B-RAF: A contributor to the melanoma phenotype.

B-RAF, a serine-threonine protein kinase, is one of the three RAF paralogs in humans. B-RAF participates in the RAS-RAF-MEK-ERK pathway, a conserved protein kinase-signalling cascade that is involved in regulating a number of critical cellular functions. Mutated B-RAF is believed to play a crucial role in the development, maintenance and progression of melanoma, where it contributes to multiple aspects of the malignant phenotype, such as cell survival, proliferation and apoptosis resistance. Indeed, it is mutated in a high proportion of melanocytic skin lesions and B-RAF mutations are preserved through melanoma progression. Despite this, the direct inhibition of B-RAF has shown little success clinically in the treatment of melanoma, presumably due to the complexity of the RAS-RAF-MEK-ERK pathway. For this reason, alternative strategies must be developed to treat oncogenic B-RAF-induced melanomas.

Cystatin B inhibition of TRAIL-induced apoptosis is associated with the protection of FLIP(L) from degradation by the E3 ligase itch in human melanoma cells.

Past studies have identified a number of distinct mechanisms that contribute to the resistance of melanoma cells against apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL). In this report we show that cystatin B is another endogenous inhibitor of TRAIL-induced apoptosis. Cystatin B-deficient melanoma cell lines established by shRNA knockdown displayed increased apoptosis that was associated with enhanced activation of caspase-8 induced by TRAIL. This was not related to the inhibitory effect of cystatin B on the lysosomal cysteine proteases, cathepsin B and L, as they did not have a role in TRAIL-induced apoptosis in most melanoma cell lines even when cystatin B was inhibited. Instead, sensitization of melanoma cells to TRAIL-induced apoptosis by inhibition of cystatin B appeared associated with decreased stability of FLIP(L) as the levels of FLIP(L) were reduced because of shortened half-life time in melanoma cells deficient in cystatin B. In contrast, over-expression of cystatin B increased the levels of FLIP(L), decreased the amount of the E3 ligase Itch associated with FLIP(L), and reduced FLIP(L) ubiquitination. Inhibition of Itch by siRNA restored the levels of FLIP(L) and blocked sensitization to TRAIL-induced apoptosis associated with deficiency in cystatin B. Taken together, these results indicate that cystatin B regulates Itch-mediated degradation of FLIP(L) and thereby TRAIL-induced apoptosis in melanoma cells.

Modeling methane production from beef cattle using linear and nonlinear approaches.

Canada is committed to reducing its greenhouse gas emissions to 6% below 1990 amounts between 2008 and 2012, and methane is one of several greenhouse gases being targeted for reduction. Methane production from ruminants is one area in which the agriculture sector can contribute to reducing our global impact. Through mathematical modeling, we can further our understanding of factors that control methane production, improve national or global greenhouse gas inventories, and investigate mitigation strategies to reduce overall emissions. The purpose of this study was to compile an extensive database of methane production values measured on beef cattle, and to generate linear and nonlinear equations to predict methane production from variables that describe the diet. Extant methane prediction equations were also evaluated. The linear equation developed with the smallest root mean square prediction error (RMSPE, % observed mean) and residual variance (RV) was Eq. I: CH(4), MJ/d=2.72 (+/-0.543) + [0.0937 (+/-0.0117) x ME intake, MJ/d] + [4.31 (+/-0.215) x Cellulose, kg/d] - [6.49 (+/-0.800) x Hemicellulose, kg/d] - [7.44 (+/-0.521) x Fat, kg/d] [RMSPE=26.9%, with 94% of mean square prediction error (MSPE) being random error; RV=1.13]. Equations based on ratios of one diet variable to another were also generated, and Eq. P, CH(4), MJ/d=2.50 (+/-0.649) - [0.367 (+/-0.0191) x (Starch:ADF)] + [0.766 (+/-0.116) x DMI, kg/d], resulted in the smallest RMSPE values among these equations (RMSPE=28.6%, with 93.6% of MSPE from random error; RV=1.35). Among the nonlinear equations developed, Eq. W, CH(4), MJ/d=10.8 (+/-1.45) x (1-e([-0.141 (+/-0.0381) x DMI, kg/d])), performed well (RMSPE=29.0%, with 93.6% of MSPE from random error; RV=3.06), as did Eq. W(3), CH(4), MJ/d=10.8 (+/-1.45) x [1-e({-[-0.034 x (NFC/NDF)+0.228] x DMI, kg/d})] (RMSPE=28.0%, with 95% of MSPE from random error). Extant equations from a previous publication by the authors performed comparably with, if not better than in some cases, the newly developed equations. Equation selection by users should be based on RV and RMSPE analysis, input variables available to the user, and the diet fed, because the equation selected must account for divergence from a "normal" diet (e.g., high-concentrate diets, high-fat diets).

