Inosine monophosphate dehydrogenase and myeloid cell maturation

In previous studies of purine ribonucleotide metabolism in the human myeloid leukemia cell line HL-60, we observed that there is a down- regulation of guanine ribonucleotide biosynthesis from the central intermediate, inosine monophosphate (IMP) and a depletion of intracellular guanosine triphosphate (GTP) and guanosine diphosphate (GDP) pools that occur during the induced maturation of these cells. We also found that inhibitors of IMP dehydrogenase, the enzyme that catalyzes the first step of guanylate synthesis from IMP, are potent inducers of HL-60 maturation. Because of these observations we specifically investigated the activity of IMP dehydrogenase in HL-60 cells and in a new inducible human myeloid leukemia cell line, RDFD2- 25, both during maintenance culture and during induced maturation of the cells. Enzyme activity was examined directly in cell extracts with a radiometric assay that measures free 3H2O formed from [2-3H] IMP during the conversion of IMP to XMP. Uninduced HL-60 and RDFD2 cells in maintenance culture were found to have high levels of IMPD activity (5.2 to 5.7 pmol IMP metabolized/10(7) cells/min) compared with normal neutrophils and monocytes that had been purified from blood (less than 1.5 pmol IMP metabolized/10(7) cells/min). However, when HL-60 and RDFD2-25 cells were induced to mature with retinoic acid (10(-6) mol/L), dimethylformamide (6 X 10(-2) mol/L), or a known IMPD inhibitor, tiazofurin (10(-6) mol/L), IMPD activity in the cells fell by 51% to 80% within three to six hours. These changes in IMPD activity preceded detectable functional and antigenic maturation of the cells by at least 12 hours and were not temporally related to changes in cellular proliferation. These findings are consistent with the concept that the regulation of myeloid cell maturation may be influenced by intracellular concentrations of guanine ribonucleotides because IMP dehydrogenase activity is known to be rate limiting for the production of these nucleotides.     

Effects of tiazofurin on guanine nucleotide binding regulatory proteins in HL-60 cells

Guanine nucleotide binding proteins (G proteins) are regulatory molecules that couple membrane receptors to effector systems such as adenylate cyclase and phospholipase C. The alpha subunits of G proteins bind to guanosine 5'-diphosphate (GDP) in the unstimulated state and guanosine 5' triphosphate (GTP) in the active state. Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide), a specific inhibitor of inosine monophosphate (IMP) dehydrogenase, decreases guanylate synthesis from IMP in HL-60 promyelocytic leukemia cells and depletes intracellular guanine nucleotide pools. This study demonstrates that treatment of HL-60 cells with tiazofurin is associated with a fourfold increase in membrane binding sites for the nonhydrolyzable analogue GDP beta S. This increase in binding sites was associated with a 3.2-fold decrease in GDP beta S binding affinity. Similar findings were obtained with GTP gamma S. These effects of tiazofurin treatment on guanine nucleotide binding were also associated with decreased adenosine diphosphate-ribosylation of specific G protein substrates by cholera and pertussis toxin. The results further demonstrate that tiazofurin treatment results in inhibition of G protein-mediated transmembrane signaling mechanisms. In this regard, stimulation of adenylate cyclase by prostaglandin E2 was inhibited by over 50% in tiazofurin-treated cells. Furthermore, tiazofurin treatment resulted in inhibition of N-formylmethionylleucylphenylalanine-induced stimulation of phospholipase C. Taken together, these results indicate that tiazofurin acts at least in part by inhibiting the ability of G proteins to function as transducers of intracellular signals.     

GM-CSF: modulation of biochemical and cytotoxic effects of tiazofurin in HL-60 cells

