Influence of cell cycle and oncogene activity upon topoisomerase II alpha expression and drug toxicity

The cell cycle, oncogenic signaling, and topoisomerase (topo) II alpha leve ls all influence sensitivity to anti-topo II drugs. Because the cell cycle and oncogenic signaling influence each other as well as topo II alpha level s, it is difficult to assess the importance of any one of these factors ind ependently of the others during drug treatment. Such information, however, is vital to an understanding of the cellular basis of drug toxicity. We, th erefore, developed a series of analytical procedures to individually assess the role of each of these factors during treatment with the anti-topo II d rug etoposide. All studies were performed with asynchronously proliferating cultures by the use of time-lapse and quantitative fluorescence staining p rocedures. To our surprise, we found that neither oncogene action nor the c ell cycle altered topo II alpha protein levels in actively cycling cells. O nly a minor population of slowly cycling cells within these cultures respon ded to constitutively active oncogenes by elevating topo II alpha productio n. Thus, it was possible to study the effects of the cell cycle and oncogen e action on drug-treated cells while topo II alpha levels remained constant , Toxicity analyses were performed with two consecutive time-lapse observat ions separated by a brief drug treatment. The cell cycle phase was determin ed from the first observation, and cell fate was determined from the second . Cells were most sensitive to drug treatment from mid-S phase through G(2) phase, with G(1) phase cells nearly threefold less sensitive. In addition, the presence of an oncogenic src gene or microinjected pas protein increas ed drug toxicity by approximately threefold in actively cycling cells and b y at least this level in the small population of slowly cycling cells. We c onclude that both cell cycle phase and oncogenic signaling influence drug t oxicity independently of alterations in topo II alpha levels.