Fludarabine induces differential expression of proteins in human leukemia and lymphoma cells.

The purine analog fludarabine (FdAMP) is widely used for chemotherapy of B-lymphoid malignancies, and multiple mechanisms of action leading to apoptosis have been proposed. We examined changes at the protein level induced in the Raji cell line (Burkitt's lymphoma) by fludarabine nucleoside (FdA). Raji cells are sensitive to FdA. Raji cells treated with FdA (3 micro M, 24 hours), accumulate multiple phosphorylated forms of p53 in the nucleus that in turn degrade to phosphorylated forms of p40. Using CD antibody microarrays to determine surface expression profiles for Raji cells treated with FdA, we found up-regulation of the following CD antigens: CD20, CD54, CD80, CD86, and CD95. FdA thus induces changes in the genetic program of the cells that might be exploited to obtain synergy with therapeutic antibodies.

Phenotypic protein profiling of different B cell sub-populations using antibody CD-microarrays.

Antibody microarrays enable extensive protein expression profiling, and provide a valuable complement to DNA microarray-based gene expression profiling. In this study, we used DotScan antibody microarrays that contain antibodies against 82 different cell surface antigens, to determine phenotypic protein expression profiles for human B cell sub-populations. We then demonstrated that the B cell protein profile can be used to delineate the relationship between normal B cells and malignant counterparts. Principle component analysis showed that the lymphomas did not cluster with the normal memory B cells or germinal centre B cells, but they did cluster with germinal centre founder cells and naïve B cells.

Parenteral administration of glutamine modulates acute phase response in postparturient dairy cows.

The objective of this study was to investigate whether administration of L-Gln would affect mediators of acute phase response in postparturient dairy cows. Twenty-four multiparous Holstein cows were blocked by the expected day of calving and randomly assigned to 1 of the 3 treatment groups (n = 8/group): 1) i.v. infusion of 10 L of 0.85% NaCl (control), 2) i.v. infusion of 106, or 3) 212 g/d of L-Gln mixed with 10 L of 0.85% NaCl solution; each treatment was given 8 h/d for each of 7 consecutive days starting on d 1 after calving. Blood samples were collected 1 wk before the expected day of parturition as well as on d 0, 7, 14, and 21 after parturition; plasma concentrations of serum amyloid A (SAA), haptoglobin, and lipopolysaccharide-binding protein were measured by ELISA, and alpha(1)-acid glycoprotein was assessed by radial immunodiffusion. Concentrations of SAA, haptoglobin, and alpha(1)-acid glycoprotein increased in control cows after parturition, reaching peak values on d 0 or 7 postpartum (60, 1,093, and 963 microg/mL, respectively). Cows infused with 106 g/d of L-Gln had greater concentrations of SAA in plasma on d 14 and 21 compared with controls (62.8 vs. 30.2 and 71.1 vs. 34.5 microg/mL, respectively). Cows infused with 212 g/d of L-Gln had greater concentrations of SAA on d 7 (82.5 vs. 53.9 microg/mL) and lower concentrations of haptoglobin on d 14 and 21 postpartum compared with controls (264 vs. 621 and 175 vs. 587 microg/mL, respectively). Cows treated with 106 and 212 g/d of L-Gln had greater plasma lipopolysaccharide-binding protein concentrations on d 7 compared with control group (50.0 and 35.6 vs. 10.8 microg/mL, respectively). There were no treatment differences with respect to milk yield and DM intake during the experimental period. In conclusion, our data indicate that i.v. administration of L-Gln modulated acute phase mediators in dairy cows after parturition and warrants further research into the mechanisms behind these effects.

