Masa-atsu Yamada

Aphidicolin does inhibit repair replication in HeLa cells.

Abstract Aphidicolin was shown to be a specific inhibitor of eukaryotic DNA polymerase α. We have examined the effect of aphidicolin on repair synthesis as well as replication of HeLa cell DNA, and found that it inhibits not only DNA replication but also UV-induced DNA repair in hydroxyurea-arabinosyl cytosine treated cells.

Localization of the replication point of mammalian cell DNA at the membrane

Abstract The replication site of DNA in HeLa S3 cells has been studied with “M-band” technique after Tremblay and others. When the cells are mixed with sodium lauroyl sarcosinate and Magnesium ion directly on the sucrose gradient and spun, virtually all the DNA is caught in the M-band, suggesting that mammalian cell DNA is attached to the nuclear membrane. After being treated with sodium lauroyl sarcosinate and sheared by a vortex-mixer, the DNA is found to have moved from the M-band to the supernatant. Pulse-labeled DNA is more resistant to shearing as compared with the bulk of DNA and chased away from the M-band during a subsequent growth. These results indicate that, as in the case of bacteria, the replication points of mammalian cell DNA are on the membrane.

Altered frequency of initiation sites of DNA replication in Werner's syndrome cells

SummaryDNA replication of cultured fibroblasts of early passage derived from Werners syndrome (adult progeria) patients and from normal subjects were compared by DNA fiber autoradiography. The frequency of replication initiation was decreased in Werners syndrome cells derived from five patients compared with that in normal cells derived from three persons of different ages. The rate of DNA chain elongation did not differ between Werners syndrome cells and normal cells.

A temperature-sensitive mutant of cultured mouse cells defective in chromosome condensation☆

Abstract A temperature-sensitive mutant, designated ts85, was isolated from a mouse mammary carcinoma cell line, FM3A. The ts85 cells grew at 33 °C (permissive temperature) with a doubling time of 18 h, which was almost the same as with wild-type cells, whereas the cell number scarcely increased at all at 39 °C (non-permissive temperature). When the ts85 cells were shifted from 33 to 39 °C, their DNA synthesis fell to below 1% of the initial value in 14 h. RNA or protein synthesis, however, was maintained at the initial levels for at least 14 h at 39 °C. Cytofluorometric analysis of asynchronous cultures and studies with synchronous cultures suggested that the bulk of the cells cultured at 39 °C for 12–18 h were arrested in late S and G2 phases. Electron microscopic observations revealed that chromatin was abnormally condensed into fragmented and compact forms, particularly around nucleoli, in about 80% of cells of an asynchronous culture incubated at 39 °C for 16 h. Cells in mitosis were not detected in such cultures and nuclear membrane and nucleoli were still intact. Such abnormal chromosome condensation was not observed in the ts85 cells at 33 °C or in wild-type cells at either temperature. Since these findings suggest that a ts gene product of ts85 cells is necessary for chromosome condensation, ts85 cells may represent a useful tool for establishing the mechanisms of chromosome condensation. The interrelationship between abnormal chromosome condensation and reduction in DNA synthesis of the ts85 cells is discussed.

Prolongation of S phase and whole cell cycle in Werner's syndrome fibroblasts

Abstract The cell cycle was determined in early passage fibroblasts from Werners syndrome and normal subjects. The average cell cycle time was prolonged in Werners syndrome cells due to changes in the duration of S phase. Using alkaline sucrose gradient sedimentation, the rate of DNA elongation was examined, and no difference was observed between Werners syndrome cells and normal cells.

Inducibility of sister-chromatid exchanges by heavy-metal ions

A number of screening tests has been developed for carcinogens in cultured mammalian cells, aimed at filling the gap between bacterial mutations and carcinogenesis. These include chromosomal aberrations, somatic cell mutations and cell transformation. Sister-chromatid exchange (SCE) is also regarded as one of the probes for the detection of carcinogens. SCE involves the reciprocal interchange of DNA between chromatids, which does not result in an overall alteration of chromosome morphology. The fluorescent plus Giemsa method of Perry and Wolff (1974) provides rapid and unequivocal detection of SCE after incorporation of 5-bromodeoxyuridine (BUdR) for 2 rounds of replication. Using this technique, Perry and Evans (1975) and Carrano et al. (1978) showed that known mutagens induce SCE at a concentration much lower than that which induces chromosomal aberrations. In this report, we examine the inducibility of SCE with many kinds of heavy-metal ions including suspected carcinogens such as As, Cr, Ni and others. Don Chinese hamster cells were grown in Dulbeccos modified Eagles medium supplemented with 10°70 fetal bovine serum. For the semi-quantitative determination of the cytotoxicity of various metal compounds, a modification of the method of Toplin (1959) was used. In practice, 4 × 104 cells were incubated with 1 ml of the growth medium plus various concentrations of metal compounds in Lab-Tek chambers (4 chambers per slide) (Lab-Tek Products, Westmont, IL). After 72 h of incubation, the cells were fixed and stained with Giemsa solution. TCIDs0 (50% inhibition dose of tissue-culture cells) was estimated by microscope or by eye. The SCE frequency was measured in Don cells by the technique described by Perry and Wolff (1974). Cells were exposed to various compounds throughout the incubation with BUdR (1 /zg/ml) for 2 cell cycles (28 h). During the last 2 h of incubation, Colcemid (0.1 #g/ml, CIBA Ltd., Basle, Switzerland) was added. Metal compounds were solubilized in sterile dimethyl sulfoxide (DMSO) at 10 mg/ml immediately

