Entry into Mitosis in Vertebrate Somatic Cells Is Guarded by a Chromosome Damage Checkpoint That Reverses the Cell Cycle When Triggered during Early but Not Late Prophase

Abstract

When vertebrate somatic cells are selectively irradiated in the nucleus during late prophase (<30 min before nuclear envelope breakdown) they progress normally through mitosis even if they contain broken chromosomes. However, if early prophase nuclei are similarly irradiated, chromosome condensation is reversed and the cells return to interphase. Thus, the G2 checkpoint that prevents entry into mitosis in response to nuclear damage ceases to function in late prophase. If one nucleus in a cell containing two early prophase nuclei is selectively irradiated, both return to interphase, and prophase cells that have been induced to returned to interphase retain a normal cytoplasmic microtubule complex. Thus, damage to an early prophase nucleus is converted into a signal that not only reverses the nuclear events of prophase, but this signal also enters the cytoplasm where it inhibits e.g., centrosome maturation and the formation of asters. Immunofluorescent analyses reveal that the irradiation-induced reversion of prophase is correlated with the dephosphorylation of histone H1, histone H3, and the MPM2 epitopes. Together, these data reveal that a checkpoint control exists in early but not late prophase in vertebrate cells that, when triggered, reverses the cell cycle by apparently downregulating existing cyclin-dependent kinase (CDK1) activity.

Regulation of mitosis in response to damaged or incompletely replicated DNA require different levels of Grapes (DrosophilaChk1)

“…These data suggest that damage incurred in late G 2 /early M cannot cause a delay in metaphase-anaphase transition, whereas the same level of radiation applied earlier in the cell cycle can. These observations are in agreement with results from mammalian cells in which doses of radiation that readily produced a G 2 /M delay if applied in interphase had no effect on mitotic progress if applied in early M (Rieder and Cole, 1998). It is possible that damage caused by radiation at G 2 /M is not recognized by the checkpoint or that the damage is sensed but that cells are unable to activate the checkpoint at such a late point in the cell cycle.…”

The p38-Mediated Stress-Activated Checkpoint: A Rapid Response System for Delaying Progression through Antephase and Entry into Mitosis

“…9 Before the commitment to mitosis is made chromosome condensation can be arrested or reversed, and entry into mitosis delayed or prevented, by a variety of insults including e.g., X-rays, 10,11 colchicine 12,13 hypothermia, 14,15 or chromosome damage. 16 One conspicuous outcome of these treatments is that the cell often decondenses its chromosomes and returns to what Bullough and Johnson (1951) termed "antephase". However, as noted above chromosome condensation is really a progressive process that starts in early G 2 .…”

Imaging the division process in living tissue culture cells

“…1; [24]). Importantly, once the commitment to mitosis is made, the cell becomes relatively refractory to radiation damage [35][36][37], although excessive illumination can still produce delays during mitosis and/or an aborted division [38].…”

“…Some cells, like PtK 1 , LLC-PK, Indian Muntjac, and primary cell cultures from humans and salamanders, are extremely sensitive to light during antephase -to the point where it becomes very difficult to follow the G 2 /M transition at reasonable framing rates even with low-light level systems (e.g., [16,36,37]; reviewed in [23]). Other cell types may be more resistant.…”

“…Most modern stage heaters employ Peltier devices that can maintain specimens at any temperature, from below 0 to 100 °C, for many hours. They can either be built at little cost (see design in Rieder and Cole [36]) or purchased commercially (e.g., the 14417 heated-stage insert; WPI, Sarasota, FL; www.wpieurope.com/microscopy/HSI.html, or several different stages from 20-20 Technologies; Wilmington, NC; www.20-20tech.com). The major problem with this approach is that the objective lens, and to a lesser extent the condenser, both act as large radiant bodies to cool the chamber, especially when oil-immersion lenses are used.…”