Inhibition of DNA synthesis in peripheral blood mononuclear cells treated with phosphatidylserines containing unsaturated acyl chains.

The immunosuppressive action of phosphatidylserine has been studied in mitogen-activated human peripheral blood mononuclear cells. The addition of phospholipid (10-60 nmol/10(6) cells) causes a dose-dependent inhibition of DNA synthesis induced by PHA, anti-CD3 mAb, allogeneic lymphocytes and tetradecanoylphorbol acetate plus ionomycin. In contrast, the interleukin-2-dependent DNA synthesis is less affected. Flow cytometric analysis and binding of radioiodinated interleukin-2 show that the phospholipid prevents the expression of interleukin-2 and transferrin receptors. Removal of monocytes by adherence does not change the action of phosphatidylserine. Furthermore, the phospholipid is equally effective in preparations depleted of CD4+ or CD8+ lymphocytes. Phosphatidylinositol partly reproduces the action of phosphatidylserine. Phosphatidic acid, phosphatidylglycerol and phosphatidylcholine are inactive. Also unsaturated phosphatidylserine analogues inhibit DNA synthesis whereas saturated phosphatidylserines do not. The data suggest that phosphatidylserine mainly affect the steps of T cell activation preceding the production of interleukin-2 and the expression of its receptor. The phosphorylserine headgroup and the unsaturated acyl chains contribute to this effect.     

Effects of protein kinase inhibitors 1(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and N-[2-guanidinoethyl]-5-isoquinolinesulfonamide hydrochloride (HA1004) on calcitriol-induced differentiation of HL-60 cells

HL-60 promyelocytic leukemia cells were induced to differentiate by 1,25-dihydroxyvitamin D3 (calcitriol) into mature monocytes. Differentiation was assessed by nitro blue tetrazolium dye reduction, nonspecific esterase activity, and DNA synthesis. Terminal differentiation of cultures induced by calcitriol (10 nM) was inhibited by 80% when cells were treated simultaneously with protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) (32 microM) and N-[2-guanidinoethyl]-5-isoquinolinesulfonamide hydrochloride (HA1004) (320 microM). The IC50 for inhibition of calcitriol-induced differentiation was approximately 15 microM for H-7 and 170 microM for HA1004. The IC50 values for H-7 and HA1004 antagonism of calcitriol-induced differentiation are quantitatively and relatively correlated to their known action to inhibit protein kinase C activity. Treatment of cells with concentrations of 0-32 microM H-7 or 0-320 microM HA1004 alone did not affect cell growth, differentiation, or trypan blue exclusion. However, higher concentrations of H7 (greater than 32 microM) and HA1004 (greater than 320 microM) were found to be cytotoxic. The data presented suggest that calcitriol-induced differentiation is antagonized by inhibitors of protein kinase and are consistent with the hypothesis that kinase C activity is required for HL-60 cell differentiation.     

Nonsteroidal antiestrogen inhibition of protein kinase C in human corpus luteum and placenta

These studies were undertaken to determine whether nonsteroidal antiestrogens would inhibit the calcium/lipid-dependent protein kinase (protein kinase C) activity in hormonally-responsive human reproductive tissues. Cytosol was prepared from human corpus luteum and term placenta. Protein kinase C activity was examined with various antiestrogens, estrogens, and catecholestrogens. The nonsteroidal antiestrogens tamoxifen, clomiphene and Z-4-hydroxytamoxifen inhibited protein kinase C in cytosol from human corpora lutea and placentae in a concentration-dependent manner. The IC50 values were 35-45 microM for tamoxifen, 58-66 microM for clomiphene, and 88 microM for hydroxytamoxifen. Protein kinase C purified 600-fold from human placenta was also inhibited by tamoxifen. The estrogens, estradiol and diethylstilbestrol (DES), and the catecholestrogens, 2-hydroxyestradiol and 4-hydroxyestradiol, had no effect on protein kinase C activity, nor were they able to prevent the inhibition of protein kinase C by the antiestrogens. Inhibition of the enzyme by the antiestrogens was competitive with phosphatidylserine and 1,2-diolein. In addition, tamoxifen inhibited enzyme activity stimulated by the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). The data suggest that the action of these antiestrogens on protein kinase C was a direct inhibition of the enzyme. Furthermore, the site of interaction showed markedly different structural specificity from that of the estrogen receptor.     

