Predictive and Prognostic Value of 18F-fluorodeoxyglucose Uptake Combined with Thymidylate Synthase Expression in Patients with Advanced Non-Small Cell Lung Cancer.

We investigated the relationship between tumor 18F-fluorodeoxyglucose (FDG) uptake on positron emission tomography/computed tomography (PET/CT) scans and thymidylate synthase (TS) expression. In addition, we evaluated the value of FDG uptake in predicting treatment response and prognosis when combined with TS expression in patients with advanced non-small cell lung cancer (NSCLC). We measured the maximum standard uptake value, metabolic tumor volume, and total lesion glycolysis (TLG) of tumor lesions on pretreatment scan in 234 patients (age: 60.1 ± 9.4 years; males: 56.4%) with stage IV non-squamous NSCLC who were enrolled in the prospective phase II clinical trial. We investigated the correlation of the parameters with TS expression and the predictive values of the parameters compared with other clinical factors. Among these parameters, TLG was the most relevant parameter that had a significant correlation with TS expression (ρ = 0.192, P = 0.008). A multivariable Cox proportional-hazards model revealed that high TLG was a significant independent predictor for treatment response (hazard ratio [HR]: 2.05; P = 0.027), progression-free survival (HR: 1.39; P = 0.043), and overall survival (HR: 1.65; P = 0.035) with other factors. In patients with advanced non-squamous NSCLC, tumor TLG on pretreatment PET/CT scan has predictive and prognostic value.

Purification, activity, and expression levels of two uridine-cytidine kinase isoforms in neuroblastoma cell lines.

Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine, cytidine, and several pyrimidine ribonucleoside analogs. We overexpressed and purified the two known isoforms of human UCK in Escherichia coli, produced a specific antibody against UCK1 and characterized the kinetic properties of UCK1 and 2. The Vmax of purified recombinant UCK2 was 22- and 8-fold higher with uridine and cytidine, respectively, compared to those observed for the purified recombinant UCK1 enzyme. The Km of UCK1 was 39- and 40-fold higher with uridine and cytidine, respectively, compared to those observed for the purified recombinant UCK2 enzyme. The UCK1 antibody showed no cross reactivity against UCK2. Our data showed that UCK1 and 2 are both expressed in several neuroblastoma cell lines, including four MYCN single copy cell lines and five MYCN amplified cell lines, with the exception that UCK1 was not expressed in SJNB8. These results indicate that UCK2 in neuroblastoma might be used as a selective target for chemotherapy using UCK2-dependent pyrimidine analogues.

Resistance to the nucleotide analogue cidofovir in HPV(+) cells: a multifactorial process involving UMP/CMP kinase 1.

Human papillomavirus (HPV) is responsible for cervical cancer, and its role in head and neck carcinoma has been reported. No drug is approved for the treatment of HPV-related diseases but cidofovir (CDV) exhibits selective antiproliferative activity. In this study, we analyzed the effects of CDV-resistance (CDVR) in two HPV(+) (SiHaCDV and HeLaCDV) and one HPV(-) (HaCaTCDV) tumor cell lines. Quantification of CDV metabolites and analysis of the sensitivity profile to chemotherapeutics was performed. Transporters expression related to multidrug-resistance (MRP2, P-gp, BCRP) was also investigated. Alterations of CDV metabolism in SiHaCDV and HeLaCDV, but not in HaCaTCDV, emerged via impairment of UMP/CMPK1 activity. Mutations (P64T and R134M) as well as down-regulation of UMP/CMPK1 expression were observed in SiHaCDV and HeLaCDV, respectively. Altered transporters expression in SiHaCDV and/or HeLaCDV, but not in HaCaTCDV, was also noted. Taken together, these results indicate that CDVR in HPV(+) tumor cells is a multifactorial process.

The structure of uridylate kinase with its substrates, showing the transition state geometry.

