8. Analysis of cultured HSCN1cl10, HSCN1cl10-AE cells and transplant recipients
  9. Results and discussion
  10. Expression of AML1-ETO in murine HPCs
  11. In vitro effects of AML1-ETO
  12. In vivo effects of AML1-ETO
  13. Acknowledgments
  14. References

2·5 × 105 HSCN1cl10 cells were deposited into a single well of a non-tissue culture 12-well plate (Becton-Dickinson, Franklin Lakes, NJ, USA) coated with fibronectin fragments, and retroviral infection with MSCVneo AML1-ETO was performed as previously described (Varnum-Finney et al, 2000). HSCN1cl10 cells are green fluorescent protein (GFP) positive, as they express the Notch intracellular domain linked to GFP.

Western blots

  1. Top of page
  2. Summary
  3. Methods and materials
  4. Cell culture
  5. Retroviral infection
  6. Western blots
  7. Competitive repopulation assay
  8. Analysis of cultured HSCN1cl10, HSCN1cl10-AE cells and transplant recipients
  9. Results and discussion
  10. Expression of AML1-ETO in murine HPCs
  11. In vitro effects of AML1-ETO
  12. In vivo effects of AML1-ETO
  13. Acknowledgments
  14. References

Cell lysates were prepared as previously described (Varnum-Finney et al, 2000) and proteins were separated using sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose. A rabbit affinity-purified polyclonal antibody was raised against a peptide mapping to an internal region of rat C/EBPα (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Immunoreactivity was detected as previously described (Varnum-Finney et al, 2000).

Analysis of cultured HSCN1cl10, HSCN1cl10-AE cells and transplant recipients

  1. Top of page
  2. Summary
  3. Methods and materials
  4. Cell culture
  5. Retroviral infection
  6. Western blots
  7. Competitive repopulation assay
  8. Analysis of cultured HSCN1cl10, HSCN1cl10-AE cells and transplant recipients
  9. Results and discussion
  10. Expression of AML1-ETO in murine HPCs
  11. In vitro effects of AML1-ETO
  12. In vivo effects of AML1-ETO
  13. Acknowledgments
  14. References

The culture and analysis of cell lines and transplant recipients were performed as previously described (Varnum-Finney et al, 2000). In transplant recipients, bone marrow aspirates were also obtained and analysed as described for the peripheral blood samples.

Expression of AML1-ETO in murine HPCs

  1. Top of page
  2. Summary
  3. Methods and materials
  4. Cell culture
  5. Retroviral infection
  6. Western blots
  7. Competitive repopulation assay
  8. Analysis of cultured HSCN1cl10, HSCN1cl10-AE cells and transplant recipients
  9. Results and discussion
  10. Expression of AML1-ETO in murine HPCs
  11. In vitro effects of AML1-ETO
  12. In vivo effects of AML1-ETO
  13. Acknowledgments
  14. References

To test the effects of AML1-ETO expression in haematopoietic precursors, the murine HSC line, HSCN1cl10, was transduced with a retrovirus expressing human AML1-ETO and the neomycin resistance gene. After selection with G418, we detected the fusion protein by Western blot analysis (data not shown). This cell line is designated hereafter as HSCN1cl10-AE.

In vitro effects of AML1-ETO

  1. Top of page
  2. Summary
  3. Methods and materials
  4. Cell culture
  5. Retroviral infection
  6. Western blots
  7. Competitive repopulation assay
  8. Analysis of cultured HSCN1cl10, HSCN1cl10-AE cells and transplant recipients
  9. Results and discussion
  10. Expression of AML1-ETO in murine HPCs
  11. In vitro effects of AML1-ETO
  12. In vivo effects of AML1-ETO
  13. Acknowledgments
  14. References

Both HSCN1cl10 and HSCN1cl10-AE cells grew continuously in liquid culture with serum-containing medium supplemented by 4GF, and remained morphologically and phenotypically immature (Varnum-Finney et al, 2000) (Fig 1A–C). As with the parental cell line, HSCN1cl10-AE cells were dependent on 4GF for survival; death occurred within 24–72 h after removal of the 4GF (data not shown). Growth curves for transduced and non-transduced cells in 4GF were similar (Fig 1A) with no significant growth advantage of the AML1-ETO-transduced cells. No differences in the expression of antigens associated with immature precursors (Sca-1 and c-kit), myeloid cells (GR-1, F4/80) or lymphoid cells (CD25, Thy1) were noted (Fig 1C and data not shown). Additionally, HSCN1cl10-AE cells resembled a ‘hand-mirror’ shape morphology in liquid culture, and cytospin preparations revealed immature blasts (Fig 1B).

