A variant P388 murine leukemic cell resistant to 4′-demethylepipodophyllotoxin-9-(4,6-O-ethylidene)-β-d-glucopyranoside (etoposide) (VP-16-213) was cloned. The variant P388/VP-16 cell line was 159-fold resistant to 4′-demethylepipodophyllotoxin-9-(4,6-O-ethylidene)-β-d-glucopyranoside and showed cross-resistance to vincristine (18.9-fold) and Adriamycin (522.9-fold), determined by comparing the 50% inhibitory concentrations in a 48-h growth inhibition assay. To identify the possible role of Ca2+-phospholipid-dependent protein kinase (protein kinase C) in this drug resistance, we studied the specific phorbol ester binding component and protein kinase C in the parent and drug-resistant sublines of P388 cells. The phorbol ester receptor, as expressed by the numbers of sites per cell, significantly decreased in P388/VP-16 (57.6% of control). Scatchard analysis revealed that the variant contained a single class of binding sites. However, no difference was observed in the dissociation constants (Kd), thereby suggesting much the same affinity of receptors between the two lines. Phorbol diester analogues inhibited [20-3H]phorbol-12,13-dibutyrate binding of both the variant and control cell lines, in a stereospecific manner and consistent with their binding potency. The activity of protein kinase C, which is related to the phorbol ester receptor, significantly decreased in the variant cell. The enzyme activity, particularly in the membrane fraction of P388/VP-16 cells, was remarkably decreased. These data suggest that the decrease in the specific phorbol diester receptor and protein kinase C in the variant cells might correlate with the pleiotropic drug resistance.

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Supported in part by a Grant-in-Aid (60440050) for scientific research from the Ministry of Education, Science, and Culture of Japan.

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