The mechanism of inhibition of DNA replication in HeLa S3 cells by the antitumor drug Ledakrin and other antitumor 1-nitro-9-aminoacridines

These studies were aimed at characterizing the capability of an antitumor DNA-damaging drug, Ledakrin, and its analogs to inhibit DNA replication in HeLa S3 cells. The studied agents are extremely potent inhibitors of [³H]thymidine incorporation in whole cells. These compounds produced also a potent dose- and time-dependent inhibition of DNA synthesis in subcellular systems derived from drug-treated cells, as found by [³H]dGTP incorporation in cellular lysates and nuclei. Experiments in which nuclei from control and drug-treated cells were supplemented with cytoplasmic fractions from either control or drug-treated cells, or with exogenous DNA, demonstrate that Ledakrin and other 1-nitro-9-aminoacridines inhibit DNA replication in HeLa S3 cells by interfering with the DNA template, while not affecting DNA polymerase(s) or other enzymes and replication factors. The negligible effect of Ledakrin added to lysates or nuclei from untreated cells suggests that metabolic activation is a prerequisite for replication inhibition by Ledakrin. Analysis of the size of newly synthesized DNA, by alkaline sucrose gradient sedimentation, indicates that Ledakrin does not inhibit the initiation of replication but does interfere with chain growth. Impairment of DNA replication by 1-nitro-9-aminoacridines seems to originate from DNA damage and to result in the inhibition of cellular growth.     

Transient Stimulation of Deoxyribonucleic Acid-Dependent Ribonucleic Acid Polymerase and Histone Acetylation in Human Embryonic Kidney Cultures Infected with Adenovirus 2 or 12: Apparent Induction of Host Ribonucleic Acid Synthesis

The synthesis of cell-specific ribonucleic acid (RNA) appeared to be stimulated in human embryonic kidney (HEK) cultures infected with adenovirus 2 or 12. Deoxyribonucleic acid (DNA)-RNA hybridization experiments revealed that by 44 to 70 hr after infection with either virus, the relative amount of pulse-labeled RNA capable of hybridizing with HEK cell DNA increased considerably; such RNA was detected in both nuclear and cytoplasmic fractions. The main increase in apparent host RNA synthesis was preceded by (i) a relatively early transient stimulation of the DNA-dependent RNA polymerase activity in isolated nuclei, and (ii) a small but consistently observed increase in the rate of acetylation of lysine-rich and arginine-rich histone fractions. The Mn²⁺-(NH₄)₂SO₄ and Mg²⁺-activated RNA polymerase reactions measured in nuclei isolated from cells infected with adenovirus 2 or 12 were stimulated at about the same time; a rapid loss of polymerase activity followed. The augmentation of the two RNA polymerase reactions found in adenovirus 12-infected cells was independent of protein synthesis. After the initial increase, the acetylation rate of histones of cells infected with adenovirus 2 or 12 declined, until late in infection it was approximately 40 to 70% of the control cell rate.     

Cytoplasmic Compartmentalization of the Protein and Ribonucleic Acid Species of Vesicular Stomatitis Virus

The cytoplasmic sites of synthesis in L cells of the protein and ribonucleic acid species of vesicular stomatitis virus were studied by polyacrylamide gel electrophoresis after fractionation of membrane and other cytoplasmic components by the Caliguiri-Tamm technique. The viral spike protein (glycoprotein G) was found primarily associated with a smooth membrane fraction which is rich in plasma membrane; the G protein was also present in fractions containing rough endoplasmic reticulum. The nonglycosylated envelope protein S (also called M) was found in the smooth membrane fractions but was more abundant in endoplasmic reticulum-enriched fractions. Longer labeling resulted in detection of nucleoprotein N, as well as other minor nucleocapsid proteins L and NS₁, in the cellular membrane fractions. The N protein appeared to be made in membrane-free cytoplasm along with progeny ribonucleic acid and later became associated with membrane containing G and S viral proteins.     

