A nucleolus-specific phosphoprotein in mouse ascites tumor cells

Analysis of proteins in nucleoli and chromatin of mouse ascites tumor cells labeled with [³²P]orthophosphate by SDS-polyacrylamide gel electrophoresis showed that a highly radioactive protein was localized in the nucleoli. This protein was purified and the final preparation appeared as a single component on hydroxylapatite column chromatography with or without SDS. This protein was found to be a nucleolus-specific phosphoprotein with a molecular weight of 120 000. The phosphate moiety in this protein turned over very rapidly whereas the protein itself was stable. When the nucleoli were disrupted by EDTA treatment, this unique protein was found as a major protein constituent of the ultracentrifugal supernatant.     

Phosphorylation of a nucleolus-specific phosphoprotein in vitro.

The cellular site and characteristics of the phosphorylation of a nucleolus-specific phosphoprotein (molecular weight, 120 000) in mouse ascites tumor cells were studied. The phosphoprotein was strongly labeled with ³²P when the isolated nucleoli were incubated with [γ-³²P]ATP in vitro. This phosphoprotein, and protein kinase for the protein phosphorylation were both purified from 0.3 M KCl soluble protein fraction of the nucleoli by hydroxylapatite and phosphocellulose column chromatographies. It was found that phosphorylation of the nucleolus-specific phosphoprotein was catalyzed selectively by a guanosine 3:5-monophosphate-dependent protein kinase in the nucleoli and the reaction product was the same phosphoprotein as the substrate used.     

DNA synthesis in detergent-treated mouse ascites sarcoma cells.

Mouse ascites sarcoma cells (SR-C3H/He cells) were made permeable to nucleoside triphosphates by treatment with nonionic detergents in a nearly isotonic condition. The permeable cells synthesized DNA in the presence of the four deoxyribonucleoside triphosphates, ATP, Mg2+, and the proper ionic environment. The optimum detergent concentration for DNA synthesis was 0.015--0.020% with Triton X-100, 0.020% with Nonidet P-40, and about 0.0025% with Brij 58. Higher concentrations of detergents were rather inhibitory to DNA synthesis. DNA synthesis in Triton-permeabilized cells was thought to be replicative, and the activity in the optimum conditions was much higher than that measured in hypotonic permeable cells or in isolated nuclei. These studies show the potential usefulness of detergent treatment for examining DNA replication in mammalian cells in vitro.     

Zinc and copper accumulation and isometallothionein induction in mouse ascites sarcoma S180A cells.

To investigate Zn and Cu accumulation and isometallothionein (iso-MT) induction in ascites-sarcoma S180A cells, 5 micrograms of Zn2+ or Cu2+/g body weight was administered to tumour-bearing mice intraperitoneally. In the tumour cells the Zn or Cu concentration increased more than in the host liver, which is the target organ for those metals; the maximum Zn or Cu level was about 2-3 times that in the host liver. The amounts of Zn-MT or Cu-MT accumulated in the tumour cells and host liver were proportional to such dose accumulation levels in the each cytosol; the maximum level of Zn-MT or Cu-MT was 4 or 2 times higher than in the host liver. MT accumulated in the tumour cells showed two subfractions (MT-1 and MT-2); the ratio of Zn (or Cu) bound to MT-1 to that bound to MT-2 in the host liver and tumour cells was 1.0 (or 1.0) and 0.7 (or 0.25) respectively, suggesting that the induction level of MT-2 in the tumour cells is more than that of MT-1. The h.p.l.c. profiles (using an anion-exchange column) of the isolated MT-1 and MT-2 subfractions from Zn-treated normal-mouse liver showed a single peak (MT-1-1) and two peaks (MT-2-1 and MT-2-2) respectively; mouse MTs were separated into three isoforms. In the ascites cells, the MT fraction obtained by a gel filtration was also separated into three isoforms; however, the amount of MT-2-1 isoform was 3 times that in the Zn-treated normal-mouse liver.     

