Copper-nuclease efficiency correlates with cytotoxicity for the 4-methoxypyrrolic natural products.

The DNA-targeting activities of the 4-methoxypyrrolic natural products, that include prodigiosin (1), tambjamine E (2), and the blue pigment (3), have been compared using fluorescence spectroscopy to study DNA binding and agarose gel electrophoresis to assess their ability to facilitate oxidative copper-promoted DNA cleavage. Fluorescence emission titration of 3 with calf-thymus DNA (CT-DNA) shows that the natural product occupies a site size (n) of ca. two base pairs and possesses an affinity constant (K) of approximately 6x10(5) x M(-1). Similar to prodigiosin (1), the blue pigment 3 was found to facilitate oxidative double-strand DNA (dsDNA) cleavage without the aid of an external reducing agent. Quantitation of ds- (n2) and ss- (n1) breaks provided n1:n2 ratios of approximately 8-12, which were significantly greater than the number expected from the accumulation of ss-breaks (approximately 120). This was contrasted by the nicking activity of tambjamine E (2), which only generates ss-breaks in the presence of copper. The superior copper-nuclease activity of 1 and 3 also correlated with their superior anticancer properties against leukemia (HL-60) cells. These results are discussed with respect to the mode of cytotoxicity by the 4-methoxypyrrolic natural products.

Design, synthesis, and biological activity of a novel non-cisplatin-type platinum-acridine pharmacophore.

Platinum-acridine conjugates were prepared from [PtCl2(ethane-1,2-diamine)] and the novel acridinylthioureas MeHNC(S)NMeAcr (6) and MeHNC(S)NMe(CH2CH2)NHAcr (15) by replacing one chloro leaving group in the cisplatin analogue with thiourea sulfur. In HL-60 leukemia cells, IC(50) values for 7 (Pt-tethered 6) and 16 (Pt-tethered 15) were 75 and 0.13 microM, respectively. In the ovarian cell lines 2008 and C13, 16 was active at micromolar concentrations and showed only partial cross-resistance with clinical cisplatin. Possible structure-activity relationships are discussed.

High affinity interleukin-3 receptor expression on blasts from patients with acute myelogenous leukemia correlates with cytotoxicity of a diphtheria toxin/IL-3 fusion protein.

Diphtheria fusion proteins are a novel class of agents for the treatment of chemotherapy resistant AML. We prepared DT(388)IL3 composed of human interleukin-3 (IL3) fused to the catalytic and translocation domain of diphtheria toxin (DT(388)) and assessed its activity on patient AML blasts. The number and affinity of IL3 receptors in circulating blasts was measured using a radiolabeled IL3 agonist (SC-65461). Ninety-two percent of patients' blasts had both high and low affinity IL3 receptors. DT(388)IL3 cytotoxicity (>1 log cell kill) was seen in nine of 25 samples (36%). There was a significant correlation between DT(388)IL3 log cell kill and blast high affinity IL3 receptor density (P=0.0044). These results show that specific high affinity IL3 binding is one factor important in the sensitivity of patients' leukemic blasts to DT(388)IL3.

Oncogene-dependent engraftment of human myeloid leukemia cells in immunosuppressed mice.

We have developed an in vivo model of differentiated human acute myeloid leukemia (AML) by retroviral infection of the cytokine-dependent AML cell line TF-1 with the v-Src oncogene. When injected either intravenously or intraperitoneally into 300 cGy irradiated SCID mice, animals formed multiple granulocytic sarcomas involving the adrenals, kidneys, lymph nodes and other organs. The mean survival time was 34+/-10 days (n = 40) after intravenous injection and 24+/-3 days (n = 5) after intraperitoneal injection of 20 million cells. The cells recovered from leukemic animals continued to express interleukin-3 receptors and remained sensitive to the diphtheria fusion protein DT388IL3. Further, these granulocytic sarcoma-derived cells grew again in irradiated SCID mice (n = 10). The cytogenetic abnormalities observed prior to inoculation in mice were stably present after in vivo passage. Similar to the results with v-Src transfected TF-1 cells, in vivo leukemic growth was observed with TF-1 cells transfected with the human granulocyte-macrophage colony-stimulating factor gene (n = 5) and with TF-1 cells recovered from subcutaneous tumors in nude mice (n = 5). In contrast, TF-1 cells expressing v-Ha-Ras (n = 5), BCR-ABL (n = 5), or activated Raf-1 (n = 44) did not grow in irradiated SCID mice. This is a unique, reproducible model for in vivo growth of a differentiated human acute myeloid leukemia and may be useful in the assessment of anti-leukemic therapeutics which have human-specific molecular targets such as the interleukin-3 receptor.

