DNA fork displacement rates in human cells

DNA fork displacement rates were measured in 20 human cell lines by a bromodeoxyuridine-313 nm photolysis technique. Cell lines included representatives of normal diploid, Fanconi's anemia, ataxia telangiectases, xeroderma pigmentosum, trisomy-21 and several transformed lines. The average value for all the cell lines was 0.53 0.08 mum/min. The average value for individual cell lines, however, displayed a 30% variation. Less than 10% of variation in the fork displacement rate appears to be due to the experimental technique; the remainder is probably due to true variation among the cell types and to culture conditions.     

Estimation of DNA fork displacement rates from isolated mammalian chromosomes

Chromosomes were isolated from Chinese hamster ovary cells that had been pulse-labeled with ³H-thymidine and bromodeoxyuridine and allowed to proceed to metaphase in the presence of colcemid. The chromosomes were then irradiated with 313 nm light and the length of the pulse-labeled DNA segments was estimated by the distribution of ³H-DNA in alkaline sucrose gradients. The average rate of fork displacement, calculated from this measurement, was 0.9 m/min, which was in excellent agreement with measurements from interphase cells. By the same method, hydroxyurea was shown to retard DNA chain growth by 50–60% in both isolated chromosomes and interphase cells.     

DNA fork displacement rates in Bloom's syndrome fibroblasts

DNA fork displacement rates were measured in three lines of Bloom's syndrome cells and in a normal diploid fibroblast line. Fork displacement rates in Bloom's cells were approx. 55–65% of the rate in normal fibroblasts.     

DNA fork displacement rate measurements in heated Chinese hamster ovary cells

DNA fork displacement rates (FDR) were measured in Chinese hamster ovary (CHO) cells heated at either 43.5 degrees C or 45.5 degrees C for various times. The inhibition of fork movement rate by heat was both time and temperature dependent, i.e., 10-20 min at 43.5 degrees C or 5 min at 45.5 degrees C was required to decrease the FDR to 20-30% of the control rate of 1 micron/min. Following heating, the reduced FDR was found to be constant for at least 75 min. The observed effects of heat on reduced rates of DNA replicon initiation and chain elongation and the increase in DNA with single-stranded regions could be explained by the heat sensitivity of the FDR. Any of these alterations in the DNA replication process may lead to many opportunities for abnormal DNA and/or protein interactions to occur which ultimately may lead to the observed formation of chromosomal aberrations.     

The rate of fork movement during DNA replication in mammalian cells

DNA fiber autoradiography was used to measure the rate of replication fork progression along replication units in human diploid cells. The rate in different replication units differs very significantly and lies within the range 0.1 to 1.2 m/min. However, no significant changes were found in the rate of fork movement along single replication units operating during long intervals of S phase. Moreover, the fork progression rate is constant in many replication units of human cells.     

The tumorigenicity of diploid and aneuploid human pluripotent stem cells

Human embryonic stem cells (HESCs) and induced pluripotent stem cells (HiPSCs) offer an immense potential as a source of cells for regenerative medicine. However, the ability of undifferentiated HESCs to produce tumors in vivo presents a major obstacle for the translation of this potential into clinical reality. Therefore, characterizing the nature of HESC-derived tumors, especially their malignant potential, is extremely important in order to evaluate the risk involved in their clinical use. Here we review recent observations on the tumorigenicity of human pluripotent stem cells. We argue that diploid, early passage, HESCs produce benign teratomas without undergoing genetic modifications. Conversely, HESCs that acquired genetic or epigenetic changes upon adaptation to in vitro culture can produce malignant teratocarcinomas. We discuss the molecular mechanisms of HESC tumorigenicity and suggest approaches to prevent tumor formation from these cells. We also discuss the differences in the tumorigenicity between mouse embryonic stem cells (MESCs) and HESCs, and suggest methodologies that may help to identify cellular markers for culture adapted HESCs.     

Changes in the rate of DNA replication fork movement during S phase in mammalian cells.

DNA fiber autoradiography was used to measure the rate of replication fork movement and the size of replication units as a function of time during the S phase of synchronized Chinese hamster ovary cells. The rate of fork movement increased by about threefold from early S to later S phase, with the most dramatic change occurring in the first hour of S phase. On the other hand, the size of replication units did not vary significantly during S phase.     

