Effect of tgfβ on human cd34+ progenitors on fibronectin at the cell cycle level

Mo A. Dao, Jan Nolta

Research output: Contribution to journalArticle

Abstract

Promising gene therapy depends on successful transaction of primitive hematopoietic stem cells (HSC). Target cells must cycle to allow integration of DMA reverse transcribed from Moloney-based retroviral vectors, yet HSC are quiescent. Fibronectin (FN) enhances transduction by promoting contact between target cells and retroviral particles (Nature Med 2:876,1996). TGFβl is a negative regulator of hematopoietic cell cycle, and reduction of TGFβl levels during transduction can increase the extent of gene transfer ( J. Exp. Med. 174: 925, 1991). In the current studies, CD34cells isolated from human marrow were transduced with retroviral supernatant in medium +/- PCS, +/- anti-TGFβ antibody with cytokines on BSA vs. FN-coated plates. The best transduction was obtained with anti-TGFβ on FN. We next examined the effect of TGFβ and FN on the signalling pathway leading to cycling of CD34+ progenitors. TGFβ suppresses CDK4 at the translation level. CDK4 associates with cyclin D1 to hyperphosphorylate Rb which then releases E2F, a transcription factor required for DNA replication. TGFβ increases the levels of P211, p27and pIS14, which block cell cycle progression by inactivating cyclin/CDK complexes. We incubated CD34+ cells in media containing cytokines on BSA vs. FN coated plates +/- TGFβ or + aTGFβ for 12-14 hours at 37. Western blots were done and probed with aCDK4, aE2F, ap15, and ap27. In the presence of TGFβ, bone marrow CD34+ progenitors expressed an increased level of p15lnM, a cyclin dependent kinase inhibitor which binds and inactivates the activity of cyclinO-cdk4 complex. CDK4 levels were decreased in mink lung epithelial cells cultured with TGFβ (Cell 74:1009, 1993). The same response was observed in primary human CD34+ cells from bone marrow, assayed 12 hours after addition of TGFβ. However, in subsequent studies examining BM CD34+ cells for 15 hours and in cord blood CD34+ cells cultured for 12 hours with TGFβ, an increase in the level of CDK4 was observed along with an increase in p15. Decreased levels of CDK inhibitors explains how neutralizing TGFβl releases hematopoietic progenitors from quiescence, increasing gene transfer.

Original languageEnglish (US)
Pages (from-to)753
Number of pages1
JournalExperimental Hematology
Volume25
Issue number8
StatePublished - 1997
Externally publishedYes

Fingerprint

Fibronectins
Cell Cycle
Hematopoietic Stem Cells
Bone Marrow
E2F Transcription Factors
Cytokines
Mink
Cyclins
Cyclin-Dependent Kinases
Cyclin D1
DNA Replication
Fetal Blood
Bone Marrow Cells
Genetic Therapy
Genes
Anti-Idiotypic Antibodies
Blood Cells
Western Blotting
Epithelial Cells
Lung

ASJC Scopus subject areas

  • Cancer Research
  • Cell Biology
  • Genetics
  • Hematology
  • Oncology
  • Transplantation

Cite this

Effect of tgfβ on human cd34+ progenitors on fibronectin at the cell cycle level. / Dao, Mo A.; Nolta, Jan.

In: Experimental Hematology, Vol. 25, No. 8, 1997, p. 753.

Research output: Contribution to journalArticle

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