Ling-Huei Yih

Effects of exposure protocols on induction of kinetochore-plus and -minus micronuclei by arsenite in diploid human fibroblasts

Arsenic, widely distributed in the environment, is a potent human carcinogen. Arsenite genotoxicity has been observed in a variety of cells and animal systems. However, the underlying mechanism is not completely clear. In this study, human fibroblasts (HFW) were treated with 1.25-10 microM arsenite for 24 h (low dose and long exposure) and 5-80 microM for 4 h (high dose and short exposure), and the arsenite accumulation, cytotoxicity, and micronucleus (MN) induction were examined. By these two different protocols, HFW cells showed equivalent levels of arsenite accumulation, but exhibited different kinetics of cell killing and different types of MN generation. Arsenite induced mainly kinetochore-positive MN (K+-MN) in HFW cells by low dose exposure whereas mainly kinetochore-negative MN (K--MN) was induced by high dose exposure. Catalase reduced both K+- and K--MN induced by these two exposure protocols. Except for the case of K+-MN induction by the high dose exposure protocol, N-acetyl-cysteine (NAC) in both low and high dose protocols was also shown to effectively reduce arsenite-induced MN. The present results imply that oxidative stress is involved in arsenite-induced MN in diploid human fibroblasts. However, different protocols for arsenite exposure may result in different cellular damage.

Induction of Centrosome Amplification during Arsenite-Induced Mitotic Arrest in CGL-2 Cells

Arsenite-induced mitotic abnormalities result in mitotic death in several cancer cell lines. However, how arsenite induces these effects is not known. We have previously shown that arsenite induces mitotic arrest, mitotic abnormalities, and mitotic death in CGL-2 cells. To further delineate the mechanism of action of arsenite, we examined its effect on centrosome duplication and the possible link between centrosome dysregulation and arsenite-induced mitotic death. Immunofluorescence staining of gamma-tubulin revealed that centrosome amplification was induced in arsenite-arrested mitotic cells but not in nocodazole-arrested cells. When S phase-enriched cells were treated with arsenite, they progressed into and arrested at mitosis and then formed supernumerary centrosomes. A further increase in arsenite-induced centrosome amplification was seen during the prolonged mitotic arrest. The arsenite-induced supernumerary centrosomes might result from uneven fragmentation of centrosome, overexpression of pericentriolar materials, and inhibition of centrosomal coalescence during mitosis. Furthermore, termination of mitotic arrest by treatment of arsenite-arrested mitotic cells with cyclin-dependent kinase 1 inhibitors or by suppression of spindle checkpoint function by small interfering RNA-mediated silencing of BubR1 or Mad2 markedly reduced the induction of centrosome amplification and mitotic death in arsenite-treated cells. These results indicate that centrosome amplification is induced in arsenite-arrested mitotic CGL-2 cells in a spindle checkpoint-dependent manner and is involved in the induction of arsenite-induced mitotic death.

Heat shock protein inhibitors, 17-DMAG and KNK437, enhance arsenic trioxide-induced mitotic apoptosis

Arsenic trioxide (ATO) has recently emerged as a promising therapeutic agent in leukemia because of its ability to induce apoptosis. However, there is no sufficient evidence to support its therapeutic use for other types of cancers. In this study, we investigated if, and how, 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG), an antagonist of heat shock protein 90 (HSP90), and KNK437, a HSP synthesis inhibitor, potentiated the cytotoxic effect of ATO. Our results showed that cotreatment with ATO and either 17-DMAG or KNK437 significantly increased ATO-induced cell death and apoptosis. siRNA-mediated attenuation of the expression of the inducible isoform of HSP70 (HSP70i) or HSP90alpha/beta also enhanced ATO-induced apoptosis. In addition, cotreatment with ATO and 17-DMAG or KNK437 significantly increased ATO-induced mitotic arrest and ATO-induced BUBR1 phosphorylation and PDS1 accumulation. Cotreatment also significantly increased the percentage of mitotic cells with abnormal mitotic spindles and promoted metaphase arrest as compared to ATO treatment alone. These results indicated that 17-DMAG or KNK437 may enhance ATO cytotoxicity by potentiating mitotic arrest and mitotic apoptosis possibly through increased activation of the spindle checkpoint.

