The protein kinases tightly bound to DNA are present in normal tissues and in regenerating liver, but strongly decreased in hepatomas

We have previously described in rat liver two protein kinases tightly bound to DNA, one is serine-specific, the other arginine-specific. In this work we show that both enzymes are present in various rat tissues and in liver from various species. Both kinase specific activities are strongly decreased in methyl-DBA-induced hepatomas and in HTC cells but not in regenerating liver after hepatectomy. This decrease is then not related to cell proliferation.     

Increased thymidylate synthase (EC 2.1.1.45) activity in normal and neoplastic proliferation

Thymidylate (dTMP) synthase (EC 2.1.1.45) activity was measured in 100,000 x g supernatant fluid with a sensitive, rapid radio assay. The activity in normal rat liver was low (0.098-0.204 nmol/hr/mg protein). dTMP synthase specific activities in rat thymus, spleen, bone marrow, testis, lung, heart, brain, kidney, and small intestine were 6297, 1842, 1500, 788, 215, 76, 61, 39 and 24%, respectively, of that of the liver. The activity in 5-day-old rat liver was 16-fold higher than in adult. dTMP synthase activity increased in rat hepatomas to 7- to 125-fold of that of normal rat liver. There was a significant correlation between the increase in synthase activity and the proliferation rates of the hepatomas. In 8 human colon carcinomas, dTMP synthase activity increased to 2.9- to 8-fold of that of normal human colon mucosa. In leukemic leukocytes from 3 leukemia patients, activity was 8- to 10-fold higher than in normal leukocytes.     

Increase in liver and kidney deoxycytidine kinase activity linked to neoplastic transformation

Deoxycytidine kinase activity in normal rat liver cytosol was low (0.8 nmol/hr/mg protein); it increased 2–26-fold in 12 lines of chemically-induced, transplantable rat hepatomas of different growth rates. The increased kinase activity correlated positively with the hepatoma growth rate. The kinase activity did not change in the regenerating liver and the activity in the differentiating, neonatal rat liver was similar to values in adult liver. Deoxycytidine kinase activity in 2 chemically-induced, transplantable rat kidney tumors was increased to twice the value found in normal renal cortex. Among 15 normal rat tissues examined the highest kinase activities were observed in thymus, bone marrow and spleen. Of the normal and malignant rat tissues tested, only testis had detectable cytidine deaminase activity.     

Choline kinase and ethanolamine kinase are separate, soluble enzymes in rat liver.

Choline kinase and ethanolamine kinase are located in the cytosol from rat liver and have been copurified more than 500-fold by affinity chromatography [P. J. Brophy and D. E. Vance (1976) FEBS Lett. 62, 123-125]. Kinetic properties of the two activities were determined. Choline kinase had a Km for choline of 0.033 mM and ethanolamine was a competitive inhibitor (Ki = 6.2 mM). Ethanolamine kinase had a Km for ethanolamine of 7.7 mM and choline was a 'mixed' type of inhibitor with a Ki of 0.037 mM. Both enzymes activities responded in a similar fashion to the adenylate energy charge. Betaine and choline phosphate partially inhibited both kinases with a 93% inhibition of the ethanolamine kinase by 5 mM choline phosphate. CTP and ethanolaminephosphate partially inhibited the ethanolamine kinase, but not the choline kinase. Other metabolites tested had negliglible effects on both kinases. The affinity-column-purified enzyme was analyzed by disc gel electrophoresis which resolved the two activities. Hence, although many of the properties of the two activities are similar, choline kinase and ethanolamine kinase must be separate enzymes. Analysis of rat liver cytosol by disc gel electrophoresis indicated four isoenzymes for choline kinase and ethanolamine kinase.     