Dna Synthesis Rate Changes During the S Phase In Mouse Epidermis

The in vivo DNA synthesis rate throughout the S phase of mouse epidermal cells was investigated. Epidermal basal cells were isolated at various times of the day from normal animals injected with [³H]TdR 30 min before sacrifice, and from pulse-labelled animals with regenerating and growth-inhibited epidermis. the cells were analysed by DNA flow cytometry combined with cell sorting. Cells from successive fractions of the S phase were sorted on glass slides and subjected to quantitative [³H]TdR autoradiography. The results confirmed the presence of unlabelled (slowly replicating) cells in the S phase, the proportion of which was circadian stage-dependent with minimum values at midnight and in the early morning. the DNA synthesis rate throughout the S phase showed a general trend with high values in the mid-fractions, a pattern which was similar in normal and in growth perturbed epidermis. In the early morning the DNA synthesis rate pattern was bimodal with maxima both in the first and second half of the S phase, with a corresponding trough in mid-S. At this time of day the cell progression rate through S is at its maximum, indicating a relationship between the overall DNA synthesis rate and the rate distribution pattern through S.     

DNA polymerase activities during erythropoiesis : Effects of erythropoietin, vinblastine, colcemid, and daunomycin

Erythropoietin stimulates DNA synthesis in the spleen of the polycythemic mouse with the maximum effect occurring approx 48 h after the hormone is administered. The increase in DNA synthesis is accompanied by morphologic evidence of increased erythropoiesis, increased ⁵⁹Fe incorporation into heme, and an increase in the activity of the cytoplasmic high molecular weight DNA polymerase (DNA polymerase-α). In contrast, the activity of the low molecular weight DNA polymerase (DNA polymerase-β) does not significantly change after administration of erythropoietin. Vinblastine, colcemid, and daunomycin prevent the effects of erythropoietin on mouse spleen, so that increases in DNA synthesis, DNA polymerase-α, and ⁵⁹Fe incorporation do not occur. DNA polymerase-α may have a short half-life in cells since its activity is barely detectable 12 to 24 h after administration of inhibitors of cellular proliferation or nucleic acid synthesis. The half-life of DNA polymerase-β may be long since it is unaffected by these inhibitors. Cytoplasmic rather than nuclear DNA polymerases appear to play a major role in erythropoietin-stimulated DNA synthesis and replication of erythroid cells.     

DNA polymerases in midgestation mouse embryo, trophoblast, and decidua

The DNA polymerases of midgestation mouse embryo, trophoblast, and decidua have been examined. A low molecular weight, nuclear. DNA-dependent polymerase (D-DNA polymerase) and a higher molecular weight cytoplasmic enzyme were found in all three cell types. A DNA polymerase which utilized the poly(A) strand of oligo(dT) · poly(A) as template (R-DNA polymerase) was also found in the three cell types. This enzyme was present both in the nucleus and the cytoplasm. All enzyme levels were highest in the rapidly dividing embryonic cells, substantially lower in the DNA replicating but nondividing trophoblast cells, and lowest in the nonreplicating, nondividing decidual cells. Our observations are consistent with the idea that the nuclear and cytoplasmic D-DNA polymerases are under coordinate control. The relationship of these enzymes to DNA synthesis in vivo is discussed.     

Nucleic Acids Synthesis of Nuclear Polyhedrosis Virus in Cultured Embryonic Cells of Silkworm