Summary. Cytokines, such as granulocyte macrophage colony stimulating factor (GM-CSF) or interleukin-3 (IL-3) recruit quiescent cells into the cell cycle and sensitize these cells towards cell cycle specific chemotherapeutic agents. We examined the in vitro effects of GM-CSF on HL-60 cells and tested its modulatory influence on biochemical and cytotoxic effects seen with tiazofurin, a potent and specific inhibitor of IMP dehydrogenase. Incubation of HL-60 cells with 500 U/ml GM-CSF for 4 d enhanced cell proliferation, which was accompanied by a significant increase in IMP dehydrogenase activity (from 2·22 in control cells to 3·70 nmol/mg/h in cells pretreated with GM-CSF). When HL-60 cells were incubated with 100 μm tiazofurin for 2 h, intracellular GTP decreased to 46% of untreated control cells. In HL-60 cells pretreated with GM-CSF, GTP pools decreased to 38% of control after incubation with tiazofurin which is 69% of the predicted value for additive effect. The MTT chemosensitivity assay yielded significantly decreased IC₅₀ values for tiazofurin in HL-60 cells, preincubated with GM-CSF (IC₅₀ decreased from 13 μ m to 10 μ m ). Therefore our results suggest that combination therapy with GM-CSF and tiazofurin may be beneficial for the treatment of refractory leukaemia patients.     

Studies of purine and tiazofurin metabolism in drug sensitive human chronic myelogenous leukemia K 562 cells

Summary Antineoplastic activity of tiazofurin (2--D-ribofuranosylthiazole-4-carboxamide) is mediated by an anabolite of the drug thiazole-4-carboxamide adenine dinucleotide (TAD), an analog of NAD which inhibits IMP dehydrogenase activity resulting in the depletion of guanylate pools and cell death. Human chronic myelogenous leukemia K 562 cells were found to be sensitive to tiazofurin with an IC₅₀ of 19.2 M. TAD content in K 562 cells (1.3 nmol/10⁹/h) was in the range found in susceptible murine and human tumor cells. Studies were conducted to relate tiazofurin toxicity with biochemical effects by examining nucleotide pools. Among the nucleotides, only guanylate pools were significantly depleted by the drug. To further study the effect of the drug on the purine nucleotide de novo and salvage biosynthetic pathways, flux of radiolabelled formate and guanine was employed. The results showed that de novo synthesis of guanylates was curtailed primarily by the drug's action without influencing adenylate biosynthesis or salvage of guanine to guanylates. These studies show that K 562 cells are sensitive to selective inhibition of de novo guanylate pathway indicating that human chronic myelogenous leukemia in blast crisis might be a good candidate for Phase II clinical trials with tiazofurin.Abbreviations ALL acute lymphoblastic leukemia - ANLL acute non lymphocytic leukemia - CML chronic myelogenous leukemia - GMP, GDP, GTP guanosine 5-mono-, di-, triphosphate - HPLC high pressure liquid chromatography - IMP inosine 5-monophosphate - IMPD IMP dehydrogenase - NAD nicotinamide-adenine-dinucleotide - NCI National Cancer Institute; - PBS phosphate buffered saline - TAD thiazole-4-carboxamide-adenine dinucleotide - TCA trichloracetic acid - TRMP tiazofurin 5-monophosphate - TRTP tiazofurin 5-triphosphate. The research work outlined in this paper was supported by United States Public Health Service grant R 35 CA 42510 awarded by the National Cancer Institute, Department of Health and Human Services, USA to G. Weber. The results will be presented at the 79th annual meeting of the American Association for Cancer Research in New Orleans, May 25–28, 1988     

Purine oversecretion in cultured murine lymphoma cells deficient in adenylosuccinate synthetase: genetic model for inherited hyperuricemia and gout.

Alterations in several specific enzymes have been associated with increased rates of purine synthesis de novo in human and other mammalian cells. However, these recognized abnormalities in humans account for only a few percent of the clinical cases of hyperuricemia and gout. We have examined in detail the rates of purine production de novo and purine excretion by normal and by mutant (AU-100) murine lymphoma T cells (S49) 80% deficient in adenylosuccinate synthetase [IMP:L-aspartate ligase (GDP-forming), EC 6.3.4.4]. The intracellular ATP concentration of the mutant cells is slightly diminished, but their GTP is increased 50% and their IMP, four-fold. Compared to wild-type cells, the AU-100 cells excrete into the culture medium 30- to 50-fold greater amounts of purine metabolites consisting mainly of inosine. Moreover, the AU-100 cell line overproduces total purines. In an AU-100-derived cell line, AU-TG50B, deficient in adenylosuccinate synthetase and hypoxanthine/guanine phosphoribosyltransferase (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), purine nucleoside excretion is increased 50- to 100-fold, and de novo synthesis is even greater than that for AU-100 cells. The overexcretion of purine metabolites by the AU-100 cells seems to be due to the primary genetic deficiency of adenylosuccinate synthetase, a deficiency that requires the cell to increase intracellular IMP in an attempt to maintain ATP levels. As a consequence of elevated IMP pools, large amounts of inosine are secreted into the culture medium. We propose that a similar primary genetic defect may account for the excessive purine excretion in some patients with dominantly inherited hyperuricemia and gout.     

