Mitotic checkpoints, MUGs and kinetochores

Abstract

The kinetochore is a specialized locus located at the centromere (primary constriction) of mammalian mitotic chromosomes and serves as an attachment site for spindle microtubules. Thus, kinetochores are essential for the correct movement, alignment and partitioning of chromosomes during mitosis. With exception of the minute centromeres of yeasts, i.e., S. cerevisiae, much remains to be learned about the molecular organization of DNA and protein components of larger, more complex centromeres/kinetochores of chromosomes of higher eukaryotic organisms. This has been due, for the most part, to the lack of reliable procedures for isolating and purifying functional kinetochores of the higher eukaryotes. Hope came unexpectedly approximately 30 y ago, when we utilized a method developed by Schlegel and Pardee1 for driving metaphase-arrested cells into mitosis prematurely, bypassing S-phase and DNA synthesis. We termed these “mitotic cells with unreplicated genomes” or MUGS, and to our surprise, when MUGs were examined by EM, we discovered numerous kinetochores that had become detached from the condensed chromatin.2 These laminar-like elements were essentially identical to kinetochore lamina or plates normally seen at the centromere of mitotic chromosomes (Fig. 1) and were mostly attached or associated with mitotic spindle microtubules. This fortuitous and unexpected discovery enabled us to ascertain that the kinetochores from metaphase chromosomes were more structurally complex than anticipated, consisting of repeated protein subunits interspersed by DNA linkers.3 Moreover, we determined that the number of detached kinetochores in each MUGs was 2–5 times greater than the actual diploid chromosome number, consistent with the notion that kinetochores were structurally repetitive. Figure 1. Electron micrographs of contiguous serial sections of normal attached kinetochore (G–K) and detached kinetochores from MUGs (M–P) are shown below. Reproduced from reference 3 with permission. Initially we were optimistic that MUGS offered a potential strategy for the purification and isolation of kinetochores from human chromosomes. However, this notion was threatened initially when MUGS were thought to be produced in only a limited number of mammalian cell lines, i.e, hamsters, rats and deer. Subsequently, however, Balczon4 found that by overexpressing cyclin A, MUGS could be readily induced in HeLa cells. In a later study, Wise and Brinkley5 reported that kinetochore fragments of MUGS, although fully detached from chromosomes, could undergo both normal prometaphase movements and equatorial alignment via spindle microtubules, even in the absence of paired sister kinetochores, as seen in normal mitosis. Therefore, it was concluded that “information” needed for proper chromosome alignment at metaphase, resides largely within the mitotic spindle per se and is not as a function of kinetochores. It was confirmed, however, that detached kinetochores of MUGS, although properly aligned on the metaphase spindle, were incapable of undergoing anaphase movement and segregation to spindle poles without attachment to chromosomes. In view of the plethora of new knowledge on the regulation of cell cycle and spindle checkpoints, it should be possible to establish a more efficient molecular rationale for MUG induction and perhaps decipher more clearly the molecular mechanisms associated with centromere fragmentation and kinetochore detachment. Although the methodology offers a logical approach for fractionation of centromere/kinetochores in human cells, could the induction of such catastrophic events in mitotic cells have potential application to cancer chemotherapy? A recent report by Beeharry et al.6 offers a reasonable rationale for such an approach. In their search for chemosensitization agents that could be useful tools for overriding cell cycle checkpoints and inducing cell death (mitotic catastrophe), these investigators re-discovered MUGs after almost 30 y of quiescence. When S-phase cells were treated with gemcitabine in combination with Chk1 inhibitors, S-phase checkpoints were overridden, and the cells displayed detached kinetochores essentially identical to those previously in our original reports. Even greater efficiency and more relevant results were obtained when topoisomerase II-mediated S-phase-arrested cell were used. Perhaps of more significance was their success in inducing MUGs in cells derived directly from primary human pancreatic tumors (EGF-1 cells). Previous studies of MUGS have all been limited to establish cells in vitro. MUGS represent manifestations of severe mitotic catastrophe, and that end-point itself, may have relevance for novel strategies in the realm of cancer chemotherapy. However, MUG technology as a strategy for isolating pure fractions of functional kinetochores needed in the construction of artificial chromosomes remains a worthwhile goal.