Cloning and expression of malarial pyrimidine enzymes.

We have cloned genes encoding three enzymes of the de novo pyrimidine pathway using genomic DNA from Plasmodium falciparum and sequence information from the Malarial Genome Project. Genes encoding dihydroorotase (reaction 3), orotate phosphoribosyltransferase (reaction 5), and OMP decarboxylase (reaction 6) have been cloned into the plasmid pET 3a or 3d with a thrombin cleavable 9xHis tag at the C-terminus and the enzymes were expressed in Escherichia coli. To overcome the toxicity of malarial OMP decarboxylase when expressed in E. coli, and the unusual codon usage of the malarial gene, a hybrid plasmid, pMICO, was constructed which expresses low levels of T7 lysozyme to inhibit T7 RNA polymerase used for recombinant expression, and extra copies of rare tRNAs. Catalytically-active OMP decarboxylase has been purified in tens of milligrams by chromatography on Ni-NTA. The gene encoding orotate phosphoribosyltransferase includes an extension of 66 amino acids from the N-terminus when compared with sequences for this enzyme from other organisms. We have found that other pyrimidine enzymes also contain unusual protein inserts. Milligram quantities of pure recombinant malarial enzymes from the pyrimidine pathway will provide targets for development of novel antimalarial drugs.

Accumulation of 5-phosphoribosyl-1-pyrophosphate in human CCRF-CEM leukaemia cells treated with antifolates.

Amido phosphoribosyltransferase (APRT) catalyzes the first step of the de novo biosynthesis of purine nucleotides, the conversion of 5-phosphoribosyl-1-pyrophosphate (PRPP) into 5-phosphoribosylamine (PRA). APRT is a valid target for development of inhibitors as anticancer drugs. We have developed a thin layer chromatographic assay for PRPP extracted from cells. Using coupling enzymes, PRPP with excess [2-14C]orotate (OA) is quantitatively converted to [2-14C]OMP and then [2-14C]UMP with hydrolysis of the PPi. The reaction products are isolated on poly(ethyleneimine)-cellulose (PEI-C) chromatograms. Human CCRF-CEM leukaemia cells growing in culture have been exposed to a number of antifolates and their effects upon cellular levels of PRPP determined. The steady-state level of PRPP measured in CCRF-CEM cells was 102+/-11 microM. Following addition of an antifolate to a culture, accumulation of PRPP in cells indicates the degree of inhibition of APRT. In human CCRF-CEM leukaemia cells, lometrexol (LTX), 2,4-diamino-6-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydro-quinazoline (PY899), methotrexate (MTX), N(alpha)(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523), piritrexim (PTX), metoprine, 2,4-diamino-6-(3,4,5-trimethoxyanilino)-methylpyrido[3,2-d]pyrimidine (PY873) and multitargeted antifolate, N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid (MTA) directly or indirectly induce inhibition of APRT indicated by time-courses for accumulation of PRPP to maximum values of 3-12-fold. These data indicate that LTX induces the most potent inhibition of APRT.

The identification, cloning and functional expression of the gene encoding orotidine 5'-monophosphate (OMP) decarboxylase from Plasmodium falciparum.

The coding region of a putative orotidine 5'-monophosphate decarboxylase gene from Plasmodium falciparum was identified in genomic data from the Malarial Genome Sequencing Project. The gene encodes a protein of 323 amino acids with a predicted molecular weight of 37.8 kDa. The gene was cloned into a bacterial expression vector and over-expressed in Escherichia coli. The recombinant protein was purified and shown to have orotidine 5'-monophosphate decarboxylase activity, confirming the identity of the gene.

