4 Mitotic Catastrophe Fiorenza Ianzini, FI, PhD, and Michael A. Mackey, MAM, PhD Summary Mitotic catastrophe (MC) is the result of premature or inappropriate entry of cells into mitosis, usually occurring because of chemical or physical stresses. MC is characterized by changes in nuclear morphology and the eventual appearance of polyploid cell progeny in affected cell populations, is markedly enhanced in cells lacking p53 function, and is the result of overaccumulation of cyclin B1 in cells delayed late in the cell cycle by the inducing agent. Thus, MC is considered to be the predominant mechanism underlying mitotic-linked cell death. Along with characteristic features associated with MC, a delayed DNA damage phenotype has been noted in these populations, suggesting a potential role for MC in mutagenesis and the acquisition of genomic instability. Although generally lethal, some cells can survive MC through mechanisms that are incompletely understood. Cytological features associated with meiotic cell division have been noted in polyploid cell populations produced through MC, a finding that might be particularly relevant in the understanding of tumor progression and that might provide a novel mechanism for the generation of quasi-diploid progeny from MC-induced polyploid cell populations. This review summarizes the literature pertaining to MC and describes current lines of research in this interesting research area. Key Words: Mitotic catastrophe; cell cycle regulation; cyclin B1; endopoly- ploid cells; mitosis; meiosis; carcinogenesis; tumor progression; delayed DNA damage; SPCC. 1. MOLECULAR MECHANISMS UNDERLYING MITOTIC CATASTROPHE 1.1. Mitotic Catastrophe is the Result of Premature Entry into Mitosis Following Abrogation of G2/M Checkpoint Function Exposure of some cell types to a broad class of agents can lead to a loss of regulation of cell division, such that cells enter into a premature mitosis, an event that culminates in a phenomenon called mitotic catastrophe (MC). MC is characterized by the aberrant From: Cancer Drug Discovery and Development Apoptosis, Senescence, and Cancer Edited by: D. A. Gewirtz, S. E. Holt and S. Grant © Humana Press Inc., Totowa, NJ 73 74 Part I / Apoptosis and Alternative Modes of Cell Death nuclear morphology observed following premature mitotic entry (1) and often results in the generation of aneuploid and polyploid cell progeny. Ultrastructural studies of HeLa cells (2,3) during long-duration hyperthermia characterized a general disorganization of cellular organelles that occurs in cells undergoing spontaneous premature chromosome condensation (SPCC). Cells undergoing SPCC can either fail to achieve cytokinesis (early-SPCC) or divide and fuse shortly thereafter (late-SPCC) (4). The early and late classifications arose based on two different observations: in the case of Chinese hamster ovary (CHO) and HeLa cells heated for up to 24 h at 415 C, cells arrest in mid-S phase and SPCC figures observed in cell preparations have the characteristic appearance of cells in S phase undergoing chromosome condensation due to mitotic factors (5), thus producing early-SPCC. Following radiation and other agents that block primarily in late S and G2 phases, the condensed chromosome morphology is more fibrillar, looking identical to the morphology obtained when a G2 phase cell is induced to prematurely enter mitosis (5); thus, these cells are termed late-SPCC. The difficulty in distinguishing SPCC from normal mitoses in some cell lines, especially late-SPCC, has lead to the use of nuclear fragmentation as an endpoint indicative of MC; studies have shown that these two quantities correlate well with SPCC (4,6). Stress-induced SPCC and subsequent MC are found following activation of cyclin B1/cdc2 kinase complex occurring while cells are delayed in S or G2 phases (4,7), indicating that stress-induced MC is the result of abrogation of cell cycle regulatory pathways, in particular the G2 checkpoint (8–10). MC has been found to occur in cells exposed to a variety of treatments, including adriamycin (11), 5-fluorouracil (12), etoposide (13), temozolomide (14), topotecan (15), camptothecin (16), combretastatin (17), paclitaxel (13,18), phytoestrogens (19), overexpression of c-H Ras (20), prolonged mild heat shock (17,21,22), UV radiation (23), methyl-methanesulfonate (23), ionizing radiation (4,24–26), and hyperthermia combined with radiation (27). Specific effects on mitotic spindle formation and mitotic microtubule stability, mediated by cyclin B1/cdc2 kinase activity, have been identified as playing an important role in MC (28,29). Furthermore, there is evidence in support of the notion that cell cycle delays play a critical role in the development of SPCC and MC. All the aforementioned studies