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Release of early human hematopoietic progenitors from quiescence by antisense transforming growth factor beta 1 or Rb
J Hatzfeld1, M L Li, E L Brown
1Laboratoire C.N.R.S. de Biologie Cellulaire et Moléculaire des Facteurs de Croissance, ICIG, Hôpital Paul-Brousse, Villejuif, France.
Abstract:
We have used antisense oligonucleotides to study the roles of transforming growth factor beta (TGF-beta) and the two antioncogenes, retinoblastoma susceptibility (Rb) and p53, in the negative regulation of proliferation of early hematopoietic cells in culture. The antisense TGF-beta sequence significantly enhanced the frequency of colony formation by multi-lineage, early erythroid, and granulomonocytic progenitors, but did not affect colony formation by late progenitors. Single cell culture and limiting dilution analysis indicated that autocrine TGF-beta is produced by a subpopulation of early progenitors. Antisense Rb but not antisense p53 yielded similar results in releasing multipotential progenitors (colony-forming unit-granulocyte/erythroid/macrophage/megakaryocyte) from quiescence. Rb antisense could partially reverse the inhibitory effect of exogenous TGF-beta. Anti-TGF-beta blocking antibodies, antisense TGF-beta, or Rb oligonucleotides all had similar effects. No additive effects were observed when these reagents were combined, suggesting a common pathway of action. Our results are consistent with the model that autocrine production of TGF-beta negatively regulates the cycling status of early hematopoietic progenitors through interaction with the Rb gene product.
Insights
Antisense oligonucleotides targeting transforming growth factor beta (TGF-beta) and retinoblastoma susceptibility (Rb) released early hematopoietic cells from quiescence. Autocrine TGF-beta negatively regulates early hematopoietic progenitor cycling via Rb.
Area of Science:
- Hematology
- Cell Biology
- Molecular Biology
Background:
- Hematopoietic stem cell proliferation is tightly regulated.
- Transforming growth factor beta (TGF-beta) and antioncogenes like retinoblastoma susceptibility (Rb) and p53 are implicated in cell cycle control.
Purpose of the Study:
- To investigate the roles of TGF-beta, Rb, and p53 in the negative regulation of early hematopoietic cell proliferation.
- To elucidate the mechanism by which autocrine TGF-beta affects progenitor cell cycling.
Main Methods:
- Use of antisense oligonucleotides to inhibit gene expression of TGF-beta, Rb, and p53.
- Colony formation assays for multi-lineage progenitors.
- Single cell culture and limiting dilution analysis to detect autocrine factors.
Main Results:
- Antisense TGF-beta and antisense Rb enhanced progenitor colony formation, while antisense p53 did not.
- Autocrine TGF-beta is produced by a subpopulation of early progenitors.
- Rb antisense partially reversed TGF-beta inhibition, suggesting a common pathway.
Conclusions:
- Autocrine TGF-beta negatively regulates early hematopoietic progenitor cycling.
- This regulation involves interaction with the Rb gene product.
- TGF-beta and Rb act through a common pathway to control hematopoietic progenitor proliferation.