Tin-protoporphyrin potentiates arsenite-induced DNA strand breaks, chromatid breaks and kinetochore-negative micronuclei in human fibroblasts

Numerous reports have shown that oxidative stress is involved in arsenite-induced genetic damage. Arsenite is also a potent inducer of heme oxygenase (HO)-1. To understand whether HO-1 could function as a cellular antioxidant and protect cells from arsenite injury, the effects of tin-protoporphyrin (SnPP), a competitive inhibitor of HO-1, on arsenite-induced genetic damage were examined in human skin fibroblasts (HFW). In the present study, we found that SnPP at 100 microM significantly potentiated arsenite-induced cytotoxicity, DNA strand breaks (assayed by alkaline single cell gel electrophoresis(SCGE)), and chromatid breaks. Although arsenite alone mainly induced kinetochore-plus micronuclei (K(+)-MN), SnPP only synergistically enhanced kinetochore-negative micronuclei (K(-)-MN). The increase in K(-)-MN by SnPP cotreatment was consistent with the increase in DNA strand breaks and chromatid breaks caused by SnPP. However, at higher arsenite doses, K(+)-MN was significantly reduced by SnPP. Pretreatment of HFW cells with hemin, an inducer of HO-1, significantly attenuated the cytotoxicity of arsenite. Therefore, the present results suggest that HO-1 induction by arsenite plays certain roles in protecting cells from arsenite-induced injury.

Requirement of a functional spindle checkpoint for arsenite-induced apoptosis.

To understand the potential influence of spindle checkpoint function in response to arsenic trioxide (ATO)‐induced apoptosis observed in cancer cell lines, we examined the correlation between activation of the spindle checkpoint and susceptibility to ATO‐induced apoptosis in 10 cancer cell lines lacking functional p53. The ability to functionally activate the spindle checkpoint in each cancer cell line was assessed by the induction of mitotic arrest after Taxol treatment. Bromodeoxyuridine (BrdU) pulse‐chase analysis of Taxol‐treated cell lines with low mitotic arrest showed that they were not arrested at mitosis but divided abnormally, confirming that spindle checkpoint activation was impaired in these cell lines. Our results demonstrate that apoptosis was significantly induced by ATO in cancer cell lines with functional activation of the spindle checkpoint and substantial induction of mitotic arrest. Cell lines with negligible mitotic arrest exhibited little ATO‐induced apoptosis. However, no such correlation was observed following treatment of cells with camptothecin, a topoisomerase I inhibitor. Furthermore, attenuation of the spindle checkpoint function by small interfering RNA‐mediated silencing of BubR1 and Mad2 in cancer cells that were susceptible to ATO‐induced mitotic arrest and apoptosis greatly reduced the induction of mitotic arrest and apoptosis by ATO and increased the formation of micronuclei or multinuclei in survived cells. The marked correlation between ATO‐induced mitotic arrest and apoptosis indicates that the induction of apoptosis by ATO was highly dependent on the functional activation of the spindle checkpoint in cancer cells lacking normal p53 function. J. Cell. Biochem. 105: 678–687, 2008.

Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest.

DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry of γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest.

HSP70 colocalizes with PLK1 at the centrosome and disturbs spindle dynamics in cells arrested in mitosis by arsenic trioxide.

Heat shock protein 70 (HSP70) has been shown to be a substrate of Polo-like kinase 1 (PLK1), and it prevents cells arrested in mitosis by arsenic trioxide (ATO) from dying. Here, we report that HSP70 participates in ATO-induced spindle elongation, which interferes with mitosis progression. Our results demonstrate that HSP70 and PLK1 colocalize at the centrosome in ATO-arrested mitotic cells. HSP70 located at the centrosome was found to be phosphorylated by PLK1 at Ser631 and Ser633. Moreover, unlike wild-type HSP70 (HSP70wt) and its phosphomimetic mutant (HSP70SS631,633DD), a phosphorylation-resistant mutant of HSP70 (HSP70SS631,633AA) failed to localize at the centrosome. ATO-induced spindle elongation was abolished in cells overexpressing HSP70SS631,633AA. Conversely, mitotic spindles in cells ectopically expressing HSP70SS631,633DD were more resistant to nocodazole-induced depolymerization than in those expressing HSP70wt or HSP70SS631,633AA. In addition, inhibition of PLK1 significantly reduced HSP70 phosphorylation and induced early onset of apoptosis in ATO-arrested mitotic cells. Taken together, our results indicate that PLK1-mediated phosphorylation and centrosomal localization of HSP70 may interfere with spindle dynamics and prevent apoptosis of ATO-arrested mitotic cells.

