concanavalin-a and 3-methoxybenzamide

6(5H)-phenanthridinone, a recently identified poly(ADP-ribose)polymerase (PARP) inhibitor, is able, at micromolar concentrations, to inhibit concanavalin A-induced lymphocyte proliferation and to potentiate the effect of gamma radiation upon murine spleen cells. When added at the onset of a mixed lymphocyte culture, this compound strongly depresses the induction of primary allogeneic (anti-H2k) cytotoxic T-lymphocytes (CTLs). Lymphokine-activated killer (LAK) induction was also found to be impaired by the PARP inhibitor. Taken together, these results clearly indicate that PARP plays a key-role in immune reactions involving cytotoxicity and that 6(5H)-phenanthridinone could be considered as a potent immunomodulator.

Topics: Acridines; Acridones; Animals; Benzamides; Cells, Cultured; Concanavalin A; Female; Gamma Rays; Immunosuppressive Agents; Interleukin-2; Isoquinolines; Killer Cells, Lymphokine-Activated; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Lymphocytes; Male; Mice; Mice, Inbred C3H; Mice, Inbred DBA; Phenanthrenes; Poly(ADP-ribose) Polymerase Inhibitors; Recombinant Proteins; Spleen; T-Lymphocytes, Cytotoxic

The previously reported extensive DNA strand breakage in resting murine splenic lymphocytes is not an artifact of the extraction or assay procedure. The benzamide inhibitors of poly(ADP ribose) synthetase (pADPRS), such as 5-methoxybenzamide (MBA), had been shown to block the strand break repair occurring within 2 h of activation of splenic lymphocytes by the mitogen concanavalin A (conA); the inhibitors also blocked early events in proliferation, such as blast formation, as well as entry into S phase. Inhibitors of pADPRS blocked lymphocyte proliferation by inhibiting the activity of this enzyme, rather than by non-specific effects. Aphidicolin, an inhibitor of alpha-polymerase, also prevented DNA strand break repair in conA-stimulated cells but, unlike MBA, did not prevent blast formation. DNA strand breaks accumulated in the presence of MBA at the same linear rate (300-400/h) in both resting and conA-treated cells. We and others had hypothesized that this accumulation was due to a continuous production of strand breaks in lymphocytes, leading to their accumulation in presence of repair inhibitors. However, incubation of the cells with aphidicolin at concentrations that inhibited repair did not result in any increase in strand breaks. The hypothesis of continuous cycling of breaks is incorrect; accumulation of breaks was due to some indirect effect of MBA, such as a possible disinhibition of an ADP-ribosylation-sensitive endonuclease described in other cell types. All of the early stages of lymphocyte proliferation, including blast transformation (but not DNA synthesis) require ADP ribosylation. Repair of DNA strand breaks is not a precondition for blast formation, though experiments involving the combined effects of MBA and aphidicolin showed that repair of the breaks is essential in order for the cells to replicate their DNA. Our data are consistent with a model suggesting that DNA strand breaks introduced into differentiated cells act as an additional safety-catch mechanism that restrains them from replicating their genetic material but not from undergoing the early stages of proliferation.

Topics: Adenosine Diphosphate Ribose; Animals; Aphidicolin; Benzamides; Cell Division; Cells, Cultured; Concanavalin A; Diterpenes; DNA; DNA Repair; Interphase; Lymphocyte Activation; Lymphocytes; Mice; Mice, Inbred BALB C; Nucleoside Diphosphate Sugars; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases