Prolonged prometaphase blocks daughter cell proliferation despite normal completion of mitosis

Abstract

The mitotic checkpoint maintains genomic stability by blocking the metaphase-anaphase transition until all kinetochores attach to spindle microtubules [1, 2]. However, some defects are not detected by this checkpoint. With low concentrations of microtubule-targeting agents, the checkpoint eventually becomes satisfied, though the spindles may be short and/or multipolar [3, 4] and the fidelity of chromosome distribution and cleavage completion are compromised. In real life, environmental toxins, radiation, or chemotherapeutic agents may lead to completed but inaccurate mitoses. It has been assumed that once the checkpoint is satisfied and cells divide, the daughter cells would proliferate regardless of prometaphase duration. However, when continuously exposed to microtubule inhibitors, untransformed cells eventually slip out of mitosis after 12-48 hr and arrest in G1 [5-8] (see also [9]). Interestingly, transient but prolonged treatments with nocodazole allow completion of mitosis, but the daughter cells arrest in interphase [10, 11] (see also [9, 12]). Here we characterize the relationship between prometaphase duration and the proliferative capacity of daughter cells. Our results reveal the existence of a mechanism that senses prometaphase duration; if prometaphase lasts >1.5 hr, this mechanism triggers a durable p38- and p53-dependent G1 arrest of the daughter cells despite normal division of their mothers.

Figure 1

A. Prometaphase spindles in RPE1 cells. (a) Control. (b) 0.08μM nocodazole. (c) 100 μM monastrol. (d) 4μM MG132 at 30 minutes after drug application. Fluorescence microscopy: DNA (blue); alpha-tubulin (green); pericentrin (red). B. Duration of mother cell prometaphase and the proliferative capacity of daughter cells; 0.08μM nocodazole. Each vertical bar represents a daughter cell and the height of the bar indicates the duration of prometaphase for its mother cell. The dark line across each vertical bar represents the time the mother cell spent in prometaphase in the presence of drug. The vertical bars are rank ordered by the duration of prometaphase for the mother cells. Daughters that proliferate are to the left of the vertical red line and those that arrest in G1 are to the right of this line. Prometaphase in control cells (cell rounding/nuclear envelope breakdown to anaphase onset) averages 18 minutes (range 9-30 minutes, n=117). C. Duration of mother cell prometaphase and the proliferative capacity of the daughter cells; 100μM monastrol. D. Duration of mother cell prometaphase and the proliferative capacity of the daughter cells. RPE1 cells were treated with 5μM Taxol for 10 minutes before mitosis. Satisfaction of the mitotic checkpoint was variable giving a range of prometaphase durations.

Figure 2

A. p53 and p21 expression in the daughters of RPE1 mother cells spending >1.5 hrs in prometaphase; 0.08μM nocodazole. Individual mother cells and their progeny were continuously followed; daughter cells were fixed 2 and 48 hours after nocodazole removal. Nine cells in each category were double labeled with anti-p53 and anti-p21 antibodies; the rest were single labeled. Table: percent daughters that expressed nuclear p53 or p21 at the indicated times. Fluorescence and phase contrast images. B. Proliferative capacity of daughter RPE1 cells cultured in dishes after their mothers were held in prometaphase for 2-6 hours by 0.08μM nocodazole. The p53+ category shows percentage of cells with BrdU incorporation and p21 expression at the indicated times after drug removal for p53 normal cells. The p53 RNAi category shows the percentages of p53 knockdown cells incorporating BrdU at the indicated times after drug removal. First image: Overlain phase contrast and BrdU fluorescence image of p53 normal cells 24 hours after drug removal. Second image panels: Phase contrast image of two fields of p53 normal cells 168 hours after drug removal showing dead cells. Third image: Overlain phase contrast and BrdU fluorescence image of p53 knockdown cells 24 hours after drug removal.

Figure 3

A. The mitotic checkpoint is promptly satisfied in MG132 treated RPE1 cells. Panel (a): Cells fixed and immunostained for the checkpoint proteins Mad2 and BubR1 30 minutes after drug application. These proteins are not detectable on centromeres and all chromosomes are aligned on the metaphase plate. Panel (b): To validate our methodology cells were treated with 0.08μM nocodazole. Mad2 and BubR1 were localized to unattached kinetochores of monooriented chromosomes but not localized to bioriented chromosomes at the metaphase plate. B. Duration of mother cell prometaphase and the proliferative capacity of their daughter cells. RPE1 cells in mitosis were held in prometaphase for an additional 10-70 minutes with 4μM MG132. In a few cases (color coded arrowheads) one daughter proliferated while its sister arrested in G1.

Figure 4

A. Duration of mother cell prometaphase and the proliferative capacity of the daughter cells when p38 activity is inhibited during prolonged prometaphase; 0.08μM nocodazole plus SB 203580. The p38 inhibitor was washed out concurrently with the nocodazole. B. Duration of mother cell prometaphase and the proliferative capacity of the daughter cells when p38 activity is inhibited after prolonged prometaphase. Cells were held in prometaphase with 0.08μM nocodazole alone and SB 203580 was continuously applied after nocodazole removal. C. Consequence of prolonged prometaphase are irreversible. We followed individual cells exposed to 0.08μM nocodazole for 6 hours. After drug removal, SB 203580 was transiently introduced for 3-18 hours to allow daughters that otherwise would have arrested in G1 to become committed to the next cell cycle. Top panel diagrammatically shows the behavior of the progeny of 43 mothers that were held in prometaphase for >1.5 hours. The number of progeny still in view is indicated. All progeny showed the same behavior. All “Granddaughters” arrested in G1 despite being born of mitoses of ~normal duration (average 24 minutes, range 10-49 minutes, from cell rounding to daughter cell flattening; controls average 27 minutes, range 18-36 minutes). Bottom panel shows behavior of the progeny of 33 same preparation mothers held in prometaphase <1.5 hours. All “Granddaughters” proliferated.