reported that delays late in the cell cycle were observed prior to MC. In SPCC and MC induced by long-duration mild heat shock of HeLa cells, blockage in G1 during the heat treatment using either cycloheximide or caffeine (both agents known to induce a G1 block in HeLa cells) results in a reduction in S delay, SPCC, and cytotoxicity (30). Inhibition of cell cycle progression following radiation exposure leads to protective effects on cell survival, which has prompted some authors to consider that repair of potentially lethal damage can occur during the arrest interval (31). It has been proposed that one of the cellular functions of mutations in the tumor-suppressor gene p53 is to promote MC as a mechanism for removing damaged cells from populations following genotoxic stress (32). The mechanisms underlying this function of p53 involve its activity as a modulator of G2 checkpoint mechanisms. 1.2. p53 is an Important Repressor of MC All living organisms have evolved mechanisms to regulate the timing of cell division within the context of the completion of DNA replication. Events associated with mitosis, such as nuclear envelope breakdown, chromosome condensation, and assembly of the mitotic apparatus are mediated through the specific phosphorylation of cellular proteins Chapter 4 / Mitotic Catastrophe 75 by large multi-protein complexes containing, in addition to other proteins, the cdc2 and cyclin B1 gene products. The activity of this complex is stimulated through the action of the cdc25C phosphatase; inhibition is mediated by wee1 and other inhibitory kinases; in both cases, changes in the phosphorylation state of key residues of cdc2 result in changes in cyclin-dependent kinase activity. Cdc25C activity is inhibited in the presence of unreplicated DNA, whereas inhibitory kinases such as wee1 are constitu- tively active. Cyclin B1 biosynthesis also contributes to the regulation of mitotic entry, as cyclin B1 levels are cell cycle regulated, with the gene being expressed only in late S and G2 phases in human cells (33); proteosome-mediated degradation of cyclin B1 begins in anaphase, resulting in undetectable levels of the protein by the time cells enter the next S phase (34). These pathways act in concert to ensure that mitosis does not commence prior to the completion of DNA replication. Other mechanisms function to inhibit entry into mitosis if DNA is damaged, resulting in a G2 block; there are many pathways involved here, some are p53-dependent, and others are not. The end result is to reduce the activity of cyclin B1/cdc2-containing complexes, and the mecha- nisms that have been elucidated demonstrate the incredible diversity at play in biological organisms. Chk1, chk2, atm, and atr gene products all contribute to the activation of p53 in response to genotoxic stress, whereas p53-mediated inhibition of cyclin B1/cdc2 activity can occur through expression of the cdk inhibitor p21, direct effects on cyclin B1 or cdc2 biosynthesis, inhibition of cdc25C nuclear transport through chk1/chk2- and 14-3-3--dependent pathways (35), or destabilization of cyclin B1/cdc2 complexes by gadd45 (36). One study (37) has also implicated chk2 in the phosphorylation- mediated inactivation of cdc25A and radiation-induced inhibition of DNA synthesis that is generally thought to be responsible for S phase delays following irradiation (38). In the absence of checkpoint activation, mitosis can occur very rapidly, provided the right activating pathways predominate over the inactivating pathways (Fig. 1). When cells are damaged or otherwise perturbed from the usual sequence of cell cycle events, many of these regulatory pathways serve to check further progress through the cell cycle, until either a sensed deficiency (e.g., incomplete DNA replication) is ameliorated or some other imbalance is fixed. This balance between inhibitory and stimulatory pathways relies on the abundance of these same regulators, as it has been demonstrated that overexpression of cyclin B1 in G2-arrested cells leads to MC in genetic manipulation studies (8), independent of the activity of the inhibitory pathways. So, under ordinary conditions, these checkpoints serve to ensure that orderly progress through the cell cycle occurs. Under extraordinary conditions, however, these regulatory pathways can become abrogated, resulting in a deviation from the expected cellular behavior. MC is just one of these deviations. 1.3. MC Has Been Associated with Overexpression of Cyclin B1 in p53-Deficient Cells Previous studies of heat- and radiation-induced MC in HeLa cells (4,7,27) have demonstrated that a common feature of the effect of the two modalities is the high accumulation of cyclin B1 that occurs prior to the appearance of MC. Further studies indicate that this phenomenon