T. Michael Dexter

Transforming growth factor-beta 1 induces apoptosis independently of p53 and selectively reduces expression of Bcl-2 in multipotent hematopoietic cells.

Transforming growth factor-β1 (TGF-β1) can inhibit cell proliferation or induce apoptosis in multipotent hematopoietic cells. To study the mechanisms of TGF-β1 action on primitive hematopoietic cells, we used the interleukin-3 (IL-3)-dependent, multipotent FDCP-Mix cell line. TGF-β1-mediated growth inhibition was observed in high concentrations of IL-3, while at lower IL-3 concentrations TGF-β1 induced apoptosis. The proapoptotic effects of TGF-β1 occur via a p53-independent pathway, since p53null FDCP-Mix demonstrated the same responses to TGF-β1. IL-3 has been suggested to enhance survival via an increase in (antiapoptotic) Bcl-xL expression. In FDCP-Mix cells, neither IL-3 nor TGF-β1 induced any change in Bcl-xL protein levels or the proapoptotic proteins Bad or Bax. However, TGF-β1 had a major effect on Bcl-2 levels, reducing them in the presence of high and low concentrations of IL-3. Overexpression of Bcl-2 in FDCP-Mix cells rescued them from TGF-β1-induced apoptosis but was incapable of inhibiting TGF-β1-mediated growth arrest. We conclude that TGF-β1-induced cell death is independent of p53 and inhibited by Bcl-2, with no effect on Bcl-xL. The significance of these results for stem cell survival in bone marrow are discussed.

Influence of growth factors and substrates on differentiation of haemopoietic stem cells

During the last few years there has been major progress in our understanding of the mechanisms underlying the regulation of haemopoietic stem cell proliferation and development. In the past 12 months, advances have been made in identifying how growth factor receptor expression is regulated in primitive haemopoietic cells, in determining the transcription factors that are associated with development, and in recognizing some of the specific molecular interactions that occur between the bone marrow stromal cells and the haemopoietic progenitor cells. These and previous studies clearly demonstrate that the environment can influence the survival, the proliferation and the differentiation of haemopoietic cells at every stage of development.

Bcr-Abl protein tyrosine kinase activity induces a loss of p53 protein that mediates a delay in myeloid differentiation.

Chronic myeloid leukaemia is a haemopoietic stem cell disorder, the hallmark of which is the expression of the Bcr-Abl Protein Tyrosine Kinase (PTK). We have previously reported that activation of a temperature sensitive Bcr-Abl PTK in the multipotent haemopoietic cell line FDCP-Mix for short periods resulted in subtle changes including, a transient suppression of apoptosis and no inhibition of differentiation. In contrast, activation of the Bcr-Abl PTK for 12 weeks results in cells that display a delay in differentiation at the early granulocyte stage. Flow cytometric analysis also indicates that the expression of cell surface differentiation markers and nuclear morphology are uncoupled. Furthermore, a significant number of the mature neutrophils display abnormal morphological features. Prolonged exposure to Bcr-Abl PTK results in interleukin-3 independent growth and decreased p53 protein levels. FDCP-Mix cells expressing a dominant negative p53 and p53null FDCP-Mix cells demonstrate that the reduction in p53 is causally related to the delay in development. Returning the cells to the restrictive temperature restores the p53 protein levels, the growth factor dependence and largely relieves the effects on development. We conclude that prolonged Bcr-Abl PTK activity within multipotent cells results in a reduction of p53 that drives a delayed and abnormal differentiation.

Apoptosis in haemopoietic progenitor cells exposed to extremely low-frequency magnetic fields

Epidemiological studies have indicated a modestly increased risk for the development of acute myeloid leukaemia in children who live close to high-voltage power-lines. Recent evidence has suggested that a common property shared by a number of known and suspected tumour promoters is their ability to block the process of apoptosis. Therefore, one possible mechanistic explanation for the apparent leukaemogenic effect of weak, low-frequency magnetic fields, such as emitted by power-lines and electrical appliances, would be their expression of tumour-promoting activity by interfering with the regulation of apoptosis in multipotent haemopoietic progenitor cells. In order to test this hypothesis, we have employed the well-characterized multipotential haemopoietic progenitor cell line FDCP-mix(A4). These cells are non-leukaemic and undergo apoptosis when deprived of appropriate growth factors such as Interleukin-3. We have tested a series of different regimes of weak, low-frequency magnetic fields: nulled fields, Ca2+-ion cyclotron resonance conditions at 50 Hz, and vertical 50 Hz fields of 6 microT(RMS), 1 mT(RMS) and 2 mT(RMS), exposing the cells for 2 hours, 24 hours, 4 days or 7 days under various culture conditions. We have not seen any significant alteration in apoptosis induced by any of the exposure regimes tested. We therefore conclude that the regulation of viability and apoptosis in FDCP-mix(A4) cells is not disturbed by weak magnetic fields of the magnitude and type indicated.

