Induction of Apoptosis and Cell Cycle-specific Change in Expression of p 53 in Normal Lymphocytes and MOLT-4 Leukemic Cells by Nitrogen Mustard 1

DNA damage in the cell activates expression of the p53 tumor suppressor gene, whose role is associated with cell arrest in G1 or apoptosis. The aim of this study was to examine the cell cycle position-related changes in expression of p5.3, as well as induction of apoptosis, in mitogen-stimubated normal human lymphocytes and in human leukemic MOLT-4 cells (which express mutated p13), following DNA damage by the alkybating agent nitrogen mustard. Measurement ofp53 expression and DNA content by flow cytometry followed by bivariate analysis of the data made it possible to correlate the drug-induced changes in p13 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 normab lymphocytes to 5 p M nitrogen mustard caused their arrest in G1, an increase in p53 expression which was maximal in such cells, and significant apoptosis in cells located beyond the arrest point (S and G2 + M cells). In contrast, neither arrest in G, nor significant apoptosis of MOLT-4 cells was seen after administration of either 0.5 or 5 p.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 G2 + M MOLT-4 cells in response to the nitrogen mustard. Although a severalfold lower level ofp53 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 G1 cells, causes cell arrest in G1, and induces apoptosis of the Received 2/16/95; revised 3/7/95; accepted 3/14/95. 1 This work was supported by National Cancer Institute Grant CA 28704, and by the ‘ ‘This Close” Foundation for Cancer Research. 2 To whom requests for reprints should be addressed, at Cancer Research Institute, New York Medical College, 100 Grasslands Road, Ebmsfomd, NY 10523. cells that either were in the S-phase at the time of drug administration and/or escaped G1 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, nor a trigger for immediate apoptosis. INTRODUCTION The p53 tumor suppressor gene is widely mutated in human tumors (1, 2). The product of this gene, p53 (wt3) is a key participant in the cellular response to DNA damage (3-5). Depending on the cell type and the extent of damage, p.53 plays a key robe either in halting cell progression through G1 on in triggering the process leading to apoptosis (6-10). It has been shown (3, 4) that the p53 protein level increases via posttmanscriptional mechanisms in response to agents that cause DNA damage. These studies, however, were done in bulk, using biochemical methods that do not allow analysis of individual cells (4) or by single parameter flow cytometry, which preeludes determination of cell cycle phase-specific changes in p53 expression (3). Thus, neither the heterogeneity of cell response nor a direct relationship of the responding cells to their position in the cell cycle could be assessed. In the present study, we have used flow cytometry combined with bivaniate p53/ DNA content analysis to address this issue. Mitogen (PHA)stimulated normal human lymphocytes were chosen as an cxample of cells expressing wt p53 and were compared to human lymphocytic leukemic MOLT-4 cells carrying mutant p.53 ( 1 1). An alkylating agent, NM, was used as the DNA damaging agent. In a previous study (12), this drug was shown to induce apoptosis of HL-60 cells indiscriminately, regardless of cell cycle