Mizoribine, an inhibitor of inosine monophosphate dehydrogenase, inhibits interleukin-6 production by freshly prepared rheumatoid synovial cells

Mizoribine, an immunosuppressive drug, has been used for treatment in organ transplantation, lupus nephritis, and rheumatoid arthritis (RA). On the basis of in vitro experiments, mizoribine has been postulated to be an inhibitor of inosine monophosphate (IMP) dehydrogenase, a pivotal enzyme in the formation of guanine ribonucleotides from IMP. To further characterize the mechanism of the antirheumatic action of this drug, we examined the effect of mizoribine on the production of interleukin (IL)-6, a major inflammatory cytokine in rheumatoid synovia, by freshly prepared rheumatoid synovial cells (RSC). Mizoribine (1.25–5 μg/ml) was able to inhibit the spontaneous production of IL-6 by fresh RSC in a dose–response fashion. The addition of guanosine monophosphate (GMP) reversed its inhibitory effects. In addition, mizoribine inhibited the enhanced production of IL-6 by the IL-1α and/or tumor necrosis factor α-stimulated RSC. Inhibition was also observed at the mRNA level, determined by Northern blot analysis. In contrast, mizoribine did not affect IL-8 production by these cells. These data suggest that mizoribine inhibits IL-6 production by fresh RSC, possibly owing to the depletion of intracellular GMP, and that this inhibitory effect of the drug on rheumatoid synovial cells may be related to its efficacy in RA. Received: April 4, 2000 / Accepted: October 19, 2000     

Changes in actin content during induced myeloid maturation of human promyelocytes

Actin is an important cytoskeletal protein; new actin synthesis occurs during differentiation of many motile cells. To better understand the process of myeloid maturation, the change in actin content during induced maturation of HL-60 human promyelocytic leukemia cells was studied. HL-60 cells induced toward myeloid maturation by a 5-day exposure to dimethylformamide showed an 86% increase in a 43,000 mol wt protein comigrating with rabbit muscle actin on dodecyl sulfate polyacrylamide gels. To further demonstrate that this was an increase in actin content, the total actin content of lysed HL-60 cells was measured by the ability of actin to inhibit DNAase I. Using this assay, actin content of HL-60 cells increased 96% during induced differentiation. The amount of incorporation of 3H-leucine into actin doubled after a 5-day exposure to dimethylformamide, suggesting the increase in actin was due primarily to new synthesis. Total new protein synthesis increased 2-7-fold during differentiation. Additional analysis of polyacrylamide gels showed increased quantities and new synthesis of a high molecular weight protein comigrating with rabbit muscle myosin. This study shows that actin content increases during myeloid maturation. It also demonstrates that the HL-60 cell line is a useful model to study both functional and biochemical events during human myeloid differentiation.     

Mechanism of deoxyadenosine-induced catabolism of adenine ribonucleotides in adenosine deaminase-inhibited human T lymphoblastoid cells

Loss of ATP accompanying accumulation of dATP has recently been reported to occur in the erythrocytes and lymphoblasts of patients with T lymphocytic leukemia during treatment with deoxycoformycin, an inhibitor of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) that causes the accumulation of deoxyadenosine. We have studied the mechanisms responsible for adenine ribonucleotide depletion in cultured human CEM T lymphoblastoid cells treated with deoxycoformycin and deoxyadenosine. Accumulation of dATP was accompanied by depletion of total soluble adenine ribonucleotides without change in the adenylate energy charge, by the route ATP --> AMP --> IMP --> inosine --> hypoxanthine; conversion of IMP to AMP and de novo purine synthesis were inhibited in these cells. ATP degradation did not occur in a mutant of CEM that was incapable of phosphorylating deoxyadenosine, or in a B cell line with very limited ability to accumulate dATP. We found that dATP and ATP were both able to stimulate markedly the deamination of AMP by lymphoblast AMP deaminase; dAMP was a poor substrate for this enzyme (K(m) = 2.4 mM, vs. 0.4 mM for AMP). Similarly, dATP as well as ATP caused marked activation of IMP dephosphorylation by a lymphoblast cytoplasmic nucleotidase. Inhibition of intracellular AMP deaminase with coformycin prevented degradation of adenine ribonucleotides without affecting dATP accumulation. We propose that ATP-dependent phosphorylation of deoxyadenosine generates ADP and AMP. Simultaneously, dATP accumulation stimulates deamination of AMP, but not dAMP, and the dephosphorylation of IMP to inosine. Coupling of AMP degradation to ATP utilization in deoxyadenosine phosphorylation maintains the adenylate energy charge despite net depletion of cellular ATP.     

