Diverse system stresses: common mechanisms of chromosome fragmentation

Abstract

Chromosome fragmentation (C-Frag) is a newly identified MCD (mitotic cell death), distinct from apoptosis and MC (mitotic catastrophe). As different molecular mechanisms can induce C-Frag, we hypothesize that the general mechanism of its induction is a system response to cellular stress. A clear link between C-Frag and diverse system stresses generated from an array of molecular mechanisms is shown. Centrosome amplification, which is also linked to diverse mechanisms of stress, is shown to occur in association with C-Frag. This led to a new model showing that diverse stresses induce common, MCD. Specifically, different cellular stresses target the integral chromosomal machinery, leading to system instability and triggering of MCD by C-Frag. This model of stress-induced cell death is also applicable to other types of cell death. The current study solves the previously confusing relationship between the diverse molecular mechanisms of chromosome pulverization, suggesting that incomplete C-Frag could serve as the initial event responsible for forms of genome chaos including chromothripsis. In addition, multiple cell death types are shown to coexist with C-Frag and it is more dominant than apoptosis at lower drug concentrations. Together, this study suggests that cell death is a diverse group of highly heterogeneous events that are linked to stress-induced system instability and evolutionary potential.

Figure 1

(a) Induction of C-Frag in H460-neo- and H460-Bcl-2-overexpressing cells by concurrent 12-h treatment with colcemid and Dox results in complete cleavage of PARP. Bcl-2-overexpressing and control cells were used to show that PARP degradation is not caused by apoptotic death in the case of C-Frag. (b–d) HCT116 14-3-3σ^−/− cells treated with 0.2 μg/ml Dox have been shown to die through MC. Seventy-two hours of treatment results in the mitotic index falling to almost 0 (orange line), whereas the percentage of multi-nucleated/multi-lobulated cells increases significantly (blue line) and the percentage of apoptotic cells rises slightly (red line) (b). During this time there is a degradation of cellular morphology as cells enlarge and die, and in agreement with previous results, by 72 h approximately 90% of cells died (c). In previous reports, the bulk of cell death was thought to be occurring in mitotic cells; however, we were unable to identify such population by cytogenomic analysis, suggesting that this death may not be attributable to mitosis. (d) Examples of C-Frag, apoptotic, and multi-lobulated/nucleated cells are shown on the left, the middle, and the right, respectively following conventional cytogenetic preparation

Figure 2

C-Frag is detectable in in vivo tumors prepared for cytogenomic analysis (a). Here, two examples of cells undergoing C-Frag from an untreated primary mouse tumor are shown (left and right panels). In the photo on the right the cell on the top left is an example of a defective mitotic figure (DMF) that is also undergoing C-Frag. The morphology of C-Frag is readily distinguishable from the normal mitotic figures of the same tumor (b)

Figure 3

The effects of various stressors on C-Frag are examined (^*P<0.05, ^**P<0.01, ^***P<0.001) (a–c). (a) C-Frag is elevated during genomic instability (pd25) (blue bar) as compared with stable genomes (pd54) (green bar, P<0.05). (b) Increasing the culture temperature of COLO-357 cells from 37 to 42°C (blue line) results in a transient increase in the frequency of C-Frag until spontaneous C-Frag falls to >6%. Following the shift of temperature back to 37°C (green line) there is another corresponding increase in the CFI, which then stabilizes at <5%. Statistical significance was calculated by comparing each time point to the 0 time point of the treatment. (c) Treatment of COLO-357 cells with specific stress inducers, including DTT (blue line), TG (red line), and TM (green line), results in varied increased C-Frag over a 24-h period. The error bars represent the standard deviation. (d) Western blot analysis of the same DTT and TG treatments show an increase in the expression of CHOP, a key protein in the ER stress response. CHOP expression increases steadily in both TG and DTT treatments from 0 to 8 h, then falls off at 24 h as cells adapt to the stress. Activated XBP1 expression changes similarly in DTT-treated cells, whereas TG treatment results in little expression. Finally, there is an increase in ER proteins bearing the ER translocation sequence KDEL from 0 to 24 h of treatment, which is indicative of ER stress (d). Concurrent treatment with colcemid and Dox to induce C-Frag in HCT116 cells results in increased centrosome amplification (e). (f) An example of centrosome amplification (right panel, arrowheads) taking place in a cell undergoing C-Frag (left panel, arrowhead). Multiple centrosomes are detectable (arrowheads), whereas in a normal neighboring cell undergoing mitosis (left panel, arrow), only two centrosomes are present (right panel, arrows). Inhibition of HSP90 function with 17-DMAG results in increased C-Frag (g). Bars represent the CFI of cells treated with or without (control – blue bar) doxorubicin at the indicated concentration for 54 h. After 24 h of treatment 100 nM 17-DMAG was added followed by the addition of colcemid 6 h prior to harvest. Doxorubicin treatment at 1 μM and 1 μg/ml (orange and purple bars respectively) would typically result in cell cycle arrest and death via apoptosis or necrosis, however 17-DMAG treatment allows cells to progress to mitosis with damage. Damaged mitotic cells are then eliminated by C-Frag. Treatment with 50 nM (red bar) and 150 nM (green bar) elevates the CFI, however many cells do not contain sufficient damage to induce C-Frag

Figure 4

Different treatment combinations result in different outcomes. Concurrent treatment of HCT116 cells with colcemid and Dox, mitomycin-C (MMC), or methotrexate (MTX) result in a varied induction of cell death by apoptosis (green bars) and C-Frag (blue bars) as judged by cytogenetic examination (a). Treatment of cells with docetaxel instead of colcemid results in mitotic arrest as expected, combinatorial treatment with 1 μg/ml Dox and either 1 μM docetaxel or 10 μM docetaxel results primarily in C-Frag or apoptosis, respectively (b). As judged by previous work, some of the values for apoptotic death observed here were lower than what has been shown previously. This is likely because of differences in cytogenomic assays as compared with molecular assays to identify apoptosis such as TUNEL, which may be more sensitive

Figure 5

A proposed model illustrating the relationship between stress and the induction of various types of cell death. When a cell faces a given stress that is strong enough to induce death, multiple factors influence the selection of the specific pathways of cell death and depend on the availability of targets (mitotic or interphase cells) and/or the availability of certain pathways