ASH: NOS1, and NOS1-Specific Inhibitors Induce CLL Cell Death

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[5139] Chronic Lymphocytic Leukemia Cells (CLL) Express the Neuronal

Isoform of Nitric Oxide Synthase (NOS1), and NOS1-Specific Inhibitors

Induce CLL Cell Death. Session Type: Publication Only

Marc C. Levesque, Youwei Chen, Bethany E. Beasley, W.

OLoughlin, Jon P. Gockerman, ph O. , Dipak K. Ghosh, J.

Brice Weinberg Medicine, Duke University and Durham VA Medical

Centers, Durham, NC, USA

Nitric oxide (NO) regulates B cell caspase-3 activity via reversible

S-nitrosylation of the active site cysteine of caspase-3. As such, NO

regulates B cell apoptosis. NO is produced by three different NO

synthases (NOS) in humans. Two of these isoforms, NOS1 (brain or

neuronal NOS (nNOS)) and NOS3 (endothelial NOS (eNOS)) produce low

levels of NO, are constitutively expressed and their activity is

primarily regulated by calcium fluxes. In contrast to the

constitutive NOS enzymes, inducible NOS (NOS2 or iNOS) is regulated

primarily at the transcriptional level and is transcribed in response

to several pro-inflammatory stimuli. Previous studies indicate that

CLL cells express NOS2 but not NOS3, and no previous studies have

examined NOS1 expression in CLL cells. The purpose of our study was

to determine whether NOS inhibitors would induce CLL cell death and

apoptosis. We screened several NOS inhibitors for their ability to

induce CLL cell death using a MTS assay and/or viability assay based

on propidium iodide exclusion. We screened non-specific NOS

inhibitors (L-NMMA, L-NAME, 2-amino-4-methyl pyridine, 2-amino-6-

methyl pyridine, 2-amino-3-methyl pyridine, S-ethyl-ITU, L-

thiocitrulline and NG-nitro-L-arginine), NOS1-specific inhibitors (AR-

R17477, vinyl-L-NIO, N-propyl-L-arginine and S-methyl-L-

thiocitrulline) and NOS2-specific inhibitors (1400W, GW274150, L-NIL

and S-methyl-ITU). The non-specific NOS inhibitors and NOS2-specific

inhibitors either did not induce CLL cell death or induced CLL cell

death with EC50 values (average concentration of compound that

induced 50% CLL cell death) in the mM range. In contrast, two of the

four NOS1-specific inhibitors induced CLL cell death at lower

concentrations (AR-R17477 EC50 = 14 1.5 uM (SEM), N = 3 and vinyl-L-

NIO EC50 = 580 160 uM, N = 3). We also determined whether CLL cells

expressed NOS1 protein and mRNA using immunoblots and RT-PCR assays,

respectively. We found that 13 of 13 CLL cell samples expressed NOS1

protein and 7 of 20 expressed NOS1 mRNA. Therefore, we believe that

NOS1 expression by CLL cells may be a regulator of CLL cell

apoptosis. Strategies designed to inhibit CLL cell nitric oxide

synthases and NO production may be effective for the treatment of CLL.

Abstract #5139 appears in Blood, Volume 102, issue 11, November 16,

2003

Keywords: Chronic lymphocytic leukemia|Nitric oxide|Neuronal nitric

oxide synthase