This study showed that nitric oxide (NO)-generating S-nitrosothiols, S-nitroso-Nacetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO), increased proliferation of BALB/c 3T3 (clone A31-1-1) fibroblasts in vitro. Treatment with SNAP at a relatively low concentration (0.005-0.02 raM) induced an increase in cell number compared to control. SNAP (0.005-0.02 raM) and GSNO (0.025-0.05 raM) both showed an increase of [3H]thymidine incorporation in exponentially growing cells in a dose-dependent manner. The maximal effect was observed at about 40 and 90% above control at 0.02 mM and 0.05 raM, respectively. The increase induced by 0.02 mM SNAP was abolished by the addition of 0.01mM oxyhemoglobin, a scavenger of NO.[3H]Thymidine incorporation in stationary cells was also increased by SNAP. In addition, 0.02raM SNAP produced a 1.8-3.2-fold increase of thymidine kinase activity in exponentially growing cells.
In the previous paper, we described the identification of two abundant mRNAs of Sarcophaga peregrina (flesh-fly) which are selectively expressed in the fat body of middle third instar larvae. One of these mRNAs was found to encode a protein with a molecular mass of about 25,000 (25-kDa protein) when translated in vitro (Tamura, H., et al. (1983) Dev. Biol. 99, 145-151). Present paper reports the nucleotide sequence of a 2.3 kb DNA containing the entire gene for the 25-kDa protein. This gene consisted of four exons and contained an open reading frame for 184 amino acids. A CAT box and a TATA box were found in the 5'-flanking sequence. A poly A addition signal of AATAAA was assigned to the non-coding region in the fourth exon. A sequence having 75% homology with SV40 enhancer core sequence was identified in the non-coding region of the first exon.
When a cachexigenic subclone (clone 20) of murine colon 26 adenocarcinoma was transplanted into female BALB/c mice, hepatic NNMT activity continued to increase until death in proportion to progressive carcass weight loss, a marker of cancer cachexia. On the other hand, noncachexigenic subclone (clone 5)-transplanted mice showed neither increase of NNMT activity nor carcass weight loss. Among cytostatic fluorinated pyrimidines, 5′ ′ ′ ′-dFUrd could inhibit the increase of NNMT activity and prevent weight loss in mice bearing clone 20. On the other hand, 2′ ′ ′ ′-dFUrd did not show these effects. 5-FUra and Tegafur inhibited the increase of NNMT activity at higher concentrations. These findings suggest that the levels of hepatic NNMT activity are closely associated with the degree of weight loss, and they appear to be a useful marker of cancer cachexia. Key words:Nicotinamide N-methyltransferase -Cancer cachexia -Liver -Colon 26 adenocarcinoma Ado-Met:NNMT (EC 2.1.1.1) catalyzes the N-methylation of nicotinamide and other pyridines.1) Ado-Met functions as a methyl donor for this reaction. NNMT is predominantly localized in the mammalian liver. 2-4)Recently, the NNMT gene was cloned and characterized. 5)Cancer cachexia is a complex syndrome characterized by body weight loss, anorexia, depletion of muscle and fat tissue, anemia and some altered blood metabolic parameters (e.g., hypoglycemia), etc. 6) Cancer cachexia is responsible for both decreased response to therapy and shortened survival. 7)Our previous study showed that hepatic NNMT activity increased after inoculation of various tumors into mice. 8)However, the mechanisms by which NNMT activity increases are not yet understood.Colon 26 adenocarcinoma is a chemically induced, murine colon-adenocarcinoma cell line.9) This tumor has been shown to induce marked cachexia (estimated in terms of carcass weight loss) in mice when the tumor mass is still relatively small, a situation similar to that found clinically. 10,11) There are two subclones of colon 26 adenocarcinoma; one is clone 20 with a remarkable cachexia-inducing potency, and the other is clone 5 without such potency. 12)In order to determine the relationship between the level of hepatic NNMT activity and the degree of cancer cachexia, we examined these parameters in a murine model using the aforementioned two clones. In the mice bearing the original colon 26 adenocarcinoma, cachexia was prevented by 5′-dFUrd in spite of the ineffectiveness of many other cytostatics.13) Thus, we examined whether levels of NNMT activity were correlated with the prevention of cachexia by 5′-dFUrd in this model. The present study showed that there was a direct correlation between levels of hepatic NNMT activity and the degree of cancer cachexia. MATERIALS AND METHODS Chemicals[ 3 H-methyl]Ado-Met (80 Ci/mmol) was purchased from Amersham International plc, Buckinghamshire, UK. Nicotinamide and 1-methylnicotinamide chloride were from Sigma, St. Louis, MO. 5′-dFUrd was donated by Nippon Roche K. K., Tokyo. 2′-dFUrd and 5-FUra we...
DNA kinase activity of rat liver nuclei was detected in situ after electrophoresis in sodium dodecyl sulfate/ polyacrylamide gel containing 5'-hydroxyl nicked DNA as DNA substrate. After renaturation of polypeptides, the gel was incubated with [Y-~~PIATP and Mg2+. An active polypeptide corresponding to MI 61 000 was observed as a radioactive band by autoradiography. The intensity of the band was proportional to the amount of the enzyme applied. The active band common to various tissues of rat was observed with the nuclear extracts, indicating that DNA kinase for rat tissues is composed of a single polypeptide of MI 61000. In contrast, T4 polynucleotide kinase (MI = 140000) showed an active polypeptide band corresponding to the subunit of M , 33 000. DNA kinase that phosphorylates 5'-hydroxyl ends of DNA has been isolated from T2-and T4-infected Escherichia coli cells [l], rat liver [2, 31 and calf thymus [4, 51. Although DNA kinase is presumed to be involved in DNA repair, DNA replication or both in co-operation with DNA ligase that joins juxtaposed 3'-hydroxyl and 5'-phosphoryl ends at nicks in duplex DNA, the physiological function remains unknown [l, 61. In comparison with the phage-induced polynucleotide kinases, mammalian DNA kinases show markedly different properties, such as restricted substrate specificity (inactive on RNA substrate), lower K,,, for ATP (2-4 pM) and acidic pH optimum (PH 5.5) [2-61. Mammalian DNA kinase is preferable for 5'-end labeling of DNA in the presence of RNA at a lower concentration of [ Y -~~P I A T P (unpublished data). We have previously purified DNA kinase from calf thymus, which is composed of a single polypeptide of MI 54000 [5], and DNA kinase partially purified from rat liver nuclei shows an apparent MI of 80000 by gel filtration [2, 31. Recently the corresponding enzyme from rat testis is reported to have a single 38-kDa polypeptide [7]. To solve the discrepancy in molecular properties of mammalian DNA kinase, we tried to adopt a new method, the 'activity gel' method (in situ assay of enzyme after SDS-PAGE) [8] for the detection of the active polypeptide of mammalian DNA kinase in crude and impure enzyme preparations. This new method to detect mammalian
Protein kinase activity in isolated nuclei from rat liver was detected in situ after electrophoresis on SDSpolyac~~a~de gel untying no exogenous protein substrate. After rena~ation of ~l~tides, the gel was incubated with [~-~aplATp and divalent cations. Among five major protein kinase activities observed as radioactive bands by autoradio~aphy, a protein k&se autophospho~lating on tyrosine (A& 30 O&l) was identified and found to be localized in the nucleus, particularly in the nuclear matrix. The intensity of the activity band representing the level of the protein-tyrosine kinase in rat liver nuclei did not appreciably change during 3-24 h after partial hepatectomy.Protein-tyrosine kinase Autophosphorylation Nuclear matrix (Rat liver)
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