Christopher J. Skidmore

Nuclear ADP-ribosylation in the chick lens during embryonic development

Nuclear ADP-ribosyltransferase is present in cells from the chick lens throughout embryonic development. The activity does not decrease when the cells become post-mitotic and commence terminal differentiation but declines slowly in both epithelia and fibre cells. At all stages studied the enzyme retains its ability to be activated by DNA strand breaks induced either by X-irradiation or by the action of an endogenous endonuclease. There is no correlation between the enzyme activity or the levels of its substrate NAD+ and the changes in DNA repair capacity which have been observed during the development of the lens.

The Relationship Between DNA Strand Breaks and ADP-Ribosylation

The nuclear NAD+:ADP-ribosyltransferase of eukaryotes (EC 2.4.2.30-ADPRT) is a remarkable enzyme. It will catalyse at least two chemically distinct reactions — the transfer of ADP-ribose to protein and the transfer of ADP-ribose to the growing chain of (ADP-ribose)n [1]. It apparently effects its own short term regulation by automodification [2, 3]. In addition it interacts strongly with DNA and is activated by DNA strand breaks [4]. These properties would seem to indicate a certain complexity of structure in the enzyme and, indeed, recent proteolytic studies [5,6] have indicated that it possesses at least three functional domains.

In Vivo Poly(ADP-ribose) Levels in Carcinogen Treated Human Promyelocytic Leukemia Cells

The cellular role of nuclear ADP-ribosylation has been analyzed traditionally by the investigation of the activity of ADP-ribosyl transferase (NAD+:protein ADP-ribosyl transferase, EC 2.4.2.30) and by the use of enzyme inhibitors (1). ADP-ribosyl transferase is activated when strand breaks are induced in DNA by alkylating agents and x-rays (2, 3) or during the process of cell differentiation (4, 5). Inhibitors of the enzyme inhibit most measures of DNA repair (3) although nicotinamide can stimulate unscheduled DNA synthesis (6). 3-Aminobenzamide in particular inhibits differentiation of myoblasts (7) and of macrophage precursor cells. It is only with the recent introduction of reliable methods for measuring the steady-state levels of poly(ADP-ribose) that it has been possible to look at ADP-ribosylation directly in vivo. We have initiated a study comparing the behavior of ADP-ribosylation following DNA damage and during differentiation. We use the human cell line HL60, promyelocytic cells which have the capacity to differentiate into either granulocyte or macrophages in culture following treatment with phorbol-12-myristate 13- acetate or retinoic acid, respectively (5). We present here initial studies in which the levels of poly(ADP-ribose) in vivo in undifferentiated HL60 cells are measured following treatment with the powerful mutagen N- methyl-N’-nitro-N’-nitrosoguanidine (MNNG). We also investigate the effect of ADP-ribosyl transferase inhibitors on the steady state level of ADP-ribosylation.

The Effect of Benzamides on the Activity of Nuclear ADP-Ribosyl Transferase and the Accumulation of Poly(ADP-Ribose) In Vivo

Investigation of the cellular role of the nuclear protein modification ADP-ribosylation has depended heavily on the use of benzamides as inhibitors of ADP-ribosyl transferase (EC 2.4.2.30) in intact cell studies. The substituted benzamides were first introduced by Whish (1) and have proved to be useful inhibitors of low toxicity and fairly high specificity. The role of ADP-ribosylation in DNA repair (2) has been strongly supported by the stimulation of the cytotoxicity of DNA-damaging agents by the benzamides.