Cellular metabolism in lymphocytes of a novel thioether-phospholipid-AZT conjugate with anti-HIV-1 activity.

We previously synthesized a thioetherphospholipid-AZT conjugate (3'-azido-3'-deoxy-5'-(1-hexadecylthio-2-methoxypropyl)-phosphothymidine, CP-102) with potent anti-HIV-1 activity and significant reduction in cell cytotoxicity compared to AZT alone. To study the cellular metabolism of the conjugate compound we synthesized a double-tritium-labeled thioetherphospholipid-AZT conjugate (3'-azido-3'-deoxy-5'-(1-[9,10-3H]-S-octadecylthio-2-O-methoxypropyl)-phosphothymidine-[methyl-3H], [3H]CP-102). The intracellular radioactive metabolic products of [3H]CP-102 treated human lymphoblastoid CEM-SS cells were analyzed by HPLC and thin-layer chromatography. Results of this investigation provide evidence that a putative intracellular lipid cleavage enzyme metabolizes [3H]CP-102 to form a thioetherdiglyceride compound that migrates with an authentic 1-S-octadecyl-2-O-methyl-thioglycerol standard on TLC. The thioetherdiglyceride metabolite did not react with the ninhydrin reagent indicating it did not contain a primary amine such as that found on serine or ethanolamine containing phospholipids. Also, the product did not contain a phosphatidic acid group based on migration characteristics in the TLC plate. The other major hydrophilic metabolite was 3'-azido-3'-deoxythymidine-[methyl-3H]-monophosphate (AZT-MP) with lesser amounts of AZT, AZT-DP and AZT-TP. In summary, the best interpretation of these data is that the thioetherphospholipid-AZT conjugate, [3H]CP-102, is cleaved by a putative intracellular lipid cleavage enzyme to release a thioetherdiglyceride compound and AZT-MP. The resulting AZT-MP was either dephosphorylated to AZT or sequentially phosphorylated to AZT-DP and, ultimately, to AZT-TP, the known inhibitory metabolite against HIV-1 reverse transcriptase. Phospholipid-nucleoside conjugates may provide a unique approach for developing anti-HIV-1 prodrugs that do not have a strict requirement for a nucleoside kinase for initial activation of the prodrug to an antiviral form.

In vitro interleukin-3 binding to leukemia cells predicts cytotoxicity of a diphtheria toxin/IL-3 fusion protein.

Patients with acute myeloid leukemia frequently develop chemotherapy resistant blasts. To overcome multidrug resistance, a diphtheria toxin fusion protein (DTIL3) was engineered by fusing the catalytic and translocation domains of diphtheria toxin (DT) to human interleukin-3 (IL-3). However, when blasts were isolated from patients and tested for colony growth inhibition by DTIL3, only a third of the samples showed sensitivity to the fusion protein. Prior to clinical development, we need to be able to identify which patients are likely to respond to therapy with DTIL3. In this report, we compared the inhibition of thymidine incorporation in human leukemia cell lines by DTIL3 to the IL-3 receptor number and affinity. We found DTIL3 was cytotoxic to four of the eight cell lines tested with half-maximal inhibition of thymidine incorporation (IC(50)) from 1 to 50 pM. The IL-3 receptor density for these cell lines ranged from 0 to 2635 receptors per cell. The dissociation constant for an IL-3 high-affinity receptor agonist was 0.5 nM for cell lines with receptors. We found a correlation for the cell lines between the presence of high-affinity IL-3 receptors and sensitivity to DTIL3 (p = 0.03). These results suggest the variability in sensitivity of patient leukemic progenitors to DTIL3 may be due in part to the presence or absence of high-affinity IL-3 receptors.

Diphtheria toxin fused to human interleukin-3 is toxic to blasts from patients with myeloid leukemias.

