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Purpose: The present study was undertaken to look for an agent or agents able to modulate the cytotoxic effect of 5-fluorouracil (FUra) and to investigate the role of serine-threonine phosphatase inhibitors on the cytotoxic effect of FUra. Methods: The cytotoxicities of FUra and protein phosphatase inhibitors (PPIs) were evaluated by two different methods: a clonogenic assay and a proliferation assay. In the clonogenic assay, cancer cells were treated with various concentration of FUra with or without PPIs for 72 h. The drug-containing medium was replaced by fresh medium, the cultures incubated for an additional 10 days, and the colonies enumerated. In the proliferation assay the cells were treated with FUra alone or in combination with PPIs for 96 h and cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay based on the uptake of the tetrazolium dye. Thymidine kinase (TK) activity was determined based on the catalytic phosphorylation of [3H]d-thymidine to [3H]dTMP. Incorporation of FUra into DNA and RNA was determined by treating the cells with [2-14C]fluorouracil for 72 h and measuring the radioactivity in the isolated DNA and RNA fractions. Results: The serine-threonine phosphatase inhibitors caliculin A (CAL), okadaic acid (OA) and microcystin-LR (MCLR) dose-dependently inhibited the growth of Clone 20 and Clone 5 cells of Colon 26 murine colon adenocarcinoma cells, human cervical cancer HeLa cells, human gastric cancer MKN 7 cells, and murine sarcoma S-180 cells in vitro. Among the compounds tested, MCLR at non-toxic concentrations was found to increase FUra incorporation into RNA and DNA in Clone 20 cells by 60% and 127%, respectively, to increase TK activity alone (twofold) as well as in combination with FUra (threefold), and to potentiate the cytotoxicity of FUra synergistically and cytospecifically in vitro. The cytotoxicity of FUra alone or in combination with MCLR, but not that of PPIs alone, was abrogated almost completely by exogenous thymidine (dThd), suggesting that inhibition of thymidylate synthetase (TS) is the growth-limiting event in the cytotoxic action of FUra even in combination with MCLR. Conclusions: The findings presented here suggest that MCLR synergistically and cytospecifically potentiates the antitumor activity of FUra with substantial improvement in the therapeutic index of FUra via enhancement of both DNA- and RNA-directed cytotoxicity.

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.

In order to investigate the pathogenesis of ossification of the posterior longitudinal ligament (OPLL) of the spine, we examined the distribution of insulin-like growth factor I (IGF-I) in the posterior longitudinal ligaments of OPLL patients, and analyzed the effects of IGF-I on the cultured spinal ligament cells. For that purpose we established eight varieties of OPLL and non-OPLL cell lines obtained from spinal ligaments of corresponding patients, respectively. In contrast to non-OPLL cases, all the OPLL cases were histologically shown to contain round-shaped cartilage-like cells in the transitional region from preossifying to ossifying ligaments, and these cells were strongly stained with an antibody for IGF-I. In the vicinity of preossifying cartilaginous tissues, ligament cells also had a rod-like appearance and were positive for IGF-I immunohistochemically. The effects of IGF-I on cultured spinal ligament cells were assayed by alkaline phosphatase (AP) activity, DNA synthesis, and the amounts of collagen produced. The number of OPLL cell lines that increased AP activity, responding to IGF-I irrespective of 1,25(OH)2D3, was significantly larger than that of non-OPLL cell lines, although IGF-I stimulated DNA and procollagen type I carboxyl-terminal peptide synthesis in most of both OPLL and non-OPLL cell lines. These data demonstrate the dominant expression of IGF-I in the posterior longitudinal ligaments of OPLL patients, and suggest that IGF-I preferentially induces osteogenic differentiation in OPLL cells rather than in non-OPLL cells. IGF-I, therefore, may be involved in the local ossification process of spinal ligaments observed in OPLL patients.

ゼオライトをアルミナ基材上に直接コーティングする手法を考案し, 膜状ゼオライト担持Ru触媒を作製した。MFIゼオライトを担体としたルテニウム系触媒について, 粉末, 円柱状に成形した触媒, アルミナボール上に製膜した膜状触媒を用いて二酸化炭素のメタン化反応を行った。それぞれの触媒について比表面積, 金属比表面積などの物性値を測定し, 二酸化炭素のメタン化反応に対する活性に与える影響を調べた。反応実験において, いずれの触媒も測定した金属比表面積が増大するにつれ転化率が増加した。コーティング操作の前に減圧下で基材にゼオライト原料溶液を含浸させることで, 緻密なゼオライト膜が合成できることを見いだした。前処理で減圧含浸操作をおこなったゼオライト膜をイオン交換した触媒は, 前処理なしの場合に比べ金属比表面積が大きく, 触媒活性の高い膜状触媒となることがわかった。

