Progress 02/01/00 to 12/31/04
Outputs
The development of mature blood cells results from the interaction of progenitor cells with signals from their environment. Although many of the signals that regulate the differentiation of progenitor cells are known, the mechanisms that limit hematopoiesis to times of need are not well understood. This project has focused on the analysis of the murine Polycythemia (Pcm) mutation. Pcm is a dominant mutation that causes erythrocytosis and leukocytosis. Analysis of hematopoiesis in these mice showed that Pcm mutant progenitors exhibited hypersensitivity to growth factors and cytokines. We interpreted these results to mean that the defect in Pcm mice is in the ability of cells to sense threshold concentrations of growth factors. Normal cells will not respond unless a certain amount of growth factor signal is present. Pcm cells respond to low concentrations of growth factors that normal cells ignore. In order to fully characterize the Pcm mutant we generated a genetic
linkage map of the locus. Our mapping localized the gene to a 250 kb interval on the proximal portion of chromosome 1. We are currently investigating candidate genes in that region. In summary, we have demonstrated that Pcm mice have a defect in the ability of hematopoietic progenitor cells to sense threshold concentrations of growth factors and we have localized the gene to a small interval on mouse chromosome 1.
Impacts
This research will identify the Polycythemia locus and characterize the mechanism by which it regulates cytokine signaling. The impact of this research is two fold. First, this research will provide basic information concerning the regulation of cell signaling in hematopoietic cells. Second, the phenotype of these mice is very similar to human myeloproliferative disease. Therefore the identification of the Polycythemia gene could identify a gene mutated in this human disease or provide information concerning the etiology these disorders.
Publications
- No publications reported this period
|
Progress 01/01/03 to 12/31/03
Outputs
The murine Polycythemia mutation causes a phenotype characterized by erythrocytosis and leukocytosis. Previous work in our laboratory has demonstrated that the mutation alters the response of progenitor cells to certain cytokines and growth factors. Specifically this defect results in hypersensitivity and an inability of cells to sense threshold concentrations of cytokines. Over the past year, the major focus of our work has been the identification of the Polycythemia gene. We have refined the genetic linkage map of the Polycythemia locus and have identified a 1.93 Mb interval that contains the genes. We are now testing a series of new genetic markers for polymorphisms to be used in the further localization of the locus. Based on the mouse genome sequence, this interval contains 24 known or predicted genes. We are now testing these genes for expression in affected cells to identify candidates for further study.
Impacts
This research will identify the Polycythemia locus and characterize the mechanism by which it regulates cytokine signaling. The impact of this research is two fold. First, this research will provide basic information concerning the regulation of cell signaling in hematopoietic cells. Second, the phenotype of these mice is very similar to human myeloproliferative disease. Therefore the identification of the Polycythemia gene could identify a gene mutated in this human disease or provide information concerning the etiology these disorders.
Publications
- No publications reported this period
|
Progress 01/01/02 to 12/31/02
Outputs
Mutation of the Polycythemia, Pcm, gene in mice causes myeloproliferative disease, which is characterized by high hematocrits and leukocytosis. This phenotype is very similar to the human myeloproliferative disease, Polycythemia vera, P. vera. Because P. vera patients exhibit increased blood viscosity and increased numbers of platelets in circulation, they are high risk for vascular complications. Initial analysis of the Pcm mutant mice did not reveal any vascular defects, such as hemorrhage or blood clots at 6-8 weeks of age, the time when the Pcm phenotype is first manifested. Recent analysis of Pcm mice one year or older showed that these mice exhibited signs of vascular complications including ischemic brain injury and hemorrhages in the lungs. In addition the older mice showed signs of splenomegely marked by lymphoid and myeloid cell proliferation. These features are often observed in P. vera patients, which is consistent with Pcm being a model system for
studying human P. vera. Our work has focused on the cloning of the Pcm gene. To that end we have mapped the Pcm gene to 20 cM interval on mouse chromosome 1. The recent publication of the mouse genome sequence has allowed us to determine that this interval is encoded by 30Mb of DNA. We are presently using single nucleotide polymorphism, SNP, markers derived form the mouse genome project to finish the high density linkage map. We are also using the mouse genome sequence to look for candidate genes that map to this interval. We have determined that 156 genes are encoded in this 30Mb interval and have begun screening for the expression of these genes in cell affected by the Pcm mutation.
Impacts
Impact: The mutations that cause human myeloproliferative diseases have not been identified. The analysis of the Pcm mutation in mice will provide insight into the mechanisms that lead to myeloproliferative disease and could in the future be used to test and develop effective therapies for these diseases.