Leukemic blasts from patients with acute phase chronic myeloid leukemic and refractory acute myeloid leukemia are highly resistant to a number of cytotoxic drugs. To overcome multi-drug resistance, we engineered a diphtheria fusion protein by fusing human interleukin-3 (IL3) to a truncated form of diphtheria toxin (DT) with a (G4S)2 linker (L), expressed and purified the recombinant protein, and tested the cytotoxicity of the DTLIL3 molecule on human leukemias and normal progenitors. The DTLIL3 construct was more cytotoxic to interleukin-3 receptor (IL3R) bearing human myeloid leukemia cell lines than receptor-negative cell lines based on assays of cytotoxicity using thymidine incorporation, growth in semi-solid medium and induction of apoptosis. Exposure of mononuclear cells to 680 pM DTLIL3 for 48 h in culture reduced the number of cells capable of forming colonies in semi-solid medium (colony-forming units leukemia) > or =10-fold in 4/11 (36%) patients with myeloid acute phase chronic myeloid leukemia (CML) and 3/9 (33%) patients with acute myeloid leukemia (AML). Normal myeloid progenitors (colony-forming unit granulocyte-macrophage) from five different donors treated and assayed under identical conditions showed intermediate sensitivity with three- to five-fold reductions in colonies. The sensitivity to DTLIL3 of leukemic progenitors from a number of acute phase CML patients suggests that this agent could have therapeutic potential for some patients with this disease.

Role of c-Jun N-terminal kinase/p38 stress signaling in 1-beta-D-arabinofuranosylcytosine-induced apoptosis.

1-beta-D-Arabinofuranosylcytosine (ara-C) induced apoptosis in HL-60 cells, which was preceded by the activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38 mitogen-activated protein kinase (MAPK). 2'-Amino-3'-methoxyflavone (PD098059) and 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) were used to inhibit the activity of ERK and p38, respectively. SEK-AL, a dominant-negative mutant of SEK1, was transfected into HL-60 cells (HL-60/SEK-AL) to assess the role of JNK/SAPK activity in apoptosis. PD098059 (25 microM) inhibited ara-C-induced caspase-3-like activity but was ineffective in altering ara-C-mediated apoptotic DNA fragmentation and clonogenicity. On the other hand, SB203580 (20 microM) inhibited ara-C-induced caspase-3-like activity, apoptotic DNA fragmentation, and clonogenicity. The inhibition of JNK1 activation in HL-60/SEK-AL cells did not block ara-C-induced apoptotic DNA fragmentation. These results suggest that ara-C-induced apoptotic DNA fragmentation and loss of clonogenicity occur through a p38-dependent pathway.

Extracellular signal-regulated kinase (ERK) activity is required for TPA-mediated inhibition of drug-induced apoptosis.

Leukemia cells respond to toxic stimuli by undergoing a form of programmed cell death known as apoptosis. However, the signaling events responsible for the execution of this form of death are poorly understood. Mitogen-activated protein kinase (MAPK) signaling cascades are involved in the cellular response to extracellular stimuli. Specifically, extracellular signal-regulated kinases (ERKs) have been associated with proliferation and differentiation, whereas the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs) have been implicated in cell arrest and death. We report the use of 12-O-tetradecanoylphorbol-13-acetate (TPA) in the inhibition of apoptosis in HL-60 cells stimulated with the JNK/SAPK activator anisomycin. This anti-apoptotic effect was accompanied by a sustained increase in ERK activity. Furthermore, the use of protein kinase C (PKC) inhibitors suggested that PKC was involved in the induction of ERK activity and in the inhibition of apoptosis by TPA since the inhibition of apoptosis was attenuated when cells were pretreated with PKC inhibitors. Lastly, we observed that the use of the MEK1 inhibitor PD98059 inhibited TPA-mediated ERK activity and abrogated the anti-apoptotic effects of TPA. However, apoptotic inhibition was not solely ERK-dependent since cells lacking JNK/SAPK stimulation did not undergo apoptosis. Therefore, we conclude that TPA inhibits the induction of apoptosis in anisomycin-treated HL-60 cells through an ERK-dependent pathway and that this effect can be reversed by the attenuation of ERK activity accompanied with the stimulation of JNK/SAPK activity.

Gemcitabine induces programmed cell death and activates protein kinase C in BG-1 human ovarian cancer cells.