Uridylate kinase from Saccharomyces cerevisiae is a member of the nucleoside monophosphate (NMP) kinase family and catalyzes the reaction ATP+NMP<==>ADP+NDP with moderate specificity for UMP. The recombinant enzyme crystallized together with two substrate molecules. The structure was solved, by multiple isomorphous replacement and solvent flattening, at 3.0 A and then refined at 2.13 A resolution. The present R-factor is 19%. Superposition onto the structure of a substrate-free adenylate kinase revealed the motions induced by substrate binding. A further superposition onto an adenylate kinase with bound P1,P5-bis(5'-adenosyl)pentaphosphate (Ap5A), a two-substrate-mimicking inhibitor, failed to explain the UMP preference of the uridylate kinase, but superimposed the nucleosides and in particular the non-transferred phosphates at the ATP- and NMP-site rather well. The coincidence of the phosphates indicate strongly that these groups assume their final positions during catalysis. This locates the transition state, which can be modeled with reasonable geometry in agreement with an in-line associative SN2 mechanism.

Camguk, Lin-2, and CASK: novel membrane-associated guanylate kinase homologs that also contain CaM kinase domains.

MAGUKs (membrane-associated guanylate kinase homologs) are proteins involved in cell junction organization, tumor suppression, and signalling. Their structure includes one or three copies of a DHR or PDZ domain (discs-large homologous region or PSD-95/SAP90, discs-large ZO-1 homologous domain), an SH3 domain, and a guanylate kinase domain. MAGUKs were classified into two subfamilies: Dlg-like with three DHR/PDZ domains and p55-like with a single DHR/PDZ domain. There is now a new subfamily whose members have a novel domain structure: a calcium/calmodulin-dependent protein kinase domain in the N-terminus as well as the DHR/PDZ, SH3 and GUK domains in the C-terminus. These new MAGUKs may regulate transmembrane molecules that bind calcium, calmodulin, or nucleotides, camguk (cmg) is a Drosophila member of this novel MAGUK subfamily; we report its sequence and domain structure.

Next-generation sequencing reveals novel rare fusion events with functional implication in prostate cancer.

Gene fusions, mainly between TMPRSS2 and ERG, are frequent early genomic rearrangements in prostate cancer (PCa). In order to discover novel genomic fusion events, we applied whole-genome paired-end sequencing to identify structural alterations present in a primary PCa patient (G089) and in a PCa cell line (PC346C). Overall, we identified over 3800 genomic rearrangements in each of the two samples as compared with the reference genome. Correcting these structural variations for polymorphisms using whole-genome sequences of 46 normal samples, the numbers of cancer-related rearrangements were 674 and 387 for G089 and PC346C, respectively. From these, 192 in G089 and 106 in PC346C affected gene structures. Exclusion of small intronic deletions left 33 intergenic breaks in G089 and 14 in PC346C. Out of these, 12 and 9 reassembled genes with the same orientation, capable of generating a feasible fusion transcript. Using PCR we validated all the reliable predicted gene fusions. Two gene fusions were in-frame: MPP5-FAM71D in PC346C and ARHGEF3-C8ORF38 in G089. Downregulation of FAM71D and MPP5-FAM71D transcripts in PC346C cells decreased proliferation; however, no effect was observed in the RWPE-1-immortalized normal prostate epithelial cells. Together, our data showed that gene rearrangements frequently occur in PCa genomes but result in a limited number of fusion transcripts. Most of these fusion transcripts do not encode in-frame fusion proteins. The unique in-frame MPP5-FAM71D fusion product is important for proliferation of PC346C cells.

Cloning and heterologous expression of Entamoeba histolytica adenylate kinase and uridylate/cytidylate kinase.