Induction of differentiation of human and murine myeloid leukemia cells in culture by tunicamycin.

Tunicamycin, an antibiotic that specifically blocks the synthesis of N-acetylglucosamine-lipid intermediates and thereby prevents glycosylation of glycoproteins, induced differentiation of both human (HL-60) and murine (M1) myeloid leukemia cell lines in culture. At 0.1-1.0 microgram/ml, it induced differentiation of both HL-60 and M1 cells, characterized by increase in phagocytic cells and changes to resemble mature myeloid cells. Fc receptors were also induced in M1 but not in HL-60 cells; induction of intracellular lysozyme activity was not detected in either HL-60 or M1 cells. With this concentration of tunicamycin, there was marked decrease in rate of incorporation of radioactive glucosamine into macromolecules and a decrease in the rate of DNA synthesis. These data show that glycosylation of cellular proteins has an important role in maintaining these myeloid leukemia cells in an undifferentiated state in culture. The results also indicate that induction of phagocytosis in both HL-60 and M1 myeloid leukemia cells and of Fc receptors in M1 cells does not require continued synthesis of the oligosaccharide portions of cellular proteins by the lipid-linked pathway.     

Actin polymerization and its relationship to locomotion and chemokinetic response in maturing human promyelocytic leukemia cells

We studied actin polymerization in the HL-60 human promyelocytic leukemia cell line during induced myeloid maturation and its relationship to the rate of locomotion (ROL). The percent G-actin (of total actin) was measured by DNAase I inhibition, F-actin was determined by fluorescence-activated cell sorter (FACS) analysis of nitrobenzoxadiazol (NBD)-phallacidin-stained cells, and ROL was measured by computer-assisted analysis of the tracks of individual cells. Uninduced HL-60 cells moved slowly (2.3 +/- 1.0 microns/min) and showed no change in ROL or in the state of actin polymerization when stimulated by formyl-methionyl-leucyl-phenylalanine (fMLP). Nonstimulated cells induced to differentiate with dimethylformamide had no change in the degree of actin polymerization but exhibited a mean (m) ROL similar to normal human polymorphonuclear leukocytes (PMN) (8.6 +/- 1.4 micron/min [HL-60 cells] v 7.8 +/- 1.8 microns/min [PMN]. When induced HL-60 cells were stimulated with fMLP, actin polymerization occurred. The F-actin content increased, as determined by FACS analysis of NBD-phallacidin-stained cells, and the percentage of G-actin decreased, as determined by a 24.5% decrease in DNAase I inhibitory activity. However, induced HL-60 cells stimulated with fMLP did not increase their mROL. These studies show that, unlike normal human PMN, chemotactic peptides can cause an intracellular biochemical change that is not associated with a chemokinetic response in induced HL-60 cells. The HL-60 cell line may be a useful model to study the development of chemotactic peptide-mediated actin polymerization during myeloid cell maturation.     