PURPOSE: Cytosine arabinoside induces apoptosis and this cell death process is influenced by protein kinase C signaling events in leukemic cells. We present findings that extend these observations to include another deoxycytidine analog, gemcitabine, which is more potent in solid tumors. METHODS AND RESULTS: Gemcitabine induced programmed cell death in BG-1 human ovarian cancer cells based on biochemical and morphologic analyses. The DNA was fragmented in BG-1 cells exposed to gemcitabine (0.5 microM, 1.0 microM and 10 microM) for 8 h, but gemcitabine treatment did not induce internucleosomal DNA degradation. Scanning and transmission electron microscopy of BG-1 cells showed morphologic changes associated with apoptosis in response to gemcitabine: membrane blebbing, the formation of apoptotic bodies and chromatin condensation. Thus, BG-1 cells undergo programmed cell death in response to gemcitabine treatment without internucleosomal DNA fragmentation. Furthermore, gemcitabine (10 microM) activated protein kinase C in BG-1 cells and the phosphorylation of the endogenous protein kinase C substrate, myristoylated alanine-rich C kinase substrate, was increased following exposure of BG-1 cells to gemcitabine for up to 6 h. Clonogenicity studies with gemcitabine in combination with various protein kinase C-modulating agents demonstrated that gemcitabine cytotoxicity was influenced by protein kinase C signaling events in BG-1 cells. Short-term (1 h) exposure to TPA (1 or 10 nM) followed by gemcitabine (0.5 microM for 4 h) did not alter the response to gemcitabine. However, a 24-h exposure to TPA followed by gemcitabine resulted in synergistic cytotoxicity, while coincubation of TPA with a PKC inhibitor (e.g. bisindolylmaleimide or calphostin-C) in this regimen abrogated the synergistic response. CONCLUSIONS: Based on our findings, it is plausible that gemcitabine therapy could be improved by modulating PKC signaling events linked to drug-induced apoptosis/cytotoxicity.

The effects of gemcitabine and TPA on PKC signaling in BG-1 human ovarian cancer cells.

Protein kinase C (PKC) signaling pathways play an important role in cell survival and anticancer drug-induced apoptosis. We observed in clonogenicity assays of BG-1 human ovarian cancer cells that gemcitabine cytotoxicity was increased synergistically when drug treatment was followed or preceded by a 24-h exposure to 10 nM 12-O-tetradecanoylphorbol-13-acetate (TPA). Coincubation of 10 nM TPA with pharmacological inhibitors of PKC abrogated the synergism of TPA and gemcitabine. These observations prompted further investigation of PKC signaling events linked to TPA and gemcitabine cytotoxicity in BG-1 cells. Because PKC isoforms are differentially expressed in various cell types, we determined that BG-1 cells express the alpha, beta, delta, epsilon, and zeta isoforms of PKC. In addition, 1-h exposures to 10 microM gemcitabine triggered cytosol to membrane translocation of PKC isoforms alpha, delta, and epsilon, indicating these isoforms were activated by gemcitabine. We also explored the PKC mechanism(s) responsible for the synergism of TPA and gemcitabine, and determined that treatment with 10 nM TPA for 24 h in BG-1 cells: 1) downregulated PKCdelta and PKCalpha, without affecting PKCepsilon, 2) did not affect cell cycle distribution into S phase. 3) increased extracellular signal-regulated kinase signaling, and 4) increased intracellular alkaline phosphatase activity, a biochemical marker of cellular differentiation. Chronic exposure (24 h) to TPA enhanced gemcitabine cytotoxicity, perhaps by inducing cellular differentiation pathways in BG-1 cells. Therefore, the use of differentiating agents in combination with gemcitabine may improve its clinical efficacy.

Induction of apoptosis by gemcitabine in BG-1 human ovarian cancer cells compared with induction by staurosporine, paclitaxel and cisplatin.

