The DNA methylation inhibitor 5-azacytidine modulates 6-thioguanine toxicity in mammalian cells.

In order to assess the effects of combining two antimetabolites used separately to treat human leukemias, we carried out an experimental study by exposing V79 Chinese hamster cells, a 6-thioguanine (6-tG)-sensitive cell line, to sequential and concurrent treatments with 5-azacytidine (5-azaC) and 6-tG. In this paper, we demonstrate that there is a clear dependency for the way in which this combination was tested. Pre-treatment with 5-azaC made V79 cells more resistant to 6-tG by a substantial reduction in 6-tG incorporation into DNA; this effect could still be detected for several cell divisions after the removal of 5-azaC, and was achieved neither by reduced cell growth nor by the induction of hypoxanthine-guanine-phosphoribosyltransferase (HGPRT-) mutants. The reverse order of treatment produced a higher toxic effect than exposure to each prodrug alone.

[The mutagenic action of gamma radiation on Chinese hamster cells. The cytogenetic characteristics of the mutants at the HPRT locus]. / Mutagennoe deistvie gamma-izlucheniia na kletki kitaiskogo khomiachka. Tsitogeneticheskaia kharakteristika mutantov po lokusu HPRT.

The peculiarities of mutation induction and cytogenetic characteristics of spontaneous and radiation-induced HPRT mutant clones have been studied. The linear-quadratic dependence of the mutation induction on radiation dose has been found. High heterogeneity of cytogenetic parameters (aneuploidy and chromosome aberration frequency) has been shown in the mutants.

[Plasmid pSVc-myc-1 induces gene mutations and chromosome aberrations in cultured Chinese hamster cells]. / Plazmidnye pSVc-myc-1 indutsiruet gennye mutatsii i khromosomnye aberratsii v kul'tiviruemykh kletkakh kitaiskogo khomiachka.

The induction of mutations to 6-thioguanine-resistance and chromosome aberrations by the plasmid pSVc-myc-1, carrying the activated cellular oncogene c-myc, isolated from a mouse plasmocytoma was studied in a cultured Chinese hamster cell line. The yield of HPRT- mutants and chromosome aberrations increased 1.6 times on the average after pSVc-myc-1 treatment. The mutagenic activity of pSVc-myc-1 was statistically significant. The role of mutagenic effects of activated cellular oncogenes in malignant transformations is discussed.

[The effect of prolonged gamma irradiation at a decreasing dose rate on survivability and gene mutation induction in cultured Chinese hamster cells]. / Vliianie protiazhennogo gamma-oblucheniia so spadaiushchei moshchnost'iu dozy na vyzhivaemost' i induktsiiu gennykh mutatsii v kul'tiviruemykh kletkakh kitaiskogo khomiachka.

Continuous gamma irradiation at decreasing dose rate was shown to be less effective than acute exposure with regard to the lethal effect and frequency of mutations of resistance to 6-thioguanine in cultured Chinese hamster cells. The cell population subjected to continuous irradiation was more radioresistant than the intact one. Lethal and genetic effects of continuous irradiation at decreasing dose rate were mainly determined by the contribution of the radiation dose received during the first 24 h of exposure.

Inhibition of metabolic coupling by metals.

Several metals were evaluated in cell cultures for their ability to inhibit metabolic coupling, the intercellular transfer of low-molecular-weight metabolites by directly connecting gap junctions. To monitor inhibition of metabolic coupling, wild-type Chinese hamster V79 cells proficient in metabolism of 6-thioguanine (6-TG) were cocultured with mutant V79 cells unable to metabolize 6-TG to its toxic metabolite (6-TG-resistant cells). In the presence of 6-TG, inhibition of metabolic coupling by the metals was manifested as increased recovery of 6-TG-resistant cells compared to recovery in untreated cocultures. The toxic metal compounds, arsenic(V) acid, mercury(II) chloride, lead(II) acetate, and nickel(II) chloride, significantly (p less than .05) increased recovery of 6-TG-resistant cells at concentrations that did not alter cell survival. However, because the increased recovery observed with nickel showed no concentration dependence, its importance may be negligible. Cadmium chloride increased 6-TG-resistant cell recovery only at a toxic concentration, and zinc sulfate failed to increase recovery. These data demonstrate that some metal compounds inhibit metabolic coupling, and suggest that inhibition of junctional communication should be further evaluated as a potential mechanism of toxicity of some metals.

Biochemical basis of the prevention of 6-thiopurine toxicity by the nucleobases, hypoxanthine and adenine.