This study was undertaken to investigate the effects of insulin-like growth factor 1 (IGF-1) and basic fibroblast growth factor (bFGF) on the DNA and matrix synthesis of cells out-grown from the anterior cruciate ligament (ACL). Five batches of ACL cells from five 8-week-old Japanese white rabbits were isolated and maintained in culture until the fifth passage. We analyzed the effects of various concentrations of IGF-1 (1–1000 ng/ml) on [3H]-thymidine uptake in the cells at the first and fifth passages, and collagen content in the cell layer at the third passage, in the presence or absence of bFGF (10ng/ml). In the absence of bFGF, IGF-1 caused a significant increase in the synthesis of DNA and collagen in the ACL cells. IGF-1 and bFGF, in combination, synergistically increased the DNA synthesis of ACL cells, whereas such synergistic enhancement was not observed in their collagen production. The amounts of [3H]-thymidine incorporated into the cells incubated with IGF-1 (500ng/ml) and bFGF (10ng/ml) combined were 1.3-1.5 times greater at first passage and 13–1.9 times greater at fifth passage than the sum of these with the growth factors used individually. Based on this in vitro finding, we consider it clinically relevant that IGF-1 and bFGF, when used together, have the capability to enhance the primary healing of ruptured ACL.

This study showed that nitric oxide (NO)-generating S-nitrosothiols, S-nitroso-N-acetylpenicillamine (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 mM) induced an increase in cell number compared to control. SNAP (0.005-0.02 mM) and GSNO (0.025-0.05 mM) 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 mM, respectively. The increase induced by 0.02 mM SNAP was abolished by the addition of 0.01 mM oxyhemoglobin, a scavenger of NO. [3H]Thymidine incorporation in stationary cells was also increased by SNAP. In addition, 0.02 mM SNAP produced a 1.8-3.2-fold increase of thymidine kinase activity in exponentially growing cells.

We have isolated a rat cDNA encoding a receptor-type protein-tyrosine-phosphatase (RTP) expressed in brain and kidney (RPTP-BK) and characterized its expression in the developing central nervous system. RPTP-BK has seven fibronectin type III-like repeats in the extracellular region and a unique catalytic phosphatase domain in the cytoplasmic region. Bacterial expression of its phosphatase domain showed that the dephosphorylation of phosphotyrosine residues was mediated by the cytoplasmic catalytic domain. Sequence comparison revealed that RPTP-BK is homologous with GLEPP1, a rabbit PTP expressed in renal glomerular epithelia, and has the same phosphatase domain as murine PTPϕ expressed in macrophages. RPTP-BK has also significant homology with Drosophila DPTP10D in the phosphatase domain, whose expression is localized exclusively in growth cones of the embryonal brains. The gene for RPTP-BK is well conserved among other species, and the expression in the brain but not in the kidney is developmentally regulated during the neonatal stage. Hybridization in situ showed that RPTP-BK is highly expressed in the postmitotic maturing neurons of the olfactory bulb, developing neocortex, hippocampus and thalamus. Because the expression of RPTP-BK in the developing neocortex is correlated with the stage of axonogenesis in cortical neurons, RPTP-BK might be crucial in neural cell development of the mammalian central nervous system.

Helicon wave excited plasma (HWP) controlled by magnetic mirrors was applied to sputtering growth of a thin yttria-stabilized zirconia (YSZ) electrolyte film with solid oxide fuel cell (SOFC). Electron energy increased as increasing mirror field coil current from 4 to 15 A and its maximum value obtained at mirror field by coil current of 15 A. Furthermore, XRD intensity of (111) cubic system of a sputtered YSZ film also increased by adding magnetic mirrors. As a result, it was found that energy property of HWP and crystal structures of a sputtered YSZ film could be improved largely by reduction of particle energy losses due to the plasma confinement effect of magnetic mirrors.

We have examined the alkaline phosphatase (AP) activity of primary calvaria-derived osteoblast-like cells from the twy (tip-toe walking Yoshimura) and normal ICR control mouse. The twy mouse displays elevated osseous formation particularly in the spine, and the pathophysiological features resemble that of human ankylosing spinal hyperostosis. In the proliferative stage of cultured bone cells, parathyroid hormone (PTH) stimulation induced the elevation of AP activity of both twy and ICR mouse-derived cells. When they reached confluence, the AP activity of ICR mouse-derived cells ceased to increase with PTH stimulation. The twy mouse-derived cells, however, continued to respond to PTH, with the enzyme activity increasing even in the confluent, stationary stage. PTH stimulation also increased the intracellular cAMP content of twy mouse-derived cells but it did not influence that of ICR mouse-derived cells in the stationary stage. Moreover, stimulation with dibutyryl cAMP, but not with phorbol myristate acetate, increased the AP activity of both twy and ICR-derived bone cells irrespective of culture conditions, either in the proliferative or in the confluent stage. These data suggest that the protein kinase A-mediated pathway plays a pivotal role in bone cells with PTH stimulation, and that the uninhibited AP activity observed in twy mouse-derived bone cells might be due to some deviating process between the PTH ligand/receptor interaction and cAMP generation.

