Enhancement of the differentiation-inducing properties of 6-thioguanine by hypoxanthine and its nucleosides in HL-60 promyelocytic leukemia cells

Previous work has shown that 6-thioguanine (TGua) is an effective inducer of differentiation of Friend and HL-60 leukemia cells which lack hypoxanthine-guanine phosphoribosyltransferase but is at best only weakly active in inducing maturation in parental wild-type cells. Studies in wild-type and mutant HL-60 cells have provided evidence that the free-base TGua is the form of this drug that induces differentiation, while the formation of TGua nucleotides leads to cytotoxicity and inhibits differentiation. To attempt to increase the potential of TGua to serve as an inducer of parental HL-60 leukemia cells, physiological purine and pyrimidine nucleosides were tested for their ability to protect HL-60 cells against TGua-induced cytotoxicity. Adenosine, deoxyadenosine, inosine, and deoxyinosine completely prevented the toxic action of the purinethiol, while guanosine and deoxyguanosine were only partially effective. The capacity of adenosine and deoxyadenosine to prevent the cytotoxicity of TGua was abolished by the inhibitor of adenosine deaminase, deoxycoformycin, implying that inosine and deoxyinosine were the active forms of the protecting agents. The protective activities of inosine and deoxyinosine appeared to depend on phosphorolysis catalyzed by purine nucleoside phosphorylase, since exogenously added hypoxanthine was as effective as inosine in reducing the cytotoxicity of the purine antimetabolite. Accumulation of TGua nucleotides in the acid-soluble fraction of HL-60 cells treated with TGua was significantly decreased by the presence of inosine. Inosine also served under these circumstances as a D-ribose 1-phosphate donor to TGua, as evidenced by its increased conversion to 6-thioguanosine. The prevention of the cytotoxicity of TGua by the simultaneous administration of hypoxanthine or its nucleosides resulted in an expression of the differentiation-inducing properties of TGua in HL-60 cells, as measured by the accumulation of nitroblue tetrazolium-positive cells. These findings support the concept that the processes of cytotoxicity and differentiation are separable events produced by different metabolic forms of the purine antimetabolite.     

Effects of adenosine 3'5'-cyclic monophosphoric acid on the morphology, growth and cell cycle of Earle's L cells

The morphology of Earle's L cells is radically altered after incubation with adenosine 3′5′-cyclic monophosphate (cyclic AMP) and cell growth is inhibited. The concentration of nucleotide required to completely inhibit cell growth depends on the initial cell density although the breakdown of cyclic AMP in the supernatant fluid to adenosine is independent of the cell density. A decrease in mitotic index is noted several hours after the addition of cyclic AMP, while a reduction in the fraction of cells in S is only observed after 2 days. Upon removal of the drug an increase in the number of cells synthetizing DNA (as detected autoradiographically) is observed well before the increase of mitotic rate. These results suggest that cells are blocked in moving through S to G2. Cells resume normal growth rates and morphology only 6–8 days after the removal of cyclic AMP, but can then be propagated indefinitely.     

Effects of 2-amino-1,3,4-thiadiazole on ribonucleotide pools of leukemia L1210 cells.

The effects of 2-amino-1,3,4-thiadiazole [aminothiadiazole (NSC 4728)] on purine and pyrimidine ribonucleotide pools of L1210 ascites cells in vivo are presented and discussed as they relate to the site of action. Within 1 hr after administration of the drug, the levels of guanosine triphosphate, guanosine diphosphate, adenosine triphosphate, and adenosine diphosphate were reduced, whereas those of inosine monophosphate (IMP) and uridine triphosphate were increased. The most pronounced effects were the lowering of guanine ribonucleotide pools and the elevation of IMP. Aminothiadiazole produced a marked inhibition (approximately 95%) of the incorporation of [8-14C]inosine into guanine nucleotides, whereas only a slight inhibition (approximately 20%) of incorporation into adenine nucleotides was observed. These results suggest that the thiadiazole (or a metabolite thereof) inhibits the conversion of IMP to guanosine monophosphate; this conclusion is reinforced by the observation that mycophenolic acid, a known inhibitor of this conversion, produced effects on ribonucleotide pools similar to those produced by aminothiadiazole. Aminothiadiazole did not inhibit IMP dehydrogenase isolated from L1210 cells. The effects of the thiadiazole on nucleotide pools were prevented by simultaneous administration of nicotinamide. Since nicotinamide is known to prevent or reverse the antileukemic activity of aminothiadiazole, it is probable that the inhibition of synthesis of guanosine monophosphate is related to the antileukemic action of this agent.     