Arsenic Trioxide Induces Abnormal Mitotic Spindles Through a PIP4KIIγ/Rho Pathway

Arsenite-induced spindle abnormalities result in mitotic cell apoptosis in several cancer cell lines, but how arsenite induces these effects is not known. Evidence to date has revealed that arsenite activates Rho guanosine triphosphatases (GTPases). Because Rho GTPases regulate spindle orientation, chromosome congression, and cytokinesis, we therefore examined the involvement of Rho GTPases and their modulators in arsenite-induced mitotic abnormalities. We demonstrated that arsenic trioxide (ATO) disrupted the positioning of bipolar mitotic spindles and induced centrosome and spindle abnormalities. ATO increased the level of the active guanosine triphosphate-bound form of Rho. Inhibition of Rho-associated protein kinases (ROCKs) by Y-27632 ameliorated ATO-induced spindle defects, mitotic arrest, and cell death. These results indicate that ATO may induce spindle abnormalities and mitotic cell death through a Rho/ROCK pathway. In addition, screening of a human kinase and phosphatase shRNA library to select genes that mediate ATO induction of spindle abnormalities resulted in the identification of phosphatidylinositol-5-phosphate 4-kinase type-2 gamma (PIP4KIIγ), a phosphatidylinositol 4,5-biphosphate (PIP2) synthesis enzyme that belongs to the phosphatidylinositol phosphate kinase (PIPK) family. Sequestration of PIP2 by ectopic overexpression of the pleckstrin homology domain of phospholipase C-δ1 protected cells from ATO-induced cell death. Furthermore, depletion of PIP4KIIγ, but not other isoforms of the PIPK family, not only reduced Rho GTPase activation in ATO-treated cells but also alleviated ATO-induced spindle defects, mitotic arrest, and mitotic cell apoptosis. Thus, our results imply that ATO induces abnormalities in mitotic spindles through a PIP4KIIγ/Rho pathway, leading to apoptosis of mitotic cells.

Suppression of sodium arsenite-potentiated cytotoxicity of ultraviolet light by cycloheximide in Chinese hamster ovary cells.

Post-treatment with sodium arsenite synergistically increased the cytotoxicity of ultraviolet (UV) light. The potentiation of UV cytotoxicity by sodium arsenite was apparently suppressed by cycloheximide (CHM), a protein synthesis inhibitor. The protective effect of CHM against sodium arsenite-potentiated UV cytotoxicity was well correlated to its activity in inhibiting the synthesis of stress proteins, particularly a small polypeptide with a molecular weight of 8500 dalton. This small stress protein was demonstrated as ubiquitin by immunoprecipitation. Our results also showed that neither ubiquitin induction nor potentiation of UV cytotoxicity by post-treatment with sodium arsenite was observed in the stationary cells. Thus, we suggested that ubiquitin is possibly involved in the action of arsenite in potentiating UV-induced cell killing.

Glyfoline induces mitotic catastrophe and apoptosis in cancer cells.

Glyfoline exhibits cytotoxic activity in vitro and antitumor activity in mice bearing murine or human solid tumors, but the underlying mechanisms are unknown. In our study, we found that glyfoline inhibited cell growth and induced accumulation of mitotic cells in human cancer cell lines. Glyfoline induced the appearance of spindle abnormalities, chromosome mis‐segregation, multipolar cell division and multiple nuclei, all of which are indicative of mitotic catastrophe. However, glyfoline did not bind to DNA and did not inhibit or stabilize tubulin polymerization, but slightly increased the resistance of mitotic spindles to nocodazole‐induced disassembly. In addition, microtubule aster formation was significantly enhanced in the extract prepared from glyfoline‐arrested mitotic cells compared to that from synchronized mitotic cells. When Eg5, a mitotic kinesin that plays an essential role in establishing mitotic spindle bipolarity, was inhibited using S‐trityl‐cysteine in glyfoline‐treated cells, formation of spindle multipolarity, multipolar cell division, and multinuclei was significantly reduced. After glyfoline‐mediated arrest of cells at mitosis, considerable poly(ADP‐ribose) polymerase degradation was induced and the number of annexin V‐positive cells significantly increased, indicating that glyfoline ultimately induces apoptosis. Small interfering RNA‐mediated silencing of the spindle checkpoint proteins BUBR1 and MAD2 markedly reduced induction of mitotic cell accumulation, but did not affect glyfoline‐induced mitotic catastrophe and apoptosis. Thus, glyfoline induces mitotic catastrophe probably by enhancing microtubule aster formation and subsequent apoptosis in cancer cells independently of spindle checkpoint function.