John Gooya

IL-3 Induces Inhibitor of DNA-Binding Protein-1 in Hemopoietic Progenitor Cells and Promotes Myeloid Cell Development

Hemopoiesis depends on the expression and regulation of transcription factors, which control the maturation of specific cell lineages. We found that the helix-loop-helix transcription factor inhibitor of DNA-binding protein 1 (Id1) is not expressed in hemopoietic stem cells (HSC), but is increased in more committed myeloid progenitors. Id1 levels decrease during neutrophil differentiation, but remain high in differentiated macrophages. Id1 is expressed at low levels or is absent in developing lymphoid or erythroid cells. Id1 expression can be induced by IL-3 in HSC during myeloid differentiation, but not by growth factors that promote erythroid and B cell development. HSC were transduced with retroviral vectors that express Id1 and were transplanted in vivo to evaluate their developmental potential. Overexpression of Id1 in HSC promotes myeloid but impairs B and erythroid cell development. Enforced expression of Id1 in committed myeloid progenitor cells inhibits granulocyte but not macrophage differentiation. Therefore, Id1 may be part of the mechanism regulating myeloid vs lymphoid/erythroid cell fates, and macrophage vs neutrophil maturation.

DNA hypomethylation caused by Lsh deletion promotes erythroleukemia development

Hematopoietic malignancies are frequently associated with DNA hypomethylation but the molecular mechanisms involved in tumor formation remain poorly understood. Here we report that mice lacking Lsh develop leukemia associated with DNA hypomethylation and oncogene activation. Lsh is a member of the SNF2 chromatin remodeling family and is required for de novo methylation of genomic DNA. Mice that received Lsh deficient hematopoietic progenitors showed severe impairment of hematopoiesis, suggesting that Lsh is necessary for normal hematopoiesis. A subset of mice developed erythroleukemia, a tumor that does not spontaneously occur in mice. Tumor tissues were CpG hypomethylated and showed a modest elevation of the transcription factor PU.1, an oncogene that is crucial for Friend virus induced erythroleukemia. Analysis of Lsh-/-hematopoietic progenitors revealed widespread DNA hypomethylation at repetitive sequences and hypomethylation at specific retroviral elements within the PU.1 gene. Wild type cells showed Lsh and Dnmt3b binding at the retroviral elements located within the PU.1 gene. On the other hand, Lsh deficient cells had no detectable Dnmt3b association suggesting that Lsh is necessary for recruitment of Dnmt3b to its target. Furthermore, Lsh-/- hematopoietic precursors showed impaired suppression of retroviral elements in the PU.1 gene, an increase of PU.1 transcripts and protein levels. Thus DNA hypomethylation caused by Lsh depletion is linked to transcriptional upregulation of retroviral elements and oncogenes such as PU.1 which in turn may promote the development of erythroleukemia in mice.

Cell-nonautonomous function of Id1 in the hematopoietic progenitor cell niche

Development of hematopoietic stem cells (HSCs) and their immediate progeny is maintained by the interaction with cells in the microenvironment. We found that hematopoiesis was dysregulated in Id1(-/-) mice. Although the frequency of HSCs in Id1(-/-) bone marrow was increased, their total numbers remained unchanged as the result of decreased bone marrow cellularity. In addition, the ability of Id1(-/-) HSCs to self-renew was normal, suggesting Id1 does not affect HSC function. Id1(-/-) progenitors showed increased cycling in vivo but not in vitro, suggesting cell nonautonomous mechanisms for the increased cycling. Id1(-/-) HSCs developed normally when transplanted into Id1(+/+) mice, whereas the development of Id1(+/+) HSCs was impaired in Id1(-/-) recipients undergoing transplantation and reproduced the hematologic features of Id1(-/-) mice, indicating that the Id1(-/-) microenvironment cannot support normal hematopoietic development. Id1(-/-) stromal cells showed altered production of cytokines in vitro, and cytokine levels were deregulated in vivo, which could account for the Id1(-/-) hematopoietic phenotypes. Thus, Id1 is required for regulating the hematopoietic progenitor cell niche but is dispensable for maintaining HSCs.

Hematopoiesis and Thymic Apoptosis Are Not Affected by the Loss of Cdk2

ABSTRACT Cell cycle regulation is essential for proper homeostasis of hematopoietic cells. Cdk2 is a major regulator of S phase entry, is activated by mitogenic cytokines, and has been suggested to be involved in antigen-induced apoptosis of T lymphocytes. The role of Cdk2 in hematopoietic cells and apoptosis in vivo has not yet been addressed. To determine whether Cdk2 plays a role in these cells, we performed multiple analyses of bone marrow cells, thymocytes, and splenocytes from Cdk2 knockout mice. We found that Cdk2 is not required in vivo to induce apoptosis in lymphocytes, a result that differs from previous pharmacological in vitro studies. Furthermore, thymocyte maturation was not affected by the lack of Cdk2. We then analyzed the hematopoietic stem cell compartment and found similar proportions of stem cells and progenitors in Cdk2−/− and wild-type animals. Knockouts of Cdk2 inhibitors (p21, p27) affect stem cell renewal, but a competitive graft experiment indicated that renewal and multilineage differentiation are normal in the absence of Cdk2. Finally, we stimulated T lymphocytes or macrophages to induce proliferation and observed normal reactivation of Cdk2−/− quiescent cells. Our results indicate that Cdk2 is not required for proliferation and differentiation of hematopoietic cells in vivo, although in vitro analyses consider Cdk2 to be a major player in proliferation and apoptosis in these cells and a potential target for therapy.

