Characterization of the metabolic forms of 6‐thioguanine responsible for cytotoxicity and induction of differentiation of HL‐60 acute promyelocytic leukemia cells

Abstract

HL-60 human acute promyelocytic leukemia cells that lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity have been developed by mutagenization and selection. These cells exhibited markedly decreased sensitivity to the cytotoxic action of 6-thioguanine (TG) and, in contrast to parental HL-60 cells, had the capacity to undergo terminal granulocytic differentiation after treatment with this purine antimetabolite. Analysis of extracellular and intracellular metabolites of TG revealed negligible metabolism of TG in these HGPRT- HL-60 cells. These findings are consistent with the concept that inhibition of cellular replication requires generation of analog nucleotide and suggest that TG itself is capable of initiation of differentiation. 6-Thioguanosine (TGuo) had limited activity, while beta-2'-deoxythioguanosine (dTGuo) was inactive, as an inducer of maturation of HGPRT- HL-60 cells. These cells converted relatively large amounts of the nucleosides to the free base TG; the simultaneous exposure of cells to 8-aminoguanosine (AGuo), an inhibitor of purine nucleoside phosphorylase activity, decreased the degradation of TGuo and dTGuo to TG and promoted the intracellular accumulation of TG nucleotides, presumably through the action of nucleoside kinase activities. In a double mutant deficient in both HGPRT and deoxycytidine kinase (DCK) activities, dTGuo was devoid of cytotoxicity and was an effective inducer of maturation. The potency of dTGuo as an inducer in this system was not significantly affected by the presence of AGuo. These results suggested that dTGuo itself was also an active initiator of maturation. Thus, induction of differentiation appeared to be due to the free base, TG, as well as its deoxynucleoside form, dTGuo, whereas the formation of TG nucleotides appeared to antagonize maturation and produce cytotoxicity.

Effect of 6-thioguanine on Chlamydia trachomatis growth in wild-type and hypoxanthine-guanine phosphoribosyltransferase-deficient cells

“…One way is that chlamydiae may well be starved for ATP and GTP because of the TGMP-induced inhibition of wild-type host cell de novo purine nucleotide biosynthesis. In agreement with earlier studies (6,17,30), we found, by high-pressure liquid chromatography analysis of acid-soluble extracts, that a substantial quantity of TGMP was formed and there was a decrease in ATP and GTP pools in TG-treated wild-type cells (data not shown). The decreased purine pools could inhibit chlamydial growth and DNA synthesis directly by depriving the parasite of nucleic acid precursors or indirectly by starving the parasite for host ATP, its only source of metabolic energy (15,25,28).…”

“…TG is at best a weak inducer of differentiation in wild-type HL-60 human acute promyleocytic leukemia cells and Friend murine erythroleukemia cells but is a highly effective inducer of maturation in HGPRT-mutant clones of these cell lines (13,17,19,31). The work of Ishiguro et al (17) confirmed that TG itself was capable of inducing the differentiation of HGPRT-HL-60 cells, while the wild-type cells metabolized the guanine analog to the expected cytotoxic nucleotides. The purine antimetabolites TG and AG are both inhibitors of and substrates for tRNA-guanine ribosyltransferase (9,10).…”

Guanine analog-induced differentiation of human promyelocytic leukemia cells and changes in queuine modification of tRNA.

“…It has been known for some time that guanine analogs can induce differentiation of HL-60 cells (7), but these same guanine analogs are also highly cytotoxic (17). Various studies showed that cells selected for 6-thioguanine resistance were deficient in HGPRT (13,14).…”

“…Since HGPRT deficiency should impair the formation of 6-thioguanine nucleotides, Gusella and Housman (14) proposed that the generation of these cytotoxic nucleotides and their subsequent incorporation into DNA are probably not involved in the differentiation sequence. Recent work by Ishiguro et al (17) verified that 6-thioguanine itself was capable of inducing the differentiation of HGPRT-deficient HL-60 cells, while wild-type HL-60 cells metabolized the guanine analog to the expected cytotoxic nucleotides. Although these studies demonstrated that 6-thioguanine was the metabolic form responsible for the induction of differentiation of HGPRT-deficient HL-60 cells, the molecular basis for this induction was not established.…”

A drug‐repositioning screen for primary pancreatic ductal adenocarcinoma cells identifies 6‐thioguanine as an effective therapeutic agent for TPMT‐low cancer cells

“…Once 6‐TG enters the cell, it is converted to 6‐thioguanine nucleotide (6‐TN), which is toxic (Ishiguro et al ., 1984). Its toxicity is mainly caused by the incorporation of 6‐TN into DNA during the S phase of the cell cycle (Lennard et al ., 1989), thereby indicating its effectiveness in cancer.…”