Pilot Project Grant Program Awardees

2008-2009 Pilot and Feasibility Grant Winners

• Natalia Ivanova, Ph.D.
  Assistant Professor of Genetics
  Identification of Molecular Regulatory Components in Hematopoietic Stem Cells
  
• Gary Kupfer, M.D.
  Associate Professor of Pediatrics and Pathology
  Section Chief of Pediatrics
  Function of Codanin, the protein defective in congenitial dyserythropoietic anemia
• Philip Askenase, M.D.
  Professor of Internal Medicine and Pathology
  Section Chief of Clinical Immunology and Allergy
  Role of RNA in a T-Cell Suppressor factor Relevant to Hematopoietic Cancers

Previous Grant Awardees:

• 2007-2008
• 2006-2007
• 2005-2006
  

Natalia Ivanova, Ph.D.
Assistant Professor of Genetics

Identification of Molecular Regulatory Components in Hematopoietic Stem Cells
Very little is known about the molecular mechanisms that regulate self-renewal and differentiation of hematopoietic stem cells (HSC). Given that exhaustive functional genetic screens in HSC are not feasible, nor are cell lines available that accurately recapitulate HSC behavior, I have chosen global gene expression analysis in primary cells followed by functional characterization of candidate regulatory gene products as an alternative approach towards the comprehensive identification of HSC regulatory mechanisms.

I have previously performed transcriptional profiling of fetal and adult HSC in order to identify genes that are specifically expressed in HSC but not in more differentiated compartments of the hematopoietic hierarchy. These studies have yielded a comprehensive molecular profile of HSC and defined sets of candidate genes that are likely to function as key regulators of self-renewal and differentiation.

The goal of this proposal is to conduct loss-of-function analyses of selected candidate genes in order to identify gene-products that are required for self-renewal of HSC. Lentiviral short hairpin RNA vectors will be used to transducer purified mouse HSC. Consequences of gene inactivation will be analyzed in vivo and in vitro, employing a broad range of assay systems. These studies are expected to define molecular components and pathways that control cell-fate decisions in HSC and will facilitate further analyses HSC regulation at multiple molecular, biochemical and physiological levels.

Gary Kupfer, M.D.
Associate Professor of Pediatrics and Pathology
Section Chief of Pediatrics

Function of Codanin, the protein defective in congenitial dyserythropoietic anemia
Congenital dyserythropoietic anemia (CDA) is a genetic disorder marked by specific red cell aplasia and multinucleated red cell precursors. Codanin, the protein defective in CDA I, has no known function but is found in heterochromatin and thought to be involved in chromosome segregation or organization. We have shown that codanin is phosphorylated at mitosis and have mapped the phosphorylation sites. In this proposal, we will further investigate the possible role for codanin in erythropoiesis.

In Aim 1, we will analyze the function of codanin at mitosis by targeting the 3’ UTR of endogenous codanin using siRNA while concomitantly expressing phosphomutant forms of codanin in the erythroleukemia cell line K562. The outcome of expression of mutant codanin will be measured by proliferation, morphology, and globin production.

In Aim 2 we will purify protein complexes of codanin that are erythroid specific. Codanin-containing protein complexes from HeLa and K562 (erythroid) cells will be purified and compared by silver stain, with unique erythroid-specific protein bands sequences by mass spec in order to isolate codanin binding protein that will lend understanding for codanin’s function.

Study of rare diseases of bone marrow failure such as CDA promises to uncover more generalizable information about hematopoiesis.

Philip Askenase, M.D.
Professor of Internal Medicine and Pathology
Section Chief of Clinical Immunology and Allergy

Role of RNA in a T-Cell Suppressor factor Relevant to Hematopoietic Cancers
Effector immunity against tumors sometimes is directed at altered self-antigens. It is often blocked by regulatory cells. Such suppressor T cells (Ts) inhibit effector responses to hematopoietic cancers, and also are relevant to bone marrow and stem cell transplantation. We work in a model murine system of immunity to hapten altered self. Here, effector cells are inhibit by specific Ts. The Ts are generation in high antigen dose tolerized mice, as might occur in cancer.

We previously described the biological activity of a macromolecular suppressor factor derived from the culture supernatant of Ts, from the high antigen dose tolerized mice. It was called a “T cell suppressor factor” or TsF, and presumed to be a protein. The Ts and TsF inhibited the in vivo-acting effector T cells. In parallel studies in vitro, we also showed that a component of the factor suppresses the responsiveness of T cells to the cytokine IL-2. We now propose that this subfactor may not be a protein, but an RNA.

Our present specific aim is to cDNA clone and identify the RNA portion of the TsF. Determine the biological properties of the cloned TsF RNA, towards eventual therapy of malignancies.