Embryos of the silkworm, Bombyx mori L., were dispersed by trypsin and the dissociated cells were cultured for infection with nuclear polyhedrosis virus (NPV) of the silkworm. The monolayer and suspension cultures were infected with NPV. RNA and DNA syntheses in the normal and NPV-infected cells were measured by incorporation of ³²P into RNA and DNA fractions. RNA and DNA syntheses in the cells after infection significantly increased over those in control cells (mock infection). The effects of actinomycin D, chloramphenicol and mitomycin C on RNA and DNA syntheses in infected cells were examined. The syntheses were inhibited by the antibiotics. It was suggested that the cellular DNA synthesis was inhibited by the viral infection, because the mitomycin C-resistant DNA synthesis was found in the normal cells but not in the infected cells treated with mitomycin C. The rate of DNA synthesis induced by NPV was immediately dropped to that of control cells by addition of chloramphenicol, while the RNA synthesis induced by NPV was not affected for 6 hr after the addition of chloramphenicol. If the antibiotic did not affect the size of precursor pools, this event suggested that the RNA polymerase concerned with viral RNA synthesis was more stable than the DNA polymerase participating in the viral DNA synthesis. The viral DNA as templates for RNA and DNA syntheses was decomposed by mitomycin C.     

Deoxyribonucleic acid poymerase of BHK-21/C13 cells. Heterogeneity, molecular asymmetry and subcellular distribution of the enzymes.

Nuclear and cytoplasmic fractions were prepared from exponentially-growing BHK-21/C13 cells; DNA polymerase was extracted from them and analysed by gel filtration and sucrose-density-gradient centrifugation. DNA polymerase I is heterogeneous comprising species covering a considerable range of molecular weights. These have been tentatively identified as four subspecies of apparent molecular weights 900000-1000000 (IA), 460000-560000 (IB), 270000-320000 (IC) and 140000-200000 (ID), as assessed by gel filtration through Sepharose 6B. DNA polymerase II has a mol.wt. of 46000 +/- 4000 as assessed by gel filtration on Sepharose 6B, and 48000 +/- 2000 as assessed by gel filtration on Sephadex G-100. Sedimentation analyses on sucrose density gradients showed that the DNA polymerase I species had sedimentation coefficients predominantly in the range 6-8 S. DNA polymerase II had predominantly a sedimentation coefficient of 3.2 S although a component with lower sedimentation coefficient was found. The lack of correlation between the molecular weights derived from gel filtration and the sedimentation coefficients is attributed to molecular asymmetry. DNA polymerase I was found to be associated predominantly with the cytoplasm although certain types of nuclear preparation contained large amounts of it. DNA polymerase II was found to be mostly if not exclusively in nuclear preparations.     

The Polymerase Activity of Mammalian DNA Pol ζ Is Specifically Required for Cell and Embryonic Viability

DNA polymerase ζ (pol ζ) is exceptionally important for maintaining genome stability. Inactivation of the Rev3l gene encoding the polymerase catalytic subunit causes a high frequency of chromosomal breaks, followed by lethality in mouse embryos and in primary cells. Yet it is not known whether the DNA polymerase activity of pol ζ is specifically essential, as the large REV3L protein also serves as a multiprotein scaffold for translesion DNA synthesis via multiple conserved structural domains. We report that Rev3l cDNA rescues the genomic instability and DNA damage sensitivity of Rev3l-null immortalized mouse fibroblast cell lines. A cDNA harboring mutations of conserved catalytic aspartate residues in the polymerase domain of REV3L could not rescue these phenotypes. To investigate the role of REV3L DNA polymerase activity in vivo, a Rev3l knock-in mouse was constructed with this polymerase-inactivating alteration. No homozygous mutant mice were produced, with lethality occurring during embryogenesis. Primary fibroblasts from mutant embryos showed growth defects, elevated DNA double-strand breaks and cisplatin sensitivity similar to Rev3l-null fibroblasts. We tested whether the severe Rev3l^-/- phenotypes could be rescued by deletion of DNA polymerase η, as has been reported with chicken DT40 cells. However, Rev3l^-/- Polh^-/- mice were inviable, and derived primary fibroblasts were as sensitive to DNA damage as Rev3l^-/- Polh^+/+ fibroblasts. Therefore, the functions of REV3L in maintaining cell viability, embryonic viability and genomic stability are directly dependent on its polymerase activity, and cannot be ameliorated by an additional deletion of pol η. These results validate and encourage the approach of targeting the DNA polymerase activity of pol ζ to sensitize tumors to DNA damaging agents.     