A protein factor which stimulated [3H]thymidine uptake into free hepatocytes prepared from normal mouse liver was detected in the ascitic fluid of gynecological cancer patients. The factor was subsequently further purified from the ascitic fluid of an endometrial cancer patient by DEAE-Sephacel, Sephadex G-150 and Phenyl-Sepharose CL-4B column chromatographies, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single protein band of 54,000 Da, designated tentatively as 54K ascitic protein (54K-AP). 54K-AP was similar to human alpha 1-antitrypsin (alpha 1-AT) in terms of SDS-PAGE and immunological behavior, but was slightly different in terms of amino acid sequence and isoelectric point. Although 54K-AP inhibited the activities of bovine trypsin and alpha-chymotrypsin as did human alpha 1-AT, 54K-AP inhibited the plasminogen activator released from human endometrial cancer Ishikawa cells more efficiently than alpha 1-AT. Because, in contrast to normal serum, the serum from the endometrial cancer patients stimulated [3H]thymidine uptake into hepatocytes, the possibility arises that 54K-AP could be produced by the cancer host as a defence mechanism against the cancer.

Nicotinamide methyltransferase (Nmd CH3transferase) activity increased in the liver of mice after i.p. transplantation of Ehrlich ascites tumor (ascitic form), but not in the liver of mice with acute inflammation induced by the i.p. administration of D-galactosamine, and it rather showed a decrease together with necrosis after carbon tetrachloride administration. When Nmd CH3transferase activity of rat hepatocytes in primary culture was investigated with the addition of dexamethasone, epidermal growth factor, transforming growth factor-beta, tumor necrosis factor-alpha and N1-methylnicotinamide (1-CH3Nmd), changes in activity were not correlated with DNA synthesis, suggesting that the increase of this enzyme activity in the tumor host liver was not directly related to liver cell proliferation. Thus, in order to make use of the increase of this enzyme activity as a tumor burden marker, a procedure for its estimation by measuring the blood level of 1-CH3Nmd, a metabolite of Nmd produced by Nmd CH3transferase, was established. The 1-CH3Nmd level in the blood of mice bearing Ehrlich ascites tumor 4 h after s.c. loading of Nmd (500 mg/kg body weight) was closely correlated with this enzyme activity in the liver (r = 0.835, P less than 0.00001) from the early to the terminal stage of tumor development. Furthermore, similar correlations were seen in the animal groups bearing various other tumors, such as s.c. implanted Ehrlich ascites tumor (solid form) and i.p. implanted sarcoma S-180, hepatoma MH-134, Yoshida ascites sarcoma and leukemia L-1210, but not solid tumors such as Lewis lung carcinoma and melanoma B-16, although almost all of the animals bearing these tumors showed a higher enzyme activity than their control normal animals.

Anabolism of 5-fluorouracil (5-FU) in the presence of uracil was examined using the cell-free extract of Ehrlich ascites tumor cells. FU-nucleoside formation from 5-FU with ribose 1-phosphate (R-1-P) or 2'-deoxyribose 1-phosphate was not readily inhibited even by the addition of uracil at 100 times higher concentration than 5-FU. FU-nucleotide formation from 5-FU with R-1-P and adenosine 5'-triphosphate or with 5-phosphoribosyl 1-pyrophosphate was slightly reduced as the concentration of uracil was increased. It was also found that 5-fluorouridine (5-FUR) was produced by "nucleoside N-ribosyltransferase," transferring a ribose moiety from uridine (UR) to 5-FU directly. This activity might play a role in the preferential formation of 5-FUR. However, 5-fluoro-2'-deoxyuridine was not produced by directly transferring a deoxyribose moiety. On the basis of several column chromatographies and characterization of kinetics, pH dependency, and response to inhibitors, the enzyme protein of the ribosyltransferase could not be distinguished from that of the phosphorylase.

Thymidine kinase (TK) is a protein closely associated with DNA replication. Here we show that, in contrast with the variation of TK activity, which changed parallel with S phase cell distribution during asynchronous culture of rat hepatoma JB1 cells, the serine residues of cytosolic TK protein was phosphorylated continuously in response to increasing G0/G1 phase cell distribution. The shifting of phosphorylation of TK protein during different cell growth conditions was further confirmed with the examination of the elution profile of cytosolic TK activity by anion-exchange high performance liquid chromatography (HPLC). HPLC analysis revealed that the rapid proliferating (62 h after asynchronous culture) rat hepatoma JB1 cells showed only the hyperphosphorylated form of cytosolic TK activity, while synchronously M phase-arrested JB1 cells showed hypo- and hyper-phosphorylated forms of cytosolic TK activity. These results suggested that the modulation of phosphorylation of cytosolic TK protein was cell cycle-dependent, and that the phosphorylation of cytosolic TK protein might be involved in the negative regulation of TK activity in vivo.