Modulation by cationic amphiphilic drugs of serine base-exchange,phosholipase d and intracellular calcium homeostasis in glioma C6 cells

We aimed to assess the effect of three drugs belonging to amphiphilic cations, imipramine, amitriptyline and propranolol, on lipid synthesis and intracellular calcium homeostasis in glioma C6 cells. Antidepressants, imipramine and amitriptyline, had a stimulatory effect on [14C]serine incorporation into phosphatidylserine. Similar effect was induced by propranolol, antidysrhythmic drug and an antagonist of beta-adrenergic receptor, but not by isoproterenol, a selective agonist of this receptor. Stimulation of serine base-exchange activity by amphiphilic cations occured at concentration as low as 5-25 microM that may be reached during clinical treatment. At much higher concentration (250 microM), those drugs also stimulated phospholipase D-mediated synthesis of [14C]phosphatidylethanol and blocked phorbol ester-induced, protein kinase C-dependent phospholipase D activity. The latter effect already occurred at low (25 microM) concentration of drugs. We have also shown that treatment of the cells with amphiphilic cations (1 mM) produced only a weak increase in the intracellular Ca2+ concentration and did not affect Ca2+ release from the intracellular stores evoked by nucleotide receptor agonists, ATP and ADP. In contrast, this treatment strongly diminished an unspecific leak of Ca2+ from the endoplasmic reticulum caused by thapsigargin and ionomycin. Mianserin, which is not cationic amphiphilic drug, did not affect phosphatidylserine synthesis and phospholipase D activity and produced heterogenous and chaotic Ca2+ responses. Our results suggest that imipramine, amitriptyline and propranolol may modulate lipid synthesis and intracellular calcium signaling independently of their action on membrane receptors, most probably by modification of the physicochemical properties of cell membranes.     

2,6-Diamino-N-([1-(1-oxotridecyl)-2-piperidinyl] methyl)hexanamide (NPC 15437): a novel inhibitor of protein kinase C interacting at the regulatory domain.

NPC 15437 is a prototype member of a new class of synthetically derived protein kinase C (PKC) inhibitors. PKC activity and binding of phorbol ester to the enzyme were inhibited by NPC 15437, with IC50 values of 19 +/- 2 microM and 23 +/- 4 microM, respectively. No inhibition of cAMP-dependent or calcium/calmodulin-dependent protein kinases was observed at concentrations of NPC 15437 up to 300 microM. To investigate the mechanism by which NPC 15437 exerts its effects, a kinetic analysis of the inhibition with respect to three activators of the enzyme, phosphatidylserine, calcium, and phorbol ester, was performed. NPC 15437 was a competitive inhibitor of the activation of PKC by phorbol ester (Ki = 5 +/- 3 microM). Stimulation of PKC alpha by phosphatidylserine was competitively inhibited by NPC 15437 (Ki = 12 +/- 4 microM). The inhibition was mixed with respect to activation by calcium. These results suggest that NPC 15437 is a selective inhibitor of PKC, interacting at the regulatory region of the enzyme. NPC 15437 inhibited PKC in intact cells, dose-dependently antagonizing the phorbol ester-induced phosphorylation of a 47-kDa protein in human platelets.     

Protein kinase C inhibits stimulation of adenylate cyclase by the histamine H2 receptor in rat parietal cells.