Purine nucleoside kinases in human T- and B-lymphoblasts

Purine nucleoside kinases in human B- and T-lymphoblasts were fractionated by DEAE-cellulose chromatography. Human B-lymphoblast cell extracts showed three peaks of nucleoside kinase activities, adenosine kinase (EC 2.7.1.20), deoxyguanosine kinase and deoxycytidine kinase (EC 2.7.1.74). However, T-lymphoblast cell extracts showed a nucleoside kinase activity which phosphorylates deoxycytidine, deoxyadenosine and deoxyguanosine, similar to deoxycytidine kinase, in addition to the three nucleoside kinases. The Km values of T-lymphoblast-specific nucleoside kinase for deoxyadenosine and deoxyguanosine, 15 and 26 microM, respectively, were smaller than those of deoxycytidine kinase, 150 and 330 microM, respectively. Deoxyadenosine phosphorylation by deoxycytidine kinase was strongly inhibited by dCTP, but the phosphorylation by T-lymphoblast-specific nucleoside kinase was only weakly inhibited by dCTP. Deoxyadenosine phosphorylating activity in B-lymphoblast extracts was more distinctly inhibited by dCTP than that in T-lymphoblast extracts.     

Tyrosine protein kinase in preneoplastic and neoplastic rat liver

When rats are subjected to chemical hepatocarcinogenesis according to the protocol of D. Solt and E. Farber ((1976) Nature (London) 263, 701-703), the liver exhibits elevated levels of tyrosine protein kinase activity as early as 3 weeks after the injection of diethylnitrosoamine. A more striking elevation in tyrosine protein kinase activity is noted in rat hepatomas induced by administration of chemical carcinogens, in particular that of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). Tyrosine protein kinase solubilized from the particulate fraction of 3'-Me-DAB-induced hepatoma has a molecular weight identical to that of p60v-src, cross-reacts with p60v-src immunologically, phosphorylates the heavy chain of anti-p60v-src IgG, and probably belongs to a family of p60c-src. The tyrosine protein kinase from the particulate fraction of normal rat liver is indistinguishable from the hepatoma kinase in these properties; thus it apparently differs only in the level of activity. Whether the liver and hepatoma kinases differ merely quantitatively or whether they differ even qualitatively, however, remains to be elucidated.     

Sequential activation of MAP kinase activator, MAP kinases, and S6 peptide kinase in intact rat liver following insulin injection.

An insulin-stimulated phosphorylation cascade was examined in rat liver after insulin injection via a portal vein by the use of immune complex kinase assays specific to the mitogen-activated protein (MAP) kinase and S6 kinase II homologue (rsk) kinase. We have prepared an antibody against the peptide consisting of a carboxyl-terminal portion of the extracellular signal-regulated kinase 1 (alpha C92), one of the MAP kinases, and an antibody against the peptide consisting of the carboxyl terminus of the mouse S6 kinase II homologue (alpha rsk(m)C). In alpha C92 immune complex assay, maximal activation of rat liver MAP kinases (approximately 4.3-fold) were observed 4.5 min after insulin injection. We also observed an insulin-stimulated MAP kinase activity (approximately 3-fold) in liver extracts from insulin-treated rat in fractions eluted from phenyl-Sepharose with 30-50% ethylene glycol. Kinase assay in myelin basic protein (MBP)-containing gel after sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by denaturation with 6 M guanidine HCl, and renaturation revealed that insulin injection stimulated the kinase activity of the 42- and 44-kDa proteins, which corresponded to the two distinct MAP kinases. In alpha rsk(m)C immune complex assay, maximal stimulation (approximately 5-fold) of the S6 peptide (Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala) kinase activity was observed 7.5 min after insulin injection. In addition, MAP kinases purified from insulin-treated rat liver were able to activate S6 peptide kinase activity in vitro in alpha rsk(m)C immunoprecipitates from untreated rat liver, accompanied by the appearance of several phosphorylated bands including a major band at 88 kDa. We also examined whether insulin injection stimulates the MAP kinase activator (Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K., and Krebs, E. G. (1991) J. Biol. Chem. 266, 4220-4227) in rat liver. Using recombinant Xenopus MAP kinase, fractions of Q-Sepharose eluted early in the NaCl gradient were found to have MAP kinase activator activity accompanied by the phosphorylation of 42-kDa recombinant Xenopus MAP kinase. From these data, we demonstrate three tiers of a cascade composed of the MAP kinase activator, MAP kinases, and an S6 peptide kinase activity in rat liver under physiological conditions in the intact animal.     

