Induction of apoptosis and cell cycle-specific change in expression of p53 in normal lymphocytes and MOLT-4 leukemic cells by nitrogen mustard

DNA damage in the cell activates expression of the p53 tumor suppressor gene, whose role is associated with cell arrest in G(1) or apoptosis, The aim of this study was to examine the cell cycle position-related changes in expression of p53, as well as induction of apoptosis, in mitogen-stimulated normal human lymphocytes and in human leukemic MOLT-4 cells (which express mutated p53), following DNA damage by the alkylating agent nitrogen mustard, Measurement of p53 expression and DNA content by flow cytometry followed by bivariate analysis of the data made it possible to correlate the drug-induced changes in p53 expression in individual cells with their cell cycle position without the need for cell synchronization. Expression of p53 was detected immunocytochemically using the AB-6 mAb, which reacts with the product of the wild-type p53 tumor suppressor gene and with most of its mutated forms, Exposure of normal lymphocytes to 5 mu M nitrogen mustard caused their arrest in G,, an increase in p53 expression which was maximal in such cells, and significant apoptosis in cells located beyond the arrest point (S and G(2) + M cells), In contrast, neither arrest in G(1) nor significant apoptosis of MOLT-4 cells was seen after administration of either 0.5 or 5 mu M nitrogen mustard for up to 24 h, although the drug reduced the rate of cell progression in the S-phase at both concentrations, Expression of p53 was highest for S and G, + M MOLT-4 cells in response to the nitrogen mustard, Although a severalfold lower level of p53 was detected in lymphocytes compared to MOLT-4 cells prior to drug treatment, the relative increase in p53 expression in response to the drug was 2-fold higher in lymphocytes, These data suggest that DNA damage caused by nitrogen mustard provides a signal that results in stabilization of wild-type p53, preferentially in G(1) cells, causes cell arrest in G(1), and induces apoptosis of the cells that either were in the S-phase at the time of drug administration and/or escaped G(1) arrest, The increase in expression of mutated p53, in response to DNA damage, is unrelated to the cell cycle position, and neither provides a signal for cell arrest in G(1) nor a trigger for immediate apoptosis.