Ann M. Mullinger

Premitotic chromosome individualization in mammalian cells depends on topoisomerase II activity.

Abstract.When DNA topoisomerase II (topo II) activity is inhibited with a non-DNA-damaging topo II inhibitor (ICRF-193), mammalian cells become checkpoint arrested in G2-phase. In this study, we analyzed chromosome structure in cells that bypassed this checkpoint. We observed a novel type of chromosome aberration, which we call Ω-figures. These are entangled chromosome regions that indicate the persistence of catenations between nonhomologous sequences. The number of Ω- figures per cell increased sharply as cells evaded the transient block imposed by the topo II-dependent checkpoint, and the presence of caffeine (a checkpoint-evading agent) potentiated this increase. Thus, the removal of nonreplicative catenations, a process that promotes chromosome individualization in G2, may be monitored by the topo II-dependent checkpoint in mammals.

DNA Repair Under Stress

SUMMARY When the excision repair process of eukaryote cells is arrested by inhibitors of repair synthesis including hydroxyurea (HU), 1-β-d-arabinofuranosylcytosine (araC) or aphidicolin, major cellular changes follow the accumulation of repair-associated DNA breaks. These changes, each of which reflects more or less severe cellular stress, include cycle delay, chromosome behaviour, fall in NAD level, the development of double-stranded DNA breaks, rapid chromosome fragmentation and cell killing. Disruption of the repair process by agents such as araC after therapeutic DNA damage may, therefore, have some potential value in cancer treatment. The extreme cellular problems associated with the artificial arrest of repair may have their subtler counterparts elsewhere, and we discuss several systems where delays in the completion of excision repair in the absence of repair synthesis inhibitors have marked repercussions on cell viability. We also show that the average completion time of an excision repair patch varies according to the state of cell culture, and that completion time is extended after treatment with insulin or following trypsin detachment. Under certain growth conditions ultraviolet irradiation followed by mitogenic stimulation results in double-stranded DNA breakage and additional cell killing, and we discuss these data in the light of protocols that have been used successfully to transform human or rodent cells in vitro. Finally, we consider whether the rejoining of DNA breaks accumulated by repair synthesis inhibitors is a valid model system for studying ligation, and show that this protocol provides an extremely sensitive assay for most incision events and, thereby, a means for discriminating between normal human cells on the one hand, and Cockayne’s Syndrome cells and their heterozygotes on the other.

Chapter 19 Human Minisegregant Cells

Publisher Summary The chapter describes methods for the production of human minisegregant cells from mitotic precursors. The aim is to assess the nature of the perturbation induced in the mitotic cell, and the roles of substances that either inhibit or promote this aberrant division. The chapter also describes the fractionation of minisegregant cells and some of their properties and helps to assess their competence to transfer information by means of cell fusion. The ability to fragment human cells by avariety of simple procedures and to produce DNA-containing minisegregants makes possible new experiments in somatic cell genetics. Reconstitution experiments between whole cells and cell fragments will form a useful contrast and complement to whole-cell fusions and should help to simplify analysis of regulatory phenomena, not least by predetermining the direction of chromosome balance in the hybrid. As minisegregants and microcells can be separated according to size and DNA content, it should be possible to control the amount of DNA introduced by these agents and therefore to construct the desired hybrid immediately.

Perturbation of mammalian cell division: human mini segregants derived from mitotic cells.

Mitotic HeLa cells can be induced to undergo abnormal cleavage and to give rise thereby to clusters of buds and/or to free mini segregants. A wide range of morphologies can be produced and the mini segregants vary considerably in size and structure. Some contain DNA which may or may not be enclosed in a nuclear membrane. This DNA in some of the mini segregants originates from dense ‘chromatin bodies’ which migrate to the periphery of the parental cell at an early stage of mitotic perturbation. The percentage of mitotic cells which undergo abnormal cleavage is affected by a number of factors including the pH of the medium, the presence of dithiothreitol or certain other thiol compounds, the absence of serum and storage of the mitotic cells at 4 °C before treatment. To date, the optimal conditions are: incubation at 37 °C in Hanks solution buffered at pH 8.0–8.4, after storage of the mitotic cells for about 3 h at 4 °C. Under these conditions about 70 % of the total mitotic population undergoes abnormal cleavage to produce clusters of mini segregants in the course of an incubation period of 3½ h. Possible mechanisms underlying the phenomenon are discussed.

Competence for assembly of sister chromatid cores is progressively acquired during S phase in mammalian cells

Condensed sister chromatids possess a protein scaffold or axial core to which loops of chromatin are attached. The sister cores are believed to be dynamic frameworks that function in the organization and condensation of chromatids. Chromosome structural proteins are implicated in the establishment of sister chromatid cohesion and in the maintenance of epigenetic phenomena. Both processes of templating are tightly linked to DNA replication itself. It is a question whether the structural basis of sister chromatid cores is templated during S phase. As cells proceed through the cell cycle, chromatid cores undergo changes in their protein composition. Cytologically, cores are first visualized at the start of prometaphase. Still, core assembly can be induced in G1 and G2 when interphase cells are fused with mitotic cells. In this study, we asked if chromatid cores are similarly able to assemble in S-phase cells. We find that the ability to assemble cores is transiently lost during local replication, then regained in chromosome regions shortly after they have been replicated. We propose that core templating occurs coincident with DNA replication and that the competence for the assembly of the sister chromatid cores is acquired shortly after passage of replication forks.