Effect of poly(ADP-ribose) formation on DNA synthesis and DNA fragmentation in nuclei of rat liver and rat ascites hepatoma AH-130 cells

To elucidate the role of poly(ADP-Rib) in the nucleus, DNA synthesis and DNA fragmentation were studied in isolated nuclei of rat liver and rat ascites hepatoma AH-130 cells. Liver and hepatoma cell nuclei formed the same amount of poly(ADP-Rib) per mg of nuclear DNA from NAD. Preincubation of liver nuclei with NAD repressed DNA polymerase activity to 30% of that of the control, but preincubation of hepatoma cell nuclei with NAD did not affect DNA polymerase activity. It was also found that incubation of liver nuclei with NAD prevented the fragmentation of nuclear DNA which occurred without NAD. Incubation of hepatoma cell nuclei with or without NAD did not result in fragmentation of DNA. The role of endonuclease in primer formation for DNA synthesis is discussed.     

Increased rate of tumor cell death caused by polyamine synthesis inhibitors

This investigation was designed to determine whether cell death plays a role in the antiproliferative action exerted by polyamine synthesis inhibitors. To estimate the rate of tumor cell death, we measured the loss of 125I from mice harboring Ehrlich ascites tumor cells in which DNA was labeled with 5-125I-iodo-2'-deoxyuridine. DL-alpha-difluoromethylornithine (0.85 mumoles/g body weight/6 h), and enzyme-activated irreversible inhibitor of ornithine decarboxylase, and methylglyoxal-bis(guanylhydrazone) (45 nmoles/g body weight/6 h), an inhibitor of S-adenosylmethionine decarboxylase, were both found to increase the rate of 125I excretion. Our data suggest that these polyamine synthesis inhibitors provoke an increase in the rate of tumor cell death beyond that normally occurring during growth, methylglyoxal-bis(guanylhydrazone) being considerably more potent than DL-alpha-difluoromethylornithine. These in vivo data were corroborated by a study where the host-mediated responses did not have to be considered. Thus, Ehrlich ascites tumor cells were adapted for suspension growth in culture and treated with methylglyoxal-bis(guanylhydrazone) or DL-alpha-difluoromethylornithine. The growth kinetics and the colony forming efficiency of the drug-treated cells clearly show that polyamine synthesis inhibitors not only slow the growth rate but also cause an increase in tumor cell death.     

Microtubule-associated protein-2 stimulates DNA synthesis catalyzed by the nuclear matrix

Microtubule-associated protein-2 (MAP-2) isolated from porcine brains stimulated DNA synthesis catalyzed by the nuclear matrix isolated from Physarum polycephalum in the presence of activated DNA as exogenous templates. The degree of the stimulation depended on the amount of the nuclear matrix, but not on that of the template. MAP-2 also stimulated DNA polymerase alpha activity solubilized from nuclei, but not DNA polymerase beta activity. These results suggest that MAP-2 stimulates DNA synthesis by interacting with the putative DNA replication machinery including DNA polymerase alpha bound to the matrix. Similar stimulation occurred in the nuclear matrix isolated from HeLa and rat ascites hepatoma cells, which strongly suggests that MAP-2 is involved in the control of DNA replication in eukaryotic cells.     

Deoxyribonucleic Acid Synthesis in Bacteriophage SPO1-Infected Bacillus subtilis I. Bacteriophage Deoxyribonucleic Acid Synthesis and Fate of Host Deoxyribonucleic Acid in Normal and Polymerase-Deficient Strains

The effect of bacteriophage SPO1 infection of Bacillus subtilis and a deoxyribonucleic acid (DNA) polymerase-deficient (pol⁻) mutant of this microorganism on the synthesis of DNA has been examined. Soon after infection, the incorporation of deoxyribonucleoside triphosphates into acid-insoluble material by cell lysates was greatly reduced. This inhibition of host DNA synthesis was not a result of host chromosome degradation nor did it appear to be due to the induction of thymidine triphosphate nucleotidohydrolase. Examination of the host chromosome for genetic linkage throughout the lytic cycle indicated that no extensive degradation occurred. After the inhibition of host DNA synthesis, a new polymerase activity arose which directed the synthesis of phage DNA. This new activity required deoxyribonucleoside triphosphates as substrates, Mg²⁺ ions, and a sulfhydryl reducing agent, and it was stimulated in the presence of adenosine triphosphate. The phage DNA polymerase, like that of its host, was associated with a fast-sedimenting cell membrane complex. The pol⁻ mutation had no effect on the synthesis of phage DNA or production of mature phage particles.     