A variety of chemotherapeutic agents induce cell death via apoptosis. We had shown previously that gemcitabine (2',2'-difluorodeoxycytidine) induced an atypical apoptosis in BG-1 human ovarian cancer cells; therefore, further studies were conducted to characterize more precisely gemcitabine-induced apoptosis in BG-1 cells compared to a general inducer of apoptosis, staurosporine. BG-1 cells exposed to 0.5, 1.0 and 10 microM gemcitabine for 8 h, or staurosporine (1.0 microM) for 6 h, exhibited high molecular weight DNA fragmentation (50 kbp); however, only staurosporine treatment produced internucleosomal DNA fragments (200 bp) in a laddered pattern on the agarose gel. Staurosporine (1.0 microM) rapidly induced phosphatidylserine plasma membrane translocation that increased linearly with time as measured by annexin V-FITC binding, and similar kinetics were observed for caspase activation by staurosporine in BG-1 cells. In contrast, 10 microM gemcitabine increased phosphatidylserine expression in a small fraction of cells (5-10%) vs. untreated controls over the course of 48 h and significant caspase activity was detected within 12 h of drug exposure. Time-lapse video microscopy of BG-1 cells exposed to 1.0 microM staurosporine or 10 microM gemcitabine for up to 72 h showed that the morphologic changes and kinetics of cell death induced by these agents differed significantly. We also evaluated the apoptosis induced by paclitaxel (a mitotic poison) and cisplatin (an agent not dependent on cell cycle functions) in BG-1 cells by these methods because these drugs are used clinically to treat ovarian cancer. Our findings demonstrate that the kinetics of apoptotic cell death induced by gemcitabine and other chemotherapeutic agents should be taken into account when designing treatment strategies for ovarian cancer.

Urinary elimination of cyclophosphamide alkylating metabolites and free thiols following two administration schedules of high-dose cyclophosphamide and mesna.

Twenty patients with a variety of neoplastic diseases were treated with preparative regimens containing high-dose cyclophosphamide (CY) administered as a 2-h infusion (60 mg/kg) for 2 days or by continuous infusion (1500 mg/m2/day) for 4 days. In patients receiving CY by 2-h infusion, the uroprotectant 2-mercaptoethane sulfonate (MESNA) was administered as an intermittent, bolus intravenous infusion (20% of CY dose) every 6 h. In patients receiving continuous infusion CY, MESNA was administrated concomitantly at an equivalent dose to CY by continuous infusion. During the first 24 h of CY administration, urine was collected at 2-h intervals and analyzed for free thiols and CY-alkylating metabolites. In patients receiving CY by short infusion and MESNA by intermittent bolus infusion, urinary concentrations of alkylating metabolites peaked at 4-8 h. During each dose of MESNA, urinary free thiols peaked at 2 h following administration but fell to pre-treatment levels at subsequent intervals. In patients receiving CY by continuous infusion, CY alkylating metabolites increased gradually over the 24-h study period while free thiols remained at a constant level during this period. With bolus administration of CY and intermittent bolus administration of MESNA every 6 h, there are periods where urinary CY-alkylating metabolites are elevated and free thiol concentrations are diminished. During continuous infusion of CY and MESNA, urinary CY alkylating metabolites reached peak concentrations at 18-22 h while the exposure of the bladder to free thiols remained constant. Recommendations are provided to increase the exposure of free thiols in the bladder when MESNA is administered by bolus or continuous infusion.

GM-CSF and asparaginase potentiate ara-C cytotoxicity in HL-60 cells.

In preparation for a clinical trial using GM-CSF on days 4-10 of sequential high-dose cytarabine (ara-C) and asparaginase (ASNase) on days 1-3 and 8-10, potential interactions between the protein synthesis inhibitor ASNase and GM-CSF were evaluated. Granulocyte-macrophage colony-stimulating factor (GM-CSF) can stimulate acute myeloid leukemia (AML) cells to proliferate in vitro and in vivo. Log phase HL-60 cells were exposed to ara-C (10 microM x 3 h) and/or ASNase (10 U/ml during the last 2 h of ara-C). Ara-C and/or ASNase was removed and cells were incubated with or without GM-CSF (10 ng/ml). After 24, 48 and 72 h of GM-CSF there was no significant difference in the S phase fraction of cells exposed to ASNase prior to GM-CSF. Soft agar cloning efficiency was determined after retreatment with ara-C +/- ASNase 24 h into the GM-CSF incubation. GM-CSF enhanced cytotoxicity for all combinations, although this effect was of borderline significance (P = 0.0621); addition of ASNase to the treatment regimen significantly (P = 0.0229) enhanced cytotoxicity without any evidence of a negative interaction with GM-CSF. In addition, ara-C metabolism was assessed during simultaneous exposure to ara-C (10 microM x 3 h) +/- ASNase (10 U/ml the last 2 h) +/- GM-CSF (10 ng/ml beginning 24 h prior to ara-C). Ara-C incorporated into DNA (P = 0.0302) and ara-CTP formation (P = 0.0084 and P = 0.0003 at 2 and 3 h timepoints, respectively) were both increased significantly by GM-CSF, with modest non-significant increases with ASNase exposures. Neither ASNase nor GM-CSF inhibited the effects of the other in this in vitro model. Therefore, when appropriately scheduled, both GM-CSF and ASNase may potentiate ara-C cytotoxicity.

