Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Haemopoiesis in vitro

Enhancement of proliferation and differentiation of erythroid progenitors by co-transduction of erythropoietin receptor and H-ras cDNAs into single CD343+ cord blood cells

Bone Marrow Transplantation volume 26, pages 817–822 (2000)Cite this article

Abstract

Our previous studies have demonstrated that retrovirus-mediated gene transduction of either the human erythropoietin receptor (EpoR) or H-ras cDNA into single purified hematopoietic progenitor (HPC), CD343+, cells from cord blood (CB) resulted in increased numbers and sizes of erythroid cell containing colonies. We therefore evaluated if there were further effects when H-ras and EpoR genes were co-transduced into the same progenitor cells. Highly purified single sorted CD343+ CB cells were transduced with retroviral vectors encoding EpoR or H-ras cDNA. At the single cell level, and in response to stimulation by a combination of growth factors, including Epo, the number of colonies formed by BFU-E and CFU-GEMM was significantly increased in cells transduced with either single H-ras or EpoR cDNA compared to mock virus-transduced cells as previously described. Increased numbers of BFU-E, but not CFU-GEMM, colonies were produced from cells simultaneously co-transduced with both EpoR and H-ras genes. Little or no growth was seen in transduced cells without exogenously added cytokines. The size of all types of colonies including CFU-GM was increased in cells transduced with H-ras and/or EpoR cDNAs, and the greatest increase was noticed in cells co-transduced with both genes. Integration and expression of either gene in individual colonies as assessed by PCR and RT-PCR analysis were 45–62% and 48–58%, respectively, with approximately 31% of the cells containing and expressing both genes. These results add to information suggesting an enhancing interacting role of H-ras and EpoR in erythroid proliferation/differentiation. Bone Marrow Transplantation (2000) 26, 817–822.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4

Similar content being viewed by others

References

  1. Morrison SJ, Shah NM, Anderson DJ . Regulatory mechanisms in stem cell biology Cell 1997 88: 287–298

    Article  CAS  Google Scholar 

  2. Koury MJ, Bondurant MC . Erythropoietin retards DNA breakdown and prevents programmed death in erythroid progenitor cells Science 1990 248: 378–381

    Article  CAS  Google Scholar 

  3. D’Andrea AD . Hematopoietic growth factors and the regulation of differentiative decisions Curr Opin Cell Biol 1994 6: 804–808

    Article  Google Scholar 

  4. Wu H, Liu X, Jaenisch R, Lodish HF . Generation of committed erythoid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor Cell 1995 3: 59–67

    Article  Google Scholar 

  5. Kieran MW, Perkins AC, Orkin SH, Zou LI . Thrombopoietin rescues in vitro erythroid colony formation from mouse embryos lacking the erythropoietin receptor Proc Natl Acad Sci USA 1996 93: 9126–9131

    Article  CAS  Google Scholar 

  6. McCormick F . Going for the GAP Curr Biol 1998 8: 673–675

    Article  Google Scholar 

  7. Wittinghofer A . Signaling transduction via Ras Biol Chem 1998 379: 933–937

    CAS  PubMed  Google Scholar 

  8. Hall A . Rho GTPases and the actin cytoskeleton Science 1998 279: 509–514

    Article  CAS  Google Scholar 

  9. McCormick F . Ras biology in atomic detail Nat Struct Biol 1996 3: 653–655

    Article  CAS  Google Scholar 

  10. Rebollo A, Marthez-A C . Ras proteins: recent advances and new functions Blood 1999 94: 2971–2980

    CAS  PubMed  Google Scholar 

  11. Balmain A, Pragnell IB . Mouse skin carcinomas induced in vivo by chemical carcinogens have a transforming Harvey-ras oncogene Nature 1983 303: 72–74

    Article  CAS  Google Scholar 

  12. Yamaguchi-Iwai Y, Satake M, Murakami Y et al. Differentiation of F9 embryonal carcinoma cells induced by the c-jun and activated c-Ha-ras oncogenes Proc Natl Acad Sci USA 1990 87: 8670–8674

    Article  CAS  Google Scholar 

  13. Lu L, Ge Y, Li Z-H et al. Retroviral transfer of the recombinant human erythopoietin receptor gene into single hematopoietic stem/progenitor cells from human cord blood increases the number of erythropoietin-dependent erythroid colonies Blood 1996 87: 525–534

    CAS  PubMed  Google Scholar 

  14. Lu L, Li Z-H, He J et al. Transduction of recombinant human erythropoietin receptor cDNA into daughter progenitors derived from single CD343+ cord blood cells changes the differentiation profile of daughter progenitors J Leuk Biol 1998 63: 389–394

    Article  CAS  Google Scholar 

  15. Lu L, Ge Y, Li Z-H et al. Influence of retroviral-mediated gene transduction of both the recombinant human erythropoietin receptor and interleukin-9 receptor genes into single CD34++CD33− or low cord blood cells on cytokine stimulated erthroid colony formation Exp Hematol 1996 24: 347–351

    CAS  PubMed  Google Scholar 

  16. Ge Y, Li Z-H, Marshall MS et al. Involvement of H-ras in erythroid differentiation of TF-1 and human umbilical cord blood CD34+++ cells Blood Cells Mol Dis 1998 24: 124–136