Elucidation of aberrant purine metabolism: application to hypoxanthine-guanine phosphoribosylstransferase- and adenosine kinase-deficient mutants, and IMP dehydrogenase- and adenosine deaminase-inhibited human lymphoblasts

We propose that the ratio of [14C]formate-labelled purine nucleosides and bases (both intra and extracellular) to nucleic acid purines provides, in exponentially growing cultures, a sensitive index for comparative studies of purine metabolism. This ratio was 4-fold greater for an HGPRT- mutant than for the parental HGPRT+ human lymphoblast line. The major components of the labelled nucleoside and base fraction were hypoxanthine and inosine. By blocking adenosine deaminase activity with coformycin we found that approx. 90% of inosine was formed directly from IMP rather than the route IMP leads to AMP leads to adenosine leads to inosine. The ratio of labelled base + nucleosides to nucleic acids was essentially unchagned for an AK- lymphoblast line and 2-fold greater than control for an HGPRT(-)-KAK- line, demonstrating that a deficiency of adenosine kinase alone has little effect on the accumulation of purine nucleosides and bases. Although adenosine was a minor component of the nucleoside and base fraction, the adenosine fraction increased from 3 to 13% with the addition of coformycin to the HGPRT(-)-AK- line. In the parental and HGPRT- lines, adenosine was shown to be primarily phosphorylated rather than deaminated at concentrations less than 5 microM. Inhibition of IMP dehydrogenase activity by mycophenolic acid caused a 12- and 3-fold increase in the rate of production of labelled base and nucleoside in the parent and HGPRT- cells respectively. These results suggest that a mutationally induced partial deficiency in the activities converting IMP to guanine nucleotides may result in an increased catabolism of IMP.     

Phospholipid transfer activities in Morris hepatomas and the specific contribution of the phosphatidylcholine exchange protein

Phospholipid transfer activities for phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine were measured in three hepatomas of increasing growth rate and degree of dedifferentiation, the hepatomas of 9633 and 7777, and compared to the activities found in normal and host liver. A 2-3-fold increase was found in the phosphatidylcholine and phosphatidylinositol transfer activities in the fast-growing 7777 hepatoma, while these activities were moderately or not increased in the 7787 and 9633 hepatomas. Phosphatidylethanolamine transfer was found to be extremely low in all three hepatomas. The possible significance of these findings with respect to the altered phospholipid content and composition of the hepatoma membranes is discussed. The contribution of the phosphatidylcholine specific exchange protein to the total phosphatidylcholine transfer activity was determined in normal and host liver and in the hepatomas 7777 and 9633 with the aid o f a phosphatidylcholine exchange protein specific antiserum. To this end a new procedure for the purification of the phosphatidylcholine exchange protein from rat liver was developed which leads to a final purification factor of 5300 and a high overall yield of 17%. In addition, this protein was chemically and immunologically characterized and its properties were compared to those of the bovine phosphatidylcholine exchange protein purified in our laboratory previously.     

Gel-based proteomics of liver cancer progression in rat

A significant challenge in proteomics biomarker research is to identify the changes that are of highest diagnostic interest, among the many unspecific aberrations associated with disease burden and inflammation. In the present study liver tissue specimens (n=18) from six experimental stages were collected from the resistant hepatocyte (RH) rat model of liver cancer and analyzed by 2D DIGE. The study included triplicates of regenerating liver, control "sham-operated" liver, three distinct premalignant stages and hepatomas. Out of 81 identified proteins two-thirds were differentially abundant in rat hepatomas compared to control rat liver and, secondly, the majority of proteins were also changed in precursor stages. This underscores the importance of adequate control samples in explorative cancer biomarker research. We confirm several proteomic changes previously identified in human hepatocellular carcinoma (HCC) and we identify novel candidate proteomic aberrations for further analysis in human HCC. In particular, increased levels of HSP70, HSP90, AKR1B1, AKR7A3, GCLM, ANXA5, VDBP, RGN and SULT1E1 were associated specifically with rat hepatomas, or with liver cancer progression in rat. In addition, we examine an integrated gel-based workflow for analysis of protein post-translational modifications (PTMs) and microtubule-association. We highlight differential PTM and localization of HSP60 as an interesting target for further analysis in liver cancer.     