Deoxycytidine kinase is phosphorylated in vitro by protein kinase C alpha.

Deoxycytidine (dCyd) kinase was effectively phosphorylated by protein kinase C. The reaction was rapid, occurring at 4 degrees C as well as at 37 degrees C and approximately 0.7 mol of phosphate could be incorporated per mol of deoxycytidine kinase. Phosphoserine was the primary amino acid to be phosphorylated. Phosphorylation of deoxycytidine kinase resulted in a 100% increase in the Vmax using dCyd as a substrate (52.16 +/- 1.3 versus 104.47 +/- 11.4 nmol/min/mg protein), and an increase in the apparent Km (2.0 +/- 0.2 microM versus 6.9 +/- 1.2 microM). The inactive antimetabolite, ara-C, is activated within a cell by deoxycytidine kinase phosphorylation of the prodrug. Recent studies have shown that ara-C activates protein kinase C in vivo [1]. Furthermore, ara-C has been shown to be metabolized to ara-CDP-choline via reversal of the cholinephosphotransferase [2] producing diglyceride, a cellular activator of protein kinase C. Thus, in situ, deoxycytidine kinase may be phosphorylated by protein kinase C with the result that self-potentiation of ara-C toxicity may occur via increased activity of deoxycytidine kinase.

Purification and characterization of deoxycytidine kinase from acute myeloid leukemia cell mitochondria.

Deoxycytidine kinase is a key anabolic enzyme for the activation of ara-C and other antitumor drugs, as well as normal purine and pyrimidine deoxynucleotides. Previously, two forms of the kinase have been identified; deoxycytidine kinase I (70 kDa) and deoxycytidine kinase II (70 kDa). Deoxycytidine kinase I utilized dCyd and ara-C as substrates, while deoxycytidine kinase II used dCyd and dThd as substrates. Deoxycytidine kinase kinase II had very low activity on ara-C as a substrate. We report a procedure for the purification of a novel deoxycytidine kinase (52 kDa) from isolated human peripheral blood leukemia cell mitochondria. This enzyme has activity similar to deoxycytidine kinase II. The enzyme was extracted from the mitochondria with digitonin (1 mg/8 mg protein) and 0.3 M NaCl, and the extract was purified by DEAE-cellulose chromatography and thymidine-Sepharose affinity chromatography. This procedure produced a near homogeneous enzyme preparation with a yield of 70%. The mitochondrial deoxycytidine kinase was localized to the outer mitochondrial membrane. The enzyme phosphorylated dCyd (Km = 17 microM), however, ara-C was not a good substrate for the mitochondrial deoxycytidine kinase. ATP was the best phosphate donor, whereas dCTP and dTTP were potent inhibitors of mitochondrial deoxycytidine kinase. In contrast, phosphorylation of ara-C by deoxycytidine kinase I utilized GTP, dGTP, or ATP as a phosphate donor.

1-beta-D-arabinofuranosylcytosine-diphosphate-choline is formed by the reversal of cholinephosphotransferase and not via cytidylyltransferase.

