Eukaryotic DNA repair is blocked at different steps by inhibitors of DNA topoisomerases and of DNA polymerases α and β

Inhibitors of (a) DNA topoisomerases (novobiocin and nalidixic acid) and of (b) eukaryotic DNA polymerases α (cytosine arabinoside) and β (dideoxythymidine) blocked different steps of DNA repair, demonstrated by the effects of the inhibitors on the relaxation of supercoiled DNA nucleoids following treatment of human cell cultures with ultraviolet light (1–3 J/m²) or MNNG (5 or 20 μM) and the subsequent restoration of the supercoiled nucleoids during repair incubation. Changes in the supercoiling of nucleoid DNA were assayed by analysis of their sedimentation profiles in 15–30% neutral sucrose gradients. Inhibition of repair by novobiocin was partially reversible; upon its removal from the culture medium, the nucleoid DNA of repairing cells became relaxed. The DNA polymerase inhibitors allowed the initial relaxation of DNA after treatment of the cells with ultraviolet or MNNG but delayed the regeneration of rapidly-sedimenting (supercoiled) nucleoid DNA for 2–4 h. Dideoxythymidine (1 mM) was more effective than cytosine arabinoside (1 μM) in producing this delay, but neither inhibitor by itself blocked repair permanently. Incubation of ultraviolet-irradiated cells with 1 μM cytosine arabinoside plus 1 mM dideoxythymidine blocked the completion of repair for 24 h, whereas incubation with 10 μM cytosine arabinoside or 5 mM dideoxythymidine produced only temporary repair delays of 2–4 h. Thus, it is likely that the two DNA polymerase inhibitors act upon separate targets and that both targets are involved in repair. It is concluded from these and from previous studies that (1) the DNA repair-sensitive target of novobiocin and nalidixic acid in vivo is not a DNA polymerase, but, rather, a DNA topoisomerase; (2) this target affects an initial step of DNA repair leading to the relaxation of supercoiled DNA; (3) the DNA polymerization step of repair may involve both α- and β-type DNA polymerases; and (4) in repair, one type of DNA polymerase may substitute for another.     

Characterization of liposomes prepared using a microemulsifier

A new type of device can prepare liposomes continuously, in large quantities and with excellent aqueous space capture efficiency. At initial lipid concentration of 300 μmol/ml these liposomes capture approx. 75% of cytosine arabinoside used as an aqueous space marker. Liposome size can be reduced by increasing the number of times the preparations are recycled through the microemulsifier. Liposomes less than 0.1 μm in diameter, as shown by electron microscopy, can be made easily. Liposomes prepared at 300 μmol/ml, composed of phosphatidylglycerol/phosphatidylcholine/cholesterol in a 0.1:0.4:0.5 molar ratio leaked less than 1% of entrapped cytosine arabinoside (Ara-C) at 4°C, and less than 10% Ara-C at 37°C plus serum, over a 48 h period. These liposomes could be useful for a number of applications including diagnostics, therapeutics and model membrane studies.     

Enhanced survival of ultraviolet-irradiated herpes simplex virus in cells exposed to antiviral agents

Enhanced survival of UV-irradiated HSV-1 is demonstrated in monkey cells exposed to inhibitors of viral DNA synthesis. Phosphonoacetic acid (PAA), adenine arabinoside (ara-A), and cytosine arabinoside (ara-C) pretreatment of infected cells is associated with concentration-dependent reactivation of UV-HSV-1. At concentrations that result in enhanced virus survival, inhibition of cell DNA synthesis is observed by either ara-A or ara-C, but not by PAA. Pretreatment of uninfected cells with acycloguanosine (ACG) is not associated with reactivation of irradiated HSV-1, and this is probably due to insufficient generation of ACG-triphosphate, the active inhibitor of viral and cell DNA synthesis.     

Induction of endoreduplication in Chinese hamsters V79 cells by cytosine arabinoside

Endoreduplication (ER) could be induced very effectively in Chinese hamster V79 cells exposed to cytosine arabinoside (1-β-D-arabinofuranosylcytosine; Ara-C). Cells were cultured for 48 hours in Ara-C containing medium. ER frequency increases rapidly after Ara-C release. About 60% of metaphase cells were endoreduplicated at 8–10 hours after release from Ara-C (5 μg/ml). Induction of ER also depends on Ara-C concentrations.     

