The interaction of dianhydrogalactitol with DNA in cultured Yoshida sarcoma cells

Using alkaline sucrose gradient sedimentation centrifugation it was found that treatment of Yoshida sarcoma cells in culture for 1 h with increasing concentrations of dianhydrogalactitol (DAG) enhanced the sedimentation rate of DNA in a dose-dependent manner. There was no difference between the amount of protein which co-sedimented with DNA released from treated and untreated cells. When DNA was extracted from the cells using a p-amino-salicylate-phenol mixture, the protein content of DNA seemed not to be affected by DAG. The possibility that DAG could form interstrand cross-linking in cellular DNA was suggested from renaturation studies. The appearance of a fast sedimenting DNA in the alkaline sucrose gradient and the evidence for a cross-linked DNA detected by renaturation technique, only appeared later than 6 h after treatment. A similar delayed effect on the depression in the rate of DNA synthesis was also observed. These data suggest that the inhibition of DNA synthesis may be related to the delayed formation of DNA interstrand cross-linked.     

Conversion of dNTP to dNMP dependent on DNA synthesis in isolated Yoshida sarcoma nuclei

Nuclei isolated from Yoshida sarcoma cells had activity for conversion of dGTP to dGMP dependent on DNA synthesis. The ratio of nucleotide generation/generation + incorporation was 0.4 ± 0.1, indicating that approx. 40% of the incorporated dGMP was excised. Two lines of evidence indicated the dependence of this activity on DNA synthesis. (1) The activity was observed only in the presence of ATP, which is essential for nuclear DNA synthesis. (2) Inhibitors of DNA synthesis, such as N-ethylmaleimide, aphidicolin, spermine and KCl, also inhibited ATP- or DNA synthesis-dependent dGMP generation. Although nuclei contain nucleoside triphosphatase (N-nucleotidase), this enzyme was not involved appreciably in DNA synthesis-dependent dGMP generation. The reason for this was explained by the following findings. (a) Inhibitors did not decrease dGMP production in the complete absence of DNA synthesis. (b) Inhibitors did not inactivate N-nucleotidase to the same degree as they inhibited DNA synthesis-dependent dGMP generation. (c) Addition of ATP reduced dGTP hydrolysis catalyzed by N-nucleotidase. (d) GDP had no appreciable effect on DNA synthesis-dependent dGMP generation, but had a diluting effect on dGMP production catalyzed by N-nucleotidase. These results show that the pathway of dGMP generation in isolated nuclei was switched on addition of ATP from a N-nucleotidase-catalyzed one to a DNA polymerase-exonuclease-catalyzed one.     

Effects of alkylating agents on the DNA replication of cultured Yoshida sarcoma cells

Logarithmically growing Yoshida sarcoma cells were treated for 1 h with low (2 decades cell kill) or high (more than 6 decades cell kill) doses of alkylating agents. Pulse and chase labelled DNA from treated cells were studied by alkaline sucrose gradient centrifugation. Nitrogen mustard (HN-2), 4-hydroperoxycyclophosphamide (CY-OOH), melphalan (L-PAM) and chlorambucil (CA) had no effect on the elongation rate of newly replicated DNA, both at low and high doses, although per cell the rate of DNA synthesis declined as inferred from the rates of [³H] thymidine incorporation compared to the increase in numbers of S phase cells in the treated populations. It is concluded that these drugs act specifically on the initiation step of the DNA replication, leaving chain elongation undisturbed. At low doses the chemically related sulphur mustard (SM) had also no effect on the maturation of new DNA but at high doses a decreased elongation rate was observed. A transient inhibition of chain growth was observed following treatment with a low dose of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). In contrast, the intercalating agent adriamycin showed a severe but delayed effect resulting in an almost complete block of the maturation.     

Studies on Ehrlich ascites tumour cells: DNA synthesis,and template activity of chromatin for DNA polymerase

Summary Chromatin obtained from Ehrlich ascites cells on different days after cell inoculation has been assayed for its template activity with added DNA polymerase 1. We have found that the template activity is 2 times higher in 7–8 day cell chromatin than in 4-day chromatin. Studies with added polylysine indicate that this increase reflects an increase in initiation sites rather than in accessibility to the enzyme. We have measured the growth fraction, mitotic index and rate of DNA chain growth in the intact cells. The results show that there is a large decrease in growth fraction with age of tumour, the number of cells dropping out of cycle approximately doubling over the period studied. The overall rate of chain growth decreases in the later stages of growth but in a small proportion of cells there is an increase in rate with fewer replicons involved in DNA synthesis. We suggest that in the ascites cells there is a decrease in level of repair and replicative enzymes with age of tumour; this would account both for the increase in initiation sites in the chromatin DNA, for the decrease in number of cells in cycle and for the overall decreased rate of chain growth.     

Effect of methyl methanesulphonate (MMS) and methylene dimethanesulphonate (MDMS) on the template activity of DNA isolated from MDMS-sensitive and -resistant Yoshida cells.