Role of p21 RAS in p210 bcr-abl transformation of murine myeloid cells

The p21 RAS product has been implicated as part of the downstream signaling of certain nonreceptor tyrosine kinase oncogenes and several growth factor receptor-ligand interactions. We have reported that the chronic myelogenous leukemia oncogene p210 bcr-abl transforms a growth- factor-dependent myeloid cell line NFS/N1.H7 to interleukin-3 (IL-3) independence. In these p210 bcr-abl-transformed cells (H7 bcr-abl.A54) and in two other murine myeloid cell lines transformed to IL-3 independence by p210 bcr-abl, endogenous p21 RAS is activated as determined by an elevated ratio of associated guanosine triphosphate (GTP)/guanosine diphosphate (GDP), assayed by thin-layer chromatography of the nucleotides eluted from p21 RAS after immunoprecipitation with the Y13-259 antibody. Treatment of p210 bcr-abl-transformed cells with a specific tyrosine kinase inhibitor herbimycin A resulted in diminished tyrosine phosphorylation of p210 bcr-abl and associated proteins, without major reduction in expression of the p210 bcr-abl protein itself. Inhibition of p210 bcr-abl-dependent tyrosine phosphorylation resulted in a reduction of active p21RAS-GTP complexes in the transformed cells, in diminished expression of the nuclear early response genes c-jun and c-fos, and in lower cellular proliferation rate. To further implicate p21 RAS in these functional events downstream of p210 bcr-abl tyrosine phosphorylation, we targeted G- protein function directly by limiting the availability of GTP with the inosine monophosphate dehydrogenase inhibitor, tiazofurin (TR). In p210 bcr-abl-transformed cells treated for 4 hours with TR, in which the levels of GTP were reduced by 50%, but GDP, guanosine monophosphate, and adenosine triphosphate (ATP) were unaffected, p210 bcr-abl tyrosine phosphorylation was at control levels. However, expression of c-fos and c-jun nuclear proto-oncogenes were strongly inhibited and p21 RAS activity was downregulated. These findings show that p210 bcr-abl transduces proliferative signals, in part, through downstream activation of p21 RAS. Furthermore, p21 RAS activity is linked to pathways that regulate c-jun and c-fos expression.     

Identification of a brefeldin A-insensitive guanine nucleotide-exchange protein for ADP-ribosylation factor in bovine brain.

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that participate in vesicular transport in the Golgi and other intracellular compartments and stimulate cholera toxin ADP-ribosyltransferase activity. ARFs are active in the GTP-bound form; hydrolysis of bound GTP to GDP, possibly with the assistance of a GTP hydrolysis (GTPase)-activating protein results in inactivation. Exchange of GDP for GTP and reactivation were shown by other workers to be enhanced by Golgi membranes in a brefeldin A-sensitive reaction, leading to the proposal that the guanine nucleotide-exchange protein (GEP) was a target of brefeldin A. In the studies reported here, a soluble GEP was partially purified from bovine brain. Exchange of nucleotide on ARFs 1 and 3, based on increased ARF activity in a toxin assay and stimulation of binding of guanosine 5'-[gamma-[35S]thio]triphosphate, was dependent on phospholipids, with phosphatidylserine being more effective than cardiolipin. GEP appeared to increase the rate of nucleotide exchange but did not affect the affinity of ARF for GTP. Whereas the crude GEP had a size of approximately 700 kDa, the partially purified GEP behaved on Ultrogel AcA 54 as a protein of 60 kDa. With purification, the GEP activity became insensitive to brefeldin A, consistent with the conclusion that, in contrast to earlier inferences, the exchange protein is not itself the target of brefeldin A.     

HL-60 cells degrade alpha-actinin to produce a fragment that promotes monocyte/macrophage maturation

An amino-terminal fragment of alpha-actinin can promote monocyte/macrophage maturation. This fragment was initially isolated from media of HL-60 myeloid leukemia cells cultured on extracellular bone marrow matrix. To determine the source of this fragment in this culture system, we investigated whether HL-60 cells grown on bone marrow stroma have increased intracellular levels of alpha-actinin that may be released into the media during cell apoptosis. HL-60 cells grown on matrix showed no evidence of increased cellular alpha-actinin compared to cells grown on plastic substrata as measured by flow cytometry. In addition, there was no evidence of increased apoptosis as determined by DNA fragmentation assays or flow cytometry. However, 100 kD alpha-actinin was found in the extracellular matrix of bone marrow stroma by Western blot analysis and immunofluorescence microscopy. The alpha-actinin content in the stroma was markedly decreased after exposure to HL-60 cells. Furthermore, lysates of HL-60 cells or of peripheral blood monocytes can degrade exogenous alpha-actinin to produce a 31 kD fragment, which promotes monocyte/macrophage maturation. We conclude that when alpha-actinin is present in the extracellular matrix, it can be modified by HL-60 cells to produce a maturation promoting 31 kD fragment.     