Inhibition of Growth by p205: A Nuclear Protein and Putative Tumor Suppressor Expressed during Myeloid Cell Differentiation

p205 belongs to a family of interferon‐inducible proteins called the IFI‐200 family, which have been implicated in the regulation of cell growth and differentiation. While p205 is induced in hematopoietic stem cells during myeloid cell differentiation, its function is not known. Therefore, the aim of this study was to determine the role of p205 in regulating proliferation in hematopoietic progenitor cells and in nonhematopoietic cell lines. We found that p205 localizes to the nucleus in hematopoietic and nonhematopoietic cell lines. Transient expression of p205 in murine IL‐3–dependent BaF3 and 32D‐C123 progenitor cell lines inhibited IL‐3–induced growth and proliferation. The closely related IFI‐200 family members, p204 and p202, similarly inhibited IL‐3–dependent progenitor cell proliferation. p205 also inhibited the proliferation and growth of normal hematopoietic progenitor cells. In nonhematopoietic cell lines, p205 and p204 expression inhibited NIH3T3 cell colony formation in vitro, and microinjection of p205 expression vectors into NIH3T3 fibroblasts inhibited serum‐induced proliferation. We have determined the functional domains of p205 necessary for activity, which were identified as the N‐terminal domain in apoptosis and interferon response (DAPIN)/PYRIN domain, and the C‐terminal retinoblastoma protein (Rb)‐binding motif. In addition, we have demonstrated that a putative ataxia telangiectasia, mutated (ATM) kinase phosphorylation site specifically regulates the activity of p205. Taken together, these data suggest that p205 is a potent cell growth regulator whose activity is mediated by its protein‐binding domains. We propose that during myelomonocytic cell differentiation, induction of p205 expression contributes to cell growth arrest, thus allowing progenitor cells to differentiate.

The Effect of C-raf Antisense Oligonucleotides on Growth Factor-Induced Proliferation of Hematopoietic Cells

While it is well established that Raf-1 kinase is activated by phosphorylation in growth factor-dependent hematopoietic cell lines stimulated with a variety of hematopoietic growth factors, little is known about the biological effects of Raf-1 activation on normal hematopoietic cells. Therefore, we examined the requirement for Raf-1 in growth factor-regulated proliferation and differentiation of hematopoietic cells using c-faf antisense oligonucleotide. Raf-1 required for the proliferation of growth factor dependent cell lines stimulated by IL-2, IL-3, G-CSF, GM-CSF and EPO that bind to the hematopoietin class of receptors. Raf-1 is also required for the proliferation of cell lines stimulated by growth factors that use the tyrosine kinase containing receptor class, including SLF and CSF-1. In addition, Raf-1 is also required for IL-6, LIF- and OSM-induced proliferation whose receptors share the gp 130 subunit. In contrast to previous results which demonstrated that IL-4 could not activate Raf-1 kinase, c-raf antisense oligonucleotides also inhibited IL-4-induced proliferation of T cell and myeloid cell lines. Using normal hematopoietic cells, c-raf antisense oligonucleotides completely suppressed the colony formation of murine hematopoietic progenitors in response to single growth factors, such as IL-3, CSF-1 or GM-CSF. Further, c-raf antisense oligonucleotides inhibited the growth of murine progenitors stimulated with synergistic combinations of growth factors (required for primitive progenitor growth) including two, three and four factor combinations. In comparison to murine hematopoietic cells, c-raf antisense oligonucleotides also inhibited both IL-3 and GM-CSF-induced colony formation of CD 34+ purified human progenitors. In addition, Raf-1 is required for the synergistic response of CD 34+ human bone marrow progenitors to multiple cytokines; however, this effect was only observed when additional antisense oligonucleotides were added to the cultures at day 7 of a 14 day assay. Finally, Raf-1 is required for the synergistic response of human Mo-7e cells and of normal human fetal liver cells to five factor combinations. Thus, Raf-1 is required to transduce growth factor-induced proliferative signals in factor-dependent progenitor cells lines for all known classes of hematopoietic growth factor receptors, and is required for the growth of normal murine and human bone marrow-derived progenitors.

Detection of growth factor receptor RNA in individual hematopoietic cells by in situ RT-PCR; comparison with RT-PCR☆

The ability to detect changes in RNA expression in single cells would greatly enhance understanding of the molecular basis of biological responses to positive and negative growth regulators. To this end, we compared expression of RNA encoding the receptors for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, leukemia inhibitory factor (LIF) and stem cell factor (SCF) in populations of primitive hematopoietic progenitors (lineage marker negative, Lin(-), and Lin(-) c-Kit(+)) by RT-PCR and in situ RT-PCR. Both Lin(-) and Lin(-) c-Kit(+) progenitors expressed all receptors by RT-PCR. However, RT-PCR could not distinguish between the possibility that all cells expressed growth factor receptor RNA, or the possibility that only a proportion of cells expressed RNA. Therefore, we used in situ RT-PCR to examine growth factor receptor mRNA expression in individual cells. In contrast to RT-PCR, we observed that only 40-80% of Lin(-) cells and 75-100% of Lin(-) c-Kit(+) cells were positive for expression of the growth factor receptor subunits, demonstrating that not all cells were receptor positive. We found that in situ RT-PCR could also be used to measure induction or repression of receptor RNA expression in these cell populations. Specifically, the percentage of cells expressing IL-6alpha receptor RNA decreased from 88% positive in freshly harvested cells to 9% in Lin(-) c-Kit(+) cells cultured in IL-3 for 18 h. Thus, in situ RT-PCR can be used to detect and quantify the number of individual cells that express growth factor receptor mRNA, and may also be useful to measure changes in expression of other endogenous genes or genes introduced by transfection and gene therapy vectors.