Detection of inhibitors of protein and nucleic acid synthesis using oocytes of Xenopus laevis microinjected with the herpes thymidine kinase gene

We have developed an assay that measures the inhibition of protein synthesis and can be used in conjunction with a whole embryo bioassay that detects the ability of a chemical to cause fetotoxicity, malformation and abnormal growth. The assay involves microinjecting the herpes thymidine kinase gene into stage 6 oocytes of Xenopus laevis then exposing the oocytes to a test compound for 18-24 h. The inhibition of thymidine kinase (TK) expression caused by an inhibitor is then measured by simple enzyme assay. Protein synthesis inhibitors such as cycloheximide, puromycin and emetine all inhibited TK synthesis. Concentrations of cycloheximide (1.4 X 10(-4) mg/ml) and puromycin (0.04 mg/ml) near the 96 h embryo LC50 inhibited thymidine kinase expression by 78% and 97%, respectively but emetine (0.01 mg/ml) had no effect. However, 0.1 mg/ml emetine inhibited TK synthesis by almost 50%. The RNA synthesis inhibitor, actinomycin D (0.013 mg/ml) inhibited TK expression by 61%. DNA synthesis inhibitors hydroxyurea (2.0 mg/ml), cytosine arabinoside (2.0 mg/ml) and ethidium bromide (0.02 mg/ml) failed to inhibit the expression of the TK gene even though these concentrations were near the 96 h embryo LC50. The whole embryo bioassay cannot differentiate the DNA synthesis inhibitors from the RNA and protein synthesis inhibitors but the oocyte assay can. This type of molecular test data can help separate classes of teratogens such as DNA synthesis inhibitors from nonteratogenic compounds such as protein synthesis inhibitors and allow the extrapolation of test data to other species.     

Small circular DNA in Drosophila melanogaster

Covalently closed small circular DNA isolated from Drosophila melanogaster is described. The small circular DNA is found in blastema stage eggs and in Schneider's cell culture line 2 and a cloned subline of line 2. It is heterogeneous in size, although the size distributions and mean sizes differ for each source. The small circular DNA from Schneider's line 2 cells ranges from 0.09-7.3 μm, with a mean contour length of 1.1 μm. This DNA has a buoyant density of 1.703 g/cc and appears to be present predominantly in the nuclear fraction of detergent-disrupted cells. The restriction enzyme EcoRI cleaves approximately 40% of the small circular DNA with a bias toward the larger size classes.Both logarithmic and stationary phase cells contain approximately 3–40 average sized small circular DNA molecules per cell, representing a maximum of 0.03% of the total cellular DNA. Exposure to cycloheximide or puromycin for 14 hr results in a 30 fold increase in the number of small circles per cell, but reduces the mean length of the circular DNA to 0.3 μm. The drug-amplified DNA has a buoyant density in the range of 1.698-1.703 g/cc. No amplification was seen in cells treated with either inhibitor for 3.5 hr. Ethidium bromide, cytosine arabinoside, β-ecdysone, and insulin all had no significant effect on the amount per cell of either small circular DNA or mitochondrial DNA.     

The repair of methyl methanesulphonate-induced lesions in the DNA of a murine lymphoma cell

The introduction of single-strand breaks into the DNA of a murine lymphoma (L5178Y) cell treated in vivo with methyl methanesulphonate (MMS) and the behaviour of these breaks on post-treatment incubation were studied. A large proportion of single-strand breaks present after MMS treatment could be repaired as shown by sedimentation in alkaline sucrose. Two inhibitors of DNA synthesis, hydroxyurea and cytosine arabinoside affected the repair process differently-hydroxyurea had only a small effect while cytosine arabinoside blocked repair and at some doses allowed further degradation of the DNA. It was also found that the level of ‘repair replication’ in the presence of cytosine arabinoside was lower than that found in the presence of hydroxyurea.     

Mitotic responses of the anterior pericardial wall of Biomphalaria glabrata (Mollusca) subjected to challenge

Using light microscope autoradiography and electron microscopy we studied the effect of juvenile hormone III (JHIII) and β-ecdysone insect molting hormone (β-ecd) on the replication of Tipula iridescent virus (TIV) in suspension cultured cells of Estigmene acrea. JHIII at a concentration of 87.5 μg/ml completely inhibited viral DNA synthesis, but upon removal of JHIII, [³H]thymidine was incorporated into the cytoplasm as detected by autoradiography and virions in developmental stages from the same cell samples were-readily seen by electron microscopy. β-ecd at a concentration of 17.5 μg/ml, unlike JHIII, permitted viral DNA synthesis in the presence of the hormone although at a reduced level when compared to TIV-infected cells. But the presence of β-ecd seemed to prevent capsid formation, although islands similar in fine structure to those of viroplastic centers were seen by electron microscopy. Once β-ecd was removed from the medium, TIV-inoculated cells appeared to synthesize new virions in a normal pattern. Both hormones inhibited host cell DNA synthesis in noninfected cells.     