Inhibition of a novel subspecies of DNA polymerase α by 2′-deoxy-2′-azidoadenosine 5′-triphosphate

DNA polymerase α₁, a subspecies of DNA polymerase α of Ehrlich ascites tumor cells, was associated with a novel RNA polymerase activity and utilized poly(dT) and single-stranded circular fd DNA as a template without added primer in the presence of ribonucleoside triphosphates and a specific stimulating factor. DNA synthesis in the above system was inhibited by the ATP analogue, 2′-deoxy-2′-azidoadenosine 5′-triphosphate more than the DNA synthesis with poly(dT)·oligo(rA) by DNA polymerase α₁ and RNA synthesis by mouse RNA polymerases I and II. Kinetic analysis showed that the analogue inhibited DNA polymerase α₁ activity on poly(dT) competitively with respect to ATP, suggesting that the analogue inhibited RNA synthesis by the associated RNA polymerase activity.     

DNA polymerases in isolated 'nuclear matrix' of Ehrlich ascites cells

Isolated nuclei from Ehrlich ascites tumor cells continue a replicative-like in vitro DNA synthesis. Polymerase alpha is the major dNTP polymerizing enzyme in nuclei. Following complete achromatinization dNTP polymerizing activities are still associated with the residual structure termed 'nuclear matrix'. In contrast to DNA synthesis in native nuclei, 'nuclear matrix' DNA synthesis is mainly due to polymerase beta-like activity.     

Effect of 5-azacytidine on DNA methylation in Ehrlich's ascites tumor cells

5-Azacytidine inhibited in vivo DNA methylation in Ehrlich's ascites tumor cells depending upon the dose at which 5-azacytidine did not inhibit DNA synthesis significantly. This drug did not inhibit DNA methylation in vitro. The DNA methylase activity in ascitic cells decreased with the increasing dose of 5-azacytidine. Hypomethylated DNA was obtained from the 5-azacytidine treated ascitic cells.     

Kinetics of the decamer - dimer dissociation of arginine decarboxylase.

Aurintricarboxylic acid inhibited replicative DNA synthesis in nucleotide-permeable mouse ascites sarcoma cells. DNA polymerase activity assayed with activated DNA template and DNA polymerase purified partially from sarcoma cells was also inhibited by aurintricarboxylic acid. The inhibition of DNA polymerase activity was probably due to the inhibitory interaction of aurintricarboxylic acid with DNA polymerase. The replicative DNA synthesis might be inhibited by aurintricarboxylic acid interacting with some essential protein component(s), such as DNA polymerase of the replication machinery.     

Production and properties of a monoclonal antibody against human epidermal growth factor

A monoclonal antibody against human epidermal growth factor (hEGF) was obtained from a mouse hybridoma cell line. The purified monoclonal antibody from the ascites fluid of a mouse injected with one of the cell lines was specific for hEGF and did not cross-react with mouse EGF (mEGF). Its Kd value for hEGF was 1.4 X 10(-9) M. This monoclonal antibody inhibited the biological activities of hEGF, including its binding to the receptor of BALB/3T3 cells and its stimulation of DNA synthesis in the cells, but did not affect the activities of mEGF. The monoclonal antibody completely inhibited DNA synthesis stimulated by human urine from a patient without a tumor, but only partially inhibited the stimulatory activity in urine from a tumor-bearing patient.     

Alterations in 3H-thymidine incorporation into DNA induced by methyl CCNU (1-(2-chloroethyl)-3-(4-methyl cyclohexyl)-1-nitrosourea) in normal and tumorous tissues in vivo.

Methyl CCNU produces a suppression of tritiated thymidine (3H-TdR) incorporation into DNA in vivo in normal bone marrow and gastrointestinal tissues which is different in magnitude and duration from that seen in L1210 ascites tumor in the same animals. This suppression and recovery pattern is not seen in animals bearing L1210 ascites tumor resistant to MeCCNU. Where a different pattern of recovery is seen between normal host target tissues and tumor, the pattern can be exploited to increase the cure rate of animals bearing advanced L1210 ascites tumor with properly spaced second doses of MeCCNU. Additional information on the potential toxicity of second doses of MeCCNU can be predicted from knowledge of the time of recovery of DNA synthesis in the normal host target tissues.     

Effect of schisanhenol on the antitumor activity of adriamycin

Schisanhenol (Sal) did not diminish the antitumor activity of adriamycin in mice bearing P388 ascites tumor. Sal did not antagonize the suppressive effect of adriamycin on DNA synthesis and cell proliferation in an L1210 ascitic tumor cell culture. Furthermore, Sal at the concentration of 0.1, 0.25, or 1 mM accelerated adriamycin-dependent DNA damage in the presence of Fe3+ in vitro. It appears that Sal was able to protect against adriamycin induced heart mitochondrial toxicity, while it did not antagonize the antitumor activity of adriamycin.     

