Establishment of a murine leukaemia cell line resistant to the growth-inhibitory effect of bryostatin 1

Abstract

Bryostatin 1 is a novel macrocyclic lactone activator of protein kinase C (PKC) which has clinical potential as an anti-cancer agent. The mechanism of action of this agent is unknown, but protein kinase C has been implicated. In order to investigate this possibility, we have developed P388 sublines resistant to bryostatin 1, by continuous challenge of the parent cell line with increasing incremental concentrations of the drug over 4 months. Cell lines were established at monthly intervals yielding four sublines: P388/BR/A, which were removed at 1 month; P388/BR/B, obtained after 2 months; P388/BR/C, obtained after 3 months; and P388/BR/D, which were established after 4 months. All four P388/BR sublines show an equal degree of resistance to the growth inhibitory effects of bryostatin 1, with a relative resistance ratio (RR) IC50 of approximately 4,000. The ability of the cytosol of cells to phosphorylate PKC-specific substrate is decreased by 41% for BR/A, 57% for BR/B 80% for BR/C and 94% for BR/D compared with the parental cell line, even when grown in the absence of bryostatin 1 for up to 4 weeks. Similar decreases are seen for cytosolic phorbol ester binding and whole-cell PKC isoenzyme expression. All four P388/BR sublines show high and equal levels of cross-resistance to the PKC activatory phorbol ester, phorbol 12-myristate 13-acetate (PMA). There is no loss of resistance to either bryostatin 1 or PMA up to 3 months after termination of exposure of the sublines to bryostatin 1. There was no significant degree of cross-resistance to daunorubicin in the bryosatin 1-resistant cell lines, P388/BR/A, B, C or D, when compared with the parent cell line, P388.