Response of mammalian ADP-ribosyl transferase to lymphocyte stimulation, mutagen treatment and cell cycling

Abstract

The inhibitors of the nuclear enzyme ADP-ribosyl transferase (ADPRT) had been shown to block the stimulation of quiescent lymphocytes with mitogens suggesting the involvement of the enzyme in the control of gene expression and cell differentiation. By means of the activity-gel assay we have analysed the intensity and the molecular mass of the catalytic bands of the enzyme at early and late times after stimulation of human lymphocytes by phytohemagglutinin. We observed that the increase in the activity of ADPRT is concurrent with the onset of DNA synthesis and is maintained for up to 10 days after lymphocyte stimulation, when DNA replication is over but the capacity to perform repair synthesis is still elevated. The analysis of ADPRT in stimulated lymphocytes by Western blots indicated that the increase in enzyme activity is due to the de novo synthesis of enzyme protein. The response of ADPRT to the treatment of human lymphocytes with DNA-damaging agents was studied at various dose-ranges, using the activity-gel technique. The results obtained indicate that dimethyl sulfate is 10 times as active as methyl methane sulfonate in stimulating ADPRT activity and that, at very high doses, the activity band of the enzyme tends to disappear. Very similar observations were obtained when Chinese hamster ovary cells were treated with the same agents, although the concentrations of the mutagens eliciting maximal ADPRT activation were 10 times higher than in human lymphocytes. When analysed by Western blots, no significant difference of the protein band of the enzyme was observed in comparing control and treated cells. This suggests that the activity-gel system can detect two different phenomena: the increase in enzyme protein, as in the case of stimulated lymphocytes, and the enzyme-activating effect of DNA-damaging agents, which occurs without changing the number of enzyme molecules. Of particular interest is the observation that mitomycin C is capable of activating ADPRT in human lymphocytes, thus suggesting that cross-linking agents are involved in promoting ADP-ribosylation reactions. We have also analysed the variations of the enzyme throughout the cell cycle in HeLa cells synchronized in S phase or in mitosis. No significant changes in the levels of the enzyme activity were revealed by the activity-gel assay during the progression of the cycle, although an overall increase of active polypeptides of larger size in concomitance with the S period was observed.

Inhibition of poly(ADP‐ribosyl)ation does not prevent lymphocyte entry into the cell cycle

“…This finding is in accordance with the observation that there are no DNA SB, requiring the intervention of ADPRT-related repair mechanisms, in quiescent lymphocytes [17]. ADPRT is induced at an early stage during mitogen-stimulated lymphocyte cultures [10], however its activity reaches maximum at around the 48-72 h [6,8]; this observation also supports the view that ADPRT is not required for early DNA repair. It has been suggested that ADPRT carries out other functions, probably related to specific changes in the chromatin structure [33][34][35].…”

“…Although the presence of DNA strand breaks (SB) appears to be a prerequisite for ADPRT activation, the role of the enzyme in DNA repair is still poorly understood [2][3][4]. Moreover, ADPRT activity has been found to increase with cell cycle progression [5][6][7][8][9][10]; this suggests that the enzyme also plays a role in physiological processes other than repair.…”

Gene expression in the cell cycle of human T lymphocytes: I. Predicted gene and protein networks

“…The difference between cycling versus resting T cells has been recently also shown by effect of the betainterferon, present in cycling T cells but not in resting T cells [Cooper et al, 2004]. Moreover expression of particular genes was shown to be cell cycle dependent, e.g., the link between ATR and p53 genes was present in cycling normal lymphocytes but absent in resting and malignant lymphocytes [Jones et al, 2004a,b] and similar effect was observed for DNA topoisomerase I [Bruno et al, 1992], ADP-ribosyl transferase [Scovassi et al, 1987], and nuclear antigen p105 [Clevenger et al, 1987].…”

Differential involvement of DNases in HeLa cell apoptosis induced by etoposide and long term-culture

“…The protocol implies SDS ± PAGE, in situ renaturation of proteins, incubation of the gel with 32 P-NAD, removal of non-incorporated substrate and autoradiography. 52,53 The radioactive band at 116 KDa represents automodified PARP.…”