Inhibition of adenosine and thymidylate kinases by bisubstrate analogs

Potential bisubstrate analogs, in which the 5'-hydroxyl group of adenosine was joined to the phosphoryl group acceptor by polyphosphoryl bridges of varying length (ApnX, where n is the number of phosphoryl groups and X is the nucleoside moiety of the acceptor), were tested as inhibitors of human liver adenosine kinase and of thymidylate kinase from peripheral blast cells of patients with acute myelocytic leukemia. Adenosine kinase was most strongly inhibited by P1,P4-(diadenosine 5')-tetraphosphate (Kd = 30 nM) and P1,P5-(diadenosine 5')-pentaphosphate (Kd = 73 nM). Thymidylate kinase was most strongly inhibited by P1-(adenosine 5')-P5-(thymidine 5')-pentaphosphate (Kd = 120 nM) and by P1(adenosine 5')-P6-(thymidine 5')-hexaphosphate (Kd = 43 nM). In these enzymes, as in adenylate and thymidylate kinases, strongest inhibition was achieved in compounds containing one or two more phosphoryl groups than the substrates combined. These results support the view that nucleoside and nucleotide kinases mediate direct transfer of phosphoryl groups from ATP to acceptors, rather than acting by a double displacement mechanism.     

Nucleoside mono- and nucleoside diphosphate kinase activities in rat liver and brain in radiation sickness

Activity of nucleoside di- and nucleoside triphosphates metabolism enzymes in tissues of rats gamma-irradiated by a dose of 30 Gy was studied 0.5, 1, 3, 6 and 24 hours after the radiation effect. It is shown that the nucleoside monophosphate kinase activity of the liver and brain is enhanced almost at all stages of the studies and the nucleoside diphosphate kinase activity is inhibited. A significant but reversible decrease of the nucleoside monophosphate kinase activity is observed in the liver 3 h later. By an end of the first day after irradiation the nucleoside mono- and nucleoside diphosphate kinase activities increase significantly both in the liver and brain.     

Cancer initiating cells or cancer stem cells in the gastrointestinal tract and liver

It has been suggested that cancer stem cells population within the solid tumor with indefinite proliferation potential drives the growth and metastasis of cancer. In literature, these malignant stem cells also named Cancer initiating cells. Cancer stem cells exhibit low rate of division and proliferation in their niche that help them to avoid chemotherapy and radiation. Epithelial cancers are believed to originate from transformation of tissue stem cells. Bone marrow-derived cells, which are frequently recruited to sites of tissue injury and inflammation, might also represent a potential source of malignancy in the gastrointestinal tract. Pancreatic cancer is one of most common cause of cancer-related death. Pancreatic cancer stem cells have been characterized recently through serial transplantation of human pancreatic cancer cells. The phenotype of Pancreatic cancer stem cells has been defined as CD24(+)CD44(+)CD326 (ESA)(+). CD133 antigen has been also suggested as a potential marker for cancer stem cell in gastrointestinal tract but recently there is also debate in this regard. More recently, other cancer stem cells in gastrointestinal tract, such as colon cancer stem cells, liver cancer stem cells, have been also characterized in their phenotype. These advances clearly will bring the new strategy in cancer treatment and control in the gastrointestinal tract. In this review, the author will discuss the current status and progress about cancer stem cell research in gastrointestinal tract and liver.     

Cloning and characterization of the multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster.

A Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) was reported to phosphorylate all four natural deoxyribonucleosides as well as several nucleoside analogs (Munch-Petersen, B., Piskur, J., and Sondergaard, L. (1998) J. Biol. Chem. 273, 3926-3931). The broad substrate specificity of this enzyme together with a high catalytic rate makes it unique among the nucleoside kinases. We have in the present study cloned the Dm-dNK cDNA, expressed the 29-kDa protein in Escherichia coli, and characterized the recombinant enzyme for the phosphorylation of nucleosides and clinically important nucleoside analogs. The recombinant enzyme preferentially phosphorylated the pyrimidine nucleosides dThd, dCyd, and dUrd, but phosphorylation of the purine nucleosides dAdo and dGuo was also efficiently catalyzed. Dm-dNK is closely related to human and herpes simplex virus deoxyribonucleoside kinases. The highest level of sequence similarity was noted with human mitochondrial thymidine kinase 2, and these enzymes also share many substrates. The cDNA cloning and characterization of Dm-dNK will be the basis for studies on the use of this multisubstrate nucleoside kinase as a suicide gene in combined gene/chemotherapy of cancer.     