image

Figure 1. In vitro characteristics of AML-ETO expression in the murine pluripotent haematopoietic cell line, HSCN1cl10 cultured in IL6, IL-11, SCF, Flt3L (4GF) versus murine GM-CSF and SCF. HSCN1cl10 and HSCN1cl10-AE cells cultured in growth medium (IMDM) and 20% fetal bovine serum supplemented with SCF, Flt3L, IL6 and IL11 (4GF) were transferred to growth medium containing either 4GF or the myeloid differentiating cytokines, SCF and GM-CSF. (A) Growth curves for HSCN1cl10 (bsl00046) and HSCN1cl10-AE (bsl00066) in 4GF and HSCN1cl10 cells (•) and HSCN1cl10-AE (bsl00001) in SCF and GM-CSF. (B) Cytospin preparations of HSCN1cl10 and HSCN1cl10-AE cells cultured in 4GF (day 0) and 5 d after transfer to SCF and GM-CSF. (C) Initial GR-1 antigen expression in HSCN1cl10 and HSCN1cl10-AE cells cultured in 4GF (day 0) and 5 d after transfer to SCF and GM-CSF. The horizontal axes represent log fluorescence intensity after staining with GR-1 and vertical axes represent cell number; numbers indicate the percentage of cells that demonstrated increased staining compared with the isotype-matched control. (D) Protein lysates were prepared from HSCN1cl10 and HSCN1cl10-AE cells cultured in 4GF and 3 d after transfer to SCF and GM-CSF. Lysates were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis, transferred to nitrocellulose and immunoblotted with an antibody for C/EBPα.

Download figure to PowerPoint

Consistent with previous studies, we found that AML1-ETO allowed early myeloid differentiation, but prevented final differentiation (Westendorf et al, 1998; Kohzaki et al, 1999; Burel et al, 2001). When the parental cell line, HSCN1cl10, was cultured with granulocyte-macrophage colony stimulating factor (GM-CSF) and SCF, these cells ceased proliferation, differentiated and died within 7–10 d. Differentiation was associated with morphological as well as phenotypic evidence of myeloid differentiation (GR-1 and F4/80 antigen expression) (Fig 1A–C). In contrast, HSCN1cl10-AE cells cultured in GM-CSF and SCF more rapidly expressed GR-1 compared with HSCN1cl10 with almost 70% of the cells expressing GR-1 by day 5 of culture (Fig 1C). In GM-CSF and SCF, HSCN1cl10-AE also grew at a rate similar to that in 4GF, whereas HSCN1cl10 ceased proliferation (Fig 1A). Furthermore, despite high levels of myeloid antigens by 5 d of culture, HSCN1cl10-AE cells retained an immature morphology (Fig 1C).

The failure of HSCN1cl10-AE cells to undergo terminal granulocytic differentiation in GM-CSF and SCF was further indicated by expression of C/EBPα, a transcription factor critical in granulocytic differentiation (Zhang et al, 1997; Pabst et al, 2001). As expected, C/EBPα protein levels increased in HSCN1cl10 cells following culture in GM-CSF and SCF for 3 d, indicating the onset of terminal differentiation. However, in the same conditions, C/EBPα protein levels decreased in HSCN1cl10-AE cells (Fig 1D), suggesting that AML1-ETO down-regulated C/EBPα expression and inhibited terminal differentiation. These findings are consistent with previous evidence of the leukaemic properties of the AML1-ETO fusion protein, namely down-regulation of C/EBPα in t(8;21) leukaemia cells (Pabst et al, 2001), a block of granulocytic differentiation, and anti-apoptotic properties (Klampfer et al, 1996; Westendorf et al, 1998; Kohzaki et al, 1999; Burel et al, 2001).

