Nuclear architecture is dramatically modified during hematopoietic differentiation, as well as during the onset and progression of leukemias. Project 2 addresses the hypothesis that fidelity of subnuclear targeting of the key hematopoietic regulatory factor AML-1 is abrogated by leukemia-related chromosomal translocations that delete specific intranuclear trafficking signals. We postulate that the misrouting of the gene regulatory factor is a dominant defect that causes deregulation of AML-mediated control of cell growth and differentiation. These investigators have shown that AML1 contains a 31 amino acid nuclear matrix targeting signal (NMTS) that facilitates trans-activation by mediating association with components of nuclear architecture which support transcription. Additionally, the applicant has demonstrated that the 8;21 chromosomal translocation in AML leukemia modifies intranuclear targeting of the AML-1 transcription factor. Thus, the specific aims are to: (i) characterize the subnuclear targeting signal in AML-1-1 by establishing the structural basis for and functional consequences of interactions with the nuclear matrix; (ii) determine the saturability and dynamics of intranuclear trafficking of AML-1 in hematopoietic cells in functional nuclear domains; (iii) assess the spatio- functional distribution of AM-1 and leukemia related AML-1 fusion proteins during cell growth and myeloid differentiation; (iv) examine the physiological consequences of subnuclear targeting for hematopoiesis by genetically ablating the NMTS in the murine AML-1 locus; and (v) define key protein/protein interactions which regulate intranuclear trafficking in hematopoietic cells by cloning the nuclear docking protein of AML-1. The results of the proposed studies will provide an in-depth understanding of the interrelationships between subnuclear targeting of gene regulatory factors, chromosomal translocations involving trafficking signals and modifications in gene expression characteristic of leukemias. GRANT-P01CA82834-01A2-0003 The long term objective of this work is to understand how centrosomes contribute to tumorigenesis. Centrosomes are poorly understood organelles required for organization of mitotic spindles and accurate segregation of chromosomes during cell division. Thus, centrosomes are critical players in the redistribution and reorganization of the genome as it is assembled into nascent nuclei following mitosis. There is no other single cellular event that has a greater impact on the quantity, composition and organization of chromatin within the nucleus. We recently made the striking observation that malignant tumors had increased levels of the centrosome protein pericentrin, abnormal centrosomes, aberrant mitotic spindles and missegregated chromosomes. Moreover, artificial elevation of pericentrin in normal cells induced nearly identical features including aneuploidy, a condition linked to tumor malignancy, metastasis and fatality. Pericentrin over-expression also appeared to abrogate the mitotic checkpoint that normally induces mitotic arrest in the presence of unattached chromosomes. Over- expressed pericentrin bound and mislocalized cytoplasmic dynein, a molecular motor known to function in spindle organization and possibly checkpoint control. Based on these observations, we propose a model in which centrosome defects contribute to tumorigenesis by causing improper segregation of the genome and creating aneuploid cells. The discovery of centromsomes as potential contributors to malignant tumor progression provides us with a unique opportunity to elucidate a novel mechanism for tumorigenesis.
The specific aims of this proposal are: 1. To determine whether proteins of the centrosome and nucleus are altered in tumors. 2. To test whether pericentrin has oncogenic potential. 3. To determine how elevated levels of pericentrin cause aneuploidy.
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