Immunophenotyping of leukemias using a cluster of differentiation antibody microarray.

Different leukemias express on their plasma membranes particular subsets of the 247 defined cluster of differentiation (CD) antigens, which may resemble those of precursor cells along the lineages of differentiation to mature myeloid and lymphoid leukocytes. The extent of use of CD antigen expression (immunophenotyping) for identification of leukemias has been constrained by the technique used, flow cytometry, which commonly specifies only three CD antigens in any one assay. Currently, leukemias and lymphomas are diagnosed using a combination of morphology, immunophenotype, cytochemistry, and karyotype. We have developed a rapid, simple procedure, which enables concurrent determination of 50 or more CD antigens on leukocytes or leukemia cells in a single analysis using a microarray of antibodies. A suspension of cells is applied to the array, and cells only bind to antibody dots for which they express the corresponding CD antigen. For patients with significantly raised leukocyte counts, the resulting dot pattern then represents the immunophenotype of those cells. For patients at earlier stages of disease, the diagnosis depends on recognition of dot patterns distinct from the background of normal leukocytes. Distinctive and reproducible dot patterns have been obtained for normal peripheral blood leukocytes, chronic lymphocytic leukemia (CLL), hairy cell leukemia, mantle cell lymphoma, acute myeloid leukemia, and T-cell acute lymphoblastic leukemia. The consensus pattern for CD antigen expression found on CLL cells taken from 20 patients in descending order of cells bound was CD44, HLA-DR, CD37, CD19, CD20, CD5, CD52, CD45RA, CD22, CD24, CD45, CD23, CD21, CD71, CD11c, and CD9. The antigens that provided the best discrimination between CLL and normal peripheral blood leukocytes were CD19, CD20, CD21, CD22, CD23, CD24, CD25, and CD37. Results obtained for the expression of 48 CD antigens from the microarray compared well with flow cytometry. The microarray enables extensive immunophenotyping, and the intact cells captured on antibody dots can be further characterized using soluble, fluorescently labeled antibodies.

Extracellular ATP-dependent suppression of proliferation and induction of differentiation of human HL-60 leukemia cells by distinct mechanisms.

Extracellular ATP suppressed the growth of HL-60 leukemia cells and induced their differentiation as revealed by N-formyl-methionyl-leucyl-phenylalanine-induced beta-glucuronidase release. ATP degraded to ADP, AMP, and adenosine, and the effect of ATP on cell growth was mimicked by these metabolites added to the cultures. The stable analog alpha,beta-methylene ATP, however, had only a weak inhibitory effect on cell growth. Adenine nucleotide-induced growth suppression was reversed by uridine, suggesting the involvement of intracellular pyrimidine starvation secondary to adenosine accumulation. Consistent with this, ATP induced intracellular starvation of pyrimidine nucleotides, and this effect was also prevented by pretreatment of cells with uridine. The order of effectiveness of ATP-induced differentiation of HL-60 cells, unlike that for growth suppression, was ATP > ADP > AMP, and adenosine had no effect. Furthermore, uridine had no effect and the stable analog, alpha,beta-methylene ATP also induced HL-60 cell differentiation, suggesting that differentiation was due to ATP per se. We tested the hypothesis that ATP-induced differentiation arises from activation of adenylyl cyclase by the novel P2Y(11) receptor using the cell-permeable inhibitor of protein kinase A, Rp-CPT-cAMPS (8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphorothioate, Rp isomer). Rp-CPT-cAMPS (1-100 microM) prevented ATP-induced differentiation of HL-60 cells as assessed by fMLP-induced beta-glucuronidase release. However, Rp-CPT-cAMPS did not prevent ATP-induced growth suppression. Taken together, the data indicate that extracellular ATP suppresses HL-60 growth and induces their differentiation by distinct mechanisms. Growth suppression arises from adenosine generation and consequent pyrimidine starvation. Differentiation arises, at least in part, from a distinct mechanism involving the activation of cell surface P2 receptors coupled to cAMP generation and activation of protein kinase A.

