In vitro effects of bryostatin 1 on the metabolism and cytotoxicity of 1-beta-D-arabinofuranosylcytosine in human leukemia cells.
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)
Grant
S
S
Division of Hematology/Oncology, Medical College of Virginia, Richmond 23298.
Boise
L
L
Westin
E
E
Howe
C
C
Pettit
G R
GR
Turner
A
A
McCrady
C
C
D000970:Antineoplastic Agents / Q000494:pharmacology*; D001085:Arabinofuranosylcytosine Triphosphate / Q000378:metabolism; D054713:Bryostatins; D002452:Cell Count; D003561:Cytarabine / Q000378:metabolism* / Q000633:toxicity; D004273:DNA, Neoplasm / Q000187:drug effects / Q000378:metabolism; D004224:Diterpenes; D006801:Humans; D007700:Kinetics; D007783:Lactones / Q000494:pharmacology*; D007942:Leukemia, Experimental / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D007951:Leukemia, Myeloid / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D018942:Macrolides; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation; D013729:Terpenes / Q000494:pharmacology; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D054713:Bryostatins; D004273:DNA, Neoplasm; D004224:Diterpenes; D007783:Lactones; D018942:Macrolides; D013729:Terpenes; D003561:Cytarabine; D001085:Arabinofuranosylcytosine Triphosphate; C011309:mezerein; D015240:Phorbol 12,13-Dibutyrate; C046785:bryostatin 1