Anti-TK monoclonal antibodies (mAbs) were raised against recombinant human cytosolic thymidine kinase (rhTK) and characterized by Western immunoblotting, enzyme-linked immunosorbent assay (ELISA) and immunostaining of tumor cells. Twenty-three clones of TK mAbs were characterized to recognize specifically not only rhTK produced by Escherichia coli but also TK subunit of 25 kDa in human lung cancer. The anti-TK mAbs reacted specifically with cytosolic TK but not with mitochondrial TK. Only one clone of the mAbs inhibited the catalytic activity of TK. By solid phase sandwich enzyme immunoassay using these mAbs, we could quantitate the cytosolic TK content in tissues. Immunohistochemical staining analysis using one of the TK mAbs showed that human lung adenocarcinoma and squamous cell carcinoma exhibited much higher staining intensity than stromal cells. These mAbs are useful for biochemical studies on the regulation of human TK in proliferating cells such as tumor cells and for diagnosis of highly proliferating tumors.

The cDNA clone encoding human thymidine kinase (hTK), was expressed in E. coli using a prokaryotic expression vector, pKK 223-3. The kinetics of the recombinant hTK (rhTK) were similar to those of cytosolic TK but not of mitochondrial TK. rhTK was highly purified in the presence of either ATP or dithiothreitol (DTT). The specific activity of rhTK purified in the presence of ATP [rhTK(ATP)] was lower than that of rhTK purified in the presence of DTT [rhTK(DTT)]. Activity of the purified rhTK(ATP) was enhanced by addition of thiols including DTT, cysteine, homocysteine and beta-mercaptoethanol but inhibited by various sulfhydryl reagents such as 5,5'-dithio-bis(2-nitrobenzoic acid). Hence, it was suggested that rhTK is a thiol-type enzyme. Apparent Mr of purified rhTK(ATP) was 100 kDa, which corresponds to the size of a tetramer (25 kDa subunit), while that of purified rhTK(DTT) was 50 kDa, the size of a dimer. The tetramer form of rhTK(ATP) was converted to the dimer by replacement of ATP by DTT. On the other hand, the dimer form of rhTK(DTT) was converted to the tetramer by addition of ATP. Thus, the catalytic activity of human cytosolic TK might be regulated by thiols as well as ATP via its polymerization status.

Thymidine kinase (TK) activity in rat hepatoma JB1 and AH136B was mainly due to cytosolic TK and was much higher than that in rat regenerating liver. Two forms of cytosolic TK, designated as TK-I and TK-II, were revealed in addition to mitochondrial TK in anion-exchange high performance liquid chromatography (HPLC) of the enzyme extract from hepatoma JB1. During incubation of the enzyme extract at 20 degrees C in the presence of phosphatase inhibitor NaF, TK-II activity remained while TK-I activity almost disappeared. Thus, TK-II appeared to be more stable than TK-I. In Western blot analysis, TK-II was much more phosphorylated and exhibiting higher specific activity than TK-I. Meanwhile, regenerating liver derived from the rat 24 h after partial hepatectomy showed only TK-I activity, which completely disappeared after incubation at 20 degrees C even in the presence of NaF. Consequently, the presence of TK-II might account for the higher TK activity in hepatomas than in regenerating liver.

Ossification of the posterior longitudinal ligament (OPLL) of the spine is characterized by heterotopic bone formation occurring in spinal ligament, causing severe compression myelopathy. In order to investigate the mechanism of OPLL development, we isolated spinal ligament cells from OPLL patients as well as non-OPLL patients, and established 10 OPLL cell lines and 7 non-OPLL cell lines, respectively. We analyzed the effects of bone morphogenetic protein-2 (BMP-2) on these cells with respect to alkaline phosphatase (AP) activity, DNA synthesis, and collagen production. BMP-2 caused a significant increase of AP activity in 4 OPLL cell lines, whereas the activity did not change in any non-OPLL cells. Among OPLL cells, BMP-2 stimulated DNA synthesis in four cell lines and procollagen type I carboxyl-terminal peptide (PICP) synthesis in five cell lines. Some non-OPLL cells also responded to BMP-2, as there was an increase of DNA synthesis in three cell lines and PICP synthesis in one cell line. These data collectively indicate that BMP-2 preferentially induces osteogenic differentiation in OPLL cells rather than in non-OPLL cells. OPLL cells, therefore, exhibit a different response to BMP-2 than non-OPLL cells, suggesting that the expression of BMP receptor(s) and/or the signal transduction initiated by BMP-2 in the spinal ligament cells of OPLL patients somewhat deviate from those in normal spinal ligament cells. Such abnormal characteristics of OPLL cells as described here provide some clues to the clarification of the pathogenesis of OPLL.

Summary Cytosolic thymidine kinase was purified 18,000-fold of the homogenate from murine Ehrlich ascites tumor, using the [p-aminophenyl 3'-dTMP]-CH-Sepharose affinity column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band of molecular weight 26,000. Two different forms, relative molecular weight 50,000 and 70,000, were found by gel f'dtration, depending on the existence of dithiothreitol, ATP and other nucleotides. These agents also stabilize and stimulate the enzyme activity. The existence of two forms was.also manifested by DEAE-Sephacel column chromatography, where the 50,000 form was eluted by 50 mM NaC1 and the 70,000 form by 400 mM NaC1.