Both methylene dimethanesulphonate (MDMS) and methyl methanesulphonate (MMS) cause the template activity of Yoshida cell DNA to decrease in a dose-dependent manner. The MDMS-resistant subline of Yoshida tumour is less sensitive in terms of DNA template activity than the MDMS-sensitive subline towards both agents. Although the difference in sensitivity is not reflected in the survival data after each agent, it does suggest that the DNA from each cell line differs in its capacity to function as an efficient template.     

The effect of dibromodulcitol on resting and dividing lymphoid cells

The effect of dibromodulcitol (DBD) on the incorporation of labelled precursors into DNA and RNA fractions of PHA-stimulated human lymphocytes and of P388F lymphoma cells at various stages of their growth was studied. Both cell systems showed sensitivity to the drug within the concentration range of 1–10 μg/ml.When DBD was added before phytohaemagglutinin (PHA), human lymphocytes showed a DNA labelling that was more affected than RNA. In contrast, by adding DBD after PHA, RNA labelling was much more inhibited than DNA. In the latter case, the decrease in DNA labelling occurred only 24 h after drug treatment whereas RNA labelling was decreased 1 h after treatment. Levels of DBD which normally produced 30% inhibition in plating efficiency of P388F lymphoma cells affected uridine-5-T incorporation to a different extent at different stages of growth of the culture. Enhanced RNA labelling occurred in early exponential stage while at later stages of growth, RNA synthesis was depressed.     

Changes in template activity and structure of nuclei from WI-38 cells in the prereplicative phase.

Quiescent confluent monolayers of WI-38 fibroblasts were stimulated to proliferate by either adding 10% fetal calf serum or by trypsinization and replating at lower density. The length of the prereplicative phase was 12 hr after serum stimulation and 18 hr after trypsinization and replating at lower density. Nuclei were isolated from WI-38 cells at different time intervals after either type of stimulation and their template activity, circular dichroism spectra, and ability to bind ethidium bromide were investigated. All these parameters were similarly increased after either type of stimulation. However, these changes, like the onset of DNA synthesis, were delayed 6 hr in cells trypsinized and replated at lower density. While there were no detectable changes in nuclear protein content after serum stimulation, at least 40% of nuclear protein, mostly nonhistone chromosomal proteins, were lost after trypsinization. The amount of nuclear proteins returned to prestimulation levels only 6-8 hr after replating. These data seem to suggest that nonhistone chromosomal proteins lost by trypsinization are essential for the entrance of WI-38 cells into the "prereplicative phase".     

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.     

Enhanced activity of tRNA-pseudouridine synthetase in Yoshida ascites sarcoma.

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 $\text{E. coli}$ 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.     

The effect of bisulfite modification on the template activity of DNA for DNA polymerase I

Cytosine residues of poly(C) and heat-denatured calf thymus DNA were transformed into 5,6-dihydrouracil-6-sulfonate (U(SO⁻₃)) residues by treatment with bisulfite. The poly(U(SO⁻₃)₂, C₃) and poly(U(SO⁻₃)₉, C₁) prepared did not form inter-base binding with either poly(A) or poly(I) as judged by the absence of hypochromicity in ultraviolet absorbance. U(SO⁻₃) residues in the DNA inactivated it to serve as template for E.coli DNA polymerase I, while the template activity was restored by conversion of the U(SO⁻₃) residues into U.     

Reconstitution of endogenous DNA synthesis in isolated nuclei and template activity of chromatin with respect to mode of inhibition by aphidicolin

We succeeded in reconstituting the endogenous nuclear DNA synthesis of the sea urchin. Endogenous DNA synthesis of isolated nuclei was reconstituted by mixing the salt-treated nuclei (chromatin exhibiting essentially no endogenous DNA synthesis) and the salt extract containing DNA polymerase-alpha. DNA synthesis in this reconstitution system showed a level of activity and a mode of inhibition by aphidicolin similar to those of the original isolated nuclei (noncompetitive with respect to dCTP). On the other hand, the inhibitory mode was competitive with respect to dCTP in DNA synthesis in the reconstituted system obtained from the chromatin and purified DNA polymerase-alpha, indicating that some other factor(s) in addition to DNA polymerase-alpha is necessary for the reconstitution with reference to the inhibitory mode of aphidicolin. We also studied the template activity of the chromatin. When chromatin was used as a template, inhibition by aphidicolin of DNA polymerase-alpha was noncompetitive and uncompetitive with respect to the template at high and low concentrations, respectively. Treatment of chromatin with 5 M urea gave urea-treated chromatin (nonhistone protein-deprived chromatin) and the extract (mainly nonhistone protein fraction). Inhibition by aphidicolin of DNA polymerase-alpha was uncompetitive with respect to the urea-treated chromatin. However, when chromatin reconstituted from the urea-treated chromatin and the extract was used as a template, the inhibitory mode by aphidicolin was similar to that with original chromatin, indicating that the nonhistone protein fraction contained factor(s) which modified the inhibitory mode of aphidicolin. Thus, the inhibitory mode of aphidicolin is a useful parameter for monitoring the resolution and reconstitution of endogenous DNA synthesis of isolated nuclei.