The action of protein kinase C on the stimulation of adenylate cyclase activity by the histamine H2 receptor was investigated in rat parietal cells. Protein kinase C was activated by preincubating cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), and adenylate cyclase activity was measured in sonicated extracts. TPA (100 nM) inhibited adenylate cyclase activity stimulated by histamine (100 nM-500 microM). This effect was related to the concentration of TPA. TPA (100 nM) enhanced the stimulation of adenylate cyclase activity by forskolin (100 microM) but had no effect on the stimulation by NaF (10 mM). In conclusion, protein kinase C inhibits stimulation of adenylate cyclase by the histamine H2 receptor. This action could be mediated by changes in the number of affinity of histamine H2 receptors or in the coupling of the receptor to the stimulatory guanine nucleotide regulatory subunit Gs.     

Effects of inhibitors of protein kinase C on the release and synthesis of histamine in rat basophilic leukemia cells (2H3).

In rat basophilic leukemia cells (2H3), a tumor analog of mast cells, the aggregation of IgE receptors results in histamine secretion and the increase in histidine decarboxylase activity which synthesizes histamine. Using inhibitors of protein kinases C, we studied the relationships between these events and protein kinase C which is activated by antigens. Histamine release is suppressed by inhibitors of protein kinase C, staurosporine, K252-a and H-7, in this decreasing order of effectiveness; and the IC50 values are 1.5 nM, 29.9 nM and 3.8 microM, respectively. The changes in the intracellular Ca concentration monitored by fura-2 fluorescence is not modified by staurosporine, although the histamine response is suppressed. Meanwhile, the increase of histidine decarboxylase was abolished by inhibitors of protein kinase C; staurosporine was the strongest, K-252a of moderate activity and H-7, the weakest, having IC50 values of 0.8 nM, 100 nM and 11.5 microM, respectively. The inhibitors of protein kinase C suppress both histamine secretion and synthesis. Therefore, the histamine synthesis may be stimulated via activation of protein kinase C to supplement the released histamine.     

2,6-Diamino-N-([1-oxotridecyl)-2-piperidinyl]methyl)hexanamide (NPC 15437): a selective inhibitor of protein kinase C.

NPC 15437 inhibited protein kinase C (PKC) activity and [3H]phorbol 12,13-dibutyrate (PDBu) binding to the enzyme in a concentration-dependent manner (IC50 values, 19 +/- 2 microM and 23 +/- 4 microM, respectively). No inhibition of cAMP-dependent protein kinase A (PKA) or calcium/calmodulin-dependent myosin light chain kinase (MLCK) was observed. A detailed kinetic analysis of the interaction of NPC 15437 and a homogeneous preparation of PKC-alpha revealed a competitive type of inhibition with respect to activation of the enzyme by both phorbol 12-myristate 13-acetate (PMA) (Ki = 5 +/- 3 microM) and phosphatidylserine (PS) (Ki = 12 +/- 4 microM). Mixed inhibition (predominantly of the non-competitive type), with respect to activation of the enzyme by calcium, was also observed. These studies indicate that NPC 15437 is a selective inhibitor of PKC, interacting at the regulatory region of the molecule. NPC 15437 inhibited phorbol ester-induced ear edema in mouse (IC50 = 175 micrograms/ear) demonstrating the ability of NPC 15437 to inhibit PKC-mediated activity in intact cells.     

Phorbol esters of different biological activities may preferentially act as mitogens of human suppressor T-cells and are equi-effective mitogens of IL-2 dependent cells

