The anthracycline antibiotic, doxorubicin, is one of the most important anticancer agents for solid tumors (Hortobagyi, 1997), and is a valuable component of intra-arterial infusion for the treatment of unresectable hepatocellular carcinoma (Acunas and Rozanes, 1999). However, despite widespread clinical use of doxorubicin, its anti-proliferative and death-inducing signaling is far from well characterized. Free radical formation and DNA damage via inhibition of topoisomerase II may be primarily responsible for its cytotoxic effects (Gewirtz, 1999). As with many other anticancer drugs, doxorubicin induces apoptosis; this may involve activation of caspases and disruption of mitochondrial membrane potential (Gamen et al., 2000). Several reports have demonstrated that various cancer cells treated with low doses (LD) of doxorubicin show a senescence-like phenotype (SLP) that resembles replicative senescence of normal cells at the morphological and enzymatic levels (Chang et al., 1999a; Wang et al., 1999). Senescent cells are generally characterized by a reduction in proliferative capacity, adoption of a flattened and enlarged cell shape, and the appearance of senescence-associated (SA)-β-galactosidase activity (Dimiri et al., 1995). Other than doxorubicin, low doses of
DNA damaging agents, including various chemotherapeutic drugs and ionizing radiation (Chang et al., 1999b), and introduction of an activated ras oncogene (Serrano et al., 1997) have been reported to induce senescence in cancer cells in a
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similar manner. However, the molecular events that trigger SLP in cancer cells are far from clear.
Another lines of evidences showed that LD of chemotherapeutic drugs (Lock and Stribinskiene, 1996), γ-radiation (Hendry and West, 1997), and activated Ras (Miranda et al., 1996) induce mitotic catastrophe. Igor Roninson attempted to define mitotic catastrophe in morphological term, namely, as a type of cell death resulting from abnormal mitosis, which usually ends in the formation of large cells with multiple micronuclei and decondensed chromatin (Roninson et al., 2001). Cells undergoing mitotic catastrophe usually do not show DNA ladder formation (He et al., 2002) or DNA breaks detectable by TUNEL staining (Chang et al., 1999b), suggesting that this death is non-apoptotic. However, some research groups regard
“mitotic catastrophe” as abnormal mitosis that leads to cell death (which can occur through necrosis or apoptosis) rather than to the cell death itself (Nitta et al., 2004;
Chu et al., 2004). Castedo et al., have recently proposed that mitotic catastrophe results from a combination of deficient cell-cycle checkpoints and cellular damage (Castedo et al., 2004a). They argued that failure to arrest the cell cycle before or at mitosis triggers an attempt of aberrant chromosome segregation, which culminates in the activation of apoptotic default pathway and cellular demise. Therefore, until now there is no consensus whether cell death through mitotic catastrophe is fundamentally different death mode from apoptosis.
p53 protein is capable of transactivating at least three genes, MDM2, GADD45, and p21 (CIP1/WAF1). p21 is thought to be the main downstream effector
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of p53 protein and is suggested to mediate p53-induced growth arrest, triggered by DNA damage. Normally, in a cell cycle, cyclin-dependent kinases (CDKs) phosphorylate proteins such as the Rb protein, allowing cells to enter the S-phase. In cells exposed to DNA-damaging agents, an arrest in the G1 phase of the cell cycle is seen. This arrest is thought to be mediated by p21, which is increased secondary to an increase in p53 protein after injury to DNA. p21 acts by binding to CDK–cyclin complexes, inhibiting their kinase activity and thereby leading to lack of phosphorylation of crucial proteins. This results in blockage of entry into the S-phase, allowing sufficient time for repair. In several studies, most of them on cell lines, p53 protein accumulation in response to DNA-damaging agents has been compared with the p21 response. A strong association has been found between DNA damage, p53 protein accumulation, and p21 expression. p21 protein expression has been detected in cells with wild-type TP53, but not in cells lacking p53 protein activity. The development of many types of human cancers (>50%) is associated with loss or mutation of p53. Furthermore, Bukholm and Nesland (Bukholm and Nesland, 2000) reported that a strong association between p21 downregulation and p53 protein accumulation and some cases with no expression of p21 and without detectable p53 protein was observed in primary human colon carcinoma. p21 also appears to induce G2 cell cycle arrest after DNA damage by directly inhibiting the activity of Cdks (Canman and Kastan, 1995; White, 1996; Levine, 1997). DNA damage is sensed by the ataxia-telangiectasia mutated protein (ATM; Perry and Kleckner, 2003; Bakkenist and Kastan, 2003). p53 is one of the key targets that are subjected to activation by
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ATM catalyzed phosphorylation (Canman et al, 1998). Activated p53, in turn, induces the expression of many proteins including p21, and is required to arrest cells at the G1 and G2 checkpoints of the cell cycle after DNA damage (Dulic et al, 1994;
Kuerbitz et al, 1992; Reed et al, 1994; Passalaris et al, 1999). These events also provide the cells with enough time to repair damaged DNA and prevent accumulation of deleterious mutations in the genome that would otherwise be subsequently transferred to daughter cells. The development of many types of human cancers (>50%) is associated with loss or mutation of p53. In particular, the loss or mutation of the p53 tumor suppressor gene has important consequences on mitotic fidelity when damaged DNA is present. In several studies, a strong association between DNA damage-induced p53 accumulation and p21 protein levels (Bukholm and Nesland, 2000) has been reported. p21 protein expression has been detected in cells harboring wild-type p53, but not in cells without functional p53. Also in primary human colon carcinoma without detectable p53 protein, p21 expression was not observed. Although several groups (Castedo et al., 2004a; Nitta et al., 2004) have reported the induction of mitotic catastrophe in p53-deficient cancer cells, involvement of p21 in mitotic catastrophe or multinucleation has not been clarified.
Chemotherapeutic agents have been reported to induce various cellular responses in cancer cells, including apoptosis, cell cycle arrest, senescence or mitotic catastrophe. However, the critical factors determining these different cellular responses are largely unknown. Recently, we have observed that doxorubicin of high dose (HD) induces apoptosis, while doxorubicin of low dose (LD) induces cell death
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through mitotic catastrophe in human hepatoma cells. In addition to the doses of the drugs, different cellular responses may be induced by the same drug of same dose, depending on the genetic backgrounds of cancer cells.
The fact that sub-apoptotic doses of the same stimuli induce SLP or cell death by mitotic catastrophe attempted us to investigate the possible link between the drug-induced SLP and cell death through mitotic catastrophe in tumor cells. Here, in part I we show that LD doxorubicin induces abnormal mitosis and SLP in Huh-7 cells, and then those cells finally die through mitotic catastrophe, accompanying the formation of multiple micronuclei and loss of membrane integrity. Many human HCC cell lines show a similar cellular response, suggesting that induction of SLP and cell death by mitotic catastrophe may be a general response to LD doxorubicin. We show for the first time that the mode of LD doxorubicin-induced cell death by mitotic catastrophe is morphologically and biochemically distinct from apoptosis induced by high doses of the same drug. In part II, we examined the role of p21 in the determination of cellular fates in response to different doses of doxorubicin. We demonstrate here that treatment with LD doxorubicin inducesd a dramatic multinucleation in p21-/- cells but not in p21+/+ cells. In contrast, treatment with MD of doxorubicin induces G2
cell cycle arrest, instead of multinucleation, both in p21+/+ and p21-/- cells.
Therefore we presents the first evidence that p21 plays a pivotal role in the blocking of multinucleation induced by LD doxorubicin but a supportive role in the attenuation of MD doxorubicin-induced multinucleation.
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