ALTERATIONS IN 3H-THYMIDINE INCORPORATION INTO DNA INDUCED BY METHYL CCNU [1-(2-CHLOROETHYL)-3-(4-METHYL CYCLOHEXYL)-1-NITROSOUREA] IN NORMAL AND TUMOROUS TISSUES IN VIVO

Methyl CCNU produces a suppression of tritiated thymidine (³H-TdR) incorporation into DNA in vivo in normal bone marrow and gastrointestinal tissues which is different in magnitude and duration from that seen in L1210 ascites tumor in the same animals. This suppression and recovery pattern is not seen in animals bearing L1210 ascites tumor resistant to MeCCNU. Where a different pattern of recovery is seen between normal host target tissues and tumor, the pattern can be exploited to increase the cure rate of animals bearing advanced L1210 ascites tumor with properly spaced second doses of MeCCNU. Additional information on the potential toxicity of second doses of MeCCNU can be predicted from knowledge of the time of recovery of DNA synthesis in the normal host target tissues.     

Purification of poly(ADP-ribose) polymerase from Ehrlich ascites tumor cells by chromatography on DNA-agarose.

Poly(ADP-ribose) polymerase with a high specific activity was obtained from Ehrlich ascites tumor cells by extraction of nuclei with 175 mM potassium phosphate, followed by chromatography on DNA-agarose. Electrophoretic analysis indicated that the preparation contained two proteins, one of which was shown to catalyze the synthesis of poly(ADP-ribose). As expected from results obtained by other workers, the synthesis was inhibited by nicotinamide and thymidine, and stimulated by DNA. Addition of histones gave inhibition of the synthesis, unless DNA was present in the reaction mixture.     

Shutoff of histone synthesis by Mengovirus infection of Ehrlich ascites tumor cells.

The histone synthesizing capacity of mengovirus-infected Ehrlich ascites tumor cells and of their corresponding postnuclear supernatants was investigated as a funcion of time post-infection. In addition, histone synthesis was compared with the synthesis of other basic host proteins under identical conditions. In the scope of mengovirus infection of Ehrlich ascites tumor cells the less complex fraction comprising basic protein, separated from the acidic proteins by carboxymethyl cellulose chromatography, can be regarded as a representative of total host protein. Histones and the remaining basic host proteins therefore are well suited as easily identifiable indicators of the host protein synthesizing potential of mengovirus-infected Ehrlich ascites tumor cells. The cessation of histone synthesis proceeds faster than the arrest of the synthesis of other basic host protein.     

Lack of correlation between thymidine kinase activity and changes of DNA synthesis with tumour age: an in vivo study in Ehrlich ascites tumour

Thymidine kinase (TK) and its isoenzymes were studied in relation to age of Ehrlich ascites tumour cells growing in vivo. Various steps of the pathway of thymidine through deoxynucleotide metabolism were studied: [3H]-thymidine cellular uptake and incorporation into DNA; the cellular nucleotide pools; and the concentration of thymidine in ascites. In addition, the proportion of cells in the various parts of the cell cycle and the bromodeoxyuridine labelling index were determined. Four isoenzymes at pI 4.1, 5.3, 6.9 and 8.3 were identified using isoelectric focusing. The TK activity declined with age of the tumour by about 90%, mostly due to a decrease of the isoenzyme at pI 8.3. However, this decline was neither related to the changes in DNA synthesis rate of the cells with tumour age, nor to the proportion of cells in S-phase or the bromodeoxyuridine (BrdU) labelling index. In contrast, the contribution of DNA synthesis via the thymidine salvage pathway relative to the total DNA synthesis increased from less than 1% at exponential growth to about 15% at plateau phase of growth. Blocking of DNA synthesis by aphidicolin did not change the TK activity. We therefore conclude that changes in TK activity and changes in cell growth are epiphenomena rather than causally related to each other. All nucleotide pools decreased with tumour age. The inhibition of TK by an increase in the deoxythymidine triphosphate pool could therefore be excluded. With a decrease of the TK activity during tumour growth, increasing amounts of TdR were excreted by the cells and accumulated in the ascites fluid. To explain our results on TK activity we propose a substrate cycle in which thymidine monophosphate supplied by de novo synthesis is dephosphorylated and is then either phosphorylated by TK to thymidine monophosphate or excreted by the cell.     

Loss of viral gene expression and retention of tumorigenicity by Abelson lymphoma cells.

Lymphomas induced by the Abelson murine leukemia virus (A-MuLV) were examined for the expression of biochemical and biological markers associated with A-MuLV transformation before and after in vivo growth in genetically distinguishable host mice. Although all tumors and clonal lines derived from them initially expressed the A-MuLV-encoded gag fusion protein p160, they ceased synthesis of this molecule after several weeks of growth in vivo as ascites tumors. Transplanted clonal lines continued to express the alloantigenic marker H-2b and the isoenzyme marker Gpi-1b of the donor tumor cells, indicating that the cells were of donor and not host origin. Examination of cellular DNA obtained from p160-positive and derivative p160-negative lines indicated that p160-negative clones had lost A-MuLV-specific proviral sequences as detected by hybridization with several probes. Although the clonal lines no longer expressed p160, they retained their malignant phenotype and continued to express the Abelson antigen, a cell surface marker associated with A-MuLV lymphomagenesis. Continued expression of the A-MuLV genome was not required for maintenance of oncogenic potential under these conditions of in vivo tumor growth.     