The actions of tetradecanoylphorbolacetate (TPA) and 12-deoxyphorbolphenylacetate-20-acetate (DPPAA) together with a phytohaemagglutinin (PHA) have been examined on the proliferative responses of human mononuclear cells (MNC) depleted of specific cell subsets by the use of monoclonal antibodies. PHA-induced proliferation was found to be reduced when monocytes/macrophages and T-helper cells were depleted from MNC, but enhanced compared with MNC responses when T-suppressor cells were depleted. In contrast, TPA- and DPPAA-induced proliferation was unchanged or slightly enhanced following macrophage/monocyte depletion, and whereas TPA-induced proliferation was largely independent of subtype constitution, the non-tumour promoting DPPAA appeared to selectively enhance proliferation of the T8+ suppressor subset. Indomethacin increased the proliferative MNC responses of phorbol esters whilst having little effect upon the PHA response, an effect antagonized by addition of PGE2. The addition of interleukin-2 (IL-2) increased the proliferative response, as well as resistance to inhibition induced by cyclosporin A and dexamethasone and partially abolished the selective actions of DPPAA and PHA. In IL-2 dependent cultures PHA induced stimulation was more sensitive to inhibition by cyclosporin than were the phorbol esters. The results suggest that, although induction of lymphocyte proliferation by phorbol esters is not a correlate for tumour promotion itself, non-promoting phorbol esters may have a more restricted ability to induce proliferation than TPA.     

Induction of cyclooxygenase-2 by bovine type I collagen in macrophages via C/EBP and CREB activation by multiple cell signaling pathways

Bovine type I collagen (Col-I) is utilized for medical purposes such as cosmetic surgery and wrinkle removal. Cyclooxygenase-2 (COX-2) plays roles in pathophysiological processes including inflammation and tumorigenesis. This study examines the effects of Col-I on the COX-2 expression and the signaling pathways in macrophages. Col-I increased the levels of COX-2 protein and mRNA in serum-stimulated Raw264.7 cells in a time- and concentration-dependent manner. Treatment of cells with Col-I increased CCAAT/enhancer binding protein (C/EBP) DNA binding. Antibody supershift experiments revealed that C/EBP DNA binding activity induced by Col-I depended largely on C/EBPbeta and C/EBPdelta. Immunocytochemistry showed that Col-I induced nuclear translocation of C/EBPbeta and C/EBPdelta, whose activation contributes to COX-2 induction. Overexpression of the dominant-negative mutant form of C/EBP abolished COX-2 induction by Col-I. Col-I also increased cyclic-AMP response element binding protein (CREB) binding to DNA. Inhibition of focal adhesion kinase (FAK) or downstream phosphoinositide 3-kinase and p70S6 kinase by specific chemical inhibitors prevented COX-2 induction by Col-I, and C/EBP and CREB from binding to their consensus DNA oligonucleotides. Experiments using chemical inhibitors or dominant-negative mutant vectors showed that the mitogen-activated protein (MAP) kinase pathways including p38-kinase and extracellular signal-regulated kinase (ERK1/2), but not c-Jun N-terminal kinase (JNK1), simultaneously regulated COX-2 induction by Col-I. This was in agreement with inhibition of Col-I-inducible C/EBP and CREB DNA binding by concomitant treatment with SB203580 and PD98059. These results provide evidence that Col-I induces COX-2 in serum-stimulated macrophages and that the multiple cell signaling pathways involving Src-focal adhesion kinase, phosphoinositide 3-kinase, and MAP kinases regulate COX-2 induction by Col-I via C/EBP and CREB activation.     

Inhibition of nicotinic responses of bovine adrenal chromaffin cells by the protein kinase C inhibitor, Ro 31-8220.