Enhanced casein kinase II activity during mouse embryogenesis. Identification of a 110-kDa phosphoprotein as the major phosphorylation product in mouse embryos and Krebs II mouse ascites tumor cells

Mouse embryos at various stages of development were used to study the relationship of protein kinase activities with normal embryogenesis. Casein kinase II (CKII) activity in developing mouse embryos shows a 3-4-fold activity increase at day 12 of gestation. Together with the CKII activity, increased phosphorylation of a 110-kDa protein is observed. Treatment of the embryo extracts with heparin, a highly specific inhibitor of CKII activity, results in a drastic reduction of the 110-kDa protein phosphorylation indicating that the protein might be a CKII-specific substrate. Rapidly proliferating mouse tumour cells also show an enhanced CKII activity. Here too, a 110-kDa phosphoprotein was the major phosphoryl acceptor. Partial proteolytic digestion shows that both proteins are identical. Other protein kinases tested (cAMP- and cGMP-dependent protein kinases) only show a basal level of enzyme activity with minor alterations throughout the different stages of embryogenesis investigated.     

Cloned complementary deoxyribonucleic acid probes for untranslated messenger ribonucleic acid components of mouse sarcoma ascites cells

Mouse sarcoma ascites cells contain several abundant mRNA species that occur to a large extent in an untranslated state. RNA preparations enriched in these species were used as starting material to construct recombinant plasmids. Cloned plasmids bearing sequences homologous to four of the untranslated mRNA species were identified by translation of hybrid-selected material. These plasmids, as well as a recombinant plasmid derived from chick alpha-actin mRNA, were used as probes for the estimation of mRNA levels in polyribosomes and in small ribonucleoprotein (RNP) particles of the ascites cells. Considerable amounts of the mRNA molecules belonging to the untranslated species were present in polyribosomes as well as in mRNPs. The actin mRNA, on the other hand, was present almost exclusively in polyribosomes. The distributions obtained by the hybridization assay resembled those estimated by translation of the same RNA preparations in cell-free systems. This indicates that the mRNA molecules of a given species engaged in translation in the cells and those present as untranslated RNP particles are equally effective in cell-free translation systems.     

Cell surface constituents of sarcoma 180 ascites tumor cells

The cell surface protein components of Sarcoma 180 ascites tumor cells have been investigated by a combination of plasma membrane isolation techniques and lactoperoxidase iodination. For plasma membrane isolation cells were homogenized in the presence or absence of Zn²⁺ and fractionated by sucrose density gradient centrifugation or a two-phase partition to give large membrane fragments or membrane envelopes. Membrane purification was monitored by phase contrast microscopy and chemical and enzyme marker assays. The membrane preparations were analyzed by acrylamide gel electrophoresis in sodium dodecylsulfate. Each preparation showed a common protein pattern of about 15 bands ranging in molecular weights from 33 000 to >300000. Two carbohydrate-containing bands were also present in all preparations. Membranes prepared with Zn²⁺ were much less fragmented and showed much greater amounts of three high molecular weight components than those prepared in the absence of Zn²⁺. This might suggest a role for these components in membrane stabilization.The tumor cells were also subjected to iodination with lactoperoxidase, followed by membrane isolation and acrylamide gel electrophoresis in sodium dodecylsulfate in order to identify polypeptides accessible to the cell surface. The major radioactive band coincided with the major carbohydrate-containing band, presumably a surface glycoprotein. A second carbohydrate-containing band showed variable labeling behavior between different cell preparations. This material had a high molecular weight, as indicated by both acrylamide gel electrophoresis and gel permeation chromatography in dodecylsulfate. Several other components are labeled to a lesser extent in the intact cell.     

Alterations in the size of the poly(a) segment in newly-synthesized messenger RNA of mouse sarcoma 180 ascites cells

The size of the poly(A) segment present at the 3-end of mRNA molecules decreases gradually after entry of the mRNA into the cytoplasm. The size reduction seems to occur equally in free and polysome-bound mRNA. It is not affected by conditions that inhibit protein synthesis, such as treatment with cycloheximide and amino acid deprivation. The latter condition leads to a substantial increase in the size of nuclear poly(A). The rate of poly(A) cleavage could determine the length of time a mRNA molecule remains functional in the cytoplasm.     

Purification and characterization of GMP synthetase from Yoshida sarcoma ascites cells

