Bryostatin 1 is a macrocyclic
lactone protein kinase C (PK-C) activator which has demonstrated promising antileukemic activity in preclinical studies. We have examined the effect of this agent on the metabolism and cytotoxicity of 1-beta-D-arabinofuranosylcytosine (
ara-C) in both log phase and high-density human promyelocytic
leukemia cells (HL-60). Exposure of low-density cells to 12.5 nM
bryostatin 1 for 24 hr prior to a 4-hr incubation with 1 or 10 microM
ara-C resulted in nearly a 2-fold increase in
ara-CTP formation. When cells were maintained under high-cell density conditions (e.g. 5 x 10(6) cells/mL) for 24 hr prior to
ara-C exposure, a 90% reduction in
ara-CTP formation and
ara-C DNA incorporation was observed. However, coincubation of high-density cells with
bryostatin 1 for 24 hr increased
ara-CTP formation 6- to 8-fold, yielding levels essentially equivalent to those achieved in low-density cells. Smaller (but still significant) increases in
ara-C DNA incorporation were also noted. Enhancement of
ara-CTP formation by
bryostatin 1 occurred over a broad
ara-C concentration range (0.1 to 100 microM), involved a temperature-dependent process, could not be mimicked by addition of hematopoietic
growth factors, and was not related to neutralization of toxic or inhibitory substances in high-density medium. Exposure of cells to
bryostatin 1 did not lead to morphologic or functional evidence of HL-60 cell maturation or an increase in cell viability, but did produce a decline in cellular proliferative activity as determined by
thymidine and
bromodeoxyuridine incorporation and cytofluorometric analysis.
Bryostatin 1 did not exert its effects in high-density cells by inhibiting
ara-C deamination or by interfering with
ara-CTP dephosphorylation, but instead appeared to act by enhancing
ara-C phosphorylation. Although cell-free extracts obtained from high-density cells exposed to
bryostatin 1 exhibited levels of
deoxycytidine kinase activity compared to controls, treated cells did display a significant decline in intracellular
dCTP levels (e.g. 0.7 vs 1.3 pmol/10(6)), and nearly a 2-fold increase in
ATP and
UTP concentrations.
Ara-CTP formation was also increased substantially by other PK-C activators including
phorbol dibutyrate and
mezerein (10-100 nM); this process was inhibited more than 70% by the PK-C inhibitor
H-7 (50 microM), but not by the PK-C inhibitors
staurosporine,
tamoxifen, and HA1004. Finally, coadministration of
ara-C and
bryostatin 1 resulted in greater than expected inhibitory effects toward HL-60 cell clonogenic growth.(ABSTRACT TRUNCATED AT 400 WORDS)