Acetyl-CoA carboxylase and fatty acid synthase activity and immunodetectable protein in adipose tissues of ruminants: effect of temperature and feeding level.

To gain insights into the regulation of fat synthesis, we have investigated the effect of cold environmental exposure and feed restriction of sheep on activity and immunodetectable protein content of acetyl-CoA carboxylase (ACC) and fatty acid synthase in adipose tissue. Subcutaneous and mesenteric adipose tissues were collected at slaughter from sheep exposed to either cold (0+/-2 degrees C) or warm (23+/-2 degrees C) environment, and given either ad libitum or restricted access to feed for three 5-wk periods. Acetyl-CoA carboxylase was isolated from frozen adipose tissue samples and activity determined as the rate of incorporation of H14CO3- into acid stable malonyl-CoA. Cold exposure and feed restriction reduced (P < .05) ACC activity in the two adipose tissue depots. Western blot analysis with peroxidase-conjugated streptavidin showed that both adipose tissue depots express a single isoform of ACC. In s.c. adipose tissue, cold exposure increased (P < .05) ACC protein abundance, which is opposite to the change in activity. However, feed restriction reduced immunodetectable ACC protein. There was no significant effect of environment or feeding level on ACC protein abundance in mesenteric tissue. Fatty acid synthase activity determined in ammonium sulfate extract by measuring the malonyl-CoA- and acetyl-CoA-dependent oxidation of NADPH was decreased (P < .05) by feed restriction in both s.c. and mesenteric tissues. Cold exposure reduced fatty acid synthase activity in s.c. but not in mesenteric tissue. There was no effect of environment on fatty acid synthase protein abundance in either adipose tissue depot. However, feed restriction significantly reduced fatty acid synthase protein abundance in the two depots. The data suggest that feed restriction and exposure of ruminants to cold environmental conditions may significantly down-regulate the activity of key lipogenic enzymes.

Effects of temperature and plane of nutrition on beta-adrenergic receptors in heart, kidney, and liver of lambs.

We determined the effects of temperature and feed intake on beta-adrenergic receptors (beta-adrenoceptors) in tissues of sheep. Twenty-four lambs were exposed during three 5-wk periods to either thermoneutral, control (W; 23+/-2 degrees C) or cold (C; 0+/-2 degrees C) temperatures and were fed either ad libitum (A) or restricted (R) levels of feed intake, resulting in four treatment groups: WA, WR, CA, and CR. Hearts, kidneys, and livers were harvested at slaughter and binding of [3H]dihydroalprenolol to plasma membrane extracts was used to determine densities (B(MAX)) and binding affinities (Kd) of beta1 and beta2 adrenoceptors. The B(MAX) values ranged from 12.10 to 201.26 and 3.38 to 12.30 fmol/mg protein for beta1 and beta2 adrenoceptors, respectively; heart and kidney had the highest and lowest values, respectively. Feed restriction reduced (P < .05) beta1 and beta2 receptor densities in heart but increased (P < .05) beta1 receptor density in kidney and liver. Cold temperature exposure reduced beta1 receptor density in heart tissue during feed restriction. The Kd values, ranging from 1.32 to 5.98 nM, were increased (P < .05) by cold exposure and feed restriction in kidney and liver. Because the effectiveness of hormones is a function of their concentrations, binding affinities, and their receptor densities, these results imply that cold temperature exposure and feed restriction could potentially reduce (in heart) and increase (in kidney and liver) metabolic responsiveness of tissues to catecholamines.

Effect of beta-adrenergic agonist L-644,969 on the impact of the thermal environment on in vitro fatty acid synthesis and lipogenic enzymes in sheep.