In order to develop new ways of tumor diagnosis and therapy, it is important to know the metabolic changes occurring while bearing the tumor, since these ways should be applied to the cancer patient, who is, after all, a tumor bearer. Various metabolic changes in the tumor host tissues, especially in the liver, have been observed to take place together with tumor-growth (Fujimura and Shimizu, 1977; Shimizu and Fujimura, 1978; 1984; Asanagi et al., 1988; Tanaka et al., 1991; Nakagawa et al., 1991; Fujimura et al., 1992a; 1992b; Hanazawa et al., 1994; Fujimura et al., 1995).

We have examined the activity of protein tyrosine phosphatase in various tumor cell lines and surgically resected human colon carcinoma specimens. The protein tyrosine phosphatase activity of rat fibroblasts increases with the transformation by v-src gene. Likewise human colon carcinoma and human neuroblastoma cell lines, in which the activation of c-src tyrosine kinase was reported, show elevated protein tyrosine phosphatase activity in most cases. We compared the protein tyrosine phosphatase activity of eight human colon cancer tissues with that of normal mucosa of the same patient. The results show that the protein tyrosine phosphatase activity in tumor tissue was increased, suggesting the biological significance of protein tyrosine phosphatase in colon cancer.

Five days after transplantation of Yoshida ascites sarcoma cells into a rat, specific activity of tRNA-pseudouridine synthetase was extremely high in the supernatant of tumor cells and moderately high in the tumor-bearing rat liver compared with normal rat liver. Enzyme assay was performed at 37°C by determining the release of tritium from heterogeneous [3H] tRNA extracted from E. coli B grown in the presence of [5,6-3H]-uracil and resulting in the increased ratio of the amount of pseudouridine to uridine residues in [3H] tRNA. Neither [5-3H]-uridine, [5,6-3H]-UTP, nor [5,6-3H]-poly U released tritium in the present assay conditions.

During the administration of the hepatocarcinogen, 2-acetylaminofluorene (AAF), to Wistar strain male rats in the diet excretion of 1-methyl-2-pyridone-5-carboxamide (2-PY) gradually increased. A plateau was reached at 21 weeks after initiation of the AAF-treatment and this increased level was maintained even after the treatment had been terminated, while the excretion of 1-methyl-4-pyridone-5-carboxamide (4-PY) decreased. All the urinary radioactivity 0–9 h after i.p. injection of 1-[Me-14C]methylnicotinamide (1-CH3Nmd) at the 20th experimental week was recovered as pyridones. In control rats only 4-PY was labeled but in AAF-treated rats both pyridones were labeled almost equally. The specific activity of 2-PY formation of 1-methylnicotinamide oxidase (1-CH3Nmd oxidase, EC 1.2.3.1) of the AAF-treated rat liver during the 16–30 weeks experimental period was 3 times higher than that of the control rat liver, while 4-PY forming enzyme activity decreased to around 20% of the control value. In control rat liver 4-PY-forming activity showed a maximum at pH 9.0 but 2-PY-forming activity continued to increase as the pH value was raised. However, in AAF-treated rat liver both 2-PY- and 4-PY-forming activities increased as pH increased. Km values for 1-CH3Nmd of 2-PY- and 4-PY-forming enzyme in control rat liver were 4.22 × 10−4 M and 6.4 × 10−5 M respectively, while in AAF-treated rat liver they were 4.63 × 10−4 M and 5.03 × 10−4 M respectively.

CDP reductase was separated from the cytosol of rat Yoshida ascites sarcoma. The precipitate, which resulted from the acidification of the cytosol by acetic acid at pH 5.2, catalyzed specifically the reduction of CDP, whereas the concurrently resulted supernatant catalyzed those of UDP, ADP and GDP. The CDP reductase showed a single peak in the pattern of the enzyme activity in DEAE-cellulose and also in Sepharose 4B column chromatography with adequate recovery of the activity.

It was revealed that thymidylate kinase was purified together with cytosolic thymidine kinase from human term placenta by p-aminophenyl thymidine-3'-phosphate-CH-Sepharose affinity column chromatography, which has been commonly used for purification of thymidine kinase. In addition, it was noted that mitochondrial thymidine kinase and nucleoside diphosphate kinase were concurrently eliminated. In the presence of ATP, cytosolic thymidine kinase and thymidylate kinase could be separated from each other by Ultrogel AcA 34 filtration, and their molecular weights were estimated to be 70,000 and 50,000, respectively. On SDS-polyacrylamide gel electrophoresis, thymidine kinase protein exhibited a band of 26,000, which was compatible with the molecular weight of the enzyme subunit calculated from its cDNA, while thymidylate kinase protein showed 24,000. Thymidylate kinase could utilize either ATP or dATP as an efficient phosphate donor, and showed substrate specificity for dTMP.

