In the presence of drugs that inhibit MT formation, the SAC cannot be satisfied, yet many cells ultimately escape mitosis and enter the next G1. Here we show that this escape is not due to adaptation pathways that suppress cyclin B/Cdk1 activity by, e.g., inhibitory phosphorylations or activation of a Cdki. Importantly, we also show that this escape is not due to the depletion of Mad2 or BubR1, which remain associated with unattached kinetochores as the SAC is bypassed. Instead, we find that in vertebrates, escaping mitosis when in the presence of a functional SAC requires proteolysis and, more specifically, the destruction of cyclin B. As first reported for treatments that stabilize MTs [9
], we also found that cyclin B levels progressively decline during a mitotic block induced by drugs that prevent MT assembly. Thus, the key issue for how a vertebrate cell escapes mitosis when it cannot satisfy the SAC is how it degrades its cyclin B during the block.
Several recent reports suggest that the checkpoint proteins Bub1 and BubR1 are degraded in HeLa in a caspase-dependent manner during a mitosis prolonged by drugs [16
]. This, by itself, would lead to checkpoint inactivation, APC-mediated cyclin B destruction, and escape from mitosis. However, these claims are not supported by our data that BubR1 remains associated with kinetochores even after cells have escaped the mitotic block (). Moreover, they are based on sorting analyses of cell populations, arrested in mitosis for 24–48 hr, in which the cells are likely dying during mitosis via apoptosis [19
]. Instead, our data appear to rule out the possibility that vertebrates escape mitosis in the absence of MTs by suddenly inactivating the SAC and thus activating the APCs near the end of the block. Indeed, when the SAC cannot be satisfied, the levels of cyclin B decline gradually and not suddenly. Furthermore, during and after escaping mitosis in the absence of MTs, the kinetochores retain high levels of checkpoint signaling proteins (e.g., Mad2 and BubR1), the APC substrate Tpx2 is not degraded, and RPE1 cells do not appear to disjoin their chromatids. Concerning the last point, it is noteworthy that in vertebrates chromatid disjunction does not occur even in untreated cells containing a fully functional spindle until ~95% of the cyclin B has been degraded [9
]. Moreover, evidence is accumulating that this event requires cyclin B destruction since it does not occur, or is considerably delayed, in non-drug-treated cells expressing a nondegradable cyclin B that have presumably satisfied the SAC [11
In lieu of checkpoint inactivation, there are two non-mutually exclusive mechanisms for how cells can escape mitosis in the presence of a functional SAC. The first is that cyclin B is slowly and constantly degraded in an APC-independent manner until it falls below the threshold for maintaining the mitotic state. For several reasons this scenario is highly unlikely. First, our data reveal that the degradation of cyclin B is specific to mitosis, since it is not seen in G2 GFP-cyclin B-expressing bystander cells. Second, the degradation of GFP-cyclin B lacking a D-box, and therefore its APC recognition site, occurs extremely slowly even in the presence of fully functional spindles (; [9
]). Presumably, in the absence of MTs, any general cyclin denaturation/degradation pathway not involving APCs would also degrade GFP-cyclin B-Δ85 at the same rate as degradable cyclin. We evaluated this idea by treating RPE1 cells expressing GFP-cyclin B with 200 nM nocodazole and 5 µM MG132, and then by asking how quickly GFP-cyclin B is degraded in the absence of proteasome function. Although this combination of drugs is lethal over prolonged periods (12–15 hr; see Figure S2
), we found that the GFP-cyclin B fluorescence intensity did not decay (and actually slowly increased) over the 5–10 hr we followed the cells (Figure S4
). Thus, when the SAC cannot be satisfied, cyclin B degradation is not due to a steady general denaturation of the protein but instead requires proteasome-mediated proteolysis. Finally, we found that cells that form rudimentary spindles in nocodazole escape mitosis significantly faster than those that cannot generate spindle MTs (; [6
]). This positive correlation between the presence of spindle MTs and an accelerated escape from mitosis is difficult to explain by a nonspecific cyclin B destruction mechanism.
The more attractive mechanism forhow cells exit mitosis in the presence of a functional SAC is simply that the SAC is not normally 100% effective at blocking all APCs from ubiquinating cyclin B. Under this condition, a constant low level of ubiquination and subsequent proteolysis would gradually drop the cyclin B level below that needed to maintain the mitotic state. This route provides a simple and elegant mechanism for ultimately overcoming the mitotic condition before the cell dies, which, at least under some conditions, may be important (e.g., although the value of this feature to multicellular organisms is not readily apparent, in plants it leads to changes in ploidy levels and new strains of commercially valuable crops). This mechanism also provides a straightforward explanation for why the nocodazole-induced mitotic delay seen in normal [23
] and some cancer [25
] cells containing abnormally low levels of Mad2 or BubR1 is greatly attenuated relative to cells containing normal levels of these checkpoint proteins: since even with normal Mad2 or BubR1 concentrations the SAC cannot prohibit a low level ubiquination of cyclin B by the APCCdc20
complex, lowering the concentration further makes the SAC even less effective at inhibiting the APCs, and the more rapidly cyclin B levels would be degraded below that needed to maintain the mitotic state.
Cells that form rudimentary spindles in response to low nocodazole concentrations are unable to satisfy the SAC as evidenced by the continuous presence of Mad2 and BubR1 on some kinetochores after they escape mitosis (). However, as noted above, such cells exit mitosis significantly faster than those that cannot generate spindle MTs (see also [6
]). Not surprisingly, we find that nocodazole-treated cells containing rudimentary spindles degrade their cyclin B much faster than cells lacking MTs(). This means that when the SAC cannot be satisfied, the mere presence of MTs somehow accelerates the destruction of cyclin B. Although the reasons for this are unknown, it is possible that APCs, which are normally associated with the spindle [29
], work more efficiently when attached to MTs. Alternatively, at the lower drug concentrations, many kinetochores become attached to spindle MTs, which makes them poor checkpoint signalers because they possess reduced amounts of checkpoint proteins (e.g., cf. ; ; see [31
]). Under this condition, the amount of the APC inhibitor ultimately generated may be significantly less than that formed in the complete absence of all MTs, and the higher residual APC activity would produce a more rapid degradation of cyclin B and a corresponding shorter delay in mitosis.