The role of benzodiazepine receptors in the induction of differentiation of HL-60 leukemia cells by benzodiazepines and purines.
A series of benzodiazepines was evaluated for their capacity to induce the differentiation of HL-60 acute promyelocytic leukemia cells. Benzodiazepines were effective initiators of maturation in the concentration range of 50 to 150 microM. The possible involvement of benzodiazepine receptors in mediating the differentiation induced by these agents was investigated. The presence of high affinity, peripheral type benzodiazepine binding sites (KD = 7.3 nM, TB = 14.5 pmol/mg protein with Ro5-4864) was demonstrated in HL-60 membranes. The occupancy of peripheral type high affinity benzodiazepine receptors by various benzodiazepines showed some correlation (r = 0.76) with their differentiation-inducing capabilities, but binding potencies were 1,000-fold higher than the concentrations required to produce differentiation. A class of benzodiazepine receptors with lower binding affinity was also detected in HL-60 membranes (KD = 28.6 microM; TB = 199 pmol/mg protein with diazepam). A higher level of correlation (r = 0.88) was demonstrated between benzodiazepine occupancy of these lower affinity receptors and the capacity to induce maturation. Significantly, benzodiazepine concentrations needed for low affinity binding and induction of differentiation were the same (25-200 microM), suggesting that low affinity benzodiazepine receptors may be involved in the induction process. We have shown that the molecular form responsible for the induction of the differentiation of HL-60 cells to mature forms by 6-thioguanine (TGua) is the free base, TGua, itself [Ishiguro, Schwartz, and Sartorelli (1984) J. Cell. Physiol., 121:383-390]. Since hypoxanthine (Hyp) and inosine (Ino) have been identified as putative endogenous ligands for high affinity benzodiazepine receptors in brain tissue, the potential involvement of benzodiazepine receptors in the differentiation of HL-60 cells by the purines was investigated. Physiological purines such as Hyp and Ino were inactive in displacing the benzodiazepines from their high and low affinity binding sites in HL-60 membranes. In contrast, TGua caused inhibition of benzodiazepine binding to high and low affinity sites. The inhibition of Ro5-4864 binding to high affinity binding sites by TGua appeared to be due to the binding of TGua to membranes through the formation of a mixed disulfide between the 6-thiopurine and protein thiols, since the inhibition was reversed by the presence of 2-mercaptoethanol. The findings suggest a possible relationship between the occupancy of benzodiazepine receptors by TGua and the induction of leukemic cell differentiation.
D001569:Benzodiazepines / Q000494:pharmacology*; D001570:Benzodiazepinones / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D006801:Humans; D007546:Isoquinolines / Q000378:metabolism; D015470:Leukemia, Myeloid, Acute / Q000473:pathology*; D011687:Purines / Q000494:pharmacology*; D011720:Pyrazoles / Q000378:metabolism; D011724:Pyridazines / Q000378:metabolism; D011963:Receptors, GABA-A / Q000502:physiology*
D001570:Benzodiazepinones; D007546:Isoquinolines; D011687:Purines; D011720:Pyrazoles; D011724:Pyridazines; D011963:Receptors, GABA-A; D001569:Benzodiazepines; C028513:4'-chlorodiazepam; C021604:CL 218872; C033830:2-phenylpyrazolo(4,3-c)quinolin-3(5H)-one; C037850:PK 11195