Rat liver chromatin was fractionated into DNA, histones and non-histone chromosomal proteins and each component was modified with N- methyl -N′- nitro -N- nitrosoguanidine or N- ethyl -N′- nitro -N- nitrosoguanidine . The radioactivity of 14C-labeled alkyl or guanidino moieties of both compounds bound significantly to both histones and non-histone chromosomal proteins and the binding of N- methyl -N′- nitro -N- nitrosoguanidine was higher than N- ethyl -N′- nitro -N- nitrosoguanidine . However the binding of both compounds to DNA was very low and its significance was hard to evaluate. All of the three components, one of which was modified, were reconstituted into chromatin, then, [3H]UMP incorporation into acid insoluble material using Escherichia coli RNA polymerase (EC 2.7.7.6) was measured. Only with the reconstituted chromatin containing histones modified either by N- methyl -N′- nitro -N- nitrosoguanidine or N- ethyl -N′- nitro -N- nitrosoguanidine , the template activity increased drastically; i.e., about 10 or 5 times higher than that with the unmodified reconstituted chromatin, respectively. However, any remarkable alteration in the electrophoretic pattern of protein fraction of the reconstituted chromatin could not be found. The results obtained in this study are discussed in the context that the modified histones could give rise to change in the mutual interaction of chromosomal components during the reconstitution of chromatin accompanied with the increase of chromatin template activity.

The effect of 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent tumor promoter, on the synthesis of proteins in BALB/c 3T3 mouse fibroblasts has been studied. When [35S]methionine-labeled proteins synthesized after the addition of TPA were analyzed by one- or two-dimensional polyacrylamide gel electrophoresis, the increased synthesis of a 32,000-dalton protein (designated p32) was noted as one of the earliest changes. The synthesis of p32 increased approximately 2-fold within 2 hr at a promoter concentration of 20 ng/ml. Phorbol 12,13-didecanoate, another potent tumor promoter, showed the same effect as TPA, whereas 4-O-methyl-TPA, which has little promoting activity, did not enhance the synthesis of p32 at the same concentration but did effect a slight increase at 1 microgram/ml. p32 differed immunologically from a major excreted protein discovered by Gottesman [Gottesman, M. M. (1978) Proc. Natl. Acad. Sci. USA 75, 2767-2771]. The increased rate of the synthesis of p32 was considered to be regulated at the transcriptional level, because the increase in synthesis of p32 was inhibited by actinomycin D, and TPA-treated cells were shown to contain a higher level of translatable mRNA coding for p32 than do control cells. A possible relationship between the increase in p32 synthesis and transformation is discussed.

Twenty four hours after intraperitoneal injection of alkylating agents, N -methyl- N′ -nitro- N -nitrosoguanidine (MNNG), N -methyl- N -nitrosourea (MNU) and methyl methanesulfonate (MMS), into rats, an increase in nicotinamide (Nmd) methyltransferase and N 1 -methylnicotinamide (1-CH 3 Nmd) oxidase activities in the liver was found. Since activities of these enzymes in the liver extracts were not stimulated directly by these agents in vitro, it is postulated that the increases in these enzyme activities are due to the induction of each enzyme.

The hydrolytic action of snake venom phosphodiesterase on poly(adenosine diphosphate ribose) was investigated in comparison with that of rat liver phosphodiesterase. The purified polymer, labeled with 32P or 14C, was partially hydrolyzed with snake venom phosphodiesterase and passed through a Sephadex G-50 column. The resulting elution profiles showed the existence of degradation products with various chain lengths, which were eluted successively after the void volume. A paper chromatogram of partially hydrolyzed 14C-labeled polymer indicated the existence of a different oligomer of 2'-(5''-phosphoribosyl)adenosine 5'-phosphate, from that found after hydrolysis with rat liver phosphodiesterase. Phosphate esters in the acid-soluble products of partial digestion of 32P-labeled polymer by snake venom phosphodiesterase were not completely susceptible to alkaline phosphomonoesterase from Escherichia coli. To confirm the endonucleolytic cleavage by snake venom phosphodiesterase, the structure of the oligomer obtained by rechromatography was examined. The oligomer was incubated with alkaline phosphomonoesterase and then this was removed and it was completely hydrolyzed by snake venom phosphodiesterase. 2'-(5''-Phosphoribosyl)adenosine, 2'-(5''-phosphoribosyl)-adenosine 5'-phosphate, and 2'-ribosyladenosine 5'-phosphate were found in the hydrolysate. From these results it is concluded that snake venom phosphodiesterase digests the polymer endonucleolytically. This is in contrast to rat liver phosphodiesterase which digests it exonucleolytically.

The direction of exonucleolytic degradation of poly(ADP-ribose) by rat liver phosphodiesterase was investigated. 1. When 14C-labeled poly(ADP-ribose) was hydrolyzed with rat liver phosphodiesterase, the concentration of 5'-AMP in the hydrolysate increased in the early stage of hydrolysis and then after 25% hydrolysis remained constant. 2. Poly(ADP-ribose) preparations labeled with 32P in two ways were prepared: poly(ADP-ribose) (I) labeled with 32P at the ribose phosphate derived from the NMN moiety of NAD and (II) labeled with 32P at the AMP portion derived from the AMP moiety of NAD. These two polymers were hydrolyzed 14 to 22% with rat liver phosphodiesterase. Partially hydrolyzed poly(ADP-ribose), named trimmed polymer, was purified by passage through a Sephadex G-50 column. Trimmed polymers were then incubated with Escherichia coli alkaline phosphomonoesterase and radioactive inorganic phosphate released was determined. Three to 6% of the radioactivity of trimmed polymer (I) was recovered as inorganic phosphate, whereas only 0.7% of the radioactivity of trimmed polymer (II) was recovered as inorganic phosphate. 3. Trimmed poly(ADP-ribose) doubly labeled with 32P at the ribose phosphate derived from the NMN moiety of NAD and with 14C in the adenine portion was prepared. After incubation with alkaline phosphomonoesterase and removal of alkaline phosphomonoesterase, it was completely hydrolyzed with venom phosphodiesterase. 2'-(Ribosyl)-5'-AMP was recovered in the hydrolysate but 2'-(5''-phosphoribosyl)-adenosine was not detected. From these results it is concluded that poly(ADP-ribose) is degraded by rat liver phosphodiesterase from the AMP terminus to the other terminus in an exonucleotic fashion.