The effect of temperature and beta-adrenergic agonist (BAA) on in vitro rates of fatty acid synthesis and catalytic activity of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) was examined in wether lambs after 5 weeks at either 0 or 20 degrees C. Feeding BAA increased (P < 0.05) rate of fatty acid synthesis by 38% in subcutaneous adipose (SC) tissue from cold-acclimated animals but the rate decreased (P < 0.05) by 27% in SC tissue from warm-acclimated animals. In mesenteric fat (MS), BAA increased (P < 0.05) fatty acid synthesis in the cold environment. In perirenal (PR) fat, rate of fatty acid synthesis was reduced (P < 0.05) by 20% by BAA in the warm but had no effect in the cold. Activity of ACC in longissimus muscle was depressed (P < 0.05) when BAA was fed in the warm environment. In adipose tissues BAA reduced (P < 0.05) ACC activity in the warm, but reduced activity in the cold was limited to SC tissue. In PR tissue FAS activity was reduced (P < 0.05) in the cold environment, while BAA increased FAS activity in the warm environment. Western blot analysis showed two isoforms of ACC with MW of 280 000 and 265 000 Da in longissimus muscle whereas only one isoform was recognized in each of Biceps femoris (280 000 Da) and adipose tissues (265 000 Da). Feeding BAA in the cold environment reduced (P < 0.05) ACC and FAS immunoprotein expression in both MS and PR adipose tissues. The studies indicate that the effect of BAA on fatty acid synthesis and lipogenic enzymes is influenced by acclimation temperature.

Divalent metal derivatives of the hamster dihydroorotase domain.

Dihydroorotase (DHOase, EC 3.5.2.3) is a zinc enzyme that catalyzes the reversible cyclization of N-carbamyl-L-aspartate to L-dihydroorotate in the third reaction of the de novo pathway for biosynthesis of pyrimidine nucleotides. The recombinant hamster DHOase domain from the trifunctional protein, CAD, was overexpressed in Escherichia coli and purified. The DHOase domain contained one bound zinc atom at the active site which was removed by dialysis against the chelator, pyridine-2,6-dicarboxylate, at pH 6.0. The apoenzyme was reconstituted with different divalent cations at pH 7.4. Co(II)-, Zn(II)-, Mn(II)-, and Cd(II)-substituted DHOases had enzymic activity, but replacement with Ni(2+), Cu(2+), Mg(2+), or Ca(2+) ions did not restore activity. Atomic absorption spectroscopy showed binding of one Co(II), Zn(II), Mn(II), Cd(II), Ni(II), or Cu(II) to the enzyme, while Mg(II) and Ca(II) were not bound. The maximal enzymic activities of the active, reconstituted DHOases were in the following order: Co(II) --> Zn(II) --> Mn(II) --> Cd(II). These metal substitutions had major effects upon values for V(max); effects upon the corresponding K(m) values were less pronounced. The pK(a) values of the Co(II)-, Mn(II)-, and Cd(II)-substituted enzymes derived from pH-rate profiles are similar to that of Zn(II)-DHOase, indicating that the derived pK(a) value of 6.56 obtained for Zn-DHOase is not due to ionization of an enzyme-metal aquo complex, but probably a histidine residue at the active site. The visible spectrum of Co(II)-substituted DHOase exhibits maxima at 520 and 570 nm with molar extinction coefficients of 195 and 210 M(-1) cm(-1), consistent with pentacoordination of Co(II) at the active site. The spectra at high and low pH are different, suggesting that the environment of the metal binding site is different at these pHs where the reverse and forward reactions, respectively, are favored.

Lack of class I major histocompatibility antigens on trophoblast of periimplantation blastocysts and term placenta in the pig.

