A Potential Role for CCAAT Enhancement Binding Protein-Epsilon (C/EBP-e) in the Regulation of Granulocytic Differentiation by G-CSF

Understanding the molecular pathways that lead to normal hematopoetic differentiation and cellular function is a topic of great medical significance, potentially affecting treatment for patients with leukemia and other myeloproliferative disorders. Granulocyte Colony Stimulating Factor (G-CSF) is of central importance in normal granulopoesis, leading to increased proliferation, faster maturation, and prolonged lifespan of committed granulocytic precursors. While the effects of G-CSF and its related receptor are well characterized, the molecular mechanisms behind its actions are little known.

Differentiation at various stages in myeloid cell lines has been associated with the up-regulation of several transcription factors, including several members of the CCAAT Enhancement Binding Protein (C/EBP) family. One member, C/EBP-e, is expressed exclusively in cells of hematopoetic origin and has been implicated in neutrophil development. In an effort to better characterize G-CSF induced neutrophil development, Dr. Link's laboratory has created several murine lines expressing genetic mutations in the G-CSF Receptor. The purpose of this study was to further this characterization by assessing the extent to which C/EBP-e expression is regulated by G-CSF.

To study myeloid differentiation, we developed a novel liquid culture system that would facilitate RNA isolation from uniformly differentiating primary bone marrow cells. Mutant G-CSF Receptor mice were injected with 5-FU and sacrificed after 2 days. Primary bone marrow cells were sterilely harvested from both femurs, and progenitors enriched using a Histopaque 1077 density gradient. These precursors were then incubated for 3 days in serum-containing media with IL-3, kit ligand, Flt-3, and thrombopoetin to prime the cells for proliferation. The cells were washed and transferred to myelocult media with kit ligand and G-CSF. As compared to a similar culture system without the cytokine pre-incubation, the new culture system better supported cell survival, proliferation, and synchronous differentiation. From a relatively homogenous population of undifferentiated precursors (>90% precursors), by day 7 the system supported a much increased population of mature pmns (>90% neutrophils). Such a system will greatly facilitate the study of C/EBP-e in primary myeloid cells.

An assay sensitive enough to detect levels of a rare transcript ion factor was needed to study G-CSF-dependent regulation of C/EBP-e. Real Time RT PCR was chosen for its sensitivity, versatility, and quantitative ability. RNA was isolated from culture system cells using the Qiagen RNeasyTM kit. Total yield was improved by precipitating the eluted RNA in ethanol, sodium acetate, and glycogen; the RNA pellet was resuspended in small volumes of RNAse-free dH₂0. DNAse treatment was employed to remove residual genomic contamination, and the RNA reverse transcribed using a standard AMV RT protocol. To avoid the difficulties of absolute quantification, Real Time PCR was used to quantify the relative expression of C/EBP-e in relation to GAPDH, a gene uniformly expressed in all cells.

Due to variability in the RNA isolation technique and RT-PCR protocol, real time analysis of in vitro tissue culture samples has been limited. Using normal RT-PCR, I have positively identified C/EBP-e transcripts from primary bone marrow cells. Real Time analysis has been hindered by residual DNA contamination and non-specific florescent labeling of C/EBP-e primer dimers. GAPDH has also proved difficult to quantify, owing to a GAPDH pseudogene that competitively inhibits cDNA amplification and detection. Switching to a C/EBP-e specific florescent probe and improving RNA isolation promises to improve real time analysis, and allow the experiment to progress to the next stage.