1. The effects of the protein kinase C inhibitor, Ro 31-8220, on the responses of cultured bovine adrenal chromaffin cells to nicotine, phorbol 12, 13-dibutyrate (PDBu) and K+ have been investigated. 2. Tyrosine hydroxylase activity was measured in situ in intact cells by measuring 14CO2 evolved following the hydroxylation and rapid decarboxylation of [14C]-tyrosine offered to the cells. Secretion of endogenous adrenaline and noradrenaline was measured by use of h.p.l.c. with electrochemical detection. Cyclic AMP levels were measured in cell extracts by RIA. 3. Ro 31-8220 produced a concentration-dependent inhibition of 300 nM PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 2 microM and complete inhibition at 10 microM. It had no effect on the responses to forskolin. 4. Ro 31-8220 produced a concentration-dependent inhibition of 5 microM nicotine-stimulated tyrosine hydroxylase activity, adrenaline and noradrenaline secretion and cellular cyclic AMP levels, with an IC50 of about 3 microM and complete inhibition by 10 microM. At concentrations up to 10 microM, Ro 31-8220 had little or no effect on the corresponding responses to 50 mm K+. 5. A structural analogue of Ro 31-8220, bisindolylmaleimide V, that lacks activity as a protein kinase C inhibitor, had no effect up to 10 microM on PDBu-stimulated tyrosine hydroxylase activity or on nicotine-stimulated cyclic AMP levels or noradrenaline secretion and only marginal inhibitory effects on nicotine-stimulated tyrosine hydroxylase activity and adrenaline secretion. 6. A structurally related protein kinase C inhibitor, bisindolylmaleimide I, inhibited PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 1 microM and complete inhibition by 3 microM, but had essentially no effect on nicotine stimulated tyrosine hydroxylase activity or catecholamine secretion. 7. The results suggest that Ro 31-8220 is not only a protein kinase C inhibitor but is also a potent inhibitor of nicotinic receptor responses in adrenal chromaffin cells by a mechanism unrelated to protein kinase C inhibition. The results are consistent with Ro 31-8220 being a nicotinic receptor antagonist.     

Stimulation by extracellular ATP and UTP of the mitogen-activated protein kinase cascade and proliferation of rat renal mesangial cells.

1. Extracellular ATP and UTP have been reported to activate a nucleotide receptor that mediates phosphoinositide and phosphatidylcholine hydrolysis by phospholipases C and D, respectively. Here we report that ATP and UTP potently stimulate mesangial cell proliferation. 2. Both nucleotides stimulate phosphorylation and activation of mitogen-activated protein kinase and a biphasic phosphorylation of the up-stream mitogen-activated protein kinase kinase. 3. When added at 100 microM, ATP gamma S, UTP and ATP were the most potent activators of mitogen-activated protein kinase. beta gamma-imido-ATP was somewhat less active and ADP and 2-methylthio-ATP caused a weak induction of enzyme activity. Activation of mitogen-activated protein kinase by both ATP and UTP is dose-dependently attenuated by the P2-receptor antagonist, suramin. 4. The protein kinase C activator 12-0-tetradecanoylphorbol 13-acetate, but not the biologically inactive 4 alpha-phorbol 12,13-didecanoate, increased mitogen-activated protein kinase activity in mesangial cells, suggesting that protein kinase C may mediate nucleotide-induced stimulation of mitogen-activated protein kinase. 5. Down-regulation of protein kinase C -alpha and -delta isoenzymes by 4 h or 8 h treatment with phorbol ester partially inhibited ATP- and UTP-triggered mitogen-activated protein kinase activation. Moreover, a 24 h treatment of mesangial cells with phorbol ester, a regimen that also causes depletion of protein kinase C-epsilon did not further reduce the level of mitogen-activated protein kinase stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of nitrogen mustards on the proliferative and Ig synthetic response of human peripheral blood lymphocytes

Maximal inhibition of pokeweed mitogen-stimulated Ig production and [3H]thymidine incorporation was shown to occur when unfractionated human peripheral blood mononuclear cells were cultured with concentrations of the nitrogen mustards melphalan, mechlorethamine or chlorambucil in the 20-100-microM range, whereas concentrations of microsome-activated cyclophosphamide (A-Cy) in the 2-mM range were required for equivalent inhibition. Around 400 microM A-Cy, IgM secretion was not inhibited, but secretion of IgA and IgG was. The [3H]thymidine incorporation of enriched populations of both large and small B and T cells all showed about 20-50-fold greater sensitivity to melphalan than to A-Cy, despite a difference of only 6-fold in alkylating activity between these drugs. Large (250 micron 3) B and T cells were only marginally more sensitive to melphalan and A-Cy than small (210 micron 3) T and B cells. Kinetic studies showed that IgG and IgA secreted by day 7 could be maximally inhibited by melphalan added as late as day 3, and IgM synthesis as late as day 2. In contrast, inhibition of Ig production by A-Cy steadily declined after the first day, especially IgM, which was no longer inhibitable by A-Cy on day 3. Inhibition of cumulative Ig production did not occur when A-Cy or melphalan was added on day 5 or later. Cell recombination experiments performed with drug pulsed and untreated monocytes plus B cells and irradiated T cells showed that inhibition of [3H]thymidine or Ig production was most striking when monocytes + B cells (rather than T cells) were exposed to melphalan in the first 16 h. When A-Cy was used in the first 16 h, inhibition of Ig production was partial and inconsistent, and inhibition of monocytes + B cell or T cell [3H]thymidine incorporation was not evident. We conclude that the nitrogen mustards melphalan and A-Cy can inhibit pokeweed mitogen-stimulated DNA synthesis by human T or B cells and Ig production in vitro, but that their mechanisms of action differ.     

