The mechanisms underlying negative regulation of human hematopoietic progenitor proliferation are still poorly understood. Normal hematopoiesis occurs in tight association with the bone marrow stroma. Adhesion of progenitors at different stages of differentiation involves different sets of cell surface receptors which recognize the extracellular matrix component fibronectin, suggesting highly specified interactions between progenitors and stroma dependent on their differentiation and/or commitment status. When grown in contact with stromal layers, primitive hematopoietic progenitors can differentiate into committed progenitors (CFC) while a fraction of primitive hematopoietic progenitors capable of initiating in vitro long-term cultures (LTC-IC) can be maintained. We have recently demonstrated that LTC-IC can be conserved to a significantly greater extent in cultures where direct contact between intact stromal layers and progenitors is not allowed. We hypothesize that direct contact between hematopoietic progenitors and stromal components exerts important, but poorly understood negative regulatory effects on primitive hematopoietic progenitors. We postulate further that this negative regulation is the result of both (A) ar relative increase in diffusible growth inhibitory factors in the stromal microenvironment upon interaction with hematopoietic progenitors and of (B) direct transmission of growth inhibitory signals between stromal components and adhesion receptors for fibronectin on LTC-IC/CFC. The studies proposed in this grant are designed to identify mechanisms underlying the negative effects exerted by the stroma on the proliferation of adherent LTC-IC.
In Specific Aim 1, single cell proliferation assays, PKH-26 labelling assays and thymidine suicide assays will be developed further to demonstrate that LTC-IC proliferation is decreased following their interaction with the BM microenvironment.
In Specific Aim 2, ELISA's of stromal supernatants and rT-PCR of stromal cells will be used to identify alterations in diffusible negative and positive cytokines produced by the stroma upon interaction with hematopoietic progenitors. The contribution of progenitor differentiation stage and hematopoietic cell density on the induction or suppression of growth regulatory molecules will be examined.
In Specific Aim 3, we will explore the nature of ligands in the stroma and adhesion receptors on LTC- IC which are responsible for transmitting negative regulatory signals to LTC-IC and compare them with receptors/ligands responsible for proliferation inhibition of more mature CFC. We will examine the effect of synthetic adhesion promoting peptides from FN, antibodies against these peptides, antibodies against integrin and other adhesion receptors on the proliferation of LTC-IC/CFC cultured in contact with stroma. These studies may provide us with in vitro models suitable for study of intra-cellular signal transduction mechanisms responsible for the induction of a quiescent state in primitive and more mature hematopoietic progenitors following their interaction with stroma.
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|Melikova, Sofya; Dylla, Scott J; Verfaillie, Catherine M (2004) Phosphatidylinositol-3-kinase activation mediates proline-rich tyrosine kinase 2 phosphorylation and recruitment to beta1-integrins in human CD34+ cells. Exp Hematol 32:1051-6|
|Salesse, S; Dylla, S J; Verfaillie, C M (2004) p210BCR/ABL-induced alteration of pre-mRNA splicing in primary human CD34+ hematopoietic progenitor cells. Leukemia 18:727-33|
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|Salesse, Stephanie; Verfaillie, Catherine M (2003) BCR/ABL-mediated increased expression of multiple known and novel genes that may contribute to the pathogenesis of chronic myelogenous leukemia. Mol Cancer Ther 2:173-82|
|Punzel, Michael; Gupta, Pankaj; Verfaillie, Catherine M (2002) The microenvironment of AFT024 cells maintains primitive human hematopoiesis by counteracting contact mediated inhibition of proliferation. Cell Commun Adhes 9:149-59|
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|Zhao, R C; Jiang, Y; Verfaillie, C M (2001) A model of human p210(bcr/ABL)-mediated chronic myelogenous leukemia by transduction of primary normal human CD34(+) cells with a BCR/ABL-containing retroviral vector. Blood 97:2406-12|
|Jiang, Y; Prosper, F; Verfaillie, C M (2000) Opposing effects of engagement of integrins and stimulation of cytokine receptors on cell cycle progression of normal human hematopoietic progenitors. Blood 95:846-54|
|Jiang, Y; Zhao, R C; Verfaillie, C M (2000) Abnormal integrin-mediated regulation of chronic myelogenous leukemia CD34+ cell proliferation: BCR/ABL up-regulates the cyclin-dependent kinase inhibitor, p27Kip, which is relocated to the cell cytoplasm and incapable of regulating cdk2 activity. Proc Natl Acad Sci U S A 97:10538-43|
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