Adenine and guanine nucleotide metabolism during platelet storage at 22 degrees C

Adenine and guanine nucleotide metabolism of platelet concentrates (PCs) was studied during storage for transfusion at 22 +/- 2 degrees C over a 7-day period using high-pressure liquid chromatography. There was a steady decrease in platelet adenosine triphosphate (ATP) and adenosine diphosphate (ADP), which was balanced quantitatively by an increase in plasma hypoxanthine. As expected, ammonia accumulated along with hypoxanthine but at a far greater rate. A fall in platelet guanosine triphosphate (GTP) and guanosine diphosphate (GDP) paralleled the fall in ATP + ADP. When adenine was present in the primary anticoagulant, it was carried over into the PC and metabolized. ATP, GTP, total adenine nucleotides, and total guanine nucleotides declined more slowly in the presence of adenine than in its absence. With adenine, the increase in hypoxanthine concentration was more rapid and quantitatively balanced the decrease in adenine and platelet ATP + ADP. Plasma xanthine rose during storage but at a rate that exceeded the decline in GTP + GDP. When platelet ATP + ADP was labeled with 14C-adenine at the initiation of storage, half of the radioactivity was transferred to hypoxanthine (45%) and GTP + GDP + xanthine (5%) by the time storage was completed. The isotopic data were consistent with the presence of a radioactive (metabolic) and a nonradioactive (storage) pool of ATP + ADP at the initiation of storage with each pool contributing approximately equally to the decline in ATP + ADP during storage. The results suggested a continuing synthesis of GTP + GDP from ATP + ADP, explaining the slower rate of fall of GTP + GDP relative to the rate of rise of plasma xanthine. Throughout storage, platelets were able to incorporate 14C-hypoxanthine into both adenine and guanine nucleotides but at a rate that was only one fourth the rate of hypoxanthine accumulation. All of these data should be helpful in improving the function and viability of PC as currently stored for 5 days, in devising methods for storage beyond 5 days, and in the development of synthetic media for PC storage.     

Induction of apoptosis in IL-3-dependent hematopoietic cell lines by guanine nucleotide depletion

Inosine 5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme that catalyzes the conversion of IMP to xanthosine monophosphate (XMP) at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides. Inhibition of IMPDH results in the depletion of guanine nucleotides, prevents cell growth by G1 arrest, and induces cell differentiation in a cell-type-specific manner. The molecular and sensing mechanisms underlying these effects are not clear. We have examined the induction of apoptosis by mycophenolic acid (MPA), a specific IMPDH inhibitor, in interleukin-3 (IL-3)-dependent murine hematopoietic cell lines. MPA treatment, at clinically relevant doses, caused apoptosis in 32D myeloid cells and in FL5.12 and BaF3 pre-B cells in the ongoing presence of IL-3. Apoptosis was completely prevented by the addition of guanosine at time points up to 12 hours, after which caspase 3 activity increased and apoptosis was not reversible. MPA treatment caused marked down-regulation of the MAP kinase kinase/extracellular regulatory kinase (MEK/Erk) pathway at 3 hours while simultaneously increasing the phosphorylation of c-Jun kinase. In addition, MPA strongly down-regulated the mammalian target of rapamcyin (mTOR) pathway, as indicated by the decreased phosphorylation of p70 S6 kinase and of 4EBP1. Inhibition of either the mitogen-activated protein kinase (MAPK) or the mTOR pathway alone by standard pharmacologic inhibitors did not induce apoptosis in IL-3-dependent cells, whereas inhibition of both pathways simulated the effects of MPA treatment. These results indicate that IMPDH inhibitors may be effective in modulating signal transduction pathways in hematopoietic cells, suggesting their usefulness in chemotherapeutic regimens for hematologic malignancies.     

Regulation of ornithine decarboxylase activity by guanine nucleotides: in vivo test in potassium-depleted Escherichia coli.

The observation that guanosine 5'-triphosphate (GTP) is an activator and guanosine 5'-diphosphate-3'-diphosphate (ppGpp) is an inhibitor of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) of Escherichia coli [E. Hölttä et al. (1974) Biochem. Biophys. Res. Commun. 59, 1104-1111] has been confirmed. The hypothesis that synthesis of both polyamine and RNA in E. coli is regulated in vivo by these nucleotides was tested in E. coli B-207. On transfer of this K+-requiring, amino-acid-deficient strigent strain from K+-medium to Na+-medium, the organism stops protein synthesis, maintains a high rate of RNA synthesis, and increases putrescine synthesis from ornithine manyfold. Under these conditions, the cells do not markedly change their contents of GTP and ppGpp. The proposed mechanism of regulation of RNA and putrescine synthesis by guanine nucleotides does not appear to explain the metabolic phenomena observed in this organism during K+ deficiency. Nevertheless, amino acid depletion in K+-medium does result in a marked increase in ppGpp.     