Characteristics of the Ca(2+)-dependent inhibition of cyclic AMP accumulation by histamine and thapsigargin in human U373 MG astrocytoma cells

1. Histamine, acting on H(1)-receptors, caused a Ca(2+)-dependent inhibition of forskolin- and isoprenaline-induced cyclic AMP accumulation in monolayers of human U373 MG cells (IC(50) 1.3+/-0.3 microM, maximum inhibition 66+/-3%). The inhibition was not reversed by the protein kinase inhibitor K-252A. 2. Thapsigargin also inhibited cyclic AMP accumulation (IC(50) 6.0+/-0.3 nM, maximum inhibition 72+/-1%). In the absence of extracellular Ca(2+) 5 microM thapsigargin caused only a 12+/-2% inhibition of cyclic AMP accumulation. 3. The inhibitory effect of 100 nM thapsigargin on forskolin-stimulated cyclic AMP accumulation was blocked by La(3+) (best-fit maximum inhibition 81+/-4%, IC(50) 125+/-8 nM). In contrast, the inhibitory action of 10 microM histamine was much less sensitive to reversal by 1 microM La(3+) (33+/-5% reversal, compared with 78+/-6% reversal of the inhibition by thapsigargin measured concurrently). However, in the presence of both thapsigargin and histamine the inhibition of cyclic AMP accumulation was reversed by 1 microM La(3+) to the same extent as the inhibition by thapsigargin alone. 4.++Thapsigargin (5 microM)+1 microM La(3+) caused only a 20+/-1% inhibition of histamine-stimulated phosphoinositide hydrolysis. 5. There was no indication from measurement of intracellular Ca(2+) of any persistent La(3+)-insensitive Ca(2+) entry component activated by histamine. 6. The results provide evidence that Ca(2+) entry is required for the inhibition by histamine and thapsigargin of drug-induced cyclic AMP accumulation in U373 MG astrocytoma cells. The differential sensitivity of the inhibitory action of the two agents to block by La(3+) suggests that more than one pathway of Ca(2+) entry is involved.     

Differential regulation of phosphatidylserine externalization and DNA fragmentation by caspases in anticancer drug-induced apoptosis of rat mammary adenocarcinoma MTLn3 cells

Caspase activation is a central event in the execution phase of apoptosis and is associated with phosphatidylserine (PS) externalization and DNA fragmentation. We investigated the role of caspase activity in anticancer drug-induced PS externalization and DNA fragmentation in MTLn3 cells. Caspase activation (DEVD-AMC cleavage) occurred in a time- and concentration-dependent manner after exposure to doxorubicin, in association with cleavage of poly(ADP) ribose polymerase and protein kinase C delta, two caspase-3 substrates. Caspase activation was closely followed by oligonucleosomal DNA fragmentation and PS externalization as determined by flow cytometric analysis. Similar observations were made for etoposide and cisplatin. Inhibition of caspases with zVAD-fmk inhibited almost completely doxorubicin-induced DNA fragmentation as well as proteolysis of protein kinase C delta. In contrast, PS externalization induced by doxorubicin was only partly affected by caspase inhibition. Flow cytometric cell sorting demonstrated that DNA fragmentation in the remaining PS positive cells after doxorubicin treatment in the presence of zVAD-fmk was fully blocked. In conclusion, these data indicate that while DNA fragmentation in anticancer drug-induced apoptosis of MTLn3 cells is fully dependent on caspase activity, PS externalization is controlled by both caspase-dependent and caspase-independent pathways.     

