Estrogen-regulated Conditional Oncoproteins: Tools to Address Open Questions in Normal Myeloid Cell Function, Normal Myeloid Differentiation, and the Genetic Basis of Differentiation Arrest in Myeloid Leukemia

Abstract

Neutrophils, monocytes and dendritic cells are effectors of innate immunity and essential coactivators in the acquired immune response. Understanding the biochemical basis of their mature cell functions, their differentiation from hematopoietic progenitors, and the mechanisms by which myeloid leukemia oncogenes block their differentiation programs, continue to be areas of active research. Four major problems limit progress in these fields. First, the biochemical analysis of mature cells is limited by the time and cost of purifying neutrophils, monocytes, or dendritic cells from wild-type and genetically modified mouse strains. Second, while immortal myeloid cell lines are used to understand the transcriptional basis of normal terminal differentiation following their treatment with differentiation-promoting agents (e.g. G-CSF, IL-6, RA, TPA), these cells contain stable defects responsible for their immortalization, and the degree to which they model normal differentiation is often incomplete. Third, these same inducible cell lines are used as model systems to determine how myeloid oncoproteins prevent differentiation; however, oncoproteins that block differentiation of marrow progenitors cultured in GM-CSF or IL-3 but permit their differentiation in response to G-CSF or RA, do not score effectively in these assays (e.g. Hoxa9, Mll-Enl). Fourth, there is no reproducible method to derive myeloid progenitor lines that execute predictable terminal differentiation to neutrophils, monocytes, or dendritic cells. Developing this type of system is needed to evaluate how myeloid gene inactivation by knockout technologies alters lineage-specific differentiation and mature cell function.

Conditional myeloid oncoproteins provide a tool to solve these research problems by providing a predictable and inexpensive means of expanding, in culture, GM-CSF– or IL-3-dependent myeloid progenitors from any genotype, and by permitting their synchronous differentiation to neutrophils, monocytes, or dendritic cells under defined culture conditions following inactivation of the conditional oncoprotein. This system of conditionally immortalizing normal bone marrow precursors provides the large numbers of normal cells required for analysis of cell biology and protein biochemistry, and further provides a model system in which to study the genetic mechanisms controlling terminal differentiation and how specific oncoproteins expressed in the cell lines prevent this differentiation program. The ability to derive conditionally-immortalized progenitor lines from knock-out mice provides cell lines for the reconstitution of knockout gene function and subsequent dissection of knockout protein function by mutational analysis. Finally, conditional myeloid cell lines can be established from both ES cells and from d10 fetal liver cells, allowing for the analysis of embryonic lethal mutants on both the maturation and terminal differentiation of mature myeloid cells. In this review, we summarize the importance and limitations of current approaches in myeloid cell research, and how estrogen-regulated conditional oncoproteins help to solve these problems.

• Estrogen-related oncoproteins
• Myeloid function
• Myeloid differentiation
• Myeloid leukemia differentiation
• DC, dendritic cells
• DMSO, dimethylsulfoxide
• ES, embryonic stem cells
• FLT3-ITD, FLT3 internal tandom duplication
• RA, retinoic acid
• TPA, tetradecanoyl phorbol myristal acetate
• MRP8, migration inhibitor related protein 4
• MRP14, migration inhibitor related protein 8
• Ly6G (GR1), leukocyte antigen 6G
• gp91phox, respiratory burst oxidase subunit 91
• Cnlp, cathelin-like protein
• NE, neutrophil elastase
• LF, lactoferrin
• MPO, myeloperoxidase
• NOP52, nucleoporin 52
• SR-A1, macrophage scavenger receptor A1
• MCSF-R, macrophage colony-stimulating factor 1
• NG, neutrophil gelatinase
• NC, neutrophil collagenase
• NOP56, nucleolar protein 56
• G-CSF, granulocyte colony-stimulating factor
• M-CSF, macrophage colony-stimulating factor
• GM-CSF, granulocyte-macrophage colony-stimulating factor
• IL-3, interleukin-3
• SCF, stem cell factor
• STAT, signal transducer and activator of transcription
• mAKRa, aldoketo reductase a
• IRF2, interferon response factor 2
• IRF8, interferon response factor 8, aka IFCSBP
• MHC, major histocompatability complex
• ICAM, integrin cell adhesion molecule
• SIGLEC, sialic acid-recognizing animal lectins of the immunoglobulin superfamily
• SMRT, silencing mediator of retinoic acid and thyroid hormone receptor
• PPAR, peroxisome proliferator-activated receptor
• Gfi-1, Growth factor independent 1