Co-incubation of human leukemia cell lines with naturally occurring nucleobases (hypoxanthine or adenine) significantly prevented the cytotoxic activity of 6-thiopurines. Extracellular hypoxanthine decreased the transport of 6-mercaptopurine into cells, but adenine had no significant effect. However, intracellular thioinosine monophosphate accumulation in the presence of 10 microM, 6-mercaptopurine was reduced to below 1% or 10% of that of the controls when 50 microM hypoxanthine or adenine was added, respectively. Finally, in adenine phosphoribosyl transferase deficient mutants, adenine provided no protective effect against 6-thiopurines, whereas hypoxanthine retained its modulating activity. These data suggest that the nucleobases compete with 6-thiopurines for the ribose-phosphate donor, 5'-phosphoribosyl-1-pyrophosphate, thus preventing the formation of active metabolites of 6-thiopurines.

Modulation of 6-thioguanine activity by guanine in human promyelocytic leukemia HL-60 cells.

The effects of guanine coadministration on the metabolism and biological activity of 6-thioguanine (6-TG) were studied in human promyelocytic leukemia cells (HL-60). Cell growth, cytotoxicity (cloning assay), and cell differentiation were measured, along with nucleotide metabolism. Guanine was efficiently salvaged by HL-60 cells; at 200 microM, guanine suppressed the formation of 6-TG mononucleotides and abolished 6-TG incorporation into nucleic acids. Similarly, guanine antagonized 6-TG cytotoxicity in a dose dependent fashion. Furthermore, guanine (200 microM) fully suppressed the 6-TG (10 microM) induced HL-60 cell differentiation, which suggests that cell differentiation at pharmacological 6-TG concentrations is dependent on the anabolism of the drug to active nucleotides. 6-TG given alone reduced GTP levels and DNA synthesis rates in HL-60 cells, while a major intracellular 6-TG metabolite, 6-thioguanosine 5'-monophosphate, accumulated to high levels (approximately 100 microM). It is suggested that accumulation of 6-thioguanosine 5'-monophosphate and a resultant partial block of the de novo biosynthesis of guanine nucleotides is responsible for 6-TG induced cell differentiation in HL-60 cells.

[RAMT cell line as a substrate for the propagation of vaccinal viral strains]. / Liniia kletok RAMT kak substrat dlia razmnozheniia vaktsinnykh shtammov virusov.

Properties of the PAMT cell line which are deficient in hypoxanthine guanyl phosphoribosiltransferase were studied. The PAMT cell line derived from African green monkey kidney tissue may be identified by selective nutrient media, contains no contaminants, is not tumorigenic, is susceptible to a wide range of viruses. Titres of poliomyelitis, tick-borne encephalitis, and carnivore plague viruses were similar in cell cultures grown in the medium with 10% serum and in those adapted to growth in the medium containing 1% serum. The properties of PAMT cell line allow it to be used in manufacture of killed virus vaccines.

Protection of normal hematopoietic stem cells from the toxicity of purine base analogs: in vivo application.

Normal murine bone marrow cells were incubated with adenosine (100 microM) for 2 hrs followed by the addition of 6-thioguanine (6-TG) (0.3 and 1.0 microgram/ml) for 1, 24, and 48 hrs, and the survival of the committed stem cells (CFUc) and pluripotential stem cells (CFUs) was compared to the survival of CFUc and CFUs which were similarly incubated without adenosine. When 0.3 microgram/ml of 6-TG was used, preincubation with adenosine increased the survival of CFUc from 25% to 80% and from 14% to 36% for 24- and 48-hr incubations, respectively (P less than or equal to 0.005). For 1 microgram/ml of 6-TG, pretreatment with adenosine increased survival from from 11% to 69% and from 5% to 24%, respectively, for 24- and 48-hr incubations (P less than or equal to 0.0005). Adenosine had a similar effect on CFUs (P less than or equal to 0.03). This effect was not seen when equimolar concentrations of hypoxanthine, inosine, or adenine were substituted for adenosine. Inoculation of adenosine into mice (500 mg/kg of body weight) 1 hr prior to injection of lethal doses of 6-TG (50 mg/kg) increased the survival of the mice from 10% to 100% and from 20% to 80%, respectively, using two administration schedules. This effect of adenosine, which, as has been previously shown, is the result of the depletion of cellular phosphoribosyl pyrophosphate pools, is potentially useful for the selective protection of normal hematopoietic cells from the toxicity of purine analogs which require activation via a phosphoribosyl pyrophosphate-dependent reaction, while therapeutic effects are produced against malignant tissue.