Autoradiographic Studies of DNA Replication in Werner’s Syndrome Cells

We have compared cultured fibroblasts of early passage derived from patients with the Werner syndrome and from normal subjects in several aspects of cell cycle time and DNA replication. The average cycle time was prolonged in Werners syndrome cells compared with normal cells because of changes in the duration of S phase. The durations of G1 and G2 were unchanged. In addition, the labeling index was lower in Werners syndrome cells, suggesting that there are two cell-cycle abnormalities in Werners syndrome cells. The cause of the prolongation of S phase was investigated by DNA fiber autoradiography and alkaline sucrose density gradient sedimentation. The rate of DNA chain elongation was not different in Werners syndrome cells from that in normal cells, but the frequency of replication initiation was decreased in Werners syndrome cells.

The mode of action of aphidicolin on DNA synthesis in isolated nuclei

Abstract Aphidicolin (a specific inhibitor of DNA polymerase-α) inhibited DNA synthesis in isolated nuclei from sea urchin embryos but ddTTP (an inhibitor of DNA polymerases-β and -γ) did not, indicating that DNA polymerase-α was responsible for DNA synthesis in isolated nuclei. DNA synthesis in isolated nuclei was inhibited by aphidicolin noncompetitively with respect to each of dNTPs indicating that properties of in situ DNA polymerase activity in isolated nuclei are different from those of the purified DNA polymerase-α which was inhibited by aphidicolin competitively with respect to dCTP and noncompetitively with respect to the other 3 dNTPs. Similar results were obtained using HeLa cell nuclei.

Decrease in uH2A (protein A24) of a mouse temperature-sensitive mutant

ts85 cell is a temperature‐sensitive mutant of cell cycle, and chromosomal protein uH2A of this mutant disappears at the non‐permissive temperature. uH2A in nucleosomes is thought to be synthesized or degradated as follows. H2A + Ubiquitin ⇆ uH2A. Up to date, the degradation of uH2A was shown to be catalyzed by uH2A lyase, however no enzymes (factors) concerning its synthesis have been elucidated. Here, we show that ATP is prerequisite for the synthesis of uH2A, and that the disappearance of uH2A at the non‐permissive temperature may be due to a reduction in the rate of synthesis rather than an increase in the rate of its degradation.

Changes in the level of poly ADP-ribosylation during a cell cycle

Abstract In order to analyze the fluctuation of the poly ADP-ribosylation level during the cell cycle of synchronously growing He La S3 cells, we have developed three different assay systems; intact and disrupted nuclear systems, and poly(ADP-ribose) polymerase in vitro system. The optimum conditions for poly ADP-ribosylation in each assay system were similar except the pH optimum. Under the conditions favoring poly ADP-ribosylation, little radioactivity incorporated into poly(ADP-ribose) was lost after termination of the poly ADP-ribosylation by addition of nicotinamide which inhibits the reactions by more than 90% in any system. In the intact nuclear system, the level of poly ADP-ribosylation increased slightly subsequent to late G2 phase with a peak at M phase. The high level of poly ADP-ribosylation in M phase was also confirmed by using selectively collected mitotic cells which were arrested in M phase by Colcemid. The level in mitotic chromosomes was 5.1-fold higher than that in the nuclei from logarithmically growing cells. Colcemid has no effect on the poly ADP-ribosylation. In the disrupted nuclear system, a relatively high level of poly ADP-ribosylation was observed during mid S-G2 phase. When poly(ADP-ribose) polymerase was extracted from the nuclei with a buffer solution containing 0.3 M KCl, more than 90% of the enzyme activity was recovered. The poly(ADP-ribose) polymerase in vitro system was dependent on both DNA and histone—10 μg each. In the enzyme system, enzyme activity was detected throughout the cell cycle and was observed to be highest in G2 phase. The high level at M phase observed in the intact nuclear system was not seen in the other two systems. Under the assay conditions, little influence of poly(ADP-ribose) degrading enzymes was noted on the level of poly ADP-ribosylation in any of the three systems. This was confirmed at various stages during the cell cycle through pulse-labeling and “chasing” by adding nicotinamide.