Cytotoxic and biochemical effects of 3,3',4,4',5,5'-hexahydroxystilbene, a novel resveratrol analog in HL-60 human promyelocytic leukemia cells

OBJECTIVE: Resveratrol (3,4',5,-trihydroxystilbene, RV), an ingredient of wine, is an inhibitor of the proliferation-linked enzyme ribonucleotide reductase (RR) and shows a broad spectrum of cytotoxic effects against human cancer cells. In order to enhance these effects, we introduced additional hydroxyl moieties into the molecule. In the present study, the activity of a novel RV analog, 3,3',4,4',5,5'-hexahydroxystilbene (M8), was investigated in HL-60 human promyelocytic leukemia cells. METHODS: Cytotoxicity of M8 alone or in combination with Ara-C was assessed employing growth inhibition assays. Effects of M8 on nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) were examined by HPLC. The apoptotic potential of M8 and RV was compared using a specific double-staining method and inhibition of TNF-alpha-induced activation of NF-kappaB was studied. Cell-cycle distribution was analyzed by FACS. RESULTS: Addition of ascorbic acid decreased the IC(50) value of M8 from 6.25 microM to 2 microM. M8 depleted dATP and dTTP pools to 41% and 21% of control values, whereas dCTP pools increased to 199% of untreated controls. In addition, TTP, ATP, CTP, and GTP concentrations were decreased while UTP concentrations increased. M8 induced apoptosis at concentrations significantly lower than RV and could remarkably inhibit the activation of NF-kappaB. M8 arrested cells in the S phase of the cell cycle while depleting cells in the G2-M phase and exhibited synergistic combination effects when applied simultaneously with Ara-C. CONCLUSION: Due to these promising results, this novel polyhydroxylated stilbene derivative might become an additional option for the treatment of leukemia and therefore deserves further preclinical and in vivo testing.     

Differential protection of normal and malignant human myeloid progenitors (CFU-GM) from Ara-C toxicity using cycloheximide

Cycloheximide, a reversible protein synthesis inhibitor, is thought to block DNA replication in normal cells by preventing synthesis of a labile protein. In animal systems, cycloheximide protects normal cells from cytotoxic S-phase specific agents, such as cytosine arabinoside (Ara-C). Malignant cells appear not to be susceptible to cycloheximide- induced cycle arrest and, subsequently, may not be protected from Ara-C cytotoxicity. The effect of cycloheximide on granulocyte/macrophage progenitors (CFU-GM) after in vitro Ara-C exposure was examined using normal human bone marrow, malignant progenitors from patients with chronic myelogenous leukemia (CML), and clonogenic cells from the human acute nonlymphocytic leukemia cell lines HL-60 and KG-1. Mononuclear or clonogenic cells were incubated for one hour with cycloheximide, followed by the addition, for three or 17 hours, of Ara-C before being plated in a methylcellulose culture system. CFU-GM survival was significantly increase if normal cells were treated with cycloheximide before Ara-C exposure. Similar cycloheximide pretreatment of CML progenitors and clonogenic HL-60 and KG-1 cells failed to protect CFU- GM from Ara-C-induced cytotoxicity.     

Synergistic growth inhibitory and differentiating effects of trimidox and tiazofurin in human promyelocytic leukemia HL-60 cells

Increased ribonucleotide reductase (RR) activity has been linked with malignant transformation and tumor cell growth. Therefore, this enzyme is considered to be an excellent target for cancer chemotherapy. We have examined the effects of a newly patented RR inhibitor, trimidox (3,4,5-trihydroxybenzohydroxamidoxime). Trimidox inhibited the growth of human promyelocytic leukemia HL-60 cells with an IC50 of 35 mumol/L. Incubation of HL-60 cells with 50 mumol/L trimidox for 24 hours decreased deoxyguanosine triphosphate (dGTP) and deoxycytidine triphosphate (dCTP) pools to 24% and 39% of control values, respectively. Incubation of HL-60 cells with 20 to 80 mumol/L trimidox even up to a period of 4 days did not alter the distribution of cells in different phases of cell cycle. Sequential incubation of HL-60 cells with trimidox (25 mumol/L) for 24 hours and then with 10 mumol/L tiazofurin (an inhibitor of inosine monophosphate dehydrogenase) for 4 days produced synergistic growth inhibitory activity, and the cell number decreased to 16% of untreated controls. When differentiation- linked cell surface marker expressions were determined in cells treated with trimidox and tiazofurin, a significantly increased fluorescence intensity was observed for the CD 11b (2.9-fold). CD 33 (1.9-fold), and HLA-D cell surface antigens. Expression of the transferrin receptor (CD71) increased 7.3-fold in cells treated with both agents, compared with untreated controls. Our results suggest that trimidox in combination with tiazofurin might be useful in the treatment of leukemia.     