We have isolated two cDNA clones encoding Entamoeba histolytica nucleotide kinases, EhAK and EhUK, expressed them in E. coli and performed functional studies of the recombinant enzymes. Nucleotide sequence analysis showed that EhAK and EhUK genes exhibited the features characteristic of E. histolytica genes, such as transcripts with relatively short 5' and 3' untranslated flanking regions containing the conserved E. histolytica transcription promoter elements located 5' to the initiation codon and a polyadenylation signal in the 3' UTR, a distinctive codon usage bias for A or T in the third position and an AT bias greater than 75% in the flanking regions of the transcripts. At the protein level, both enzymes belong to the short variant nucleoside monophosphate (NMP) kinases, which lack a 29amino acid LID region present in the long variant isoenzymes. EhAK was 30-38% identical to the members of the adenylate kinase (AK) family while EhUK was more similar (48-49% identity) to UMP/CMP kinases. Both enzymes used ATP as preferred phosphate-group donor but each one exhibited strict specificity for the acceptor NMP, EhAK for AMP and EhUK for the pyrimidine nucleoside monophosphates UMP and CMP. Biochemical characterization of the enzymes and phylogenetic reconstruction showed that EhUK is an authentic and well conserved member of the UMP/CMP kinase group while EhAK is the most divergent member known of the AK1 isoenzymes.

Deoxynucleoside anabolic enzyme levels in acute myelocytic leukemia and chronic lymphocytic leukemia cells.

The deoxynucleoside kinase reaction is often rate-limiting in the anabolism of pharmacologically active anti-cancer nucleosides. The levels of thymidine kinase (TK), deoxycytidine kinase, deoxyguanosine kinase (dGK), and thymidylate kinase were determined in leukocyte extracts from patients with chronic lymphocytic leukemia (CLL) and acute myelocytic leukemia (AML). The extracts from AML patients showed significantly higher TK activity than the ones from CLL patients. There were no differences in the levels of the other three kinases. In the case of dGK, the determinations were carried out with both an immunoblotting assay and selective enzyme activity measurements.

Comparison of two standardisation methods in real-time quantitative RT-PCR to follow Staphylococcus aureus genes expression during in vitro growth.

By real-time quantitative PCR (RTQ-PCR), two different standardisation methods were used to quantify expression of three target genes (RNAII and RNAIII transcripts of agr locus and ica transcript of icaADBC locus): (i) a relative quantification, using a transcript of three housekeeping genes (gyrase A, gyrA; guanylate kinase, gmk and 16S rRNA, 16S) as internal standard, and (ii) an absolute quantification, using cloned sequences of the target genes in known concentrations as external standards. To determine the efficiency and reliability of these two methods, the gene expressions were studied during the growth of a clinical isolate of Staphylococcus aureus. Between 3 and 20 h after inoculation, target gene transcription was analysed using LightCycler Apparatus, LC Data Analysis software and RelQuant software for relative quantification (Roche). For all target genes, the expression profiles obtained with gyrA or gmk as internal standards remained almost identical. However, these profiles varied between each other depending on the standard gene. Due to their important expression variations during growth phases, these two housekeeping genes seem inappropriate to be used as internal standards. The absolute quantification of the three transcripts of interest gave results similar to their relative quantification expressed versus 16S rRNA. Therefore, our study suggests the suitable use of 16S rRNA as internal standard in RTQ-PCR quantification of staphylococcal gene expression during the stationary phase of growth.

Modulation of human UMP/CMP kinase affects activation and cellular sensitivity of deoxycytidine analogs.

Deoxycytidine analogs are an important class of clinically active antiviral and anticancer agents. The stepwise phosphorylation of these analogs to triphosphate metabolites is crucial for biological action. Human UMP/CMP kinase (UMP/CMPK; cytidylate kinase; EC 2.7.4.14) is thought to be responsible for phosphorylation of UMP, CMP, and dCMP and may also play an important role in the activation of pyrimidine analogs. However, no evidence has verified this notion in intact cells. In this study we explored the functional roles of UMP/CMPK in natural pyrimidine synthesis and metabolism of deoxycytidine analogs, as well as 5-FU in HeLa S3 and HCT8 cells. The amounts of UMP/CMPK protein in different cell lines correlated with UMP, CMP, and dCMP kinase activities and amounts of UMP/CMPK RNA. Modulation of UMP/CMPK by overexpression or down-regulation had no impact on natural pyrimidine nucleotides and cell growth. However, down-regulating UMP/CMPK expression by siRNA led to a decrease in the formation of the triphosphate metabolites, resulting in cellular resistance to these analogs. More diphosphate and triphosphate metabolites of deoxycytidine analogs were detected and cellular sensitivity to these agents was increased in the UMP/CMPK-overexpressing cells. This study indicates that the second step enzyme (UMP/CMPK) is responsible for the phosphorylation of pyrimidine analogs and also has an impact on cellular sensitivity to these analogs in those cell lines.