Publications
- No publications reported this period
|
Progress 01/01/01 to 12/31/01
Outputs
Analysis of mice with mutations in the Polycythemia (Pcm) locus exhibit a chronic overproduction of mature myeloid cells. This phenotype in many ways resembles the symptoms of the human myeloproliferative disease Polycythemia vera, P vera, which makes Pcm mice an excellent model system for studying P. vera. Previous work from my lab has demonstrated that Pcm bone marrow cells are hypersensitive to growth factors and cytokines. We have extended this work by analyzing the biochemical events that mediate signaling downstream of the Interleukin 3 (IL-3) receptor in primary mast cell cultures. We have demonstrated that Pcm mutant mice have a defect in the mechanism that determines the threshold for activation of signaling pathways. Further analysis has detected alterations in receptor phosphorylation following cytokine stimulation, which suggests that the Pcm mutation causes a defect in a receptor proximal signaling event. We are presently pursuing the characterization of
receptor activation and have identified a candidate signaling pathway that may play a role in this process. We are also employing a position cloning approach to identify the Pcm gene. Previously, we mapped the Pcm locus to the proximal portion of mouse chromosome 1. We are generating a high-resolution genetic linkage map of this region of chromosome 1. To that end we have collected approximately 700 progeny and are analyzing the segregation of markers in this data set. In addition, we are taking advantage of the recent advances in the human genome project and the mouse genome sequencing project to identify candidate genes that map to the genetic interval identified in our linkage mapping. We are presently analyzing two candidate genes, both of which appear to be involved in regulating receptor proximal signaling events in hematopoietic cells. In conclusion, our work has identified the initial biochemical defect in cytokine receptor signaling caused by the Pcm mutation and has further
localized the gene on mouse chromosome 1.
Impacts
This research will provide insight into the cause of the human myeloproliferative disease Polycythemia vera. P. vera affects approximately 1 in 100,000 people over the age of 60 each year. The characterization of the Polycythemia mutation in mice will also provide insight in to the molecular mechanisms that regulate hematopoiesis, which in may be later exploited to design novel therapies for human hematopoietic disorders like anemia and leukemia.
Publications
- Craici, A.C. 2001. Mutation of the murine Polycythemia (Pcm) locus causes alterations in cytokine signaling resulting in myeloproliferative disease. M.S. Thesis. The Pennsylvania State University, University Park, PA. 85 pp.
|
Progress 01/01/00 to 12/31/00
Outputs
Mutation of the murine Polycythemia (Pcm) gene results in a phenotype characterized by the chronic overproduction of erythrocytes, neutrophils, granulocytes and B lymphocytes. In many ways this phenotype is similar to the pathology exhibited by patients suffering from the human myeloproliferative disease, Polycythemia vera (P. vera). Because of these similarities, we propose that Pcm is a model system for studying P. vera. To that end we are characterizing the Pcm gene in two ways. First, we have analyzed the response of Pcm bone marrow cells to different cytokines in in vitro colony assays. The Pcm bone marrow cells exhibited a hypersensitive response to erythropoietin (Epo), Stem cell Factor (SCF) and Interleukin-3 (IL-3) in erythroid colony assays and to IL-3 in myeloid colony assays. Furthermore, we observed that cultures of mast cells derived from Pcm bone marrow proliferated at greater rate in response to IL-3 stimulation than control mast cells. Taken together
these results suggested that the Pcm mutation affects a common signaling intermediate that regulates how the threshold is set for receptor dependent activation of intracellular signaling pathways. We have pursued this possibility by analyzing the activation of signaling pathways downstream of the Epo receptor (EpoR) and the IL-3 Receptor (IL-3R). Our initial results demonstrate that the activation of the MAP Kinase signaling pathway by either the IL-3R or the EpoR is abnormal in Pcm cells. In both cases the activation of MAP Kinase was hypersensitive in the PCM cells, which suggests that the mechanism that regulates the threshold for activating the MAP kinase pathway is defective in Pcm mutant mice. We are presently continuing our analysis of the signaling in Pcm cells by analyzing the effects of the Pcm mutation on other signaling pathways that are activated by the EpoR and IL-3R and by determining the exact molecular defect in the MAP Kinase pathway. The second aspect of this
project is the identification of the Pcm gene. We are employing a positional cloning approach. Previously, we mapped the Pcm locus to the proximal portion of mouse chromosome 1. We are generating a high-resolution genetic linkage map of this region of chromosome 1. At the present time we have mapped the Pcm locus to a 2.2 cM interval. In addition to our linkage mapping, we are using two PCR based techniques to isolate new polymorphic genetic markers tightly linked to Pcm. The first technique is representational difference analysis (RDA), which is a subtractive PCR technique designed to identify restriction fragment length polymorphisms (RFLPs) that are present only in the Pcm mutant DNA. The second technique is Amplified Fragment length Polymorphisms (AFLP). This technique allows you to analyze 100's of RFLPs simultaneously in order to identify markers that are tightly linked to Pcm. In summary, we have made progress in identifying the biochemical defect associated with the Pcm
mutation and in mapping the Pcm locus to a small interval on chromosome 1.
Impacts
This research will provide insight into the cause of the human myeloproliferative disease Polycythemia vera. P. vera affects approximately 1 in 100,000 people over the age of 60 each year. The characterization of the Polycythemia mutation in mice will also provide insight in to the molecular mechanisms that regulate hematopoiesis, which in may be later exploited to design novel therapies for human hematopoietic disorders like anemia and leukemia.
Publications
- No publications reported this period
|
|