Granulocyte-macrophage colony-stimulating factor/interleukin-3 fusion protein (pIXY 321) enhances high-dose Ara-C-induced programmed cell death or apoptosis in human myeloid leukemia cells.

High dose Ara-C (HIDAC) induces programmed cell death (PCD) or apoptosis in vitro in human myeloid leukemia cells, which correlates with the inhibition of their clonogenic survival. Hematopoietic growth factors (HGFs) granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) have been demonstrated to enhance the metabolism and cytotoxic effects of HIDAC against leukemic progenitor cells. We examined the effect of pIXY 321 (a GM-CSF/IL-3 fusion protein) on HIDAC-induced PCD and related gene expressions as well as HIDAC-mediated colony growth inhibition of human myeloid leukemia cells. Unlike the previously described effects of HGFs on normal bone marrow progenitor cells, exposure to pIXY 321 alone for up to 24 hours did not suppress PCD in HL-60 or KG-1 cells. However, exposure to pIXY 321 for 20 hours followed by a combined treatment with Ara-C plus pIXY 321 for 4 or 24 hours versus treatment with Ara-C alone significantly enhanced the oligonucleosomal DNA fragmentation characteristic of PCD. This was temporally associated with a marked induction of c-jun expression and a significant decrease in BCL-2. In addition, the treatment with pIXY 321 plus HIDAC versus HIDAC alone produced a significantly greater inhibition of HL-60 colony growth. These findings highlight an additional mechanism of HIDAC-induced leukemic cell death that is augmented by cotreatment with pIXY 321 and may contribute toward an improved antileukemic activity of HIDAC.     

Resveratrol,a Natural Product Derived from Grapes,Is a New Inducer of Differentiation in Human Myeloid Leukemias

A natural product, resveratrol (3,4,40-trihydroxy-trans-stilbene), a phytoalexin found in grapes and other food products, is known as a cancer chemopreventive agent. We studied the in vitro biological activity of this compound by examining its effect on proliferation and differentiation in myeloid leukemia cell lines (HL-60, NB4, U937,THP-1, ML-1, Kasumi-1) and fresh samples from 17 patients with acute myeloid leukemia. Resveratrol (20 microM, 4 days) alone inhibited the growth in liquid culture of each of the 6 cell lines. Resveratrol (10 microM) enhanced the expression of adhesion molecules (CD11a, CD11b, CD18, CD54) in each of the cell lines except for Kasumi-1. Moreover, resveratrol (25 microM, 4 days) induced 37% of U937 cells to produce superoxide as measured by the ability to reduce nitroblue tetrazolium (NBT). The combination of resveratrol (10 microM) and all-trans-retinoic acid (ATRA) (50 nM, 4 days) induced 95% of the NB4 cells to become NBT-positive, whereas <1% and 12% of the cells became positive for NBT after a similar exposure to either resveratrol or ATRA alone, respectively. In U937 cells exposed to resveratrol (25 microM, 3 days), the binding activity of nuclear factor-kappaB (NFkappaB) protein was suppressed. Eight of 19 samples of fresh acute leukemia cells reduced NBT after exposure to resveratrol (20 microM, 4 days). Taken together, these findings show that resveratrol inhibits proliferation and induces differentiation of myeloid leukemia cells.     