In vivo effects of AML1-ETO

  1. Top of page
  2. Summary
  3. Methods and materials
  4. Cell culture
  5. Retroviral infection
  6. Western blots
  7. Competitive repopulation assay
  8. Analysis of cultured HSCN1cl10, HSCN1cl10-AE cells and transplant recipients
  9. Results and discussion
  10. Expression of AML1-ETO in murine HPCs
  11. In vitro effects of AML1-ETO
  12. In vivo effects of AML1-ETO
  13. Acknowledgments
  14. References

To further assess the in vivo effects and leukaemogenic potential of HSCN1cl10-AE cells, we injected 5 × 106 HSCN1cl10-AE cells or HSCN1cl10 cells intravenously along with 1 × 105 congenic Ly 5.1 bone marrow cells into lethally irradiated (1000 cGy) mice (C57Bl/6.SJL-Ly5.1-Pep3b) in two separate experiments. There were five mice per group (HSCN1cl10-AE and HSCN1cl10) in the first experiment and six mice per group in the second. HSCN1cl10-AE cells consistently showed a higher percentage of multilineage bone marrow engraftment compared with the parental cells based on fluorescence-activated cell sorting analysis of GFP positive cells in the bone marrow (data not shown). None of the mice developed myeloid leukaemias during the first 4 months. This absence of early myeloid leukaemia suggests that AML1-ETO is insufficient for the development of rapid myeloid leukaemias and probably requires time for additional events to occur. However, two of 10 mice that received HSCN1cl10-AE cells (1/4 and 1/6 mice in the two experiments) developed T-cell leukaemias. In contrast, none of the mice that received HSCN1cl10 cells developed T-cell leukaemias. As T-cell leukaemia is an expected consequence of Notch activation, this result raises the possibility of co-operation between Notch signalling and AML1-ETO. Moreover, by 6 months post-transplant, six of the remaining eight mice transplanted with HSCN1cl10-AE developed undifferentiated leukaemias, that were kit+ and lin− (Fig 2A). This was in contrast to mice transplanted with HSCN1cl10 cells, which were all electively killed at 8 months (Fig 2B).

image

Figure 2. In vivo characteristics of HSCN1cl10 and HSCN1cl10-AE cells. Bone marrow aspirates were obtained at regular time points from recipients of HSCN1cl10 and HSCN1cl10-AE cells and FACS analysis was performed to determine the percentage of GFP (donor cells) as well as markers of immature (Sca, kit), myeloid (F4/80, GR1) and lymphoid (CD19, B220, CD3, Thy1) antigens. (A) FACS analysis of bone marrow aspirate in a representative mouse that received HSCN1cl10-AE cells. (B) Survival analysis of two independent transplant experiments.

Download figure to PowerPoint

We have therefore developed a cell line expressing the AML1-ETO fusion protein that displays in vitro and in vivo properties consistent with a preleukaemic state, which provides opportunities for the further study of the AML1-ETO chimaeric protein against the background of pluripotent precursor cells, which is presumably more reflective of the in vivo development of this leukaemia. The lack of early in vivo myeloid leukaemias, but development of non-lymphoid leukaemias after 4 months is consistent with the acquisition of secondary co-operating mutations. Thus, the HSCN1cl10-AE cell line permits the detection of oncogenic properties of leukaemogenic translocations and represents a potential platform for the introduction and rapid screening of candidate genes thought to co-operate with the fusion protein in developing frank leukaemia, thereby avoiding the use of more cumbersome, time-consuming animal models.

Acknowledgments

  1. Top of page
  2. Summary
  3. Methods and materials
  4. Cell culture
  5. Retroviral infection
  6. Western blots
  7. Competitive repopulation assay
  8. Analysis of cultured HSCN1cl10, HSCN1cl10-AE cells and transplant recipients
  9. Results and discussion
  10. Expression of AML1-ETO in murine HPCs
  11. In vitro effects of AML1-ETO
  12. In vivo effects of AML1-ETO
  13. Acknowledgments
  14. References

We thank Dr James Downing for providing the MSCVneo AML1-ETO plasmid and Barbara Varnum-Finney for her help and suggestions in preparing the manuscript. This study was supported by HL54881, CA009351, ACS FM Kirby Clinical Research Professorship.

