D. Bruce Wasson

DNA strand breaks, NAD metabolism, and programmed cell death

An intimate relationship exists between DNA single-strand breaks, NAD metabolism, and cell viability in quiescent human lymphocytes. Under steady-state conditions, resting lymphocytes continually break and rejoin DNA. The balanced DNA excision-repair process is accompanied by a proportional consumption of NAD for poly(ADP-ribose) synthesis. However, lymphocytes have a limited capacity to resynthesize NAD from nicotinamide. An increase in DNA strand break formation in lymphocytes, or a block in DNA repair, accelerates poly(ADP-ribose) formation and may induce lethal NAD and ATP depletion. In this way, the level of DNA single-strand breaks in the lymphocyte nucleus is linked to the metabolic activity of the cytoplasm. The programmed removal of lymphocytes (and perhaps of other cells) with damaged DNA, may represent a novel physiologic function for poly(ADP-ribose)-dependent NAD cycling.

Programmed cell death and adenine deoxynucleotide metabolism in human lymphocytes

Agents that cause the accumulation of DNA strand breaks are directly cytotoxic to non-dividing normal human peripheral blood lymphocytes, and to chronic lymphocytic leukemia (CLL) cells. Activation of poly(ADP-ribose) polymerase (ADPRP), and the resultant consumption of NAD, play an essential role in mediating the toxicity of these agents. Human peripheral blood lymphocytes contain a substantial number of alkali-sensitive DNA sites, reflecting ongoing DNA strand breakage and repair. However, resting lymphocytes have a limited capacity to synthesize NAD. Pulse-chase experiments indicate that approximately 75% of their NAD turnover is due to ADPRP activity. Exposure of the cells in vitro to deoxyadenosine, or to 2-chlorodeoxyadenosine (CdA, an adenosine deaminase resistant deoxyadenosine congener), caused an increase in DNA strand breaks, rapid NAD consumption, ATP depletion and cell death. Supplementation of the medium with inhibitors of poly(ADP-ribose) polymerase blocks the fall in cellular NAD and ATP, and protects the lymphocytes from the toxicity of DNA damaging agents. Slowly dividing malignant lymphocytes from patients with CLL are also susceptible to lethal NAD depletion following DNA damage. 2-chlorodeoxyadenosine (CdA) induced massive DNA strand break formation in CLL cells in vitro and a fall in NAD and ATP pools. In an initial clinical trial, several CLL patients, and two patients with hairy cell leukemia, have responded to treatment with CdA, with minimal toxicity. Thus, the suicidal activation of ADPRP in response to DNA damage has been rationally exploited in the treatment of chronic lymphoid malignancies.

Biochemical Basis for Deoxyadenosine and 2-Chlorodeoxyadenosine Toxicity to Resting Human Lymphocytes

An Inherited deficiency of adenosine deaminase (ADA; adenosine aminohydrolase EC 3, 5, 4, 4) results in a combined immunodeficiency disease (1). The selective lymphopenia seen in ADA deficient children has been attributed to the toxic effects of deoxyadenosine (dAdo) metabolites (2). Micromolar concentrations of dAdo are toxic in vitro to ADA-inhibited human resting peripheral blood lymphocytes (PBL). The ADA-resistent dAdo congener, 2-chlorodeoxyadenosine (CdA) is similarly cytotoxic to resting human T cells (3). Since the toxic mechanisms of dAdo have not been clearly elucidated, we have examined the metabolic changes that follow exposure of PBL to dAdo plus deoxycoformycin (dCF), as well as to CdA (4).

Activity of 2-Chloro-2′-Deoxyadenosine in Chronic Lymphocytic Leukemia, Hairy Cell Leukemia, and Autoimmune Hemolytic Anemia

Interest in the pathways of nucleotide metabolism in human lymphocytes was stimulated by the serendipitous observations of Giblett, et al., who found that two inborn errors of purine nucleoside metabolism cause severe impairment of lymphocyte function, while sparing other cell types (1, 2). A genetic deficiency of adenosine deaminase engenders a combined immunodeficiency syndrome (1). A similar deficiency in purine nucleoside phosphorylase is associated with a selective cellular immune deficit (2).