CGP57148B (STI-571) induces differentiation and apoptosis and sensitizes Bcr-Abl-positive human leukemia cells to apoptosis due to antileukemic drugs

The differentiation and apoptosis-sensitizing effects of the Bcr-Abl-specific tyrosine kinase inhibitor CGP57148B, also known as STI-571, were determined in human Bcr-Abl-positive HL-60/Bcr-Abl and K562 cells. First, the results demonstrate that the ectopic expression of the p185 Bcr-Abl fusion protein induced hemoglobin in the acute myeloid leukemia (AML) HL-60 cells. Exposure to low-dose cytosine arabinoside (Ara-C; 10 nmol/L) increased hemoglobin levels in HL-60/Bcr-Abl and in the chronic myeloid leukemia (CML) blast crisis K562 cells, which express the p210 Bcr-Abl protein. As compared with HL-60/neo, HL-60/Bcr-Abl and K562 cells were resistant to apoptosis induced by Ara-C, doxorubicin, or tumor necrosis factor-alpha (TNF-alpha), which was associated with reduced processing of caspase-8 and Bid protein and decreased cytosolic accumulation of cytochrome c (cyt c). Exposure to CGP57148B alone increased hemoglobin levels and CD11b expression and induced apoptosis of HL-60/Bcr-Abl and K562 cells. CGP57148B treatment down-regulated antiapoptotic XIAP, cIAP1, and Bcl-x(L), without affecting Bcl-2, Bax, Apaf-1, Fas (CD95), Fas ligand, Abl, and Bcr-Abl levels. CGP57148B also inhibited constitutively active Akt kinase and NFkappaB in Bcr-Abl-positive cells. Attenuation of NFkappaB activity by ectopic expression of transdominant repressor of IkappaB sensitized HL-60/Bcr-Abl and K562 cells to TNF-alpha but not to apoptosis induced by Ara-C or doxorubicin. Importantly, cotreatment with CGP57148B significantly increased Ara-C- or doxorubicin-induced apoptosis of HL-60/Bcr-Abl and K562 cells. This was associated with greater cytosolic accumulation of cyt c and PARP cleavage activity of caspase-3. These in vitro data indicate that combinations of CGP57148B and antileukemic drugs such as Ara-C may have improved in vivo efficacy against Bcr-Abl-positive acute leukemia.     

Imatinib mesylate selectively influences the cellular metabolism of cytarabine in BCR/ABL negative leukemia cell lines and normal CD34+ progenitor cells

STI-571 (Imatinib/Glivec) has been shown to have synergism with various chemotherapeutic agents including cytosine arabinoside (Ara-C) in BCR/ABL positive leukemia cells. The antiproliferative and proapopotic effects of STI-571 in these experiments are mainly explained by its ability to specifically block the fusion-protein BCR/ABL which has a constitutively active tyrosine kinase activity. We investigated the effects of STI-571 in combination with Ara-C on BCR/ABL negative leukemia cell lines and CD34+ hematopoietic progenitor cells in-vitro. Raji, HL-60, K562, Kasumi and KG1a leukemia cells and CD34+ cells from healthy donors were incubated with 5-20 microg/ml Ara-C for 5 h alone or in combination with 10 microg/ml STI-571. Intracellular levels of Ara-CTP measured by HPLC were increased 1.5-3 fold in leukemia cells with most promiment effects in HL-60, Kasumi and Raji cells. In HL-60 cells a linear correlation between the concentration of STI-571 (1-10 microg/ml) and the subsequent levels of Ara-CTP was observed. A linear increase of Ara-CTP could be induced by increasing the incubation time with STI-571 from 2-6 h with a ceiling effect after 8 h. In contrast coincubation of mononuclear cells or purified CD34+ cells with STI-571 at therapeutic concentrations lead to decreased intracellular levels of Ara-CTP. The synergism between Ara-C and STI-571 was even more pronounced in Raji and HL-60 cells when 300 ng/ml G-CSF were added at the beginning of the culture period. Intracellular measurements of STI-571 revealed no decreased or increased levels of the compound when increasing Ara-C concentrations were used. Our findings indicate that STI-571 can have significant impact on nucleoside metabolism in malignant and non-malignant hematopoietic cells. Further investigations will have to show whether theses effects can lead to increased cytotoxicity in primary blasts of patients with acute leukemia.     

Effect of antimetabolites and thymidine blockage on the induction of differentiation of HL-60 cells by retinoic acid or 1 alpha,25-dihydroxyvitamin D3

Induction of differentiation of the HL-60 human promyelocytic leukemia cell line by retinoic acid or 1 alpha,25(OH)2D3 was analyzed under the condition in which cellular DNA synthesis was inhibited by several antimetabolites or blocked by thymidine. The results demonstrate that differentiation occurs in the absence of DNA synthesis and that some inhibitors of DNA synthesis may enhance the differentiation of HL-60 cells by the above inducers. Among the antimetabolites used, the enhancement of induction of differentiation by hydroxyurea was shown to be more effective than that of Ara-C or aphidicolin. The effect of thymidine blockage was similar to that of hydroxyurea. These different effects may be due to the different points at which the cell cycle is blocked by these agents. These results seem to be common in both the differentiation of the granulocytic line induced by retinoic acid and of the macrophage line induced by 1 alpha,25(OH)2D3. The present study also suggests that combination treatment with the inhibitor of DNA synthesis and the inducer of differentiation could be beneficial in the clinical therapy of leukemia. The mode of action of clinical low-dose Ara-C treatment is also discussed.     