The radioactive acid-insoluble material, Product I, which was formed by rat-liver nuclear preparation from labeled NAD or labeled ATP with nicotinamide mononucleotide, has been partially purified. It has a peak at 3 S with sucrose density-gradient centrifugation, and a density of around 1.57 with Cs 2 SO 4 equilibrium density-gradient centrifugation. Product I was converted into the acid-soluble form by snake-venom phosphodiesterase. Product I was not digested by micrococcal nuclease, spleen phosphodiesterase, potato phosphodiesterase, potato nucleotide pyrophosphatase, spleen NAD nucleosidase, or trypsin. The radioactive acid-soluble substance, Product II, which was released by venom-phosphodiesterase digestion, was isolated from a Dowex 1 formate column. Product II showed absorption maxima at 261, 259, and 258 mμ under alkaline neutral and acidic conditions, respectively. The molar ratio of adenine (as adenosine), ribose, phosphorus and periodate consumption was 1:2:2:1. All phosphates were converted to inorganic phosphate by acid-phosphatase digestion. Structures of Products I and II were proposed.

A method was developed for isolation and purification of radioactive poly ADP-ribose formed by a nuclear enzyme from rat liver. The procedure involved: (a) Pronase digestion and phenol extraction, (b) pancreatic RNase and pancreatic DNase digestion, (c) a first gel filtration through a Sephadex G-50 column, (d) micrococcal nuclease and spleen phosphodiesterase digestion, and (e) a second gel filtration. The over-all recovery of acid-insoluble radioactivity in the procedure was more than 50% and one-third of the acid-insoluble radioactivity was eluted just after the void volume from the Sephadex G-50 column. Increase of the specific radioactivity (counts per min per OD260) and decrease of the ratio of absorption at 280 mµ to that at 260 mµ were used as criteria of purity of poly ADP-ribose. At the final step of purification, the specific radioactivity of poly ADP-ribose was 80-fold that of the crude extract after Pronase digestion and phenol extraction. The specific activity of purified poly ADP-ribose and that of α-32P-ATP used as a precursor were 1.26 x 105 cpm per OD260 and 0.99 x 105 cpm per OD260, respectively. The A280:A260 ratio of absorption of purified poly ADP-ribose at the final step was 0.26. Purified poly ADP-ribose was completely hydrolyzed by snake venom phosphodiesterase and did not yield any nucleotide other than 5'-AMP and PR-AMP on Dowex 1 column chromatography. Experiments with 14C-ATP and NMN with a 32P-labeled whole nuclear preparation showed that poly ADP-ribose after the second gel filtration was devoid of 32P radioactivity. This indicates that poly ADP-ribose at this step was already free from contaminating RNA and DNA.

A series of N-alkyl-N-nitrosoureas, which produce both alkylation and carbamoylation, was found to induce neurite formation in mouse neuroblastoma N-18 cells just as does N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Related compounds, which produce only carbamoylation or nitroguanidination, did not induce morphological differentiation, while methyl methanesulfonate (MMS) and dimethyl sulfate, which produce only alkylation, did. MNNG, methyl nitrosourea and MMS were more effective in the induction of differentiation than their corresponding ethyl derivatives. These data suggested that alkylation contributed to the induction of morphological differentiation in N-18 cells.

The activity of thymidylate synthetase in the liver of the ddY strain male mouse increased transitorily according to the increase in tumor cell number at maximum 7-9 days after ip transplantation of Ehrlich ascites tumor. The enzyme was able to be purified from the tumor host mouse liver or from the normal mouse liver in the same manner as from tumor cells using Affi-Gel blue and methotrexate-Sepharose 4B affinity column chromatography. The three enzyme preparations obtained were purified at 27,000-38,000-, and 8,000-fold, and yielded total activities of 11, 3, and 16% of these homogenates, respectively. These preparations were similar in molecular weight to the whole enzyme (67,000) and its subunit (34,000), optimum pH, and Km values either for deoxyuridine 5'-monophosphate or tetrahydrofolate in the presence of formaldehyde. Furthermore, the amount of 5-fluoro-2'-deoxyuridine 5'-monophosphate forming the ternary complex with the enzyme and tetrahydrofolate paralleled the enzyme activities in the cytosol fractions of the three tissues. The characteristics of the tumor host liver enzyme were similar to those of the proliferating tissues, the Ehrlich ascites tumor.

