pyrimidinones and Leukemia--T-Cell

Purine nucleoside phosphorylase (PNP) was recognized more than 30 years ago as a potential target for the treatment of patients with T-cell malignancies when an inherited deficiency of PNP was reported to be associated with a profound T-cell lymphopenia. The biochemical basis for this T-cell deficiency was subsequently shown to be related to the accumulation of plasma 2'-deoxyguanosine (dGuo) and intracellular dGuo triphosphate (dGTP). These observations have led to a search for PNP inhibitors that would be useful clinically in the management of T cell-derived malignancies. The most potent inhibitor of PNP described to date is forodesine, a rationally designed, transition-state analogue inhibitor. The preclinical and clinical pharmacology of forodesine showed its effectiveness in inhibiting PNP and augmenting dGuo levels in plasma. Increased dGTP concentrations in leukemia cells of different lineages provides strong support for the potential use of this agent in the treatment of patients with hematologic malignancies of both T- and B-cell origin.

Topics: Biosynthetic Pathways; Clinical Trials as Topic; Deoxyguanine Nucleotides; Deoxyguanosine; Humans; Leukemia, T-Cell; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; T-Lymphocytes

Newborns with a genetic deficiency of purine nucleoside phosphorylase (PNP) are normal, but exhibit a specific T-cell immunodeficiency during the first years of development. All other cell and organ systems remain functional. The biological significance of human PNP is degradation of deoxyguanosine, and apoptosis of T-cells occurs as a consequence of the accumulation of deoxyguanosine in the circulation, and dGTP in the cells. Control of T-cell proliferation is desirable in T-cell cancers, autoimmune diseases, and tissue transplant rejection. The search for powerful inhibitors of PNP as anti-T-cell agents has culminated in the immucillins. These inhibitors have been developed from knowledge of the transition state structure for the reactions catalyzed by PNP, and inhibit with picomolar dissociation constants. Immucillin-H (Imm-H) causes deoxyguanosine-dependent apoptosis of rapidly dividing human T-cells, but not other cell types. Human T-cell leukemia cells, and stimulated normal T-cells are both highly sensitive to the combination of Imm-H to block PNP and deoxyguanosine. Deoxyguanosine is the cytotoxin, and Imm-H alone has low toxicity. Single doses of Imm-H to mice cause accumulation of deoxyguanosine in the blood, and its administration prolongs the life of immunodeficient mice in a human T-cell tissue xenograft model. Immucillins are capable of providing complete control of in vivo PNP levels and hold promise for treatment of proliferative T-cell disorders.

Topics: Animals; Antineoplastic Agents; Drug Design; Enzyme Inhibitors; Humans; Leukemia, T-Cell; Mice; Nucleosides; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Static Electricity; T-Lymphocytes; Transplantation, Heterologous

Immucillin-H (BCX-1777, forodesine) is a transition state analogue and potent inhibitor of PNP that shows promise as a specific agent against activated human T-cells and T-cell leukemias. The immunosuppressive or antileukemic effects of Immucillin-H (ImmH) in cultured cells require co-administration with deoxyguanosine (dGuo) to attain therapeutic levels of intracellular dGTP. In this study we investigated the requirements for sensitivity and resistance to ImmH and dGuo. (3)H-ImmH transport assays demonstrated that the equilibrative nucleoside transporters (ENT1 and ENT2) facilitated the uptake of ImmH in human leukemia CCRF-CEM cells whereas (3)H-dGuo uptake was primarily dependent upon concentrative nucleoside transporters (CNTs). Analysis of lysates from ImmH-resistant CCRF-CEM-AraC-8D cells demonstrated undetectable deoxycytidine kinase (dCK) activity, suggesting that dCK and not deoxyguanosine kinase (dGK) was the rate-limiting enzyme for phosphorylation of dGuo in these cells. Examination of ImmH cytotoxicity in a hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient cell line CCRF-CEM-AraC-8C, demonstrated enhanced sensitivity to low concentrations of ImmH and dGuo. RT-PCR and sequencing of HGPRT from the HGPRT-deficient CCRF-CEM-AraC-8C cells identified an Exon 8 deletion mutation in this enzyme. Thus these studies show that specific nucleoside transporters are required for ImmH cytotoxicity and predict that ImmH may be more cytotoxic to 6-thioguanine (6-TG) or 6-thiopurine-resistant leukemia cells caused by HGPRT deficiency.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Deoxycytidine Kinase; Deoxyguanosine; Drug Screening Assays, Antitumor; Humans; Hypoxanthine Phosphoribosyltransferase; Leukemia, T-Cell; Models, Biological; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones

Effects of sphingosine on Ca2+ mobilization in the human Jurkat T cell line were examined. Sphingosine increased the cytoplasmic Ca2+ concentration ([Ca2+]i) in a dose-dependent manner with an ED50 of around 8 microM. Sphingosine and OKT3, a CD3 monoclonal antibody, transiently increased [Ca2+]i, which declined to the resting level in the absence of extracellular Ca2+. Under the same conditions, pretreatment with sphingosine inhibited but did not abolish an increase in [Ca2+]i induced by the subsequent addition of OKT3 and vice versa. However, pretreatment with sphingosine did not affect an increase in [Ca2+]i induced by OKT3 in the presence of Ca2+. OKT3 increased IP3 formation, but sphingosine did not affect the level of IP3 by itself nor did it cause IP3 formation induced by OKT3. In permeabilized Jurkat cells, the addition of IP3 released Ca2+ from nonmitochondrial intracellular stores, but the addition of sphingosine did not. Sphingosine, stearylamine, and psychosine increased [Ca2+]i and diacylglycerol (DG) kinase activation; however, ceramide did not, whereas sphingosine 1-phosphate slightly activated DG kinase without elevation of [Ca2+]i. Pretreatment with R59022, a DG kinase inhibitor, abolished the peak but did not affect the sustained response to [Ca2+]i to sphingosine. Phosphatidic acid (PA) elevated [Ca2+]i, after which it declined to a resting level even in the presence of extracellular Ca2+. In accordance with this, PA did not stimulate 45Ca2+ uptake into cells, but sphingosine and OKT3 did. Pretreatment with PA partially inhibited a rise in [Ca2+]i induced by the subsequent addition of sphingosine and vice versa in the absence of extracellular Ca2+. Under similar conditions, pretreatment with PA affected an elevation of [Ca2+]i induced by OKT3 less, after which the subsequent addition of sphingosine did not increase [Ca2+]i. In permeabilized Jurkat cells, the addition of IP3 did not release Ca2+, but PA did in the presence of heparin. Pretreatment with thapsigargin, a microsomal Ca2+-ATPase inhibitor, abolished the rises of [Ca2+]i induced by the subsequent addition of sphingosine, OKT3, and PA in the absence of extracellular Ca2+. The present results suggest that at least two kinds of intracellular Ca2+ stores exist in Jurkat cells, both of which are IP3- and PA-sensitive, and that sphingosine mobilizes Ca2+ from both stores in an IP3-independent manner. Furthermore, the IP3- but not the PA-sensitive intracellular Ca2+ store seems to reg

Topics: Amines; Calcium; Cell Line; Ceramides; Diacylglycerol Kinase; Diglycerides; Enzyme Inhibitors; Humans; Inositol 1,4,5-Trisphosphate; Kinetics; Leukemia, T-Cell; Models, Biological; Muromonab-CD3; Phosphatidic Acids; Phosphotransferases (Alcohol Group Acceptor); Psychosine; Pyrimidinones; Sphingosine; Terpenes; Thapsigargin; Thiazoles; Tumor Cells, Cultured