Synergistic activity of purine metabolism inhibitors in cultured human tumor cells

Cytotoxic effects of 3-deazaguanosine (3-DGUO) result from the inhibition of DNA synthesis and incorporation of the drug into DNA. Synergistic antiproliferative effects of a combination of 3-deazaguanosine and 2-beta-D-ribofuranosylthiazole-4-carboxamide, a potent inhibitor of inosine monophosphate dehydrogenase, was observed in human tumor cells. Inosine reversed the antiproliferative effects of the 3-deazaguanosine but not 2-beta-D-ribofuranosylthiazole-4-carboxamide. 3-Deazaguanosine monophosphate was shown to inhibit the activity of the de novo purine synthesis enzyme, 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide transformylase. The data suggested a cytotoxic effect of 3-DGUO associated with the inhibition of de novo purine synthesis by drug nucleotides, an effect which may account for the synergistic action noted.     

Biochemical properties of cyclic nucleotide phosphodiesterase in metastasizing and nonmetastasizing rat mammary carcinomas.

The biochemical properties of cyclic nucleotide phosphodiesterases in a nonmetastasizing and a spontaneously metastasizing rat mammary carcinoma were compared. The phosphooiesterases in both tumors had a pH optimum of around 8.0 and preferentially hydrolysed cyclic purine nucleotides. The rate of hydrolysis of purine nucleotides in the nonmetastasizing tumor was two times higher than in the metastasizing tumor, but the rate of pyrimidine nucleotide hydrolysis was equal in both tumors. Theophylline, caffeine, and D,L-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro20-1724) inhibited the enzyme activity in both tumors; the percent inhibition was the same by each inhibitor. The cyclic nucleotie phosphodiesterase activity in either tumor was stimulated by Mg++, Mn++, and Co++ and suppressed by Ca++, Zn,++, and Ni++. EDTA inhibited the activity below the basal level (activity in the absence of added cation), an this inhibition could be recovered up to the basal level by an equimolar quantity of either Mn++ or Mg++. Further stimulation of the enzyme activity with increasing concentrations of divalent cations was observed only with Mn++. Similar effects were observe with ethylene glycol bis(beta-aminoethyl ether)-tn,n-tetraacetic acid. The stimulatory cations affected both the low and high Michaelis constant (tkm) enzymes in these tumors by increasing the maximum velocity. In the low Km enzyme, the Km was also slightly increased. Neither guanosine 3',5'-cyclic monophosphate nor adenosine 3',5'-cyclic monophosphate had any effect on the hydrolysis of the other at physiologic levels.     

Mechanism of adenosine triphosphate catabolism induced by deoxyadenosine and by nucleoside analogues in adenosine deaminase-inhibited human erythrocytes

The mechanism of the depletion of ATP, recorded in the erythrocytes of adenosine deaminase-deficient children and of leukemia patients treated with deoxycoformycin, was investigated in normal human erythrocytes treated with this inhibitor of adenosine deaminase. Deoxyadenosine, which accumulates in both clinical conditions, provoked a dose-dependent accumulation of dATP, depletion of ATP, and increases in the production of inosine plus hypoxanthine. Concomitantly, there was an increase of AMP and IMP, but not of adenosine, indicating that catabolism proceeded by way of AMP deaminase. A series of nucleoside analogues (9-beta-D-arabinofuranosyladenine, N6-methyladenosine, 6-methylmercaptopurine ribonucleoside, tubercidin, ribavirin, and N-1-ribosyl-5-aminoimidazole-4-carboxamide riboside) also stimulated adenine nucleotide catabolism and increased AMP and IMP to various extents. The effects of deoxyadenosine and of the nucleoside analogues were prevented by 5'-iodotubercidin, an inhibitor of adenosine kinase. Strikingly, they were reversed if the inhibitor was added after the accumulation of nucleotide analogues and initiation of adenine nucleotide catabolism. Further analyses revealed linear relationships between the rate of phosphorylation of deoxyadenosine and nucleoside analogues and the increase in AMP and between the elevation of the latter above a threshold concentration of 10 microM and the rate of adenine nucleotide catabolism. Kinetic studies with purified erythrocytic AMP deaminase, at physiological concentrations of its effectors, showed that the enzyme is nearly inactive up to 10 microM AMP and increases in activity above this threshold. We conclude that the main mechanism whereby deoxyadenosine and nucleoside analogues stimulate catabolism of adenine nucleotides by way of AMP deaminase in erythrocytes is elevation of AMP, secondary to the phosphorylation of the nucleosides.     

