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.

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.

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.

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.

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.

Relationship between the catalytic sites of human bifunctional IMP synthase.

BACKGROUND AND AIMS: The bifunctional enzyme, IMP synthase, contains 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase and IMP cyclohydrolase activities and catalyses the ninth and tenth reactions of the pathway for de novo biosynthesis of purine nucleotides (AICAR-->FAICAR-->IMP). The spatial relationship between the two active sites on IMP synthase has been investigated along with the possibility that the intermediate, FAICAR, may be channelled between the two sites. METHODS: The two catalytic activities and the overall reaction (AICAR-->FAICAR-->IMP) were assayed using 3H-labelled AICAR or FAICAR with isolation of the reaction products by thin-layer chromatography. RESULTS: Inhibition constants for the interactions of six purine nucleoside 5'-monophosphate derivatives with AICAR transformylase and IMP cyclohydrolase were 24- to 820-fold higher for the transformylase. N-ethylmaleimide inactivated IMP cyclohydrolase but not AICAR transformylase. The rate of IMP synthesis from AICAR was consistent with a high local concentration of FAICAR at the cyclohydrolase site but addition of exogenous unlabelled FAICAR reduced the amount of [3H]AICAR formed from [3H]AICAR indicating that the channelling of FAICAR was not absolute. CONCLUSION: The AICAR transformylase and IMP cyclohydrolase active sites of IMP synthase are distinct but sufficiently close for the FAICAR produced by a transformylase site to be preferentially utilized as a substrate by a cyclohydrolase site on the same molecule if dimeric, bifunctional IMP synthase.

Comparative effects of cladribine, fludarabine and pentostatin on nucleotide metabolism in T- and B-cell lines.

BACKGROUND AND AIMS: the purine nucleoside analogues cladribine (CdA), fludarabine (F-Ara-AMP) and pentostatin (dCf), are effective therapy for a range of T- and B-cell lymphoid malignancies. The effects upon nucleotide metabolism in human CCRF-CEM T-cell leukaemia and Raji B-cell lymphoma cell lines of these drugs have been compared to assess possible mechanisms of cytotoxicity. METHODS: Leukaemia cells were exposed to a purine nucleoside analogue and perchloric acid extracts were analysed by HPLC for 2'-deoxynucleoside-5'-triphosphates (dNTPs), nucleoside-5'-triphosphates (NTPs) and drug metabolites. RESULTS: After addition of a purine nucleoside analogue, CdA-TP and F-Ara-ATP accumulate in cells while the levels of dCf-TP formed were not detectable by ultra-violet absorbance. In response to accumulating concentrations of drug triphosphate, the cellular levels of dNTPs initially decrease (0-4 h), then accumulate above their initial levels (4-10 h) before slowly declining beyond 10 h. NTPs also accumulate during the period 4-10 h before declining at later times. CONCLUSION: The temporal effects on the levels of dNTPs and NTPs of the 3 purine nucleoside analogues are similar against CCRF-CEM and Raji cells. However, CdA induces major depletions of dTTP, dGTP and dATP in CCRF-CEM cells and F-Ara-A induces a major accumulation of dATP in Raji cells.

Indirect inhibition by antibiotics of nucleotide and deoxynucleotide biosynthesis in Plasmodium falciparum.

The effects of the antibiotics, doxycycline, azithromycin, ciprofloxacin and chloramphenicol, upon levels of nucleoside-5'-triphosphates (NTPs) and 2'-deoxynucleoside-5'-triphosphates (dNTPs) have been compared in the malarial parasite, Plasmodium falciparum, and in human CCRF-CEM leukemia cells. All 4 antibiotics had more severe effects upon levels of NTPs and dNTPs in P. falciparum compared with leukemia cells providing an explanation for their selective toxicity against malaria and their utility as antimalarial drugs. In bacteria, the first 3 drugs inhibit protein synthesis while ciprofloxacin inhibits topoisomerase II. The observed depletions of NTPs and dNTPs would be a secondary effect of the drug but may result in death of the parasite.

Reversal of vinblastine transport by chlorpromazine in membrane vesicles from multidrug-resistant human CCRF-CEM leukaemia cells.

The mechanism of action of 2-chlorpromazine (2-chloro-10-(3-dimethylaminopropyl)-phenothiazine) as a reversal agent for P-glycoprotein-mediated multidrug resistance was investigated using inside out-orientated membrane vesicles prepared from vinblastine-resistant human CCRF-CEM leukaemia cells (VBL1000). 2-Chlorpromazine (10 microM) completely inhibited ATP-dependent P-glycoprotein-mediated vinblastine accumulation in the vesicles. Whereas in the absence of added ligands VBL transport was described by a hyperbolic function of vinblastine concentration, in the presence of 2-chlorpromazine vinblastine transport was a sigmoidal function. 2-Chlorpromazine was shown previously [Syed SK, Christopherson RI and Roufogalis BD (1996) Biochem Mol Biol Int 39: 687-696] to be actively transported into vesicles from multidrug-resistant cells. Colchicine (10 microM) and phenoxybenzamine (10 microM) blocked vinblastine transport but had no effect on 2-chlorpromazine transport into vesicles. The results were consistent with a two-state concerted model in which P-glycoprotein exists in two conformational states, P(A) and P(B), where 2-chlorpromazine is transported by the conformer, P(A), and vinblastine by the conformer, P(B). In the presence of 2-chlorpromazine, the conformer P(A) predominates and vinblastine transport is inhibited. Addition of 2-chlorpromazine during the steady state of vinblastine accumulation blocked uptake and resulted in enhanced vinblastine efflux from the vesicles. The findings were similar when vinblastine was added at the steady state of 2-chlorpromazine transport. We propose a minimal kinetic model whereby in these preloaded vesicles the complex VV.P(A).CC is formed, where two internal binding sites of P-glycoprotein (P(A)) are occupied by vinblastine (V) and the two external sites are occupied by 2-chlorpromazine (C). When the two binding sites on both the inside and outside of P-glycoprotein are saturated with ligands vinblastine is effluxed at a very rapid rate, and vice versa when vesicles are preloaded with 2-chlorpromazine and vinblastine is added outside. These unexpected observations and the concerted model developed provide an alternative mechanism of action for reversal agents that sensitize multidrug-resistant cancer cells to anti-cancer drugs.