Membrane-associated nucleoside diphosphate kinase from rat liver. Purification, characterization, and comparison with cytosolic enzyme

Previous studies from this laboratory have proposed that membrane-associated nucleoside diphosphate kinase (m-NDP kinase) may play a role in regulation of adenylate cyclase by channeling GTP, an essential cofactor of adenylate cyclase regulation, into GTP-binding protein (Gs) in a hormone-dependent manner. To understand the true role of m-NDP kinase, in the present study, the m-NDP kinase was solubilized and purified to apparent homogeneity from rat liver purified plasma membranes and characterized in comparison with the cytosolic enzyme purified from the same tissue (s-NDP kinase). Some physical properties determined were: molecular weight (monomer), 18,300; sedimentation coefficient (s20,w), 6.2 S; isoelectric point (pI), 6.0. These values and kinetic parameters of the m-NDP kinase were almost identical to those of the s-NDP kinase whose characteristics were more extensively studied. A peptide mapping study of the 125I-labeled m- and s-NDP kinases gave essentially identical patterns. Polyclonal antibodies against the s-NDP kinase, which also cross-reacted with the m-NDP kinase, were prepared. Immunoblotting studies with the affinity-purified antibodies revealed that the monomer molecular weight of the purified m- and s-NDP kinases was identical to the values of unpurified enzymes present in membranes and crude extract. These results demonstrate that the purified m-NDP kinase underwent no remarkable modification during solubilization and purification, and that the m- and s-NDP kinases are quite similar in protein structure, if at all different. The physiological relevance of the m-NDP kinase in relation to the adenylate cyclase system is discussed.     

The Human Nm23/Nucleoside Diphosphate Kinases

Biochemical experiments over the past 40 years have shown that nucleoside diphosphate(NDP) kinase activity, which catalyzes phosphoryl transfer from a nucleoside triphosphate toa nucleoside diphosphate, is ubiquitously found in organisms from bacteria to human. Overthe past 10 years, eight human genes of the nm23/NDP kinase family have been discoveredthat can be separated into two groups based on analysis of their sequences. In addition tocatalysis, which may not be exhibited by all isoforms, evidence for regulatory roles has comerecently from the discovery of the genes nm23 and awd, which encode NDP kinases and areinvolved in tumor metastasis and Drosophila development, respectively. Current work showsthat the human NDP kinase genes are differentially expressed in tissues and that their productsare targeted to different subcellular locations. This suggests that Nm23/NDP kinases possessdifferent, but specific, functions within the cell, depending on their localization. The roles ofNDP kinases in metabolic pathways and nucleic acid synthesis are discussed.     

Bystander effects of nucleoside analogs phosphorylated in the cytosol or mitochondria

The efficiency of nucleoside kinase suicide gene therapy for cancer is highly dependent on "bystander" cell killing, i.e., the transfer of cytotoxic phosphorylated nucleoside analogs to cells adjacent to those expressing the suicide enzyme. We have recently studied the possible use of mitochondrial nucleoside kinases as suicide genes. In the present study, we investigated if nucleoside analogs phosphorylated in the mitochondrial matrix cause bystander killing. We used deoxycytidine kinase-deficient Chinese hamster ovary cells reconstituted with deoxycytidine kinase targeted to either the cytosol or mitochondria matrix and determined the bystander cell killing when these cells were incubated with the nucleoside analogs 1-beta-D-arabinofuranosylcytosine and 2',2'-difluorodeoxycytidine. A bystander effect occurred when nucleoside analogs were phosphorylated in the cytosol, but not when these compounds were phosphorylated in the mitochondria. These findings suggest that nucleoside kinases targeted to the mitochondrial matrix have limited use in suicide gene therapy when efficient bystander cell killing is required.     