Association of thymidylate kinase activity with pyrimidine deoxyribonucleoside kinase induced by herpes simplex virus.

Thymidine kinase derived from LMTK+ does not exhibit thymidylate kinase activity. However, protein isolated by affinity column chromatography from thymidine kinase-deficient mouse cells (LMTK-) infected by herpes simplex virus type 1 shows thymidylate kinase activity in addition to thymidine kinase and deoxycytidine kinase activities. The virus-induced multifunctional enzyme has a molecular weight of 85,000, whereas the molecular weight of thymidylate kinase from uninfected LMTK- mouse cells is 71,000. The virus-induced enzyme has a Km for thymidine of 0.8 micromolar, and for thymidylate of 25 micromolar, and for thymidylate of 25 micromolar; the ratio of Vmax for thymidylate kinase to thymidine kinase is 1.7. When subjected to isoelectric focusing, thymidylate kinase activity is not separated from thymidine kinase activity, and even though four peaks of activity are observed they have a constant ratio of thymidylate kinase to thymidine kinase activity. The isoelectric points (pI) of these four peaks are 4.8, 5.8, 6.2, and 6.6, respectively. Thymidylate kinase, derived from uninfected cells when subjected to isoelectric focusing, separates into a major component with an isoelectric point at pH 8.2 and a minor component at pH 7.7. Although thymidine and thymidylate kinase activities derived from the virus-infected cells cannot be separated either by affinity column chromatography, glycerol density gradient centrifugation, or isoelectric focusing, there is a differential rate of inactivation when the enzyme is subjected to incubation at 37 degrees, with thymidylate kinase activity being more labile than thymidine kinase activity.

Slow switchover from host RNA synthesis to bacteriophage RNA synthesis after infection of Escherichia coli with a T4 mutant defective in the bacteriophage T4-induced unfolding of the host nucleoid.

Most, if not all, host RNA synthesis was shut off after infection of Escherichia coli strain B/5 with a bacteriophage T4 multiple mutant defective in the abilities to induce (i) unfolding of the host nucleoid (unf-), (ii) nuclear disruption (ndd-), and (iii) host DNA degradation (denA-, denB-). The shutoff of host RNA synthesis and turn-on of phage RNA synthesis were slower after infection of E. coli with unf- phage than after infection with unf+ phage. This delay in the switchover from host RNA synthesis to phage RNA synthesis in unf- infections did not result in a measurable delay in the onset of nuclear disruption, deoxyribonucleoside monophosphate kinase synthesis, or DNA synthesis. unf39 did not complement alc (allows late transcription on cytosine-containing DNA) mutants, supporting the proposal of Sirotkin et al. [Nature (London) 265:28-32, 1977] that alc and unf are possibly the same gene.

A mammalian cell mutant with temperature-sensitive thymidine kinase.

We have isolated a mutant clone from mouse FM3A cells with temperature-sensitive defects both in cytokinesis and in thymidine kinase enzyme activity. The clone, designated tsCl.B59, was isolated after mutagenesis at 33 degrees C followed by exposure to cytosine arabinoside at 39 degrees C. It was derived from a thymidine kinase deficient, 5-bromodeoxyuridine-resistant clone (S-BUCl.42) which was originally derived from wild-type clone H-5 of FM3A cells. The temperature-sensitive mutant clone grows normally at 33 degrees C, but not at 39 degrees C, where it exhibits an increased frequency of multinucleate cells due to defective cytokinesis. Unlike the parental S-BUCl.42 cells, which have negligible thymidine kinase activity and are unable to incorporate 3H-thymidine, the mutant in corporates substantial amounts of 3H-thymidine at 33 degrees C, although its thymidine kinase activity remains lower than that of wild-type H-5 cells. When cultures of tsCl.B59 cells are transferred to 39 degrees C, incorporation of 3H-thymidine decreases markedly. The decrease has been shown to be due to thermolability of the thymidine kinase in tsCl.B59 cells.