The virus-specific RNA species in free and membrane-bound polyribosomes of transformed cells replicating murine sarcoma-leukemia viruses

Ribonucleic acids extracted from polyribosomes of cells replicating murine sarcoma-leukemia viruses (M-MSV(MLV)) were resolved by electrophoresis on 2.5% polyacrylamide gels. Virus-specific RNA was detected by hybridization of RNA in the gel fractions with the ³H-DNA product of the viral RNA-directed DNA polymerase. The postmicrosomal supernatant and the free polyribosomes contained one peak of virus-specific RNA with a molecular weight of about 2.9 × 10⁶ (35S). In contrast, the microsomes and the membrane-bound polyribosomes contained two peaks of virus-specific RNA in approximately equal amounts with molecular weights of 2.9 × 10⁶ (35S) and 1.5 × 10⁶ (approximately 20S). The high molecular weight viral RNA species might serve as polycistronic mRNA for the synthesis of large polypeptides that are cleaved to form the smaller viral proteins.     

Activities of DNA polymerases and RNA polymerases detected in situ in growing and differentiating cells of root cortex

Summary Activities of DNA polymerases and RNA polymerases were studied by autoradiographic methods in growing and differentiating root cortex cells ofZea mays — a species in which endomitosis occurs — andTulipa kaufmanniana — in which this process does not occur. InTulipa kaufmanniana, the highest activity of DNA polymerase appears in the nuclei of meristematic zone during the S phase of the cell cycle. InZea mays, endomitotic replication of DNA occurs in all growth and differentiation zones and the activity of DNA polymerase in the nuclei is similar to that in the meristematic zone. In both species, nuclear RNA synthesis, measured with³H uridine incorporation, is highest in the meristematic zone and declines steadily with development. Activity of nuclear RNA polymerase is present in all developmental zones in both species and is similar to that in the meristematic zone.³H uridine incorporation into nucleoli decreases markedly in both species, whereas the activity of nucleolar RNA polymerase remains at a high level in all root segments inZea mays and decreases slightly inTulipa kaufmanniana.It is argued that the differences between the incorporation of³H uridine and that or³H UMP may be caused by a reduction of the pool of endogenous ribonucleoside triphosphates. Marked activities of DNA polymerase and RNA polymerase in cytoplasm are possibly related to the growth and division of plastids and mitochondria.     

THE NATURE OF THE INCREASED RATE OF DNA SYNTHESIS IN SCRAPIE-AFFECTED MOUSE BRAIN

—In vivo studies have been made of the incorporation of radioactive thymidine into acid insoluble components of control and scrapie-affected mouse brain. Experiments with hot trichloroacetic acid extracts of brain and with purified preparations of DNA have confirmed that there is an increased rate of DNA synthesis in scrapie brain which is entirely associated with nuclei. No increase was found in the rate of DNA synthesis in cytoplasmic fractions of scrapie brain. Hydroxyapatite chromatography of heat denatured and renatured DNA suggests that in scrapie brain there is a similar increase in the rates of synthesis of the poorly, moderately and highly reiterated (i.e. satellite) species of nuclear DNA. Experiments involving brain dissection indicate that the increased rate of DNA synthesis in scrapie does not take place exclusively in the subependymal layer of the lateral ventricles. From these and previously reported studies using radioautographic techniques it is concluded that the increased DNA synthesis in scrapie brain is not associated specifically with cells undergoing mitosis.     

Biochemical aspects of cardiac muscle differentiation. Deoxyribonucleic acid synthesis and nuclear and cytoplasmic deoxyribonucleic acid polymerase activity.