Cyclic nucleotide concentrations in 1,2-dimethylhydrazine induced rat colon adenocarcinoma.

The intracellular concentrations of adenosine 3',5'-cyclic monophosphate (cAMP) in colonic tumors induced in adult male Holtzman rats by 1,2-dimethylhydrazine (DMH) were found to be 1/2 the concentration found in normal large bowel tissue. Intracellular concentrations of guanosine 3'-5'-cyclic monophosphate (cGMP) in the neoplastic cells were twice the normal colon level. The concentrations of these two cyclic nucleotides were relatively constant throughout the normal colon. Thus, the anomalous tumor cyclic nucleotide concentrations are attributed to the specific cell population of the lesion and not to the site of development within the colon.     

Enzyme activities controlling adenosine levels in normal and neoplastic tissues

Adenosine is known to be associated with effects such as inhibition of immune response, coronary vasodilation, stimulation of angiogenesis, and inhibition of inflammatory reactions. Some authors suggest that adenosine may also have similar functions in tumor tissues. Tissue levels of adenosine are under close regulation by different enzymes acting at different levels. Adenosine is produced from AMP by the action of 5′-nucleotidase (5′-NT) and is converted back into AMP by adenosine kinase (AK) or into inosine by adenosine deaminase (ADA). Inosine is converted into purine catabolites by purine nucleoside phosphorylase (PNP), whereas AMP is converted into ADP and ATP by adenylate kinase (MK). The aim of this study was to analyze the activities of the above enzymes in fragments of neoplastic and apparently normal mucosa, obtained less than 5 cm and at least 10 cm from tumors, in 40 patients with colorectal cancer. The results showed much higher activities of ADA, AK, 5′-NT, and PNP in tumor tissue than in neighboring mucosa (p>0.01 for ADA, AK, and PNP; p>0.05 for 5′-NT), suggesting that the activities of purine metabolizing enzymes increase to cope with accelerated purine metabolism in cancerous tissue. The simultaneous increase in ADA and 5′-NT activities might be a physiological attempt by cancer cells to provide more substrate to accelerate salvage pathway activity.     

Initiation-promotion skin carcinogenesis: inhibition by cyclic and non-cyclic nucleotides.

The effect of nucleotides on initiation-promotion skin carcinogenesis in Swiss mice was investigated. Cyclic AMP was given before initiation with DMBA, between initiation and promotion, and at the same time as promotion with croton oil. Cyclic AMP was more effective in inhibiting tumor development when injected at the same as promotion with croton oil. 5'-adenosine-monophosphate (5'-AMP) and cyclic GMP were as effective as cyclic AMP in inhibiting tumor development under these conditions. However, adenosine, dibutyryl-cyclic AMP and 5'-guanosine-monophosphate (5'-GMP) were ineffective.     