Isolation and characterization of amphotericin B-resistant cell lines in Chinese hamster cells

Amphotericin B is a polyene macrolide antibiotic which interacts specifically with steroids in mammalian cell membranes. Amphotericin B-resistant (AMB^r) lines of stable phenotype have been isolated from cultured Chinese hamster (V79) cells. Three AMB^r clones (AMB^r-1, -2 and -3) isolated independently after treatment with nitrosoguanidine were resistant to ≥150 μg/ml of the antibiotic, while DNA synthesis as well as the colony-forming ability of the parental V79 cells was blocked by >80% of control in the presence of 20–50 μg/ml amphotericin B. The AMB^r cell line also exhibited increased resistance to other polyene macrolide antibiotics such as nystatin and pentamycin. Other agents, however, such as cytosine arabinoside or ricin, blocked DNA synthesis in AMB^r cells to the same extent as in V79 cells. The amphotericin B resistance phenotype was stably retained even after AMB^r cells were cultured in the absence of the drug for over 200 generations. The content of free cholesterol or its esters was significantly decreased in all three resistant clones. Furthermore, cholesterol synthesis from acetate as well as mevalonate was partly defective in AMB^r cells, compared with that in V79 cells.     

A requirement for DNA synthesis in foetal hepatocyte differentiation

In hepatocyte cultures derived from 15-day-old foetal rats, the appearance of the liver (L) form of pyruvate kinase is blocked when cytosine arabinoside is added on the 2nd day of culture. When added on the 3rd day of culture, the inhibitor of DNA synthesis does not prevent the appearance of the enzyme. If cytosine arabinoside is added on the 2nd day of culture and removed on the 4th day, the enzyme is detected by the 6th day of culture. The specificity of the action of cytosine arabinoside for the L form of pyruvate kinase is in contrast with the lack of effect observed on total protein synthesis and the activity of the embryonic (M2) form of the enzyme.     

5''-Methylthioadenosine in urine from normal subjects and cancer patients

A new type of device can prepare liposomes continuously, in large quantities and with excellent aqueous space capture efficiency. At initial lipid concentration of 300 mumol/ml these liposomes capture approx. 75% of cytosine arabinoside used as an aqueous space marker. Liposome size can be reduced by increasing the number of times the preparations are recycled through the microemulsifier. Liposomes less than 0.1 micron in diameter, as shown by electron microscopy, can be made easily. Liposomes prepared at 300 mumol/ml, composed of phosphatidylglycerol/phosphatidylcholine/cholesterol in a 0.1:0.4:0.5 molar ratio leaked less than 1% of entrapped cytosine arabinoside (Ara-C) at 4 degrees C, and less than 10% Ara-C at 37 degrees C plus serum, over a 48 h period. These liposomes could be useful for a number of applications including diagnostics, therapeutics and model membrane studies.     

Activation of a well behaved cell cycle in araC-treated V79 cells by caffeine

0.3–1.0 σmM araC (cytosine arabinoside) treatment of V79 cells produced inhibition of multiplication of cells which was accompanied by a large increase of cell size. In presence of 1–2 mM caffeine the inhibition of cell proliferation due to araC treatment was substantially reduced and cell-size increase was prevented; caffeine did not influence the uptake of araC by V79 cells. Flow microfluorometric analysis showed that caffeine induced a wave of cell cycle progression in 0.3 μM araC-treated cells. The cell cycle activated by caffeine in 0.3 μM araC-treated cells was largely well behaved; this was indicated by the fact that (1) prior to cell division cells achieved a tetraploid DNA content and (2) following cell division they had diploid DNA content as a result of which DNA homeostasis was maintained. At 1.0 μM araC concentration, however, extreme micronucleation was observed which gave rise to a substantial fraction of micronuclei with < G1 DNA content.     

Isolation and cell cycle analysis of temperature-sensitive mutants from chinese hamster cells

HeLa cell mitochondria were allowed to incorporate ³H-thymidine in a cell free system and the effect of ethidium bromide, cytosine arabinoside and cytosine arabinoside triphosphate on the labeling of mitochondrial DNA was studied. The labeled products, isolated by sedimentation velocity in CsCl-ethidium bromide two-step gradients, showed similar sedimentation profiles as in vivo labeled mtDNA. Cytosine arabinoside triphosphate and ethidium bromide strongly inhibited the labeling of mitochondrial DNA, whereas cytosine arabinoside appeared to be much less effective. Tritiated deoxycytidine was found to be incorporated by isolated mitochondria, whereas cytosine arabinoside was shown to enter the mitochondrial acid-soluble pool but not to be incorporated in acid-insoluble form. These results are in agreement with the previously reported findings of in vivo experiments.     