Thrombin is the major serum factor stimulating phosphoinositide turnover, but not DNA synthesis in human neuroblastoma SH-EP cells.

Fetal calf serum stimulates both phosphoinositide turnover and DNA synthesis in SH-EP cells. The phosphoinositide turnover-stimulating activity of serum is largely (70%) reduced in the presence of hirudin, a blocker of thrombin activity. Yet, hirudin does not alter the ability of serum to stimulate DNA synthesis. Purified alpha-thrombin is a potent (EC50, 35 pM) stimulator of phosphoinositide turnover in SH-EP cells, but induces DNA synthesis only at much higher concentrations (10 nM-1 microM). Thus, serum thrombin accounts for most of the ability of serum to stimulate phosphoinositide hydrolysis, but not for the effect of serum on cell division, since the concentration of thrombin in serum is not sufficient to induce DNA synthesis. These data suggest that hydrolysis of inositol lipids may not be the main signalling event mediating the mitogenic effects of alpha-thrombin.     

Mechanism and mode of action of 5-iodo-2-pyrimidinone 2'-deoxyribonucleoside, a potent anti-herpes simplex virus compound, in herpes simplex virus-infected cells.

The anti-herpes simplex virus type 2 (-HSV-2) action of 5-iodo-2-pyrimidinone deoxyribonucleoside (IPdR) was found to be exerted through inhibition of HSV DNA synthesis. The inhibition of viral DNA synthesis was not caused by inhibition of the synthesis of HSV-2-specified proteins or HSV-2 mRNA species involved with viral DNA synthesis or by depletion of deoxynucleotides. The inhibition of viral DNA synthesis may be due to damage to the DNA template in the nuclei or to an action at the DNA replication complex, because nuclei isolated from HSV-2-infected cells treated with IPdR could not support DNA synthesis in vitro. Moreover, the addition of exogenous template to the reaction enabled nuclear DNA synthesis to occur at the level of control. The major cellular metabolite of IPdR in HeLa S3 cells infected with HSV-2 was IPdR monophosphate, which was formed through virally specified kinase. Attempts to either identify or synthesize IPdR diphosphate and triphosphate were unsuccessful. The accumulation of IPdR monophosphate was dependent on the extracellular concentration of IPdR. IPdR monophosphate did not have any inhibitory effect on nuclear DNA synthesis, even at 200 microM. Thus, the action of IPdR could be due to an unidentified metabolite of IPdR or the depletion of a cellular metabolite that is essential for viral DNA synthesis.     

Activation of deoxycytidine kinase by inhibition of DNA synthesis in human lymphocytes

Deoxycytidine kinase (dCK, EC.2.7.1.74) is a key enzyme in the intracellular metabolism of 2-chlorodeoxyadenosine, 1-beta-D-arabinofuranosylcytosine, difluorodeoxycytidine, and other drugs used in chemotherapy of different leukaemias and solid tumours. Recently, stimulation of dCK activity was shown by these analogues and by other genotoxic agents such as etoposide and NaF, all of which cause severe inhibition of DNA synthesis in cell cultures. Here we describe that direct inhibition of DNA polymerases by aphidicolin stimulated dCK activity in normal lymphocytes and acute myeloid leukaemic cells, as well as in HL 60 promyelocytic cell cultures. Increased dCK activity was not due to new protein synthesis under our conditions, as measured by immunoblotting. Partial purification by diethylaminoethyl-Sephadex chromatography revealed that the activated form of dCK survived purification procedure. Moreover, it was possible to inactivate purified dCK preparations by recombinant protein phosphatase with Ser/Thr/Tyr dephosphorylating activity. These data suggest that the activation of dCK may be due to phosphorylation, and that deoxynucleoside salvage is promoted during inhibition of DNA synthesis in human lymphocytes.     