DNA-fork displacement rates in cultured mammalian cells with mutations in ribonucleotide reductase

DNA-replication fork displacement rates were measured in mouse S49 lymphosarcoma cell lines and in derivatives of those cell lines. One of the derivatives lacks dCMP deaminase activity and two others bear defined mutations in ribonucleotide reductase. We also examined a revertant cell line that was selected from one of the ribonucleotide reductase mutants and has regained normal ribonucleotide reductase activity. Our results show a correlation between decreased fork-displacement rates and alterations in ribonucleotide reductase, suggesting a possible involvement of this enzyme in the replication apparatus.     

Radiation-induced aberration in aneuploid vs. diploid swine leukocytes

Swine leukocytes were cultured for 48 h after receiving γ-ray exposures up to 400 R. Cells with one or two less than the diploid number of centromeres consistently contained more chromosome deletions than did diploid cells. This effect was not evident for dicentric and ring aberrations. Both aneuploid frequencies and the proportion of aberrations in aneuploid cells increased in irradiated samples but showed no dose-response relationship.     

gamma-Glutamyltransferase in human diploid fibroblasts and other mammalian cells.

gamma-Glutamyltransferase was determined in WI-38 human diploid fibroblasts and compared to enzyme levels determined in several other mammalian cell lines including: fibroblast-like cells from human skin, tibia and foreskin; epithelial-like cells from human, bovine and monkey kidney; and transformed cells (Chinese hamster ovary, HeLa S3 and SV-40 transformed WI-38). Transformed cells had the lowest activity found followed in increasing order by fibroblasts, human and bovine epithelial cells and monkey kidney epithelial cells. The enzyme isolated from the plasma membrane of WI-38 cells, like the enzyme from kidney and brain, was found to be irreversibly inhibited by iodoacetamide, reversibly by serine-borate, and had a strong specificity for certain amino acids. The possibility exists that gamma-glutamyltransferase could be involved in transport of amino acids into cells in culture; and glutamine, used in media, is an excellent substrate for the enzyme.     

Difference between diploid and aneuploid Chinese hamster cells in replication at mid-S-phase

Following partial synchronization of the heteroploid Chinese hamster cell line V-79 and of normal diploid lung fibroblasts of the Chinese hamster in culture, their DNA replication during S-phase was compared by means of a BrdU-incorporation/thymidine pulse technique and Hoechst-Giemsa differential staining of metaphase chromosomes. This comparison indirectly shows the S-phase of the heteroploid cells of V-79 to be 2 h shorter than the diploid cell S-phase. When the thymidine pulse is applied to diploid lung fibroblasts at mid-S-phase, differential staining colours metaphase chromosomes a pale blue. Performing the corresponding experiment with V-79 cells, neither a pale blue nor dark red staining is obtained, but rather an intermediate shade, showing prominently dark staining regions in parts. The pause in DNA synthesis observed at mid-S-phase of the diploid Chinese hamster lung fibroblasts seems to be omitted at mid-S-phase of the V-79 cells.     

Establishment of a replication fork barrier following induction of DNA binding in mammalian cells

Understanding the mechanisms that lead to replication fork blocks (RFB) and the means to bypass them is important given the threat that they represent for genome stability if inappropriately handled. Here, to study this issue in mammals, we use integrated arrays of the LacO and/or TetO as a tractable system to follow in time a process in an individual cell and at a single locus. Importantly, we show that induction of the binding by LacI and TetR proteins, and not the presence of the repeats, is key to form the RFB. We find that the binding of the proteins to the arrays during replication causes a prolonged persistence of replication foci at the site. This, in turn, induces a local DNA damage repair (DDR) response, with the recruitment of proteins involved in double-strand break (DSB) repair such as TOPBP1 and 53BP1, and the phosphorylation of H2AX. Furthermore, the appearance of micronuclei and DNA bridges after mitosis is consistent with an incomplete replication. We discuss how the many DNA binding proteins encountered during replication can be dealt with and the consequences of incomplete replication. Future studies exploiting this type of system should help analyze how an RFB, along with bypass mechanisms, are controlled in order to maintain genome integrity.     

Cell cycles in human diploid and aneuploid strains

Summary Autoradiographic investigation of the cell cycle of 12 diploid and 12 abnormal human fibroblast strains was carried out. Two cell strains (trisomy 7 and monosomy 21) derived from spontaneous abortuses showed prolongation of the G₂ period accompanied by the shortening of the S period. Other cytogenetically abnormal embryonic strains (trisomy 9, 14 and triploidy) did not deviate from the diploid pattern. Three cell strains (LHC-1-70, LHC-6-70, LHC-411) derived from the patients with karyotypes 47,XXX, 47,XY,+18 and 46,XX,5p—respectivly had embryonic types of proliferation with a short G₂ period. In two other strains from the patients with Down's syndrom the G₂ period was prolonged. There was no statistically significant difference in the parameters of the cell cycle between the control and the strains derived from a patient with Klinefelter syndrom and from a male patient with karyotype 46,XX. The modificatory effect of the chromosomal abnormalities on the parameters of cell cycle is discussed.