Exposure to Extremely Low Frequency Magnetic Fields Has No Effect on Growth Rate or Clonogenic Potential of Multipotential Haemopoietic Progenitor Cells

Recent reports indicate an increased risk of acute myeloid leukaemia in children exposed to extremely low frequency magnetic fields (ELFMFs) emitted by high voltage power lines, suggesting that ELFMFs may act as weak tumour promoters. We have investigated possible interactions of weak ELFMFs with primitive haemopoietic cells in vitro using the multipotential progenitor cell line FDCP-mix(A4). We have determined the proliferative activity and clonogenic potential of cells under both optimal and sub-optimal growth conditions and exposed to either ambient laboratory ELFMFs or three other ELFMF regimes representative of those produced by high voltage power lines: nulled fields, Ca2+-ion cyclotron resonance conditions at 50 Hz, and vertical 50 Hz fields of 6 muT(RMS). Using exposures of 1, 4, 7 and 21 days, we found no significant alteration of growth rate, cell-cycle state or clonogenic efficiency indicating that neither the proliferation nor self-renewal of multipotential FDCP-mix(A4) cells was perturbed.

Serum-free culture of enriched murine haemopoietic stem cells. I: Effect of haemopoietic growth factors on proliferation.

Using a population of cells highly enriched for multipotential day 12 spleen colony forming cells (CFU-S) (termed the FACS-BM population), and a serum-free culture system, the requirements for development of multipotential cell have been investigated and compared to previous results using serum containing cultures. In both serum-free and serum supplemented cultures interleukin-3 (IL-3) was a potent colony stimulating factor, although it was more effective in serum free conditions. However, colony stimulation by granulocyte-macrophage colony stimulating factor (GM-CSF) and macrophage-colony stimulating factor (M-CSF) was markedly reduced in the absence of serum. Significantly, the ability of interleukin-1 (IL-1) and granulocyte-colony stimulating factor (G-CSF) to synergise with these two growth factors was retained in serum-free conditions, indicating that these growth factors act directly on the FACS-BM without serum co-factors. Furthermore synergistic interactions between IL-3 plus IL-1, and IL-3 plus M-CSF were only manifest in serum-free conditions. The significance of these results in relation to the ability of these growth factors to act directly on multipotential cells is discussed.

Biological Effects of Retroviral Transfection of the Murine Interleukin-3 Gene into FDCP-Mix Cells

Several genetic changes are believed to be necessary to convert normal haemopoietic cells into leukaemic cells. These changes are associated with various biological events including immortalisation, clonal selection, changes in growth factor requirements and stromal cell dependence and changes in the balance of self-renewal and differentiation. Within the haemopoietic system, a variety of growth factors are now known to play a critical role in recruiting proliferation and development of the most primitive multipotent stem cells and the lineage-restricted progenitor cells. Some of these growth factors are biologically restricted and are able to recruit only the lineage-restricted progenitor cells (e.g. M-CSF, G-CSF and erythropoietin); others show a broader range of activities and can stimulate growth and development of multipotent stem cells and facilitate their development into several cell lineages (Dexter 1987). An example of the latter class of growth factor is Interleukin-3 (IL3).

Serum-free culture of enriched murine haemopoietic stem cells. II: Effects of growth factors and haemin on development.

A serum-free culture system was used to determine the effects of growth factors on the clonogenic development of a population of cells highly enriched for multipotential day 12 spleen colony forming cells (CFU-S) (FACS-BM). Under these conditions, interleukin-3 (IL-3) was found to be primarily a proliferative stimulus, the progenitor cells developing in the clonal assay systems produced colonies of morphologically undifferentiated cells for up to 20 days. No such induction of proliferation without maturation was observed with other growth factors (eg. granulocyte-macrophage colony stimulating factor (GM-CSF)). However, combinations of IL-3 plus secondary growth factors such as GM-CSF, macrophage colony stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF) or interleukin-1 (IL-1) led to the formation of colonies containing mature haemopoietic cells of the granulocytic, megakaryocytic or monocytic lineages. In contrast, erythroid development did not occur unless the protoporphyrin, haemin, was added to the cultures. Under these conditions mature erythroid cells were produced in cultures containing either IL-3 or GM-CSF (with or without erythropoietin (epo)). In replating experiments it was determined that the FACS-BM cells were able to generate large numbers of clonogenic cells for up to 30-40 free cultures. Such cultures, therefore, may be useful for investigating the biological and basis of the generation of clonogenic cells and of haemopoietic cell differentiation and development in response to growth factors.

Culturing Primitive Hemopoietic Cells

The maintenance of normal primitive hemopoietic cell types in culture for long periods can be achieved either by culture of hemopoietic cells in association with bone marrow-derived stromal cells, as in long-term cultures, or by establishing factor-dependent primitive hemopoietic cell lines (FDCP-Mix cell lines). The long-term culture technique and a reproducible method for establishing FDCP-Mix cell lines will be described here (1,2).

The Hox-2.4 gene is not involved in the generation of IL-3 dependent multipotent FDCP-mix cell lines.

The establishment of IL-3-dependent multipotent progenitor cell lines from Hox-2.4-expressing bone marrow cells suggests that homeobox genes may contribute to immortalization of early myeloid cells. A survey of 20 independently derived multipotent IL-3-dependent cell lines established from either src-virus-infected long-term bone marrow cultures (FDCP-mix) or Multi-CSF-virus (M3MuV)-infected bone marrow revealed that Hox-2.4 was not expressed in any of these cell lines. In addition DNA rearrangements were not observed. We conclude that activation of Hox-2.4 is not an obligatory event in the immortalization of early myeloid cells.