Role of IMP-selective 5'-nucleotidase (cN-II) in hematological malignancies

Cytotoxic nucleoside analogs (NA) are important in the treatment of hematologic malignancies. The NA in routine clinical use include the pyrimidine analog cytosine arabinoside (ara-c), which is extensively used in the treatment of acute leukemias, and the purine analogs, cladribine and fludarabine. These drugs have mostly been used in the treatment of low grade hematological malignancies. NA become therapeutically effective only after phosporylation to the triphosphate level. The 5'-nucleotidases (5'-NTs) dephosphorylate the monophosphate form of NA and, therefore, may affect the pharmacological activity of these antimetabolites in the clinic. Several 5'-NTs attached to membranes or present in the cytosol or in mitochondria are present in mammalian cells. cN-II, an IMP-selective 5'-NT, participates in the regulation of purine deoxyribonucleotide metabolism. cN-II opposes the action of the salvage enzymes by dephosphorylating purine nucleoside mononphosphates to purine nucleosides. Due to its phosphotransferase activity, cN-II can also phosphorylate inosine and 2',3'-dideoxyribonucleosides utilizing IMP as a phosphate donor. The observation that cytosolic cN-II is able to phosphorylate purine nucleosides has initiated studies on its potential participation in the metabolism of anticancer agents and in the development of cN-II inhibitory substances. In this review, we highlight the current knowledge concerning cN-II activity and regulation of intracellular deoxyribonucleotide pools and it role in hematological malignancies.     

Effect of ischemia on nucleosides and bases in rat liver and hepatoma 3924A

A high-pressure liquid chromatographic method was developed which achieved a separation and quantitation of 20 biologically important nucleosides and bases. The concentrations of pyrimidine nucleosides and bases, namely deoxycytidine, cytosine, cytidine, uracil, and uridine (22.6, 10.1, 5.2, 2.9, and 2.4 nmol/ml, respectively) were high in plasma, whereas purine nucleosides and bases were present in concentrations less than 2.5 nmol/ml. In erythrocytes, the pools of xanthine, hypoxanthine, and xanthosine were 32-, 27-, and 22-fold larger, respectively, whereas cytidine, uridine, and deoxycytidine were only 21, 12, and 5% of plasma concentrations. The results suggest a compartmental system for transport of some of the purine and pyrimidine nucleosides and bases in the whole blood. Studies on the effect of ischemia on nucleoside and base pools in rat liver indicated marked increases within 30 s in the concentrations of adenine, adenosine, inosine, hypoxanthine, uridine, and xanthine, whereas in hepatoma the effects were less pronounced. By 2 and 5 min ischemia these perturbations were most marked in both liver and hepatoma. These results indicate a need for rapid freeze-clamp preparation of tissue samples to obtain precise and repeatable results in the determination of tissue nucleoside and nucleobase concentrations.     

Immunotherapy with levamisole: early decrease of cAMP levels in lymphocytes from treated cancer patients.

Lymphocyte cyclic nucleotide content was studied before and after Levamisole administration to cancer patients. Twelve patients with disseminated cancer received 100 mg/m2 on two consecutive days; nine comparable patients with disseminated cancer served as controls. Endogenous cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were measured in lymphocytes before, 24, and 48 hours after ingestion of the first dose of Levamisole. A statistically significant decline in lymphocyte cAMP level was observed after drug administration and no significant changes were noted in cGMP levels. Further studies will be necessary to correlate this biochemical change in cyclic nucleotides with modulation of the functional level of cellular immune mechanisms.     

Differential sensitivity of normal and chemically transformed epithelial cells to cholera toxin

We have been studying the regulation of growth by cyclic adenosine 3':5'-monophosphate (cyclic AMP) and other factors in untransformed (K16) and chemically transformed (W8) rat liver epithelial cells. Initially, we found that 8-bromocyclic adenosine 3':5'-monophosphate was a more potent inhibitor of cell replication in K16 than in W8 cells. In addition, the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX) caused marked growth inhibition in K16 but not in W8 cells. Through the use of cholera toxin (CT) with or without MIX, we elevated intracellular cyclic AMP levels in a quantifiable fashion. With CT alone or combined with MIX, we observed a dose-dependent morphological change in W8 cells, which consisted of extensive "process" formation. K16 morphology was not altered at any concentration of CT +/- MIX tested. K16 cell growth was only marginally inhibited by CT alone, but markedly inhibited by CT plus MIX. W8 cell growth was moderately inhibited by CT alone or combined with MIX. Analysis of cyclic AMP levels revealed that, at all concentrations of CT +/- MIX and at all time periods tested, W8 cells produced significantly more cyclic AMP than K16 cells. It appears that morphological changes and growth inhibition are not necessarily linked and that MIX may inhibit K16 cell replication by means other than its ability to increase intracellular cyclic AMP levels.     