References

  1. Top of page
  2. Summary
  3. Methods and materials
  4. Cell culture
  5. Retroviral infection
  6. Western blots
  7. Competitive repopulation assay
  8. Analysis of cultured HSCN1cl10, HSCN1cl10-AE cells and transplant recipients
  9. Results and discussion
  10. Expression of AML1-ETO in murine HPCs
  11. In vitro effects of AML1-ETO
  12. In vivo effects of AML1-ETO
  13. Acknowledgments
  14. References
  • Burel, S.A., Harakawa, N., Zhou, L., Pabst, T., Tenen, D.G. & Zhang, D.E. (2001) Dichotomy of AML1-ETO functions: growth arrest versus block of differentiation. Molecular and Cellular Biology, 21, 55775590.
  • Klampfer, L., Zhang, J., Zelenetz, A.O., Uchida, H. & Nimer, S.D. (1996) The AML1/ETO fusion protein activates transcription of BCL-2. Proceedings of the National Academy of Sciences of the United States of America, 93, 14 05914 064.
  • Kohzaki, H., Ito, K., Huang, G., Wee, H.J., Murakami, Y. & Ito, Y. (1999) Block of granulocytic differentiation of 32Dcl3 cells by AML1/ETO(MTG8) but not by highly expressed Bcl-2. Oncogene, 18, 40554062.
  • Nucifora, G. & Rowley, J.D. (1995) AML1 and the 8;21 and 3;21 translocations in acute and chronic myeloid leukemia. Blood, 86, 114.
  • Okuda, T., Cai, Z., Yang, S., Lenny, N., Lyu, C.J., van Deursen, J.M., Harada, H. & Downing, J.R. (1998) Expression of a knocked-in AML1-ETO leukemia gene inhibits the establishment of normal definitive hematopoiesis and directly generates dysplastic hematopoietic progenitors. Blood, 91, 31343143.
  • Pabst, T., Mueller, B.U., Harakawa, N., Schoch, C., Haferlach, T., Behre, G., Hiddemann, W., Zhang, D.E. & Tenen, D.G. (2001) AML1-ETO downregulates the granulocytic differentiation factor C/EBPalpha in t(8;21) myeloid leukemia. Nature Medicine, 7, 444451.
  • Rhoades, K.L., Hetherington, C.J., Harakawa, N., Yergeau, D.A., Zhou, L., Liu, L.Q., Little, M.T., Tenen, D.G. & Zhang, D.E. (2000) Analysis of the role of AML1-ETO in leukemogenesis, using an inducible transgenic mouse model. Blood, 96, 21082115.
  • Varnum-Finney, B., Xu, L., Brashem-Stein, C., Nourigat, C., Flowers, D., Bakkour, S., Pear, W.S. & Bernstein, I.D. (2000) Pluripotent, cytokine-dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling. Nature Medicine, 6, 12781281.
  • Westendorf, J.J., Yamamoto, C.M., Lenny, N., Downing, J.R., Selsted, M.E. & Hiebert, S.W. (1998) The t(8;21) fusion product, AML-1-ETO, associates with C/EBP-alpha, inhibits C/EBP-alpha-dependent transcription, and blocks granulocytic differentiation. Molecular and Cellular Biology, 18, 322333.
  • Yergeau, D.A., Hetherington, C.J., Wang, Q., Zhang, P., Sharpe, A.H., Binder, M., Marin-Padilla, M., Tenen, D.G., Speck, N.A. & Zhang, D.E. (1997) Embryonic lethality and impairment of haematopoiesis in mice heterozygous for an AML1-ETO fusion gene. Nature Genetics, 15, 303306.
  • Yuan, Y., Zhou, L., Miyamoto, T., Iwasaki, H., Harakawa, N., Hetherington, C.J., Burel, S.A., Lagasse, E., Weissman, I.L., Akashi, K. & Zhang, D.E. (2001) AML1-ETO expression is directly involved in the development of acute myeloid leukemia in the presence of additional mutations. Proceedings of the National Academy of Sciences of the United States of America, 98, 10 39810 403.
  • Zhang, D.E., Zhang, P., Wang, N.D., Hetherington, C.J., Darlington, G.J. & Tenen, D.G. (1997) Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein α-deficient mice. Proceedings of the National Academy of Sciences of the United States of America, 94, 569574.