DNA synthesis and DNA polymerase activity have been measured in terminally differentiating cardiac muscle of the rat. Incorporation of [3H]thymidine into DNA essentially ceases by the 17th day of postnatal development. Cardiac muscle of neonatal rats contains at least two molecular species of DNA polymerase: a 3.5 S DNA polymerase that can be extracted from nuclei with 0.2 m potassium phosphate and a 6 to 8 S soluble cytoplasmic DNA polymerase. The nuclear DNA polymerase in crude extracts has a pH optimum of 9.0 and is more active with native DNA than with denatured DNA as the primer-template. The cytoplasmic DNA polymerase in crude extracts has a pH optimum of 7.5 and is more active with denatured DNA. The activity of the 6 to 8 S cytoplasmic DNA polymerase decreases 80-fold from day 1 to day 17 after birth, which correlates temporally with the reduced rate of DNA synthesis. The activity of the 3.5 S nuclear DNA polymerase remains relatively constant throughout postnatal development. Mixing experiments (assay of neonatal enzyme extracts with adult enzyme extracts) gave additive results, suggesting that the decline in 6 to 8 S DNA polymerase activity apparently is not due to the presence of absence of soluble activators or inhibitors at different times during development. These studies may provide a system which can be used to investigate the control of DNA synthesis and cellular proliferation during the terminal stages of cardiac muscle differentiation.     

Intracellular localization and metabolism of DNA polymerase α in human cells visualized with monoclonal antibody

Indirect immunofluorescence microscopy with monoclonal antibody against DNA polymerase α revealed the intranuclear localization of DNA polymerase α in G1, S, and G2 phases of transformed human cells, and dispersed cytoplasmic distribution during mitosis. In the quiescent, G0 phase of normal human skin fibroblasts or lymphocytes, the α-enzyme was barely detectable by either immunofluorescence or enzyme activity. By exposing cells to proliferation stimuli, however, DNA polymerase a appeared in the nuclei just prior to onset of DNA synthesis, increased rapidly during S phase, reached the maximum level at late S and G2 phases, and was then redistributed to the daughter cells through mitosis. It was also found that the increase in the amount of DNA polymerase a by proliferation stimuli was not affected by inhibition of DNA synthesis with aphidicolin or hydroxyurea.     

Diurnal variations in the number of granulosa cells synthesizing DNA in relation to follicle size in the mouse ovary

Summary Autoradiographs of mouse ovaries prepared after pulse-labelling with ³H-thymidine at 4-hourly intervals over a 24-hour period show, that in the early stages of follicle development there is a diurnal variation in the number of granulosa cells synthesizing DNA. This is not found in later stages.The diurnal variations are correlated with low proliferation rates of the cell populations and with growth fractions below one.It is concluded, that diurnal variations in the number of granulosa cells synthesizing DNA are due to variations in the growth fractions of the granulosa cell populations.     

Requirement of a Cytoplasmic Fraction for Synthesis of SV40 Deoxyribonucleic Acid in Isolated Nuclei*.

About 50% of the SV40 DNA in the process of replication (sv40(ri) dna) completed replication in lysates of infected BSC-1 cells by conversion to covalently closed, superhelical SV40 DNA (SV40(I) DNA). Fractionation of the lysate into nuclear and cytoplasmic components blocked 99% of the synthesis of SV40(I) DNA in the purified nuclei. The reconstituted system, made by adding back the cytoplasmic fraction before incubation at 30 degrees, completely restored the in vitro level of SV40(I) DNA synthesis. Preliminary characterization of the activity found in the cytoplasmic fraction suggested it was a soluble, heat-labile protein (or proteins) with a minimum molecular weight of about 30,000 and an active sulfhydryl group. The activity was present in both infected and uninfected monkey cells, and at a lower level in mouse, hamster, and human cell lines. Neither serum starvation nor cycloheximide treatment of cells diminished the activity in the cytoplasmic fraction. Purified cytoplasmic DNA polymerase from KB cells did not substitute for the cytoplasmic fraction which was required for elongation of newly synthesized DNA strands. In the absence of the cytoplasmic fraction, conversion of 4 S DNA into longer strands was inhibited, and SV40(RI) DNA appeared to be broken specifically at the replication forks.