Uridine diphosphate (UDP) reductase was isolated in the supernatant fraction obtained after the acidification of the cytosol of Ehrlich ascites tumor cells, and was found insensitive to 10 mM hydroxyurea. However, cytidine diphosphate (CDP) reductase, being separated concurrently in the precipitate fraction, was readily inhibited. In the cytosol fraction of either Ehrlich ascites tumor, Yoshida ascites sarcoma or regenerating rat liver after partial hepatectomy, UDP reduction activity, in contrast to CDP reduction activity, is not sensitive to hydroxyurea.

Yoshida ascites sarcoma-bearing rats excreted significantly higher quantities of deoxycytidine and pseudouridine in urine than normal rats, with a peak 5 days after transplantation of the tumor cells, and excretion of cytidine peaking 3 or 4 days later. The contribution by the injected [14C]orotic acid to labeling of urinary deoxycytidine was 32 and 3 times higher than that by [14C]uridine or [14C]cytidine, respectively. Urinary pseudouridine was also labeled 5–6 times greater by [14C]orotic acid than by [14C]uridine or [14C]cytidine. The labeling in pseudouridine was as high as that in deoxycytidine by either [14C]orotic acid or [14C]cytidine and was about 10 times higher by [14C]uridine. Neither [14C]uracil nor [3H]thymidine resulted in any labeling of either nucleoside. [6-3H]Uridine resulted in radioactivity of urinary pseudouridine, whereas [5-3H]uridine did not. The extent of labeling by the injected [14C]orotic acid of urinary deoxycytidine and pseudouridine was almost constant, at least for several days around maximal excretion of nucleosides; this was true for each injection made 1, 3 or 5 days after tumor transplantation. This study suggests that an increase of urinary deoxycytidine and pseudouridine could be derived from not only the tumor cells but also from the host liver and that urinary pseudouridine could be synthesized by rearranging the ribose in a uridine molecule, i.e., by transferring the ribose from the nitrogen 1 position of uracil to the carbon 5 position.

Summary Synthesis in a cell-free system of aldolase A under the direction of either polysomes or mRNA prepared from rat ascites hepatoma AH 7974 cells has been demonstrated. Polysomes prepared with the aid of Nonidet P-40 were found to be of high template activities when incubated with pH 5 fraction of rat liver. Polysomal RNA purified on poly(U)-Sepharose showed very high template activities in a wheat germ cell-free system. When the released products from ribosomes in both systems were analyzed by immunoprecipitation by anti-aldolase A antibody, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their radioactivities appeared as a single major peak which co-migrated with authentic aldolase A subunits.

Publisher Summary This chapter discusses the polymerization of the adenosine 5'-diphosphate-ribose moiety of nicotinamide adenine dinucleotide (NAD). The polymerization is catalyzed by certain aggregated enzyme preparations. This enzymatic polymerization is accompanied by a release of the nicotinamide moiety of NAD. No energy source is required for the polymerization as nicotinamide inhibits the reaction. Under optimal conditions, about 30% of NAD added as a substrate is converted to poly (ADP-Rib) adenosine diphosphate ribose. Poly (ADP-Rib) can be purified by the following steps: acetate buffer-ethanol precipitation; pronase digestion; phenol extraction; pancreatic RNase and DNase digestion; and gel filtration on Sephadex. For further purification, digestion with micrococcal nuclease and spleen phosphodiesterase is applied. Enzymatic formation of ADP-ribose polymer involves enzyme preparation. To follow the enzymatic formation poly (ADP-Rib), the use of NAD labeled with radioactive isotope is recommended. Ado (P)-Rib-P is purified from the phosphodiesterase hydrolyzate of polymer on Dowex l-X8 (200–400 mesh) formate column chromatography. The properties of poly (ADP-Rib) are also discussed.

[8- 14 C]ATP was incorporated by a rat-liver nuclear preparation into acid-insoluble material in the presence of NMN. Experiments using variously labeled ATP and NMN or NAD revealed that the ADP-ribose moiety of NAD was incorporated into the acid-insoluble reaction product. NAD was formed from ATP in the presence of NMN and was an immediate precursor of the acid-insoluble reaction product. The reaction involving the incorporation of NAD had an optimum pH of 8 and required neither a divalent cation nor a sulfhydryl compound. The reaction was effectively inhibited by nicotinamide but not by isonicotinic acid hydrazide. The contribution of NADase in the nuclei towards this enzymatic activity was suggested.

WE reported1 that the subcutaneous injection of a potent mutagen2, N-methyl-N′-nitro-N-nitrosoguanidine (NG), into rats induced many transplantable sarcomas at the injected loci. Schoental3 reported the occurrence of squamous cell carcinoma in the fore-stomach of rats to which NG was administered by stomach tube. Druckrey et al.4 also described the production of sarcoma in rats by the subcutaneous injection of NG. The careinogenicity of this mutagen seemed to be well documented.