Expression of IMP dehydrogenase in differentiating HL-60 cells

Addition of mycophenolic acid to cultures of HL-60 cells results in a decreased cellular level of guanine nucleotides and the induction of cell differentiation. During the early stages of this induction, steady-state levels of cellular IMP dehydrogenase (IMPDH), messenger RNA (mRNA), and protein are increased, perhaps because of cellular compensation for the inhibition of IMPDH activity. The subsequent decrease in IMPH mRNA and protein levels after several days of treatment suggests a change in the control of IMPDH expression. In contrast to the pattern of increased IMPDH expression observed in the mycophenolic acid-treated cells, treatment of HL-60 cells with two other inducers of differentiation, namely retinoic acid and phorbol 12-myristate 13-acetate, resulted in stable or decreased levels of cellular IMPDH mRNA and protein. However, the kinetics of this expression were different. These results suggest that a number of factors influence the regulation of IMPDH expression during the induction of HL-60 cell differentiation, including the nature of the inducer. A decrease in the cellular IMPDH activity was observed for all of the inducers, suggesting that this decreased activity may be a determining factor in the acquisition of a mature phenotype in the HL-60 cells.     

cGMP-induced differentiation of the promyelocytic cell line HL-60.

cGMP is a second messenger that mediates numerous metabolic events; in the present work a role in myeloid cell differentiation was demonstrated. Nitroprusside and NaNO2, which activate cytosolic guanylate cyclase and increase the intracellular cGMP concentration, induced granulocytic differentiation of the human promyelocytic cell line HL-60; differentiation was measured by acquisition of the OKM1 antigen, morphological changes, and nitroblue tetrazolium reduction. When theophylline, a phosphodiesterase inhibitor, which by itself induced modest differentiation, was added to nitroprusside or NaNO2, differentiation increased in an additive fashion. The degree of differentiation correlated with the increase in the intracellular cGMP concentration. 8-Bromoguanosine 3',5'-cyclic monophosphate, a membrane-permeable cGMP analogue, also induced differentiation of HL-60 cells but was much more effective in the presence of theophylline, with the two agents interacting synergistically. The effect of theophylline in these studies could not be attributed to increasing the intracellular cAMP concentration. Dimethyl sulfoxide, and established inducer of differentiation of HL-60 cells, markedly enhanced the differentiation induced by nitroprusside and NaNO2.     

Modulation of brain Na+ channels by a G-protein-coupled pathway.

Na+ channels in acutely dissociated rat hippocampal neurons and in Chinese hamster ovary (CHO) cells transfected with a cDNA encoding the alpha subunit of rat brain type IIA Na+ channel (CNaIIA-1 cells) are modulated by guanine nucleotide binding protein (G protein)-coupled pathways under conditions of whole-cell voltage clamp. Activation of G proteins by 0.2-0.5 mM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), a nonhydrolyzable GTP analog, increased Na+ currents recorded in both cell types. The increase in current amplitude was caused by an 8- to 10-mV negative shift in the voltage dependence of both activation and inactivation. The effects of G-protein activators were blocked by treatment with pertussis toxin or guanosine 5'-[beta-thio]diphosphate (GDP[beta S]), a nonhydrolyzable GDP analog, but not by cholera toxin. GDP[beta S] (2 mM) alone had effects opposite those of GTP[gamma S], shifting Na(+)-channel gating 8-10 mV toward more-positive membrane potentials and suggesting that basal activation of G proteins in the absence of stimulation is sufficient to modulate Na+ channels. In CNaIIA-1 cells, thrombin, which activates pertussis toxin-sensitive G proteins in CHO cells, caused a further negative shift in the voltage dependence of Na(+)-channel activation and inactivation beyond that observed with GTP alone. The results in CNaIIA-1 cells indicate that the alpha subunit of the Na+ channel alone is sufficient to mediate G protein effects on gating. The modulation of Na+ channels via a G-protein-coupled pathway acting on Na(+)-channel alpha subunits may regulate electrical excitability through integration of different G-protein-coupled synaptic inputs.