Influence of Cell Cycle and Oncogene Activity upon Topoisomerase IIα Expression and Drug Toxicity

Topoisomerase (topo) II enzymes function to unknot and decatenate covalently closed circles of DNA. In mammalian cells a requirement for type II topo has been suggested in many aspects of DNA metabolism including replication and recombination (41). There are two isozymes of topo II with molecular masses of 170 and 180 kDa, termed topo IIα and topo IIβ (7). While the biochemical activities of the two proteins are closely related, their cellular distribution and expression characteristics differ greatly. topo IIα is expressed at low levels in quiescent cells and is induced when cells are stimulated to enter the growth phase (32). As cells become contact inhibited, the levels of topo IIα are dramatically reduced (2, 22, 24). On the other hand, topo IIβ expression does not correlate with proliferative status, remaining at constant levels in quiescent and proliferating cells (32, 39). topo IIα is known to be expressed at high levels in tumors at both the protein and mRNA levels (19). This elevation is identified in a wide variety of tumors and is due in part to the increased growth fraction (3, 11, 17, 26). The expression of topo IIα is also increased as cells pass through the cell cycle (15), although reports vary as to the extent. In NIH 3T3 cells transformed by oncogenic ras (42) topo IIα levels increase, suggesting that oncogenic signaling might directly stimulate the topo IIα promoter, leading to increased protein levels in transformed cells. In support of this possibility, we found that oncogenic Ras is able to increase the expression of a reporter plasmid containing the basal topo IIα promoter driving a luciferase reporter gene (6). This increase, which requires ERK and JNK activities, was independent of cell cycle position (6). Anti-topo II drugs are used clinically against a wide range of tumors (18) and target both isotypes of topo II, although a broad range of evidence indicates that topo IIα is the primary target (4). A number of studies have identified a close relationship between topo IIα levels and drug sensitivity (9, 12, 14, 17, 30, 31). Similarly, drug resistance is commonly correlated with decreased topo IIα levels (10, 28). On the other hand, factors other than topo IIα levels have been found to influence toxicity, including drug uptake, topo IIα phosphorylation (13, 16), and cellular factors involved in activation of cell death pathways (25, 28). In order to better understand the interplay of cellular factors involved in controlling cellular response to drug treatment, we designed a series of experiments to analyze the roles of the cell cycle, oncogene action, and topo IIα levels independently of one another during treatment with an anti-topo II drug. While the level of topo IIα within the cell is believed to play a central role in determining drug toxicity (10, 36, 38, 40, 44), the considerations discussed above suggest that these levels might be altered by cell cycle position and oncogene activity. At the same time, these two physiological factors might directly influence drug toxicity independently of alterations in topo IIα levels (1, 23, 43). The challenge, therefore, is to separate the influence of the cell cycle and oncogene action on drug toxicity directly from their ability to alter topo IIα levels, and thereby to influence drug toxicity indirectly. To address this question, we have developed a means to study the cell cycle expression characteristics of topo IIα in the presence and absence of oncogenic signaling. In addition, we have developed a means to determine cell cycle-related drug toxicity. The technical approach used relied on studies of continuously cycling cells. No attempt was made to synchronize cells in the cell cycle; rather, the cell cycle position of each individual cell was determined using either quantitative fluorescence analysis of DNA or time-lapse observations to determine the timing of mitoses (37). This technical approach has proven effective in previous studies of cyclin D1, where the expression characteristics of this protein were accurately determined in each cell cycle phase, together with the dependence of this expression on cellular Ras activity (20, 21). In the present study the same approach was utilized to determine the effect of the cell cycle and oncogene activity on the expression levels of topo IIα. We were somewhat surprised to learn that neither of these factors altered topo IIα protein expression levels in actively cycling cells. It was, therefore, possible to accurately determine the influence of cell cycle position and oncogene activity on the outcome of drug treatment without concern for topo IIα protein expression levels.