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.     

Activation of caspases and induction of apoptosis by novel ribonucleotide reductase inhibitors amidox and didox

OBJECTIVE: Amidox and didox are two polyhydroxy-substituted benzohydroxamic acid derivatives that belong to a new class of ribonucleotide reductase (RR) inhibitors. RR is the rate-limiting enzyme for de novo deoxyribonucleotide synthesis, and its activity is significantly increased in tumor cells in proportion to the proliferation rate. Therefore, RR is a target for antitumor therapy. MATERIALS AND METHODS: HL-60 and K562 leukemia cells were treated with increasing doses of amidox and didox. Thereafter, the mode of cytotoxic drug action was determined by Hoechst 33258/propidium iodide (HO/PI) double staining, annexin binding, DNA fragmentation, and caspase activation. This was correlated to the decrease in dNTP levels. Staining with HO/PI and binding of fluorescein isothiocyanate-conjugated annexin V to externalized phosphatidylserine were used to quantify apoptosis. RESULTS: Low doses of amidox or didox resulted in an increase of apoptotic HL-60 cells within 48 hours. Higher doses (50 microM amidox or 250 microM didox) led to rapid induction of apoptosis, which could be detected as early as 4 hours after treatment. After 48 hours with these concentrations, almost 100% of the HL-60 cells died by apoptosis without an increase in necrosis. K562 cells were found to be resistant to amidox but not to didox. In HL-60 cells, upstream caspase 8 is processed in response to didox, whereas caspases 8 and 9 are processed upon amidox treatment. Didox-induced apoptosis, but not amidox-induced apoptosis, can be correlated with the decrease in dNTP levels.The results suggests that amidox induces several apoptosis mechanisms in HL-60 cells. In contrast, only caspase 9 is activated by didox in K562 cells, and because amidox hardly induces apoptosis in this cell line, no caspase cleavage is observed. CONCLUSIONS: Didox triggers distinct apoptosis pathways in HL-60 and K562 cells.     

The roles of bioactive sphingolipids in resveratrol-induced apoptosis in <Emphasis Type="SmallCaps">HL60</Emphasis> acute myeloid leukemia cells

Purpose  

Acute promyelocytic leukemia results from a translocation between 15 and 17 chromosomes that produce PML/RARα fusion protein. PML/RARα inhibits differentiation of myeloid precursor cells at stem cell level. Resveratrol is a phytoalexin that exerts cytotoxic effects on cancer cells. Ceramides have crucial roles in cell growth, proliferation, differentiation, drug resistance, and apoptosis. In this study, we examined the possible cytotoxic effects of resveratrol on acute myeloid leukemia cells and determined the roles of ceramide-metabolizing genes in resveratrol-induced apoptosis, in addition to investigating the possibility of increasing the sensitivity of HL60 cells to resveratrol by manipulating sphingolipids.     

Antileukemic drugs increase death receptor 5 levels and enhance Apo-2L-induced apoptosis of human acute leukemia cells