DNA-synthesizing enzymes in breast cancer (thymidine kinase, thymidylate synthase and thymidylate kinase): association with flow cytometric S-phase fraction and relative prognostic importance in node-negative premenopausal patients.

S-phase fraction (SPF) is a reference for cell-kinetic analysis. In this study, the links between SPF and the essential enzymes participating in the pyrimidine synthesis were investigated in breast cancer and their relationships with the natural history of the disease were compared. We measured thymidine kinase (TK) for salvage synthesis, thymidylate synthase (TS) for de novo synthesis and thymidylate kinase (TMK), which is required for both pathways. Our study population consisted of 211 premenopausal women with node-negative tumors. SPF was assessed prospectively by flow cytometry, whereas enzyme activities were measured retrospectively in cytosols using radioenzymatic methods. Among the enzymes analyzed, only TK demonstrated a strong correlation with SPF (r(s) = 0.59). In univariate analysis, high SPF and high levels of TK were associated with increased risk of developing distant recurrences (p < 0.001). Correlations with other prognostic factors (histological grade, steroid receptors, DNA ploidy status, urokinase plasminogen activator and plasminogen activator inhibitor type 1) confirmed a parallel association of SPF and TK with the most aggressive tumors. In contrast, TS and TMK were not associated with prognosis. After adjustment for SPF, the risk of relapse increased significantly with TK values. Subgroup analysis showed that additional information was provided by TK in the tumors with low SPF. When urokinase plasminogen activator (uPA) was a candidate variable in multivariate analysis, TK remained significant. Combined with SPF and uPA, TK could be useful to define premenopausal node-negative patients with rapidly proliferating tumors at a high risk of metastatic disease.

Cloning and expression of the essential gene for guanylate kinase from yeast.

Guanylate kinase catalyzes the reversible transfer of the terminal phosphoryl group of ATP to the acceptor molecule GMP. Detailed analysis of the in vivo function of this enzyme has been limited by the lack of any genetic data. Using oligonucleotides based on amino acid sequence information of the yeast enzyme, the Saccharomyces cerevisiae gene, GUK1, was isolated and characterized. The gene is present in single copy and maps to chromosome IV. Insertional mutagenesis of the GUK1 locus caused recessive lethality, indicating that this enzyme is necessary for vegetative cell growth. Using inducible expression systems, guanylate kinase was produced in large amounts both in S. cerevisiae and in Escherichia coli.

Role of potassium in the synthesis and decay of two specific bacteriophage T4 messages.

The role of K+ in the in vivo metabolism of specific phage T4 messengers was studied. By using a mutant of Escherichia coli defective in its ability to accumulate K+ from the growth medium, it was possible to rapidly deplete cells of their intracellular K+ and in this way determine K+-dependent reactions in vivo. The rate constants for accumulation, synthesis, and decay of the early enzymes deoxynucleotide kinase and alpha-glucosyl transferase were determined. It was shown that there is a very close association between mRNA synthesis and its decay, indicating that a mechanism may be present in the cell that can regulate the concentration of these RNAs. Since the mRNA's for these enzymes are very stable in cells depleted of K+, K+ depletion may be a useful method for the isolation of functional T4 mRNA.

Molecular characterization of the murine thymidylate kinase gene.

Thymidylate kinase (TMK) catalyzes an essential reaction of converting dTMP to dTDP, leading to formation of the DNA precursor dTTP. Unlike the preceding enzymes, thymidine kinase and thymdylate synthase, little is known about regulation of TMK after mammalian cells exit from quiescence and enter the cell cycle. In this study, cDNA of murine TMK was isolated and characterized. Murine TMK gene expression in nontransformed BALB/c 3T3 cells was shown to be regulated during Gø to S-phase transit at both mRNA and enzyme activity levels. Its timing was distinctive from that of thymidine kinase and thymdylate synthase. In contrast, the regulation of TMK in response to serum growth factor stimulation was abolished in cells transformed by either SV40 or Kirsten viruses, and tmk expression became constitutively elevated. This finding is in concordance with previous results showing that transformed cells exhibit more relaxed control than normal cells in their initiation of DNA replication.