Control of Ehrlich cell division by zinc

The nutritional requirement for zinc in the proliferation of normal and malignant cells has been demonstrated in a number of animal studies. A distinction is made between the effect of zinc status upon the host during carcinogenesis and tumor growth. The present studies focus on the Ehrlich ascites tumor in mice fed a semipurified zinc-deficient diet along with defined concentration of zinc in the drinking water. This model of zinc deficiency is compared with others in which chelating agents are used to create zinc-deficient conditions or the microorganismEuglena gracilis is examined in a defined zinc-deficient medium. It is reported here that Ehrlich cells remain quiescent for several weeks in severely deficient mice, suggesting their restriction to a G₁ or G₀ state of the cell cycle. The kinetics of thymidine and uridine uptake and incorporation into DNA and RNA in Zn-normal and Zn-deficient tumors is consistent with the inhibition of thymidine kinase and DNA polymerase in the Zn-deprived system, but with little effect on RNA synthesis. The concentration of metabolites of these labeled nucleosides in Ehrlich cells is also consistent with a primary effect upon thymidine kinase. Although the ascites fluid Zn is depressed in Zn deficiency, total cellular zinc and its distribution among cell fractions is not significantly affected. It is suggested that these effects are specific in nature and not the result of a general lack of zinc for zinc metalloproteins and other binding sites in the cell.     

Cytotoxicity and mode of action of vanada- and niobatricarbadecaboranyl monohalide complexes in human HL-60 promyelocytic leukemia cells

Vanada- and niobatricarbadecaboranyl monohalide complexes proved to be potent cytotoxic agents against murine and human leukemia and lymphoma growth as well as HeLa suspended uterine carcinoma. The vanada complex reduced the growth of KB nasopharynx, Hepe liver, HCT-8 ileum and 1-A9 ovary solid carcinomas. A mode of action study in human HL-60 promyelocytic leukemia cells showed that DNA and purine de novo syntheses were significantly inhibited with suppression of the regulatory enzymes activities of DNA polymerase alpha and PRPP-amido transferase. There was moderate inhibition of RNA synthesis and m-RNA polymerase activity. These complexes did not inhibit human topoisomerase I or II activity, although the niobium complex nicked the DNA. The complexes did activate caspases 3, 6 and 9 which are linked to apoptosis programmed cell death. These vanada- and niobatricarbadecaboranyl monohalide complexes appear to be more specific in their effects on leukemia cell metabolism than other sandwich complexes which have broad effects on multiple enzymes.     

Repair of benzo[a]pyrene-initiated DNA damage in human cells requires activation of DNA polymerase alpha

Normal human fibroblasts treated with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) yielded DNA polymerase alpha with elevated levels of activity, incorporated [3H]thymidine as a function of unscheduled DNA synthesis, and exhibited restoration of normal DNA-strand length as a function of unscheduled DNA synthesis. Lipoprotein-deficient fibroblasts treated with BPDE did not show elevated levels of DNA polymerase alpha activity, exhibited minimal [3H]thymidine incorporation, and had fragmented DNA after 24 h of repair in the absence of lipoprotein or phosphatidylinositol supplementation. When DNA polymerase beta activity was inhibited, cells with normal lipoprotein uptake exhibited [3H]thymidine incorporation into BPDE-damaged DNA but did not show an increase in DNA-strand length. DNA polymerase alpha activity and [3H]thymidine incorporation in lipoprotein-deficient fibroblasts increased to normal levels when the cells were permeabilized and low-density lipoproteins or phosphatidylinositol were introduced into the cells. DNA polymerase alpha isolated from normal human fibroblasts, but not from lipoprotein-deficient fibroblasts, showed increased specific activity after the cells were treated with BPDE. When BPDE-treated lipoprotein-deficient fibroblasts were permeabilized and 32P-ATP was introduced into the cells along with lipoproteins, 32P-labeled DNA polymerase alpha with significantly increased specific activity was isolated from the cells. These data suggest that treatment of human fibroblasts with BPDE initiates unscheduled DNA synthesis, as a function of DNA excision repair, which is correlated with increased activity of DNA polymerase alpha, and that increased DNA polymerase alpha activity may be correlated with phosphorylation of the enzyme in a reaction that is stimulated by low-density lipoprotein or by the lipoprotein component, phosphatidylinositol.