Thymidine and thymidylate kinases from the scallop Mizuhopecten yessoensis gonads

Thymidine and thymidylate kinases were isolated from the gonads of scallop Mizuhopecten yessoensis. The enzymes were purified 537- and 100-fold, respectively, and were free of phosphatase and ATPase impurities. Ions of bivalent metals and ATP were necessary for both the nucleoside and nucleotide kinase activities; the pH optimum fall into the range of 7.5-8.5. KCl and NaCl at a concentration of up to 100 mM had no inhibiting effect on the activities of these scallop enzymes. Thymidine kinase catalyzed thymidine, and, at a lower rate, deoxycytidine phosphorylations did not utilize ribo- and deoxyribonucleosides, as well as pyrimidine ribonucleosides, as a phosphate acceptor. Thymidylate kinase phosphorylated TMP and dCMP with an efficiency of about 30%. In addition to ATP, these enzymes can also utilize with different efficiencies dATP, dGTP, GTP, UTP, and CTP as a donor of phosphate groups. Thymidine kinase activity was inhibited by TMP, TTP, and dCTP.     

Photoaffinity labelling of nucleoside-transport proteins in plasma membranes isolated from rat and guinea-pig liver.

Nitrobenzylthioinosine (NBMPR) was employed as a probe of the nucleoside transporters from rat and guinea-pig liver. Purified liver plasma membranes prepared on self-generating Percoll density gradients exhibited 16-fold (rat) and 10-fold (guinea pig) higher [3H]NBMPR-binding activities than in crude liver homogenates (3.69 and 14.7 pmol/mg of protein for rat and guinea-pig liver membranes respectively, and 0.23 and 1.47 pmol/mg of protein for crude liver homogenates respectively). Binding to membranes from both species was saturable (apparent Kd 0.14 and 0.63 nM for rat and guinea-pig membranes respectively) and inhibited by uridine, adenosine, nitrobenzylthioguanosine (NBTGR) and dilazep. Uridine was an apparent competitive inhibitor of high-affinity NBMPR binding to rat membranes (apparent Ki 1.5 mM). There was a marked species difference with respect to dipyridamole inhibition of NBMPR binding (50% inhibition at 0.2 and greater than 100 microM for guinea-pig and rat respectively). These results are consistent with a role of NBMPR-binding proteins in liver nucleoside transport. Exposure of rat and guinea pig membranes to high-intensity u.v. light in the presence of [3H]NBMPR resulted in the selective radio-labelling of membrane proteins which migrated on sodium dodecyl sulphate/polyacrylamide gels with apparent Mr values in the same range as that of the human erythrocyte nucleoside transporter (45 000-66 000). Covalent labelling of these proteins was abolished when photolysis was performed in the presence of non-radio-active NBTGR as competing ligand.     

Novel role of 3-phosphoglycerate kinase,a glycolytic enzyme,in the activation of L-nucleoside analogs,a new class of anticancer and antiviral agents

l-Nucleoside analogs are a new class of clinically active antiviral and anticancer agents. The phosphorylation of these analogs from diphosphate to triphosphate metabolites is crucial for their biological action. We studied the role of 3-phosphoglycerate kinase, a glycolytic enzyme, in the metabolism of l-nucleoside analogs, using small interfering RNAs to down-regulate the amount of this enzyme in HelaS3 and 2.2.15 cells, chosen as models for studying the impact of the enzyme on the anticancer and antihepatitis B virus activities of these analogs. Decrease in the expression of 3-phosphoglycerate kinase led to a corresponding decrease in the formation of the triphosphate metabolites of l-nucleoside analogs (but not d-nucleoside analogs), resulting in detrimental effects on their activity. The enzyme is important for generating as well as maintaining the steady state levels of l-nucleotides in the cells, thereby playing a key role in the activity of l-nucleoside analogs against human immunodeficiency virus, hepatitis B virus, and cancer. This study also indicates a structure-based distinction in the metabolism of l- and d-nucleoside analogs, disputing the classic notion that nucleoside diphosphate kinases are responsible for the phosphorylation of all classes of nucleoside analog diphosphates.