[Hereditary changes in gene activity as 1 of the causes of phenotypic heterogeneity in 8-azaguanine-resistant Chinese hamster cells (CHO-K1)]. / Nasleduemye izmeneniia gennoi aktivnosti kak odna iz prichin fenotipicheskoi geterogennosti ustoichivykh k 8-azaguaninu kletok kitaiskogo khomiachka (CHO-K1).

Eight Chinese hamster clones (CHO-K1) growing at the 30 mg/ml concentration of 8-azaguanine (AG) were studied. Clones were differentiated by their resistance to AG and to 6-thioguanine, by their plating efficiency on HAT medium, and by the level of hypoxantine incorporation in cells. The differences in phenotypic properties were shown to be associated with variability in hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity. HPRT Michaelis constant (KM) for hypoxanthine and phosphoribosylpyrophosphate, and maximal reaction rate (Vm) offered considerable differences between all the resistant clones and sensitive cells. The only possible reason of these differences is a change in the HPRT coding locus. According to the results of the analysis of B15-4b-4 subclones, phenotypic and HPRT activity differences are also connected with each other; however, all subclones have the same KM of HPRT as that of the parental clone. So, differences in HPRT activity (and in Vm) may reflect changes in the HPRT content in cells of subclones. Hence, phenotypic heterogeneity of AG-resistant clones is determined by the interaction of mutational changes in the HPRT locus, and hereditable changes of genetic activity, responsible for variation of HPRT quantity in cells.

[Patterns in the x-ray induction of mutations in resistance to 5-thioguanine in Chinese hamster cell cultures studied using cysteamine]. / Izuchenie zakonomernostei induktsii rentgenovymi luchami mutatsii rezistentnosti k 6-tioguaninu v kul'turakh kletok kitaiskogo khomiachka v usloviiakh primeneniia tsisteamina.

X-ray induction of 5-thioguanine resistance mutations and the protective effect of cysteamine were studied in cultured cells of chinese hamster clone 431. Expression of mutations was studied for 8--10 days of post-irradiated growth, when cells surviving radiation at doses 200, 400, 600, 800 R were passing from 3 to 12 generations. The maximum frequency of induced mutations was found after 3-5 generations. After reaching the maximum, the number of registered mutations decreased with the number of cell generations. Pre-irradiated treatment with 5mM cysteamine decreased induction of mutations 2--4 times when using doses 200--800 R. The frequency of mutations induced by mere radiation increased non-linearly with increasing doses, while in the presence of cysteamine the linear dose dependence was observed.

Biochemical approaches to enhancement of antitumor drug selectivity: selective protection of cells from 6-thioguanine and 6-mercaptopurine by adenosine.

The cytotoxicity of 6-thioguanine and 6-mercaptopurine to cultured lymphoblasts and fibroblasts was strongly antagonized by pretreatment of the cells with 100 microM adenosine. Administration of adenosine 2 hours after the antipurine agent did not cause antagonism. In two rat hepatoma cell lines, adenosine pretreatment did not protect cells from the antipurines. Treatment of lymphoblasts or fibroblasts with 100 microM adenosine gave increases up to 150% in cellular ATP and ADP and decreases greater than 80% in UTP and UDP. In the hepatoma lines, adenine nucleotides did not increase by greater than 45%, and uridine nucleotides did not decrease by greater than 40% following adenosine treatment. The selective protection of the normal cells from 6-thioguanine and 6-mercaptopurine was probably the consequence of phosphoribosylpyrophosphate (PRPP) depletion, since adenosine pretreatment decreased PRPP pools by greater than 90% in the normal cells but by only 30% in the malignant hepatoma cells. In the absence of PRPP the antipurines would not be metabolically activated. The selectivity of the adenosine and antipurine combinations was probably attributable to the low activity of adenosine kinase and high activities of adenosine deaminase and PRPP synthetase characteristic of malignant hepatomas.

6-thioguanine: antimitotic effect on human lymphocytes in vitro prevented by adenine.

The action of 6-thioguanine on the division of PHA-stimulated human lymphocytes in vitro has been investigated and shown to have a definite antimitotic effect which is prevented by adenine but not by guanine or hypoxanthine. It is suggested that in this test system 6-TG exerts its main inhibitory action on phosphoribosylpyrophosphate amidotransferase.