In present studies, treatment with tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL, also known as Apo-2 ligand [Apo-2L]) is shown to induce apoptosis of the human acute leukemia HL-60, U937, and Jurkat cells in a dose-dependent manner, with the maximum effect seen following treatment of Jurkat cells with 0.25 microg/mL of Apo-2L (95.0% +/- 3.5% of apoptotic cells). Susceptibility of these acute leukemia cell types, which are known to lack p53(wt) function, did not appear to correlate with the levels of the apoptosis-signaling death receptors (DRs) of Apo-2L, ie, DR4 and DR5; decoy receptors (DcR1 and 2); FLAME-1 (cFLIP); or proteins in the inhibitors of apoptosis proteins (IAP) family. Apo-2L-induced apoptosis was associated with the processing of caspase-8, Bid, and the cytosolic accumulation of cytochrome c as well as the processing of caspase-9 and caspase-3. Apo-2L-induced apoptosis was significantly inhibited in HL-60 cells that overexpressed Bcl-2 or Bcl-x(L). Cotreatment with either a caspase-8 or a caspase-9 inhibitor suppressed Apo-2L-induced apoptosis. Treatment of human leukemic cells with etoposide, Ara-C, or doxorubicin increased DR5 but not DR4, Fas, DcR1, DcR2, Fas ligand, or Apo-2L levels. Importantly, sequential treatment of HL-60 cells with etoposide, Ara-C, or doxorubicin followed by Apo-2L induced significantly more apoptosis than treatment with Apo-2L, etoposide, doxorubicin, or Ara-C alone, or cotreatment with Apo-2L and the antileukemic drugs, or treatment with the reverse sequence of Apo-2L followed by one of the antileukemic drugs. These findings indicate that treatment with etoposide, Ara-C, or doxorubicin up-regulates DR5 levels in a p53-independent manner and sensitizes human acute leukemia cells to Apo-2L-induced apoptosis. (Blood. 2000;96:3900-3906)     

High-dose mitoxantrone induces programmed cell death or apoptosis in human myeloid leukemia cells

Mitoxantrone has been shown in vitro to exhibit a steep dose-response relationship with respect to the clonogenic survival of acute myeloid leukemia cells. In this report, we show that 1-hour exposure of human myeloid leukemia HL-60 and KG-1 cells to mitoxantrone concentrations ranging between 0.1 and 10.0 mumol/L induced internucleosomal DNA fragmentation of approximately 200-bp integer multiples, characteristic of cells undergoing programmed cell death (PCD) or apoptosis. Mitoxantrone-mediated PCD was associated with a steep inhibition of the clonogenic survival of the leukemic cells. In addition, intracellularly, mitoxantrone-induced PCD was associated with a marked induction of c-jun and significant repression of c-myc and BCL-2 oncogenes. Pretreatment with the protein kinase C stimulator phorbol myristate acetate enhanced mitoxantrone-induced internucleosomal DNA fragmentation, whereas protein kinase C inhibitors staurosporine and H7 had no effect. These findings suggest that PCD is a potential mechanism underlying the steep dose-response relationship of mitoxantrone to the inhibition of clonogenic survival of acute myeloid leukemia cells.     

The tetrapeptide AcSDKP, an inhibitor of the cell-cycle status for normal human hematopoietic progenitors, has no effect on leukemic cells.

The tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) inhibits the entry into DNA synthesis of murine spleen colony-forming units (CFU-S) and protects these cells during chemotherapy. This synthetic peptide also inhibits the growth of normal human marrow progenitors granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E) and decreases their percentage in DNA synthesis at nanomolar concentration. In view of its clinical application as a marrow protector, we have investigated its effects on malignant cells. Studies were carried out on HL-60 cells and on fresh leukemic cells from patients with either chronic myeloid leukemia (CML) or acute myeloid leukemia (AML). Results showed that AcSDKP, whatever the doses used, did not modify the proliferation of both HL-60 cells and AML cells even when enhanced by stimulating factors such as interleukin 3 or granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, no change in the number and the percentage in S-phase of both HL-60 clonogenic cells and CML progenitors was observed. Our data clearly demonstrate that the tetrapeptide AcSDKP was ineffective on leukemic cells and therefore by acting selectively on normal progenitors represents a potent therapeutical agent for the protection of normal bone marrow progenitors during chemotherapy.     

Resveratrol selectively inhibits leukemia cells: a prospective agent for ex vivo bone marrow purging