Cell line which is temperature-sensitive for cytokinesis

We have isolated several cell lines which are temperature-sensitive for growth. One of these appears to be temperature-sensitive for cytokinesis. It was isolated from a Syrian hamster cell line by selecting cells which were not killed by 1 μg/ml cytosine arabinoside at 39° but which grew normally at 31°. It shows an increased proportion of binucleate cells when shifted to the non-permissive temperature and time-lapse photomicroscopy shows that a high proportion of attempted mitoses fail at 39°, apparently at the stage of cytokinesis. The cells which have failed to complete mitosis reattach to the plate and have two normal-size nuclei but otherwise behave normally.     

Micellar electrokinetic capillary chromatography quantification of cytosine arabinoside and its metabolite,uracil arabinoside,in human serum

Cytosine arabinoside (Ara-C) is widely used to induce remission in adult granulocytic leukemia. High doses can be infused in refractory leukemia or in relapse. After injection, Ara-C is quickly metabolized to uracil arabinoside (Ara-U), the main inactive metabolite. We here described a micellar electrokinetic capillary chromatography (MECC) method to simultaneously determine Ara-C/Ara-U in human serum using 6-O-methylguanine as an internal standard. The assay was linear from 6.25 to 200 μg/ml with a quantification limit between 3 and 6 μg/ml. The analytical precision was satisfactory between 2 and 4.3% (within-run) and 3.7 and 7.3% (between-runs). This assay was applied to the analysis of serum from acute granulocytic leukemia patient treated by high doses cytarabine (3 g/m² body surface).     

Transformation by Murine Sarcoma Virus: Fixation (Deoxyribonucleic Acid Synthesis) and Development

Transformation of rat embryo cells by murine sarcoma virus (MSV) was contingent upon synthesis of deoxyribonucleic acid (DNA) during the first 12 hr of infection. Inhibition of DNA synthesis by thymidine (20 mm) or cytosine arabinoside (0.1 mm) resulted in the protection of cells from transformation by MSV. Transient suppression of DNA synthesis prior to infection or after a 12-hr delay had little effect on subsequent transformation, emphasizing the critical time period in in which DNA synthesis was necessary for intracellular fixation of the viral genome. These results are similar to those previously described for Rous sarcoma virus. Development of transformed cells after viral fixation was shown to be influenced by cellular density. Under conditions which allowed fixation of virus in confluent cellular monolayers, less than 20% of these cells developed into transformed foci.     

Aberrant double replication of segments of chromosomal DNA following DNA synthesis inhibition by cytosine arabinoside.

Using two different cell lines growing logarithmically, it is demonstrated that inhibition of DNA synthesis by cytosine arabinoside leads to a disruption of the sequence of replication of the chromosomal DNA. After release of the inhibition, some DNA synthesis is reinitiated in DNA segments replicated earlier in that S phase, leading to double replication of some DNA segments. This was directly demonstrated by showing that, following removal of the inhibitor, DNA was synthesized off template strands which had themselves been synthesized only 1–2 h before the addition of the inhibitor.     

Induction of myogenic differentiation in serum-free medium does not require DNA synthesis

Cells of the myogenic rat cell line L6 can be obtained as a confluent, quiescent population of undifferentiated myoblasts after growth in F12 medium supplemented with fetal calf serum. Myogenic differentiation can be induced in these cells by changing to Dulbecco's modified Eagle's (DME) medium containing insulin as the only protein component. Labeling of the cells with [3H]thymidine demonstrates that this induction of fusion occurs in the absence of DNA synthesis in about 85% of the cells. This result was confirmed using cytosine arabinoside: fusion of quiescent L6 cells was induced in the presence of this inhibitor of DNA synthesis. The myotubes formed in DME + insulin medium, with or without cytosine arabinoside, synthesize or accumulate proteins characteristic of differentiated muscle cells including myosin heavy and light chains, alpha-actin, alpha- and beta-tropomyosins, and the acetylcholine receptor. These experiments represent a direct demonstration that DNA synthesis is not required for the induction of myogenic differentiation in undifferentiated quiescent cells.