GMP synthetase was found in the cytosolic fraction of Yoshida sarcoma ascites cells. However, prolonged centrifugation resulted in precipitation of the enzyme. On sucrose density gradient centrifugation of a crude extract of Yoshida sarcoma ascites cells, a part of this enzyme showed high sedimentability at low ionic strength. On the basis of these observations, GMP synthetase was purified from Yoshida sarcoma ascites cells by means of procedures including centrifugal fractionation. The purified enzyme was shown to be homogeneous on SDS-polyacrylamide gel electrophoresis and isoelectric focusing in polyacrylamide gel. The molecular weight of the GMP synthetase was estimated to be 78,000 by SDS-polyacrylamide gel electrophoresis and gel filtration on Sephadex G-150. Its isoelectric point was estimated to be 5.6. The Km values of this enzyme for XMP, ATP, and glutamine were calculated to be 4.6, 120, and 300 microM, respectively. Although ammonia could substitute for glutamine as a donor of the amino group, the Km value was as high as 120 mM, indicating that it cannot be considered to be a physiological substrate. This enzyme showed high activity only in the presence of Mg2+, and very low activity in the presence of other divalent cations. Inhibition by nucleoside monophosphates was not significant. The enzyme required reduced sulfhydryl compounds for its activity.     

RNA synthesis in the presence of transfer RNa by DNA-dependent RNA polymerase II from mouse ascites sarcoma cells

RNA polymerase II from mouse sarcoma cells catalyzed the incorporation of UMP into an acid-insoluble fraction in the presence of tRNA. This reaction was not affected by DNase or actinomycin D but was inhibited by α-amanitin. This reaction was dependent on nucleoside triphosphate and manganese ions. RNA synthesized in the presence of tRNA could be digested with RNase A. These results suggest that the RNA synthesis by RNA polymerase II from mouse sarcoma is dependent on the presence of tRNA.     

Adriamycin-induced changes in the surface membrane of sarcoma 180 ascites cells

Adriamycin increases (a) the rate of agglutination of Sarcoma 180 cells by concanavalin A after brief exposure of 2–3 h and (b) membrane fluidity as measured by ESR within 30 min of exposure at concentrations of the anthracycline of 10^?7–10^?5 M. The effect of adriamycin on agglutination is not due to an increase in the number of surface receptors for concanavalin A, since the extent of binding of the lectin is not altered by adriamycin and no change occurs in the rate of occupancy of the concanavalin A binding sites by the lectin in cells treated with the antibiotic. The order parameter, a measurement of membrane fluidity, decreases in cells exposed to adriamycin and is dose-related. The results indicate that adriamycin can induce changes in the surface membrane of Sarcoma 180 cells within a brief period of exposure to a low but cytotoxic level of this agent.     

Altered processing of a major secreted phosphoprotein correlates with tumorigenicity in Rous sarcoma virus-transformed mammalian cells.

Anchorage-independent growth is highly correlated with neoplastic growth in vivo, and the retinoids (vitamin A and its analogs) inhibit this property in a wide variety of oncogenically transformed cells. We report here that retinoic acid-treated Rous sarcoma virus-transformed rat (RR1022) and vole (SR-1T) cells, which show reversible loss of anchorage-independent growth and assume nontransformed morphology, secrete a major 69-kilodalton phosphoprotein (pp69) instead of the 62-kilodalton phosphoprotein (pp62) secreted by their untreated counterparts. As determined by V8 protease mapping and by two-dimensional electrophoretic analysis, this 69-kilodalton polypeptide was indistinguishable from the pp69 released by nontransformed normal rat kidney cells. Neither retinoic acid-treated RR1022 cells nor normal rat kidney cells secreted pp62, and retinoic acid treatment did not have any significant effect on the synthesis, subcellular localization, or phosphokinase activity of pp60src. Furthermore, treatment with retinoic acid did not alter the synthesis of the transformation-specific 53-kilodalton phosphoprotein (p53) and secretion of the transforming growth factors in RR1022 cells. Our studies showed that there is a clear correlation between the release of pp69 or pp62 and the ability of cells to grow in vitro with or without anchorage. This may provide an important clue for elucidating specific biochemical events involved in anchorage regulation of growth.     

DNA ligase from mouse Ehrlich ascites tumor cells

The molecular (Mr = 120,000; s20, w = 5S) and catalytic properties (Km (ATP) = 3 microM; Km (nicked DNA) = 0.2 microM; Km (Mg2+) = 3 mM) of DNA ligase from mouse Ehrlich ascites tumor cells are similar to those of the enzymes from calf thymus and rodent liver. The activity level of DNA ligase from the tumor cells is about 10-fold higher than that from mouse liver. Immunochemical titration of DNA ligase with antibodies against the calf thymus enzyme showed that the higher level of DNA ligase activity in the tumor cells is due to an increase in enzyme quantity and not to elevation of the catalytic efficiency of the enzyme molecule. These results suggest that there is little apparent difference between the qualities of DNA ligases from the tumor cells and normal tissues of rodents and calf.