Bing Lim - US grants

The central goal of this proposal is to investigate the cellular and molecular events during the early stages of primitive stem cell commitment to the hematopoietic lineages. We have studied murine totipotent embryonal stem (ES) cells in methyl-cellulose culture and observed the emergence of erythroid cells and macrophages in clonally expanding ES colonies. We have also isolated two murine cDNA clones from the library of a murine multipotential hematopoietic cell line (FDCP-Mix) which cross hybridize with two human hematopoietic specific genes, vav and HS-1, whose functions are unknown. Our specific aims are to determine when vav and HS-1 are expressed in the early stages of hematopoietic stem cell differentiation and how perturbation of their expression affect hematopoiesis. Therefore, we propose: 1) to define the optimal growth conditions for ES cell derived hematopoietic cells using various cytokines including the recently identified stem cell factor, SCF-1; 2) to characterize and sequence the murine cDNA clones we have isolated, determine their homology to human clones and evaluate by RNA analysis the specificity-of their expression in murine hematopoietic cells; 3) to generate polyclonal antibodies to vav and HS-1 using fusion proteins and use the antibodies to further characterize their protein by SDS polyacrylamide gel analysis of hematopoietic cells and employ immunocytochemical techniques to localize and establish the kinetics of protein expression in ES cell derived hematopoietic cells and 4) to use ,anti-sense RNA vectors and anti-sense oligonucleotides to block the expression of these proteins in ES cells in order to understand their function in hematopoietic differentiation. These experiments should 1) ascertain the usefulness of such an in vitro model system for future studies in hematopoiesis, 2) shed light on the function and significance of hematopoietic specific genes, 3) contribute to the investigations of pathological changes in hematopoietic cells and 4) be relevant to our understanding of the ,fundamental process of cellular differentiation.

DESCRIPTION (provided by applicant): This is a proposal to continue the investigation of the role of RhoGTPases and their regulatory proteins in hematopoiesis, hematopoietic cell function and hematopoietic malignancies. The RhoGTPases form a large family of proteins that mediate an extensive array of fundamental cellular function including cell division, cell shape, intra-cellular trafficking, cytoskeletal organization, gene transcription, apoptosis, cellular transformation and metastasis. Recently a new member of the RhoGTPase family have been discovered named RhoH, that is expressed specifically only in hematopoietic cells. RhoH was first identified as a fusion protein with bcl6 in non-random translocations involving the RhoH gene at chromosome 4p13 in some cases of lyrnphoma and myeloma. Even more significant is the recent finding that a high frequency of RhoH mutations exist in the Diffuse Large B-Cell Lymphomas (DLCL). This is the first example of the involvement of a RhoGTPase in hematological malignancies, and the clinical importance of the Rho family of proteins. The pathological role of these mutations remain to be clarified. Important clues derived from recent surprising finding showed that RhoH functions as a potent inhibitor of other RhoGTPases. Consistent with RhoH being an "inhibitory" protein is the recent discovery that a cellular basal level of RhoH is crucial for maintaining T cells in the non-adherant inactive state. RhoH represents a new paradigm for the functional modulation of the RhoGTPases and other related small G-proteins. An important question is what does RhoH do, why does it function differently compared to other RhoGTPases and what is RhoH role in lymphomagenesis? We shall focus our effort to answer the following questions:Aim1) What is the cellular function of RhoH? ; Aim2) What is the mechanism of RhoH inhibitory function?; Aim3) What are the consequences of RhoH mutations in lymphoma? Knowledge from this project will bring new insigths about an important class of proteins that are increasingly implicated in carcinogenesis and cancer progression. The study will also allow us to determine if RhoH is a meaningful variable for prognostication in sub-groups of DLCL and if it can be a useful target for anti-cancer drug development.

DESCRIPTION: (Investigator's abstract) Proteolysis is a fundamental mechanism in all living organisms that provides a sensitive way for titrating levels of active proteins essential for maintaining homeostasis and diverse critical cellular functions. The molecular components of the proteolytic machinery are incompletely understood. The central goal of this proposal is to achieve a better understanding of the intricacies of this machinery through the investigation of a novel lysosomal membrane protein named LAPTm5. In murine animals, LAPTm5 is specifically expressed in hematopoietic cells, and in human, it is preferentially expressed at high levels in hematopoietic cells. The protein was found to bind to ubiquinated proteins. There is accumulating evidence that ubiquination of proteins is an important signal for proteolysis in lysosomes. LAPTm5 has also been found to alter the level of IkB in lysosomes. IkBs are the key regulators of the activation of NF-kB, a major transcriptional factor responsible for regulating an extensive list of important genes including those involved in immune and inflammatory response, cell adhesion, stress response proliferation and differentiation. The process by which IkB is degraded and regulated is therefore an area of intensive research. The immediate goal of this proposal is to investigate the function of LAPTm5 from several approaches indicated by these findings. Aim 1 will determine if LAPTm5 can affect the NF-kB system by analyzing if it regulates IkB degradation in lysosomes, whether it can activate NF-kB and whether it has an influence on the responsiveness of the NF-kB system. Aim 2 will examine the role of LAPTm5 in hematopoietic cell development by using the model of in vitro differentiation of murine embryonal stem cells into hematopoietic cells. The effect of a constitutive expression of the gene and a disruption of the gene by homologous recombination will be analyzed. Aim 3 will use protein chemistry techniques, including 2-D gel analysis to determine the identity of the ubiquinated proteins that bind with LAPTm5. A direct in situ binding assay with purified lysosomes will also be used to identify binding proteins. New findings about this unusual gene will contribute to our knowledge about the molecular basis of the functions of proteolytic organelles and their relevance to hematopoietic cells. The proposal also may lead to new insight about the regulation of a key transcriptional factor that plays a significant role in many human diseases.

DESCRIPTION (provided by applicant): The central goal of this proposal is to exploit the use of a new mutant murine strain to advance the understanding of autoimmune disorders. A new line of mice has been derived in which animals develop a severe generalized lymphadenopathy together with autoimmune glomerulonephritis and hyperimmunoglobulinemia. Significantly, the animals produce auto antibodies against double-stranded DNA and Sm antigen, both of which are specific markers for Systemic Lupus Erythematosus (SLE). Immune function studies showed a combination of severe lymphoid dysfunction and developmental defect not seen in other murine autoimmune disease models. The disease is passed with a Mendelian frequency consistent with a recessive mutation of an autosomal gene. Therefore, the disease arose from a spontaneous mutation of a gene which we have named lag (lymphoproliferative autoimmune glomerulonephropathy). Using chromosomal satellite markers to scan the murine genome, preliminary data indicate that a putative locus for the lag gene is the telomeric end of chromosome 2. This is not a region that has been linked before to autoimmune disease. The goal of this proposal is to exploit this remarkable new murine model to learn about autoimmune disease. In Specific Aim 1, we will map the location of the gene and identify the lag gene by combining positional cloning with a candidate gene approach. In Aim 2 we will characterize the disease process for the lag phenotype and identify the cells causing the disease. In Aim 3 we will examine in detail the effect of the lag mutation on T cell development. In Aim 4 we will investigate how the lag mutation affects T cell function. To support these studies, various TCRxlag transgenic animals will be generated to help the study of lymphocyte development, function and signaling. We anticipate that our proposal to study this murine model carefully will contribute a significant amount of new information for understanding the diverse genetic and molecular bases of autoimmune diseases. The identification of new genes and new pathways may uncover new targets for the development of drugs to suppress the immune system in a specific way, instead of globally. The discovery of new disease genes may also be very useful in the management, care and diagnosis of the large number of patients with autoimmune diseases.