Abstract
Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4)-deficient patients recently were found to have abnormally high levels of dATP, a negative allosteric effector of ribonucleotide reductase (ribonucleoside-diphosphate reductase, 2'-deoxyribonucleoside-diphosphate:oxidized thioredoxin 2'-oxidoreductase, EC 1.17.4.1). Therefore it was proposed that the immunodeficiency associated with adenosine deaminase deficiency is mediated through inhibition of ribonucleotide reductase and hence DNA replication. HeLa cells, treated with an adenosine deaminase inhibitor, erythro-9(2-hydroxy-3-nonyl)adenine, and deoxyadenosine to mimic the adenosine deaminase-deficient state, were monitored to determine directly the effects on ribonucleotide reductase activity and levels. A low concentration of erythro-9-(2-hydroxy-3-nonyl)adenine, which did not inhibit cell growth, nevertheless retarded the cells in G2 + M phase of the cell cycle and increased reductase activity. Reductase activity was also elevated in cells treated with a low level of deoxyadenosine which did not affect the cell cycle or cell growth. However, ribonucleotide reductase activity was reduced to one-half of the control value in cells treated with either enough deoxyadenosine to inhibit cell growth or with a combination of erythro-9(2-hydroxy-3-nonyl)adenine and deoxyadenosine, each at concentrations which individually do not inhibit cell growth. Removal of deoxynucleotides, particularly dATP, from these extracts increased ribonucleotide reductase activity to several-fold higher than control values. The reduced activity of ribonucleotide reductase in the simulated adenosine deaminase-deficient HeLa cells provides direct evidence for the thesis that adenosine deaminase deficiency disease is mediated through elevated levels of dATP which inhibit ribonucleotide reductase. In addition, the cell cycle patterns and ribonucleotide reductase levels suggest that the regulatory substance(s) that controls the level of ribonucleotide reductase is not operative until the late S or G2 phase of the cell cycle.
Highlights
EC 3.5.4A)-deficient patients recently were found to of immunodeficiency whichinvohes impairment of both huhave abnormally high levels of dATP, a negative allo- moral and cell-mediated immune functions [1,2]
The biochemical mechanism for the immunological dysfunction which occurs with adenosine deaminase deficiency disease has been subject to debate until recent reports of greatly elevated dATP levels in adenosine deaminase-deficient patients [4,5].These observations have led to a change in research emphasis from ATP to dATP, paowerful negative allosteric effector of ribonucleotide reductase
Deoxyadenosine has been recognized as a toxic agent to a variety of cell lines for two decades, and its effect is highly potentiated by the adenosine deaminase inhibitors, EHNA and 2'-deoxycoformycin [8,9,10,11,12,25]
Summary
EXPERIMENTALPROCEDURES (Fig. IC), preventing the cells from entering G2+ M. Addition of EHNA in combination with deoxyadenosine, each at a concentration which individually does not inhibit cell growth (10 p~ and 100 p ~ re,spectively), results in a complete block of cell growth similar to that found using 1 monitoring the conversion of ['HICDP to ['HIdCDP with theuse of a method originally developed by Reichard et al [18] and modified by Elford et al [19]. Cell Cycle Analysis-Of each experimental cell suspensioncontaining 3 to 6 X 1 0 cells/&, 10 ml were removed, washed, fixed with tide ReductaseLevels-A 2-fold increase inthe specific activity of ribonucleotide reductase was found in extracts of HeLa cells grown in the presence of 10 p~ EHNA compared to untreated cells (TableI). Cells grown with 100 VM deoxyadenosine exhibited nearly a 2-fold increase in ribonucleotide reductase activity, no effect on cell replication or the cell cycle was observed at this low concentration.
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