Analysis of myeloid cell development at the RNA and protein level

Presentation

Sherman M. Weissman
Sterling Professor of Genetics and Medicine
Yale University School of Medicine
295 Congress Avenue, 336 BCMM
New Haven, CT 06510
telephone: (203) 737-2282
fax: (203) 737-2286
email: barbara.gramenos@yale.edu
prestype: Platform
presenter: Sherman M . Weissman

Zheng Lian¹, Shigeru Yamaga¹, Yasuhiro Tsukahara¹, Yuval Kluger², Dov Greenbaum¹, Mark Gerstein², Nancy Berliner³, Peter Newburger⁴, Sherman M. Weissman^1
1Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, ²Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511, ³Department of Genetics and Medicine, New Haven, CT 06510, ⁴Department of Pediatrics, University of Massachusetts Medical School, Worcester, MA 01605

Polymorphonuclear cells are a highly differentiated post-mitotic cell population that differentiates through a series of steps from a common hematopoietic precursor. Mature PMN can be readily isolated from the blood of normal individuals in amounts sufficient for biochemical analysis. Model systems for inducing myeloid differentiation or polymorphonuclear cell development in vitro also exist. One favorable system is the MPRO murine promyelocytic cell line. In this line development is blocked by a dominant negative retinoic acid receptor. Upon addition of all trans retinoic acid, the cells differentiate over about four days into essentially mature neutrophils.

To analyze the processes of neutrophil differentiation and activation we have studied RNA expression in normal resting and activated neutrophils and in developing MPRO cells, using both gel display methods and oligonucleotide chip analysis. In addition, we have compared RNA patterns in less differentiated hematopoietic cells including hematopoietic stem cells and cells differentiated into other hematopoietic lineages. To complement this approach we have analyzed protein expression in the myeloid cells, using two-dimensional gel electrophoresis with a variety of pH range isoelectric focusing dimensions, and protein identification by MALDI-TOF mass spectroscopy. Technically, there are well known limitations in both the detection and quantitation of proteins by the 2D methods, and there is only a moderate correlation between mRNA levels or the changes in mRNA levels and the corresponding protein levels.

Analysis of mRNA levels show interesting changes in the expression of genes for certain transcription factors, some but not all of which had been previously implicated in myeloid development. To further investigate these we are attempting 2D gel electrophoresis of transcription factor enriched protein preparations from myeloid cells and also exploring introduction of factors into developing cells by the use of “Trojan Horse” peptides.