Fredericamycin is an antibiotic product of Streptomyces griseus that exhibits modest antitumor activity in vivo and in vitro. Because of its unique structure and the absence of a clearly defined mechanism of action, we examined the effects of this compound on L1210 cells in culture as well as on several enzymes that bind to DNA. Fredericamycin exhibits an IC50 of 4.4 microM toward L1210 cells, and its cytotoxicity is a function of the time of exposure as well as drug dose. No DNA breakage was observed in L1210 cells or isolated nuclei following exposure to highly lethal concentrations of fredericamycin. As a first step toward understanding its mechanism of action, we examined the effect of fredericamycin on several enzymes involved in DNA metabolism. The catalytic activity of both DNA topoisomerases I and II were totally inhibited by fredericamycin concentrations of 4.4 and 7.4 microM, respectively. Fredericamycin blocked etoposide-stimulated DNA cleavage by topoisomerase II both in vitro and in isolated nuclei. In addition, the drug inhibits DNA polymerase a in vitro, exhibiting an IC50 of 93 microM. These diverse actions of fredericamycin do not enable us to draw conclusions regarding its mechanism of antitumor effect but clearly identify it as a compound of pharmacologic interest.
The nuclear enzyme, topoisomerase II, is the major site of action for cancer chemotherapy agents such as etoposide, teniposide, and a variety of intercalating agents. These compounds cause the enzyme to cleave DNA, forming a DNA-protein complex that may be a key step leading to cell death. It is apparently unique as a chemotherapy target, since drug potency diminishes with decreasing enzyme activity. It was thus of interest to examine the topoisomerase content and drug-induced DNA cleavage in freshly obtained human leukemia cells and to compare the obtained data with the results of similar studies performed in well-characterized human leukemia cell lines. The human T-lymphoblast line, CCRF-CEM, was more than 100-fold more sensitive to the DNA-cleavage effect of etoposide than the cells of the 13 leukemic patients examined. One of the leukemia lines (HL-60) and a lymphoblastoid line (RPMI-7666) were somewhat less sensitive than cells of the CCRF-CEM cells, but were still 10-fold more sensitive than the patients studied. The relative insensitivity of the freshly obtained cells could not be accounted for by differences with respect to drug uptake but were associated with markedly reduced topoisomerase-II content as assayed by immunoblotting using a mouse polyclonal serum against topoisomerase II. Heterogeneity was observed in the sensitivities of patients' cells with respect to both drug-induced DNA cleavage and enzyme content. The observed differences between cultured cell lines and patients' cells may have been related to their proliferative status. Etoposide potency in normal resting lymphocytes resembles that observed in circulating leukemia cells. However, following mitogenesis with phytohemagglutinin and interleukin-2, proliferating lymphocytes become as sensitive to etoposide as cultured cell lines with regard to DNA cleavage. This effect was accompanied by an increase in topoisomerase-II content. Our data thus support the hypothesis that topoisomerase-II content may be an important determinant of cell sensitivity to certain classes of chemotherapy agents. Efforts to stimulate topoisomerase-II content may improve the therapeutic efficacy of these drugs.
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