In this study, the pattern of expression of class I major histocompatibility (MHC) antigens and mRNA on periimplantation blastocysts and term placental tissue was determined for the pig. Class I MHC antigens could not be detected immunohistochemically either on extra-embryonic membranes or on the embryonic portion of Day 14, 16, 22, and 25 blastocysts. Nor could class I MHC antigens be detected on the outer trophoblast epithelium and inner endodermal surface of the chorioallantoic membrane or on the outer and inner surfaces of the amnion at term. However, MHC class I antigens were detected on the vascular mesoderm found in both the chorion and amnion at term, and in Day 25 extra-embryonic membranes. Uterine endometrial cells and tissues and maternal peripheral blood leukocytes stained strongly for class I MHC antigens. There was a large difference in the intensity of class I MHC mRNA signal, detected by Northern blot analysis, in embryo/fetus-derived tissues compared to that in maternal tissues. The embryos appeared to express even less class I MHC mRNA than did the extra-embryonic membranes. In addition, in situ hybridization of Day 16 blastocysts indicated class I MHC mRNA to be ubiquitously expressed at low levels in embryos and extra-embryonic tissues compared to uterine endometrial tissue controls. Taken together, these results indicate that class I MHC antigens are either not expressed on the surface of the extra-embryonic/fetal membranes during gestation in the pig or are expressed at very low levels, and that specific mRNA is expressed at correspondingly low levels.

Synthesis and enzymic evaluation of 4-mercapto-6-oxo-1, 4-azaphosphinane-2-carboxylic acid 4-oxide as an inhibitor of mammalian dihydroorotase.

The design, synthesis, and enzymic evaluation of cis- and trans-4-mercapto-6-oxo-1,4-azaphosphinane-2-carboxylic acid 4-oxide 5 against mammalian dihydroorotase is presented. The design strategy for 5 was based on the strong affinity of phosphinothioic acids for zinc and that 5 also resembles the postulated tetrahedral transition state for the enzyme-catalyzed reaction. The synthesis of 5 utilized a novel protection/deprotection sequence upon 4-hydroxy-6-oxo-1, 4-azaphosphinane-2-carboxylic acid 4-oxide 4, followed by incorporation of alpha-phenyl benzenemethanethiol and exhaustive deprotection to afford 5 in 40% overall yield from 4. The activities of both isomers of 5 as inhibitors of mammalian dihydroorotase were marginally greater than that of the parent phosphinic acid 4, indicating a weak binding enhancement due to the phosphinothioic acid moiety.

Metabolic effects of thiopurine derivatives against human CCRF-CEM leukaemia cells.

UNLABELLED: BACKGROUND and aims. To compare the metabolic effects induced by the anticancer drugs, 6-mercaptopurine (6-MP), 6-thioguanine (6-TG) and 6-methylmercaptopurine riboside (MMPR), which may inhibit the de novo biosynthesis of purine nucleotides or be mis-incorporated into DNA or RNA. METHODS: Leukaemia cells were grown in culture, exposed to a thiopurine and cell extracts were analyzed for NTPs, dNTPs, drug metabolites and P-Rib-PP. RESULTS: In leukaemia cells, 6-MP was converted to MPR-MP, thio-XMP, thio-GMP, thio-GDP and thio-GTP. Metabolites of 6-TG included thio-XMP, thio-GMP, thio-GDP and thio-GTP, while MMPR-MP was the only major metabolite of MMPR, MMPR (25 microM, 4 h) induced a 16-fold increase in P-Rib-PP and 6-MP (25 microM, 4 h) induced a delayed 5.2-fold increase. MPR-MP, thio-GMP and MMPR-MP are inhibitors of amido phosphoribosyltransferase from leukaemia cells with Ki values of 114 +/- 7.10 microM, 6.20 +/- 2.10 microM and 3.09 +/- 0.30 microM, respectively. CONCLUSION: The nucleoside-5'-monophosphate derivatives of the 3 thiopurines inhibit amido phosphoribosyltransferase in growing leukaemia cells but there is also an initial inhibition of the further conversion of IMP in the pathway. In growing cells, MMPR acts solely as an inhibitor of de novo purine biosynthesis while 6-TG and to a lesser extent, 6-MP, are converted to significant concentrations of di- and tri-phosphate derivatives which may have other mechanisms of cytotoxicity.