Nitric oxide (NO) inhibits growth, induces differentiation and apoptosis in acute myeloid leukemia (AML) cells. Using the technique of Representational Difference Analysis of cDNA (RDA), I have identified a novel gene that I have named regulated by nitric oxide or rno. rno is upregulated by NO in AML cells and is expressed exclusively in hematopoietic cells. A cDNA clone of rno was obtained from a normal leukocyte library. The predicted sequence of rno shows that it belongs to the family of leucine rich repeat proteins and has a high degree of homology to the human ribonuclease inhibitor. PCR and sequencing also revealed that there are at least 3 isoforms of rno. Expression of rno in AML cells inhibits growth, and induces differentiation and apoptosis. The HYPOTHESIS for this proposal is that NO affects the growth and differentiation of AML cells by modulating the expression of specific genes. rno could be involved in mediating the effects of NO on hematopoietic cells.
The SPECIFIC AIMS are:
AIM 1 -Clone and sequence the complete cDNA for rno isoforms: This will be done by screening a cDNA library made from RNA obtained from normal human peripheral blood leukocytes.
AIM 2 -Demonstrate that rno affects the growth and differentiation of hematopoietic cells. This will be accomplished by transfecting rno into HL-60 cells and determining the effect of rno expression on cell growth and differentiation. The subcellular distribution of rno will be determined using a rno Green Fluorescent Protein fusion gene. rno will be expressed in E. coli and rno protein will be purified to study its effects on RNAse function. Antisera to rno will be raised for further functional studies. rno expression will be determined in normal hematopoietic cells at different stages of differentiation.
AIM 3 -Identify genes that are modulated by rno: rno will be cloned in an expression vector with an inducible promoter. The vector will be transfected into HL-60 cells. By using cDNA array technology, gene expression differences between rno expressing and non-expressing cells will be identified.
AIM 4 -Determine the effect of rno expression on AML cell growth in vivo. I will use a human AML xenograft model in NOD/SCID mice. The animals will be inoculated with HL-60 cells that have been transfected with an inducible rno expression vector. Induction of rno expression in the leukemia cells in vivo will be done by treatment of the animals with Tetracycline. The effect of rno expression on leukemia cell growth in vivo will be determined. This work will help understand the mechanism by which NO affects hematopoietic cell growth and differentiation and may constitute the basis for the development of novel strategies for the treatment of AML.