Pivotal Role of Poly(ADP-Ribose) Polymerase Activation in the Pathogenesis of Immunodeficiency and in the Therapy of Chronic Lymphocytic Leukemia

Activation of poly(ADP-ribose) polymerase plays an essential role in mediating the toxic effects of DNA damage in non-dividing lymphocytes. In cells with extensive DNA damage, excessive poly(ADP-ribose) polymerase activity can exhaust the available NAD pool, leading to a depletion of cellular ATP and to toxic perturbations of intermediary metabolism (1). Various agents known to damage DNA have been shown in vitro to elicit the biochemical sequelae of poly(ADP-ribose) polymerase activity in resting human lymphocytes (2). For example, exposure of lymphocytes to xanthine oxidase plus hypoxanthine causes massive DNA damage and a lethal fall in cellular NAD and ATP levels, attributable to excessive poly(ADP-ribose) polymerase activity (3). This model system suggests that poly(ADP-ribose) polymerase activation may contribute to immune dysfunction in certain chronic inflammatory states, in which stimulated neutrophils release toxic oxygen species.

Genetic Analysis of Deoxyadenosine Toxicity in Dividing Human Lymphoblasts

An Inherited deficiency of adenosine deaminase (ADA) Impairs specifically the development of the human lymphoid system (reviewed In 1). In ADA deficient children, plasma deoxyadenosine (dAdo) concentrations reach 1-2 μM. Micromolar concentrations of dAdo are toxic toward Immature human T-lymphocytes, and toward T-Iymphoblastoid cell lines grown In medium supplemented with deoxycoformycin, a tight binding ADA Inhibitor.1 Compared to other cell types, human T-lymphoblasts preferentially phosphorylate dAdo and accumulate dATP.

19 2-HALO-2|[prime]|,3|[prime]|-DIDEOXYADENOSINES: METABOLICALLY STABLE DIDEOXYNUCLEOSIDES WITH ACTIVITY AGAINST THE HUMAN IMMUNODEFICIENCY VIRUS (HIV)

2′,3′-dideoxyadenosine (ddA) has activity against the human immunodeficiency virus-1 (HIV), but is rapidly catabolized by human T cells, even when adenosine deaminase is inhibited by deoxycoformycin. To overcome this problem, we developed a simple method to synthesize the 2-fluoro-, 2-chloro-, and 2-bromo-derivatives of ddA. The isolated 2-halo-ddA derivatives were not deaminated significantly by cultured T lymphoblasts, which converted the dideoxynucleosides to the respective 5′-monophosphate, 5′- diphosphate, and 5′- triphosphate metabolites. At concentrations lower than those producing cytotoxicity in uninfected cells (3-10 μM), the 2-halo-ddA derivatives inhibited the cytopathic effects of HIV toward T lymphoblasts, and retarded viral replication. Experiments with a deoxycytidine kinase deficient mutant CEM T cell line showed that this enzyme was necessary for the phosphorylation and anti-HIV activity of the 2-halo-ddA derivatives. Thus, the 2-halo-ddA congeners, in contrast to ddA itself, are not degraded by T lymphocytes, and represent promising compounds for in vivo chemotherapy of HIV infection.

BIOCHEMICAL BASIS FOR DEOXYADENOSINE AND 2-CHLORODEOXYADENOSINE TOXICITY TO BESTING HDMAN LYMPHOCYTES: 187

Deoxyadenosine (dAdo) is toxic at micromolar concentrations to adenosine deaminase inhibited resting human peripheral blood lymphocytes. 2-chlorodeoxyadenosine (CdA), a metabolically resistant dAdo congener, exhibits similar properties. Four hours of exposure to dAdo or CdA induced the accumulation of strand breaks in the DNA of normal resting lymphocytes, as measured by a DNA unwinding assay. The DNA damage was followed by consumption of NAD, probably mediated by increased poly(ADP-ribose) synthesis. The addition of 1-5mM nicotinamide prevented the dAdo and CdA triggered fall in NAD levels, and rendered the resting lymphocytes resistant to the toxic effects of both compounds. Both dAdo and CdA inhibited the repair of radiation induced DNA damage in resting lymphocytes, by impeding DNA polymerization. CdA was 100 fold more potent than dAdo. These results suggest that (i) a slow rate of DNA polymerization is required to maintain DNA integrity in resting human lymphocytes, (ii) dAdo and CdA inhibit polymerization, and cause DNA strand breaks to accumulate, (iii) the strand breakage triggers poly(ADP-ribose) synthesis, and causes lethal NAD depletion.