Single cell tracking reveals that Msh2 is a key component of an early-acting DNA damage-activated G2 checkpoint

Abstract

Dysfunction of cell-cycle checkpoints in DNA mismatch repair (MMR)-deficient cells in response to DNA damage has implications for anticancer therapy and genetic instability. We have studied the cell-cycle effects of MMR deficiency (Msh2) in primary mouse embryonic fibroblasts (MEFs) exposed to cisplatin (10 lm  1 h) using time-lapse microscopy. Kinetic responses of MEFs from different embryos and passage ages varied, but we report a consistent drug-induced inhibition of mitotic entry (approx. 50%). There was a loss of an early-acting (o5 h) delay in G2 to M transition in Msh2 À/À cells, although a later-acting G2 arrest was apparently normal. This suggests that Msh2 primarily acts to delay mitotic entry of cells already in G2, that is, DNA damage incurred during G2 does not influence the cell once committed to mitotic traverse. Irrespective of Msh2 status, cisplatin treatment and the incurred DNA damage did not effect mitotic traverse or show any evidence for early (within 24 h) cell death. The results indicate that Msh2 À/À status can result in the premature commitment to mitosis of a cell subpopulation, determined by the fraction residing in G2 at the time of damage induction. The findings suggest a new route to MMR-driven genetic instability that does not rely primarily on the integrity of the late-acting checkpoint.

Impact of overexpression of metallothionein-1 on cell cycle progression and zinc toxicity

“…CHO-K1 and CHO-K1 MT-1 2.20 cultures were established in multiwell dishes and transferred to a time-lapse instrument (28,29) designed to capture transmission-phase images from multiwell plates. The Axiovert 100 microscope (Carl Zeiss, Welwyn Garden City, UK) was fitted with an incubator to maintain 37°C/5% CO2 (Solent Scientific, Portsmouth, UK), and images were captured using an ORCA-ER charge-coupled device camera (Hamamatsu, Reading, UK).…”

“…Each cell in the field was tracked, and the time to event and duration recorded. The time-lapse event curves were processed and analyzed as previously described (28,29) for rodent cells.…”

Cell-population tracking using quantum dots in flow cytometry

“…Traditionally tracking studies have been done using time-lapse microscope imaging techniques to visualize the process of cell dynamics. However this is a time intensive method and so limits analysis to a few hundred cells at most [4,5]. The heterogeneity of cell populations is such that statistics dictate that these studies must be scaled up to measurements on several thousands of cells if realistic models of cell behavior are to be obtained.…”

Mitotic Bypass Via An Occult Cell Cycle Phase Following DNA Topoisomerase II Inhibition In p53 Functional Human Tumor Cells

“…The multitag time-lapse event curves were processed appropriately and visualized in graphical plots as previously described. 25,26 Time-laspe microscopy: tracking eGFP-cyclin B1 profiles. The principles have been described previously.…”

“…Both increased and reduced levels o�� parental cell ���� contents are apparent in clones arising ��rom �CR����� e�posed cultures. (C) �imelapse imaging (C) �imelapse imaging �imelapse imaging 25 o�� changes in cyclin B��e��P reporter e�pression during cell cycle progression to mitosis in control cells (upper sequence) or into mitotic �ypass (up to 70% events; lower sequence) in the presence o�� 2 mg/ml �CR�����. �o evidence o�� a commitment to mitosis �e��ore cyclin B� switch�o���� (lower sequence).…”