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Zusammenfassung Der Zellcyclus von 12 normal diploiden und 12 abnormen menschlichen Fibroblasten-Zellinien wurde untersucht. Zwei Zellinien (Trisomie 7 und Monosomie 21) zeigten eine Verlängerung der G₂-Phase und eine Verkürzung der S-Phase. Zellinien (Trisomie 9, 14 und Triploidie) wichen nicht von der Norm ab. Drei Zellinien (47,XXX; 47,XY,+18; 46,XX,5p-) zeigten eine Verkürzung von G₂. G₂ war dagegen verlängert bei zwei Zellinien von Down-Syndrom. Keine Abweichung fand sich bei einem Klinefelter-Patienten und einem männlichen Probanden mit 46,XX.

     

DNA repair endonuclease activity during synchronous growth of diploid human fibroblasts.

DNA-repair endonuclease activity in response to UV-induced DNA damage was quantified in diploid human fibroblasts after synchronizing cell cultures to selected stages of the cell cycle. Incubation of irradiated cells with aphidicolin, an inhibitor of DNA polymerases alpha and delta, delayed the sealing of repair patches and allowed estimation of rates of strand incision by the repair endonuclease. The apparent Vmax for endonucleolytic incision and Km for substrate utilization were determined by Lineweaver-Burk and Eadie-Hofstee analyses. For cells passing through G1, S or G2, Vmax for reparative incision was, respectively, 7.6, 8.4 and 8.4 breaks/10(10) Da per min, suggesting that there was little variation in incision activity during these cell-cycle phases. The Km values of 2.4-3.1 J/m2 for these cells indicate that the nucleotidyl DNA excision-repair pathway operates with maximal effectiveness after low fluences of UV that are in the shoulder region of survival curves. Fibroblasts in mitosis demonstrated a severe attenuation of reparative incision. Rates of incision were 11% of those seen in G2 cells. Disruption of nuclear structure during mitosis may reduce the effective concentration of endonuclease in the vicinity of damaged chromatin. The extreme condensation of chromatin during mitosis also may restrict the accessibility of reparative endonuclease to sites of DNA damage. Confluence-arrested fibroblasts in G0 expressed endonuclease activity with Vmax of 5.5 breaks/10(10) Da per min and a Km of 5.5 J/m2. The greater condensation of chromatin in quiescent cells may restrict the accessibility of endonuclease to dimers and so explain the elevated Km. When fibroblasts were synchronized by serum-deprivation, little variation in reparative endonuclease activity was discerned as released cells transited from early G1 through late G1 and early S. Proliferating fibroblasts in G1 were shown to express comparatively high numbers of reparative incision events in the absence of aphidicolin which was normally used to inhibit DNA polymerases and hold repair patches open. It was calculated that in G0, S and G2 phase cells, single-strand breaks at sites of repair remained open for 30, 19 and 14 sec, respectively. In G1 phase cells, repair sites remained open for 126 sec. Addition of deoxyribonucleosides to G1 cells reduced this time to 42 sec suggesting that the slower rate of synthesis and ligation of repair patches in G1 was due to a relative deficiency of deoxyribonucleotidyl precursors for DNA polymerase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pattern of DNA segregation in multipolar anatelophases of different ploidy in euploid and aneuploid mammalian cells cultivated in vitro

A microdensitometrical analysis of multipolar anatolophases of cuploid and aneuploid mammalian cells in vitro has been made in order to study the quantitative distribution of DNA to the poles.In the tripolar anatelophases of aneuploid Chinese hamster cells (obtained using colchicine) DNA is distributed to poles in an almost random way. In the spontaneous tripolar anatelophases of euploid cells of Rhesus, DNA is distributed to the poles in whole number multiples of 1c. The following distributions were observed in tetraploid cells: 14 cases of 3 : 3 : 2 and 3 cases of 4 : 2 : 2. In triploid cells, 6 cases of 2 : 2 : 2 and 1 case of 3 : 2 : 1.Thus we have deduced that each ploidy degree has a preferential distribution of sets and in contrast to the original hypothesis, random segregation of the sets is not the common occurrence. Hypotheses have been made to explain the origin of multipolar mitosis and the preferential distribution of chromatid sets to the poles. The results would confirm the existence of haploid sets of chromatids and would explain the appearance of triploid, pentaploid and hexaploid cells in tissues and cell cultures.