Ex vivo purging of contaminating tumor cells may reduce the incidence of relapse in patients undergoing bone marrow transplantation. In this study we demonstrate that resveratrol, a phytoalexin with anti-oxidant and chemopreventive activity, exhibits anti-leukemic activity against mouse (32Dp210, L1210) and human (U937, HL-60) leukemic cell lines by inhibiting cell proliferation. Long-term exposure to resveratrol also inhibits the clonal growth of normal hematopoietic progenitor cells but at a higher IC50 of resveratrol than that for most of the leukemia cell lines tested. The inhibitory effect of resveratrol on hematopoietic progenitors is partially reversible, whereas the effect on leukemia cells is largely irreversible. The inhibition of leukemia cells by resveratrol involves nucleosomal DNA fragmentation (apoptosis). On the other hand, resveratrol does not induce or enhance spontaneously occurring apoptotic death in normal hematopoietic progenitor cells. In vivo experiments performed with untreated and resveratrol-treated bone marrow showed comparable hematopoietic reconstitution in lethally irradiated mice (10 Gy) as determined by survival, hematologic recovery, and the number of hematopoietic progenitor cells present in the marrow of reconstituted animals. Taken together, these results indicate the potential use of resveratrol for ex vivo pharmacological purging of leukemia cells from bone marrow autografts without significant loss in the hematopoietic activity of progenitor cells.     

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.     

The effect of pyrimidine nucleosides on adenosine-induced apoptosis in HL-60 cells

Among several nucleosides, adenosine is the only one to induce typical apoptotic cell death in human promyelocytic leukemia HL-60 cells. Intracellularly transported adenosine seemed to be required for the induction of apoptosis, since dipyridamole, which inhibits the transport of adenosine, strongly suppressed apoptosis, and 8-phenyltheophylline, a receptor antagonist, did not affect the adenosine-induced effect. The viability of adenosine-treated HL-60 cells was partially recovered by supplementation with a pyrimidine nucleoside, uridine or thymidine. Cytidine or deoxycytidine had no effect on the growth and survival of adenosine-treated cells, while uridine or thymidine inhibited adenosine-induced intracellular DNA fragmentation. These results suggest that the quantitative adjustment of purine and pyrimidine nucleosides might play an important role in the adenosine-induced apoptosis of HL-60 cells. The reduction of c-Myc expression in adenosine-treated cells was prevented by uridine or thymidine. These observations suggest that the expression of c-Myc might be related to an intracellular sensing system for the quantitative balance of nucleosides or nucleotides. Received: 14 January 1998 / Accepted: 11 May 1998     

Effect of Granulocyte Colony-Stimulating Factor on Chemotherapeutic Activity of Cytosine Arabinoside in Acute Leukemic Cell Lines

Recent studies have shown the presence of receptors for granulocyte colony-stimulating factor (G-CSF) on lymphoid leukemic cells. To determine the effect of G-CSF on chemotherapeutic activity of cytosine arabinoside (Ara-C) on lymphoid as well as myeloid leukemic cells, we evaluated cell counts, apoptosis, and growth inhibition in HL-60, KG-1, Molt-4, Jijoye, and CCRF-CEM cell lines after incubation with Ara-C (0.1 and 1 micromol/L) and/or 5 ng/mL G-CSE G-CSF potentiated the effect of Ara-C on 2 of 3 lymphoid leukemic cell lines (Molt-4 and Jijoye), whereas it decreased the apoptosis and the effect of Ara-C on myeloid cell lines (HL-60 and KG-1).     

Effects of thymidine and hydroxyurea on the metabolism and cytotoxicity of 1-B-D arabinofuranosylcytosine in highly resistant human leukemia cells

We have examined the effect of the ribonucleotide reductase inhibitors thymidine (dThd) and hydroxyurea (HU) on the metabolism and cytotoxicity of high concentrations (10 to 100 mumol/L) of cytosine arabinoside (Ara-C) in a deoxycytidine kinase-deficient, highly Ara-C-resistant human promyelocytic leukemic cell subline (HL-60/Ara-C). Administration of dThd or HU (0.5 to 3 mmol/L) in conjunction with high concentrations of Ara-C either sequentially or simultaneously lead to enhanced (and in some cases a supra-additive) inhibition of HL-60/Ara-C suspension culture growth and soft agar colony formation. In addition, exposure of cells to 0.5 mmol/L dThd or HU for 48 hours significantly increased the percentage of HL-60/Ara-C in S-phase. In contrast to previous studies involving low-dose Ara-C administration, we were unable to detect dThd- or HU-mediated increments in Ara-CTP formation in HL-60/Ara-C cells treated with high-dose Ara-C despite reductions in intracellular dCTP pools as great as 65%. However, dThd or HU did increase Ara-C incorporation into newly synthesized DNA. These studies show that administration of dThd or HU in conjunction with high concentrations of Ara-C results in an enhanced antiproliferative effect of Ara-C against Ara-C-resistant leukemic cells deficient in deoxycytidine kinase.