Insulin enhances glucocorticoid receptor-mediated induction of gene expression independent of a specific insulin response element.

We have established a system in which we observe a synergistic interaction between insulin and glucocorticoids. This includes chimeric genes constructed to contain synthetic glucocorticoid-responsive elements, 5' of the HSV thymidine kinase promoter and the chloramphenicol acetyltransferase reporter gene. The magnitude of induction of gene expression by glucocorticoid was dependent on the number of GREs. Insulin alone had virtually no effect on the expression of any of these genes but together with dexamethasone acted in a synergistic manner. This synergy diminished as the number of GREs in the promoter increased. The synergy is independent of promoter sequences other than the GREs and a functional TATAA box. Three different approaches demonstrate that the effect of insulin is not directly on the glucocorticoid signal transduction pathway. Insulin does not change the dose-response relationship for dexamethasone. The effect of insulin is independent of the intracellular concentration of glucocorticoid receptor. The effect is independent of any specific domain of the glucocorticoid receptor. The target of insulin action is likely to be part of the normal host cell transcriptional initiation complex or a putative adaptor molecule.

Escherichia coli thymidylate kinase: molecular cloning, nucleotide sequence, and genetic organization of the corresponding tmk locus.

Thymidylate kinase (dTMP kinase; EC 2.7.4.9) catalyzes the phosphorylation of dTMP to form dTDP in both de novo and salvage pathways of dTTP synthesis. The nucleotide sequence of the tmk gene encoding this essential Escherichia coli enzyme is the last one among all the E. coli nucleoside and nucleotide kinase genes which has not yet been reported. By subcloning the 24.0-min region where the tmk gene has been previously mapped from the lambda phage 236 (E9G1) of the Kohara E. coli genomic library (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987), we precisely located tmk between acpP and holB genes. Here we report the nucleotide sequence of tmk, including the end portion of an upstream open reading frame (ORF 340) of unknown function that may be cotranscribed with the pabC gene. The tmk gene was located clockwise of and just upstream of the holB gene. Our sequencing data allowed the filling in of the unsequenced gap between the acpP and holB genes within the 24-min region of the E. coli chromosome. Identification of this region as the E. coli tmk gene was confirmed by functional complementation of a yeast dTMP kinase temperature-sensitive mutant and by in vitro enzyme assay of the thymidylate kinase activity in cell extracts of E. coli by use of tmk-overproducing plasmids. The deduced amino acid sequence of the E. coli tmk gene showed significant similarity to the sequences of the thymidylate kinases of vertebrates, yeasts, and viruses as well as two uncharacterized proteins of bacteria belonging to Bacillus and Haemophilus species.

Human T-cell leukemia virus type 1 Tax oncoprotein induces and interacts with a multi-PDZ domain protein, MAGI-3.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL), whereas the closely related virus HTLV-2 has not been associated with such malignant conditions. HTLV-1 Tax1 oncoprotein transforms a rat fibroblast cell line (Rat-1) much more efficiently than does HTLV-2 Tax2. By using a differential display analysis, we isolated MAGI-3 as a Tax1-inducible gene in Rat-1 cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that Tax1 induced MAGI-3 in Rat-1 cells. MAGI-3 has multiple PDZ domains and interacted with Tax1 but not Tax2 in 293T cells. The interaction of Tax1 with MAGI-3 was dependent on a PDZ domain-binding motif, which is missing in Tax2. The interaction of Tax1 with MAGI-3 altered their respective subcellular localization, and moreover, the interaction correlated well with the high transforming activities of Tax1 in Rat-1 cells relative to Tax2. MAGI-3 mRNA and the allied MAGI-1, but not MAGI-2, were expressed in HTLV-1-infected T-cell lines. Our results suggest that the interaction of Tax1 and MAGI-3 alters their respective biological activities, which may play a role in transformation by Tax1 as well as in the pathogenesis of HTLV-1-associated diseases.