    Article  CAS  Google Scholar 

  17. Lu L, Heinrich MC, Wang L-S et al. Retroviral mediated gene transduction of c-kit into single hematopoietic progenitor cells from cord blood enhances erythroid colony formation and decreases sensitivity to inhibition by tumor necrosis factor-α and transforming growth factor-β1 Blood 1999 94: 2319–2332

    CAS  PubMed  Google Scholar 

  18. Lu L, Xiao M, Shen R-N et al. Enrichment, characterization, and responsiveness of single primitive CD34+++ human umbilical cord blood hematopoietic progenitors with high proliferative and replating potential Blood 1993 81: 41–48

    CAS  PubMed  Google Scholar 

  19. Lu L, Xiao M, Clapp DW et al. High efficiency retroviral mediated gene transduction into single isolated immature and replatable CD34+++ hematopoietic stem/progenitor cells from human umbilical cord blood J Exp Med 1993 178: 2089–2096

    Article  CAS  Google Scholar 

  20. Roberts TM . A signal chain of events Nature (London) 1992 360: 534–535

    Article  CAS  Google Scholar 

  21. Marshall CJ . Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation Cell 1995 80: 179–185

    Article  CAS  Google Scholar 

  22. Denhardt DT . Signal-transducing protein phosphorylation cascades mediated by ras/rho proteins in the mammalian cell: the potential for mutiplex signaling Biochem J 1996 318: 729–747

    Article  CAS  Google Scholar 

  23. Barber DL, Corless CN, Xia K et al. Erythropoietin activates Raf-1 by an shc-independent pathway in CTLL-Epo-R cells Blood 1997 89: 55–64

    CAS  PubMed  Google Scholar 

  24. Stokoe D, Macdonald SG, Cadwallader K et al. Activation of Raf as a result of recruitment to the plasma membrane Science 1994 264: 1463–1467

    Article  CAS  Google Scholar 

  25. Hunter T . Cytokine connections Nature (London) 1993 366: 114–116

    Article  CAS  Google Scholar 

  26. Carroll MP, Spivak JL, McMahon M et al. Erythropoietin induces Raf-1 activation and Raf-1 is required for erythropoietin-mediated proliferation J Biol Chem 1991 266: 14964–14969

    CAS  PubMed  Google Scholar 

  27. Zmuidzinas A, Mamon HJ, Roberts TM, Smith KA . Interleukin-2-triggered Raf-1 expression, phosphorylation, and associated kinase activity increase through G1 and S phases in CD3-stimulated primary human T cells Mol Cell Biol 1991 11: 2794–2803

    Article  CAS  Google Scholar 

  28. Iwatsuki K, Endo T, Misawa H et al. STAT5 activation correlates with erythropoietin receptor-mediated erythroid differentiation of an erythroleukemia cell line J Biol Chem 1997 272: 8149–8152

    Article  CAS  Google Scholar 

  29. Wakao H, Chida D, Damen JE et al. A possible involvement of Stat5 in erythropoietin-induced hemoglobin synthesis Biochem Biophys Res Commun 1997 234: 198–205

    Article  CAS  Google Scholar 

  30. Chida D, Miura O, Yoshimura A, Miyajima A . Role of cytokine signaling molecules in erythroid differentiation of mouse fetal liver hematopoietic cells: functional analysis of signaling molecules by retrovirus-mediated expression Blood 1999 93: 1567–1578

    CAS  PubMed  Google Scholar 

  31. Xia K, Mukhopadhyay NK, Inhorn RC et al. The cytokine-activated tyrosine kinase JAK2 activates Raf-1 in a p21ras-dependent manner Proc Natl Acad Sci USA 1996 93: 11681–11686

    Article  CAS  Google Scholar 

  32. Nagai T, Igarashi K, Akasaaka J-e et al. Regulation of NF-E2 activity in erythroleukemia cell differentiation J Biol Chem 1998 273: 5358–5365

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by Public Health Service Grants RO1 HL 56416 and Ro1 DK 53674 from the National Institute of Health to HEB, grants from the Phi Beta Psi Sorority to LL.

Author information

Authors and Affiliations

  1. Department of Microbiology/Immunology, Indiana University School of Medicine, Indianapolis, IN, USA

    L Lu, Y Ge, Z-H Li, M-S Dai & HE Broxmeyer

  2. Department of Medicine (Hematology/Oncology), Indiana University School of Medicine, Indianapolis, IN, USA

    L Lu & HE Broxmeyer

  3. Department of the Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN, USA

    L Lu, Y Ge, Z-H Li, M-S Dai & HE Broxmeyer

  4. The Walther Cancer Institute, Indianapolis, IN, USA

    L Lu & HE Broxmeyer

Authors
  1. L Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Y Ge
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Z-H Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M-S Dai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. HE Broxmeyer
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lu, L., Ge, Y., Li, ZH. et al. Enhancement of proliferation and differentiation of erythroid progenitors by co-transduction of erythropoietin receptor and H-ras cDNAs into single CD343+ cord blood cells. Bone Marrow Transplant 26, 817–822 (2000). https://doi.org/10.1038/sj.bmt.1702620

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.bmt.1702620

Keywords

Search

Advanced search

Quick links