Amidine analogue of chlorambucil is a stronger inhibitor of protein and DNA synthesis in breast cancer MCF-7 cells than is the parent drug

A novel amidine analogue of chlorambucil-N-(2-(4-(4-bis(2-chloroethyl)aminophenyl)butyryl)aminoethyl)-5-(4-amidinophenyl)-2-furancarboxamide hydrochloride (AB(1)) and the parent drug were compared for their effects on collagen and DNA biosynthesis in breast cancer MCF-7 cells. IC(50) values for chlorambucil and AB(1) for collagen biosynthesis were found to be about 33 and 13 microM, respectively. The greater potency of AB(1) to suppress collagen synthesis was found to be accompanied by a stronger compared with chlorambucil inhibition of prolidase activity and expression. The phenomenon was related to inhibition of beta(1)-integrin and IGF-I receptor-mediated signaling caused by this compound. The expression of beta(1)-integrin receptor, as well as Src, son of sevenless protein (SOS) and phosphorylated mitogen activated protein (MAP) kinases (MAPK), extracellular-signal-regulated kinase 1 (ERK(1)) and kinase 2 (ERK(2)) but not focal adhesion kinase pp125(FAK) (FAK), Shc, and Grb-2 was significantly decreased in cells incubated for 24 h with 10 microM AB(1) compared to the control, whereas in the same conditions chlorambucil did not evoke any changes in expression of all these signaling proteins, as shown by Western immunoblot analysis. Furthermore, AB(1) induced a stronger down-regulation of the expression of IGF-I receptor and evoked a higher antiproliferative effect. During 12 and 24 h of incubation AB(1) decreased DNA biosynthesis by about 33 % and 51 % of the control, whereas chlorambucil decreased it by about 19 % and 35 %, respectively. These data suggest that the amidine analogue of chlorambucil is a stronger inhibitor of protein and DNA synthesis in MCF-7 cells than is the parent drug.     

Structure-activity relationships of phenothiazines and related drugs for inhibition of protein kinase C

Phenothiazines are known to inhibit the activity of protein kinase C. To identify structural features that determine inhibitory activity against the enzyme, we utilized a semiautomated assay [Anal. Biochem. 187:84-88 (1990)] to compare the potency of greater than 50 phenothiazines and related compounds. Potency was decreased by trifluoro substitution at position 2 on the phenothiazine nucleus and increased by quinoid structures on the nucleus. An alkyl bridge of at least three carbons connecting the terminal amine to the nucleus was required for activity. Primary amines and unsubstituted piperazines were the most potent amino side chains. We selected 7,8-dihydroxychlorpromazine (DHCP) (IC50 = 8.3 microM) and 2-chloro-9-(3-[1-piperazinyl]propylidene)thioxanthene (N751) (IC50 = 14 microM) for further study because of their potency and distinct structural features. Under standard (vesicle) assay conditions, DHCP was noncompetitive with respect to phosphatidylserine and a mixed-type inhibitor with respect to ATP. N751 was competitive with respect to phosphatidylserine and noncompetitive with respect to ATP. Using the mixed micelle assay, DHCP was a competitive inhibitor with respect to both phosphatidylserine and ATP. DHCP was selective for protein kinase C compared with cAMP-dependent protein kinase, calmodulin-dependent protein kinase type II, and casein kinase. N751 was more potent against protein kinase C compared with cAMP-dependent protein kinase and casein kinase but less potent against protein kinase C compared with calmodulin-dependent protein kinase type II. DHCP was analyzed for its ability to inhibit different isoenzymes of protein kinase C, and no significant isozyme selectivity was detected. These data provide important information for the rational design of more potent and selective inhibitors of protein kinase C.