An experimental trial of cyclic nucleotides on multicellular spheroids derived from human brain tumours

The effects of cyclic nucleotides, dibutyryl cyclic adenosine monophosphate and dibutyryl cyclic guanosine monophosphate (db-cAMP and db-cGMP), on the growth rate of multicellular tumour spheroids were evaluated by comparing the growth delay and colony forming efficiency in vitro. Multicellular tumour spheroids were derived directly from human brain tumours. To compare the chemotherapeutic effect of cyclic nucleotides, CCNU was used as a known effective cytotoxic drug on malignant gliomas.Significant growth delay was obtained by db-cAMP (p<0.001) while CCNU was tumouricidal rather then producing a delay in growth.of the tumpur spheroids. Db-cGMP found not to be effective in decreasing the growth rate of the tumour spheroids in vitro (p>0.2).The role of cyclic nucleotides in brain tumours is discussed on a review basis. address for offprints     

Morphological differentiation of cultured mouse glioblastoma cells induced by dibutyryl cyclic adenosine monophosphate.

A culture line of mouse glioblastoma cells changed morphologically to differentiated astrocyte-like cells when cultured in medium with dibutyryl cyclic adenosine monophosphate and theophylline. Morphological alteration occurred within only 5 hr when 3 mM dibutyryl cyclic adenosine monophosphate and 1 mM theophylline were used, and in 1 day when 1 mM theophylline were used. Cells showing this morphological change reverted completely to immature cells when they were transferred to medium without these two chemicals. Addition of 1 or 3 mM dibutyryl cyclic guanosine monophosphate with 1 mM theophylline to the medium also induced development of cytoplasmic processes from these cells and the cells became stellate, although the cytoplasmic processes were not as long or as numerous as those induced by dibutyryl cyclic adenosine monophosphate, and the altered cells could not be referred to as differentiated glia cells. Sodium butyrate induced morphological alterations similar to those induced by dibutyryl cyclic guanosine monophosphate, but fewer cells showed these alterations. Addition of cyclic adenosine monophosphate or cyclic guanosine monophosphate in the presence of theophylline or addition of theophylline alone did not induce morphological changes of the cells.     

Croton oil- and benzo(a)pyrene-induced changes in cyclic adenosine 3':5'-monophosphate and cyclic guanosine 3':5'-monophosphate phosphodiesterase activities in mouse epidermis.

The topical application of croton oil, benzo(a)pyrene, acetic acid, and 12-O-tetradecanoyl-phorbol-13-acetate to mouse skin caused an increase in the activity of epidermal low-affinity cyclic adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterase. The increase was most pronounced with croton oil, began between 4 and 6 hr after application of this material, and was maintained for at least 48 hr. The activity of cyclic guanosine 3':5'-monophosphate phosphodiesterase was also increased by treatment with croton oil or 12-O-tetradecanoyl-phorbol-13-acetate, but detailed time courses were not obtained. Increased activity was observed in both the soluble fractions and the washed particulate fractions of epidermis. Fractionation of soluble extracts from acetone-treated epidermis on DEAE-cellulose columns showed the presence of enzymes with specificity for both cyclic AMP and cyclic guanosine 3':5'-monophosphate, together with a peak catalyzing the hydrolysis of both cyclic AMP and cyclic guanosine 3':5'-monophosphate. The activity of this latter nonspecific activity was selectively increased following treatment with croton oil. The increase in cyclic AMP phosphodiesterase activity was partially abolished by multiple injections of cycloheximide, suggesting that new protein synthesis was involved. Injection of the alpha-receptor antagonist phentolamine abolished a croton oil-induced rise in epidermal cyclic AMP levels and decreased the induction of cyclic AMP phosphodiesterase activity. From these results it was concluded that the increase in enzyme activity was induced by cyclic AMP.     

