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Effect of Phorbol Ester Treatment on Drug-induced, Topoisomerase II-mediated DNA Cleavage in Human Leukemia Cells¹

Departments of Medical Oncology [L. A. Z., D. C., M. H., J. M., L. E. S.] and Clinical Immunology and Biologic Therapy [M. B.], Division of Medicine, The University of Texas, M. D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas 77030

Tumor-promoting phorbol esters such as phorbol 12-myristate 13-acetate (PMA) induce the monocytoid differentiation of HL-60 human leukemia cells. The cellular receptor for PMA is protein kinase C. However, cellular events distal to protein kinase C phosphorylation are also critical steps toward differentiation. These events may include specific programs of oncogene transcription that have been associated with phorbol ester-induced leukemic cell differentiation. Recently, it has been found that topoisomerase II could be activated by protein kinase C-mediated serine phosphorylation and that PMA treatment of HL-60 cells enhanced extractable topoisomerase II from these cells. Additionally, topoisomerase II-reactive antineoplastic drugs could block PMA-induced differentiation of HL-60. This enzyme has been implicated in gene regulation, and drug-induced, topoisomerase II-mediated DNA cleavage sites have been identified within cellular oncogenes. Thus, topoisomerase II could play a critical role in the signal transduction cascade leading from PMA-protein kinase interaction to monocytoid differentiation. We have examined this relationship between topoisomerase II and PMA-induced differentiation through measurements of drug-induced, topoisomerase II-mediated DNA cleavage (via alkaline elution) in PMA-treated HL-60 cells. Etoposide-induced DNA cleavage was reduced 10-fold in HL-60 cells treated with 10 nM PMA for 24 h. Neither dimethyl sulfoxide (which produces granulocytoid differentiation) nor non-differentiation-inducing phorbol esters could produce this effect. The decreased cleavage was not due to a PMA-induced inhibition of cell-associated etoposide and was demonstrable in nuclei isolated from PMA-treated cells. The decrease was not simply related to decreased cellular proliferation rate as reflected in the inhibition of DNA synthesis because conditions leading to marked inhibition of DNA synthesis did not necessarily inhibit etoposide-induced DNA cleavage. By contrast, lower concentrations of PMA inhibited etoposide-mediated DNA cleavage disproportionately compared with PMA effects on DNA synthesis. Interestingly, PMA reduced cleavage induced by the topoisomerase II-reactive DNA intercalator 4'-(9-acridinylamino)methanesulfon-m-anisidide by 2-fold, suggesting that specific drug-DNA interactions could partially overcome the PMA-induced effect that resulted in decreased etoposide-induced, topoisomerase II-mediated DNA cleavage. Nuclear proteins in 0.35 M NaCl extracts from untreated or PMA-treated HL-60 cells were virtually identical in topoisomerase II activity and in topoisomerase II-associated drug sensitivity. This suggested that it was PMA-induced changes in cellular chromatin rather than in topoisomerase II itself that resulted in an altered chromatin-drug or chromatin-topoisomerase II interaction. This chromatin structural change, however, could not be demonstrated to have occurred within the c-myc oncogene despite the marked transcriptional down-regulation of c-myc following PMA treatment. However, prior work had suggested that the transcriptional inactivation of this gene may not be accompanied by alterations in chromatin structure. This system is particularly useful in defining whether specific nuclear events, such as altered programs of gene transcription, are mechanistically related to differentiation and whether topoisomerase II may have a role therein.

¹ This work was supported by USPHS Grant CA40090 from the National Cancer Institute, Grant CH-324 from the American Cancer Society, NIH Grant RR5511-23, and a gift to the M. D. Anderson Annual Fund for the Chemotherapy Research Program from Mr. Henry C. Beck, Jr., of Dallas, TX.

² To whom requests for reprints should be addressed, at Box 52, 1515 Holcombe Blvd., Houston, TX 77030.

Received 3/23/88. Revised 6/29/88. Revised 8/25/88. Accepted 9/ 1/88.

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