In an effort to identify the pathway leading to the formation of 1-beta-D-arabinofuranosylcytosine-diphosphate (ara-CDP)-choline from 1-beta-D-arabinofuranosylcytosine (ara-C) treatment of cultured cells, as well as of cells obtained from leukemia patients, we probed the enzymatic steps involved in the CDP-choline pathway for phosphatidylcholine biosynthesis. Ara-C-triphosphate was not a substrate for CTP:phosphocholine cytidylyltransferase activity under the conditions employed, whereas CTP and dCTP were utilized to form CDP-choline and dCDP-choline, respectively. When presented together, ara-C-triphosphate and CTP inhibited the enzymatic conversion of CTP to CDP-choline in the presence of phosphocholine, with a Ki of 6 mM. Since CTP:phosphocholine cytidylyltransferase did not appear to be responsible for the increased levels of ara-CDP-choline, we next studied the other enzyme in the pathway for phosphatidylcholine synthesis that could form ara-CDP-choline, CDP-choline:1,2-diacylglycerol cholinephosphotransferase. CDP-choline:1,2-diacylglycerol cholinephosphotransferase activity present in microsomes isolated from L5178Y murine leukemia cells exhibited a reversal of its normal catalytic activity, using CMP and 1-beta-D-arabinofuranosylcytosine-monophosphate (ara-CMP) along with phosphatidylcholine to produce either CDP-choline or ara-CDP-choline, plus diradylglycerol. The Vmax and Km values for CMP were 0.78 +/- 0.04 nmol/min/mg and 340 +/- 20 microM, respectively, whereas the Vmax and Km for ara-CMP were 0.22 +/- 0.06 nmol/min/mg and 1410 +/- 540 microM, respectively. A Ki value of 3 mM was obtained for ara-CMP under the cell-free assay conditions used. These results indicate that ara-CDP-choline most likely arises from a reversal of the CDP-choline:1,2-diacylglycerol cholinephosphotransferase utilizing ara-CMP, rather than from the catalysis of ara-C-triphosphate plus phosphocholine to ara-CDP-choline by CTP:phosphocholine cytidylyltransferase. It is speculated that this mechanism may explain, in part, the rapid cellular lysis observed with high dose ara-C therapy.

Protein kinase C regulates the stimulated accumulation of 3-phosphorylated phosphoinositides in platelets.

We have shown that platelets stimulated with thrombin or guanosine 5'-[gamma-thio]triphosphate (GTP[S]), both of which activate phospholipase C and protein kinase C (PKC), show enhancement of 3-phosphorylated phosphoinositide accumulation (3-PPI). We now report the following. (1) Inhibition of thrombin- or GTP[S]-stimulated PKC by pseudo-substrate peptide (RFARK) added to permeabilized platelets markedly inhibits 3-PPI, whereas the serine/threonine phosphatase inhibitor, okadaic acid, promotes 3-PPI. PKC activity, insufficient in itself for fully activating 3-PPI, appears crucial to receptor and post-receptor stimulation of 3-PPI, even when tyrosine phosphorylation is unimpaired. (2) Alteration of Gi by ADP-ribosylation only slightly affects the stimulation of 3-PPI by thrombin, and activation of the G-protein Gi by adrenaline has no effect on 3-PPI. (3) Inhibition of PKC blocks activated secretion of platelet-derived growth factor (PDGF). However, PDGF cannot promote platelet 3-PPI, and thus cannot account for the inhibitory effects of RFARK on 3-PPI.

Hydrolysis of neutral lipid substrates by rat hepatic lipase.

Rat hepatic lipase, an enzyme whose involvement in the catabolism of lipoproteins remains poorly defined, has both neutral lipid and phospholipid hydrolyzing activity. We determined the substrate specificity of hepatic lipase for 1-oleoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, and 1,3-dioleoyl-sn-glycerol in the Triton X-100 mixed micellar state, and compared these results to those obtained previously in our laboratory for the phospholipid substrates phosphatidic acid (PA), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). Vmax values were determined by diluting the substrate concentration in the surface of the micelle by Triton X-100. The Vmax values obtained were 144 mumol/min/mg for 1-oleoyl-sn-glycerol, 163 mumol/min/mg for 1,2-dioleoyl-sn-glycerol, and 145 mumol/min/mg for 1,3-dioleoyl-sn-glycerol. These values were higher than those obtained earlier for phospholipids which were 67 mumol/min/mg for PA, 50 mumol/min/mg for PE and 4 mumol/min/mg for PC. In addition, the mole fraction of lipid substrate at half maximal velocity (K) in the surface dilution plot was lower for the neutral lipid substrates as compared to those obtained for the phospholipid substrates. When the hydrolysis of 1,3-dioleoyl-sn-glycerol mixed micelles was studied as a function of time, cleavage at the sn-1 and sn-3 positions occurred at the same rate, suggesting that hepatic lipase is not stereoselective with respect to 1,3-diacyl-sn-glycerol substrates.(ABSTRACT TRUNCATED AT 250 WORDS)