CLL cell apoptosis induced by nitric oxide synthase inhibitors: correlation with lipid solubility and NOS1 dissociation constant.

Nitric oxide synthase (NOS) inhibitors induce chronic lymphocytic leukemia (CLL) cell apoptosis and have potential as CLL therapeutics. We determined the half-maximal concentration (ED(50)) of 22 NOS inhibitors that induced CLL cell death in vitro. There was a direct correlation of the NOS1 (but not NOS2) dissociation constant (K(d)) and the hydrophobicity partitioning coefficient of each NOS inhibitor and its ED(50). NOS inhibitors that bound tightly to CLL cell NOS1 and were hydrophobic potently induced CLL cell death. CLL cell RNA and protein analyses confirmed CLL cell NOS1 expression. Our studies permit the rational selection of NOS inhibitors for testing as CLL therapeutics.

Chronic lymphocytic leukemia cell CD38 expression and inducible nitric oxide synthase expression are associated with serum IL-4 levels.

B cell chronic lymphocytic leukemia (CLL) CD38 expression is variable and may predict outcome. Inducible nitric oxide synthase (NOS2) expression regulates CLL cell apoptosis. IL-4 and IFN-gamma regulate B cell CD38 expression and NOS2 expression. We compared IL-4 and IFN-gamma serum levels between CLL patients and normal individuals, and determined whether serum IL-4 and IFN-gamma levels correlated with CLL cell CD38 expression and NOS enzyme activity. IL-4 levels, but not IFN-gamma levels, differed between normal individuals and CLL patients. Furthermore, there was an association of IL-4 levels, but not IFN-gamma levels, with CD38 and NOS2 expression in CLL patients.

Inhibition of nitric oxide synthase by cobalamins and cobinamides.

Cobalamins are important cofactors for methionine synthase and methylmalonyl-CoA mutase. Certain corrins also bind nitric oxide (NO), quenching its bioactivity. To determine if corrins would inhibit NO synthase (NOS), we measured their effects on -L-[(14)C]arginine-to-L-[(14)C]citrulline conversion by NOS1, NOS2, and NOS3. Hydroxocobalamin (OH-Cbl), cobinamide, and dicyanocobinamide (CN(2)-Cbi) potently inhibited all isoforms, whereas cyanocobalamin, methylcobalamin, and adenosylcobalamin had much less effect. OH-Cbl and CN(2)-Cbi prevented binding of the oxygen analog carbon monoxide (CO) to the reduced NOS1 and NOS2 heme active site. CN(2)-Cbi did not react directly with NO or CO. Spectral perturbation analysis showed that CN(2)-Cbi interacted directly with the purified NOS1 oxygenase domain. NOS inhibition by corrins was rapid and not reversed by dialysis with L-arginine or tetrahydrobiopterin. Molecular modeling indicated that corrins could access the unusually large heme- and substrate-binding pocket of NOS. Best fits were obtained in the "base-off" conformation of the lower axial dimethylbenzimidazole ligand. CN(2)-Cbi inhibited interferon-gamma-activated Raw264.7 mouse macrophage NO production. We show for the first time that certain corrins directly inhibit NOS, suggesting that these agents (or their derivatives) may have pharmacological utility. Endogenous cobalamins and cobinamides might play important roles in regulating NOS activity under normal and pathological conditions.

Progressive immunoglobulin gene mutations in chronic lymphocytic leukemia: evidence for antigen-driven intraclonal diversification.

Somatic mutations of immunoglobulin genes characterize mature memory B cells, and intraclonal B-cell diversification is typically associated with expansion of B-cell clones with greater affinity for antigen (antigen drive). Evidence for a role of antigen in progression of intraclonal chronic lymphocytic leukemia (CLL) cell diversification in patients with mutated immunoglobulin genes has not been previously presented. We performed a single-cell analysis of immunoglobulin heavy and light chains in 6 patients with somatically mutated CLL-cell immunoglobulin genes and identified 2 patients with multiple related (oligoclonal) subgroups of CLL cells. We constructed genealogic trees of these oligoclonal CLL-cell subgroups and assessed the effects of immunoglobulin somatic mutations on the ratios of replacement and silent amino acid changes in the framework and antigen-binding regions (CDRs) of the immunoglobulin heavy and light chains from each oligoclonal CLL-cell population. In one subject, the amino acid changes were consistent with an antigen-driven progression of clonally related CLL-cell populations. In the other subject, intraclonal diversification was associated with immunoglobulin amino acid changes that would have likely lessened antigen affinity. Taken together, these studies support the hypothesis that in some CLL cases intraclonal diversification is dependent on antigen interactions with immunoglobulin receptors.

Clinical and molecular predictors of disease severity and survival in chronic lymphocytic leukemia.

Several parameters may predict disease severity and overall survival in chronic lymphocytic leukemia (CLL). The purpose of our study of 190 CLL patients was to compare immunoglobulin heavy chain variable region (IgV(H)) mutation status, cytogenetic abnormalities, and leukemia cell CD38 and Zap-70 to older, traditional parameters. We also wanted to construct a simple, inexpensive prognosis score that would significantly predict TTT and survival in patients at the time of diagnosis and help practicing clinicians. In univariate analyses, patients with higher clinical stage, higher leukocyte count at diagnosis, shorter leukocyte doubling time, elevated serum lactate dehydrogenase (LDH), unmutated immunoglobulin heavy chain variable region (IgV(H)) genes, and higher CD38 had a shorter overall survival and time-to-treatment (TTT). CLL cell Zap-70 expression was higher in patients with unmutated IgV(H), and those with higher Zap-70 tended to have shorter survival. IgV(H)4-34 or IgV(H)1-69 was the most common IgV(H) genes used (16 and 12%, respectively). Of those with IgV(H)1-69, 86% had unmutated IgV(H) and had a significantly shorter TTT. A cytogenetic abnormality was noted in 71% of the patients tested. Patients with 11q22 del and 17p13 del or complex abnormalities were significantly more likely to have unmutated IgV(H). We found that a prognostic score constructed using modified Rai stage, cellular CD38, and serum LDH (parameters easily obtained clinically) significantly predicted TTT and survival in patients at the time of diagnosis and performed as well or better than models using the newer markers.