All-trans retinoic acid promotes neutrophilic differentiation of several myeloid cell lines and induces complete remission in patients with acute promyelocytic leukemia (APL). In APL, the gene of retinoic acid receptor (RAR-alpha) is translocated to a new locus PML, resulting in the generation and expression of a fusion gene PML-RAR-alpha. However, the exact roles of RAR-alpha and PML-RAR-alpha in the development of neutrophils and leukemia are unclear.
The specific aims of this proposal are: (1) To determine If retinoic acid (RA) and retinoic acid receptor-alpha (RAR-alpha) play an essential role in the differentiation of neutrophils; (2) To investigate the biological consequences of the expression of the fusion gene PML-RAR-alpha in myeloid cells. Three approaches will be used to determine if RA and RAR-alpha play a crucial role in the differentiation of neutrophils. First, we will utilize a retroviral vector to express a """"""""dominant negative"""""""" RAR-alpha in a hematopoietic cell line capable of differentiation into neutrophils and macrophages. If RAR-alpha is essential to the differentiation of neutrophils, suppression of its function by the dominant negative mutant should disrupt the differentiation process. Second, we will use the same retroviral vector to transduce and express the dominant negative RAR-alpha in mouse hematopoietic stem cells to study its effect on hematopoiesis in vivo. Third, a serum-free culture system will be used to determine the requirement of RA in the development of neutrophils. To investigate the biological consequences of the expression of PML-RAR-alpha fusion gene in myeloid cells, we will employ two strategies. First, a retroviral vector harboring the fusion gene will be used to infect myeloid cell lines capable of neutrophilic differentiation in response to RA to determine whether this fusion gene exhibits dominant negative activity. Second, we will infect murine bone marrow stem cells with the same retrovirus and transplant the stem cells into recipient mice to study the leukemogenic potential of the fusion gene. The applicant's long-term research interest is the molecular control of the differentiation of normal and neoplastic neutrophils. In summary, the proposed studies allow us to gain more insights into the roles of RA and RAR-alpha in hematopoiesis.