Clinical observation on the changes in plasma cyclic nucleotides in patients with leukemia before and after treatment and their relation to cell-mediated immunity

Level of plasma cyclic nucleotides was determined in 47 normal subjects and 63 patients with leukemia. Follow-up was carried out in 20 of these patients. The relation between the level of cyclic nucleotides and cell-mediated immunity was studied. The results: There was no significant difference in cyclic adenosine monophosphate (cAMP) between the normal subjects and leukemic patients, whereas cyclic guanosine monophosphate (cGMP) level was markedly elevated and cAMP/cGMP ratio was obviously reduced in the untreated and unrelieved patients. There was no significant difference in the various types of leukemia. In patients with complete remission (CR) after treatment, their cyclic nucleotides were similar to those in normal subjects, but they remained abnormal in patients without clinical remission after treatment. In CR patients treated regularly for half a year, the cGMP level when first diagnosed was lower than that of patients who died soon or were unrelieved. Plasma cGMP level was negatively correlated to E-rosette forming cell (E-RFC) and cAMP/cGMP ratio was positively correlated to E-RFC. These results suggest that the plasma cGMP be used as additional parameter to monitor the treatment response and prognosis. Reduced E-RFC in leukemia may be related to the elevated cGMP and/or lowered cAMP/cGMP ratio.     

Effects of cyclic-nucleotide derivatives on the growth of human colonic carcinoma xenografts and on cell production in the rat colonic crypt epithelium

Previous studies have shown that various amine hormones are able to influence the growth rate of human colorectal carcinomas propagated as xenografts in immune-deprived mice, and it is now well known that the effects of many amine and other hormones are mediated by cyclic nucleotides, acting as second messengers within cells. In the present study the influence of various derivatives of cyclic adenosine monophosphate and cyclic guanosine monophosphate on the growth of two different lines of colorectal cancer growing in immune-deprived mice, and on the cell production rate in the colonic crypt epithelium of the rat, was assessed. Growth of each tumour line, as well as crypt-cell production, was suppressed by treatment wit N6O2' dibutyryl and N6 monobutyryl derivatives of cyclic adenosine monophosphate. Dibutyryl cyclic guanosine monophosphate, on the other hand, was found to promote the growth of Tumour HXK4 and to promote crypt cell production, but to have no significant effect on Tumour HXM2.     

Solubilized nuclear DNA-dependent RNA polymerases from normal rat mammary glands and from transplantable R-35 rat mammary tumors.

The three major nuclear DNA-dependent RNA polymerases (enzymes I, II and III) were present in nuclear extracts from transplantable R-35 rat mammary tumors. Except for somewhat less enzyme III, their relative distribution resembled that of nuclear extracts from late-pregnant rats. When enzyme II from normal tissue extracts was incubated for RNA synthesis with cyclic AMP, inhibition was frequently observed, but this occurred less often with nuclear extracts from the R-35 tumor. In some experiments with both normal and tumor tissue, cyclic AMP and cyclic GMP increased the apparent activity of nucleolar enzyme Ib and nucleoplasmic enzyme II, respectively. Nuclear extracts from both normal and tumor tissue contain proteins which bind radioactive cyclic AMP and cyclic GMP. Their patterns of binding were not identical. These results are consistent with the following hypothesis: altered binding by the tumor of cyclic nucleotides to putative nuclear 'r-gulatory' proteins (e.g. protein kinase subunits, or possibly other high affinity cyclic nucleotide-binding proteins unrelated to protein kinases) contributes to atative nuclear 'regularory' proteins (e.g. protein kinase subunits, or possibly other high affinity cyclic nucleotide-binding proteins unrelated to protein kinases) contributes to and may be responsible for some of the differences in response to cyclic nucleotides that were observed. It is possible that such defects occur in other tumors, or even represent a fundamental defect in all cancer cells. Several explanations for these results are discussed.     

Nucleotide-induced inhibition of surface sialyl transferase activity on cultured Burkitt's lymphoma cells.

Sialyl transferase activity was demonstrated on the surfaces of intact, cultured lymphoblastoid cells (RAJI) derived from a Burkitt's lymphoma. Pretreatment of the cells with neuraminidase increased the labeled sialoprotein by severalfold. A nunber of nucleotides were effective in decreasing the amount of sialoprotein assembly. CMP was the most effective inhibitor. UMP, AMP, and GMP were also inhibitory, but to a lesser degree. The diphosphate derivatives were similarly inhibitory, but generally less active than their monophosphate counterparts. The cyclic nucleotides were the least effective of all nucleotides tested; cCMP and cAMP showed a small degree of activity, whereas cUMP and cGMP were without effect. These studies indicated that a number of noncyclic and cyclic nucleotides can influence the activity of the sialyl transferase system.