Delta/Jagged-mediated Notch signaling induces the differentiation of agr2-positive epidermal mucous cells in zebrafish embryos.

Teleosts live in aquatic habitats, where they encounter ionic and acid-base fluctuations as well as infectious pathogens. To protect from these external challenges, the teleost epidermis is composed of living cells, including keratinocytes and ionocytes that maintain body fluid ionic homeostasis, and mucous cells that secret mucus. While ionocyte progenitors are known to be specified by Delta-Notch-mediated lateral inhibition during late gastrulation and early segmentation, it remains unclear how epidermal mucous cells (EMCs) are differentiated and maintained. Here, we show that Delta/Jagged-mediated activation of Notch signaling induces the differentiation of agr2-positive (agr2+) EMCs in zebrafish embryos during segmentation. We demonstrated that agr2+ EMCs contain cytoplasmic secretory granules and express muc5.1 and muc5.2. Reductions in agr2+ EMC number were observed in mib mutants and notch3 MOs-injected notch1a mutants, while increases in agr2+ cell number were detected in notch1a- and X-Su(H)/ANK-overexpressing embryos. Treatment with γ-secretase inhibitors further revealed that Notch signaling is required during bud to 15 hpf for the differentiation of agr2+ EMCs. Increased agr2+ EMC numbers were also observed in jag1a-, jag1b-, jag2a- and dlc-overexpressing, but not jag2b-overexpressing embryos. Meanwhile, reductions in agr2+ EMC numbers were detected in jag1a morphants, jag1b mutants, jag2a mutants and dlc morphants, but not jag2b mutants. Reduced numbers of pvalb8-positive epidermal cells were also observed in mib or jag2a mutants and jag1a or jag1b morphants, while increased pvalb8-positive epidermal cell numbers were detected in notch1a-overexpressing, but not dlc-overexpressing embryos. BrdU labeling further revealed that the agr2+ EMC population is maintained by proliferation. Cell lineage experiments showed that agr2+ EMCs are derived from the same ectodermal precursors as keratinocytes or ionocytes. Together, our results indicate that specification of agr2+ EMCs in zebrafish embryos is induced by DeltaC/Jagged-dependent activation of Notch1a/3 signaling, and the cell population is maintained by proliferation.

HSP70 regulates the function of mitotic centrosomes.

To establish a functional bipolar mitotic spindle, the centrosome expands and matures, acquiring enhanced activities for microtubule (MT) nucleation and assembly at the onset of mitosis. However, the regulatory mechanisms of centrosome maturation and MT assembly from the matured centrosome are largely unknown. In this study, we showed that heat shock protein (HSP) 70 considerably accumulates at the mitotic centrosome during prometaphase to metaphase and is required for bipolar spindle assembly. Inhibition or depletion of HSP70 impaired the function of mitotic centrosome and disrupted MT nucleation and polymerization from the spindle pole, and may thus result in formation of abnormal mitotic spindles. In addition, HSP70 may associate with NEDD1 and γ-tubulin, two pericentriolar material (PCM) components essential for centrosome maturation and MT nucleation. Loss of HSP70 function disrupted the interaction between NEDD1 and γ-tubulin, and reduced their accumulation at the mitotic centrosome. Our results thus demonstrate a role for HSP70 in regulating centrosome integrity during mitosis, and indicate that HSP70 is required for the maintenance of a functional mitotic centrosome that supports the assembly of a bipolar mitotic spindle.

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 (HSP70(wt)) and its phosphomimetic mutant (HSP70(SS631,633DD)), a phosphorylation-resistant mutant of HSP70 (HSP70(SS631,633AA)) failed to localize at the centrosome. ATO-induced spindle elongation was abolished in cells overexpressing HSP70(SS631,633AA). Conversely, mitotic spindles in cells ectopically expressing HSP70(SS631,633DD) were more resistant to nocodazole-induced depolymerization than in those expressing HSP70(wt) or HSP70(SS631,633AA). 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.

NPRL2 is required for proliferation of oncogenic Ras-transformed bronchial epithelial cells.

Nitrogen permease regulator-like 2 (NPRL2/TUSC4) is known to exert both tumor-suppressing and oncogenic effects in different types of cancers, suggesting that its actions are context dependent. Here, we delineated the molecular and functional effects of NPRL2 in malignantly transformed bronchial epithelial cells. To do so, we depleted NPRL2 in oncogenic HRas-transduced and malignantly transformed human bronchial epithelial (BEAS2B), Ras-AI-T2 cells. Intriguingly, depletion of NPRL2 in these cells induced activation of mTORC1 downstream signaling, inhibited autophagy, and impaired Ras-AI-T2 cell proliferation both in vitro and in vivo. These results suggest that NPRL2 is required for oncogenic HRas-induced cell transformation. Depletion of NPRL2 increased levels of the DNA damage marker γH2AX, the cell cycle inhibitors p21 and p27, and the apoptosis marker cleaved-PARP. These NPRL2-depleted cells first accumulated at G1 and G2, and later exhibited signs of mitotic catastrophe, which implied that NPRL2 depletion may be detrimental to oncogenic HRas-transformed cells. Additionally, NPRL2 depletion reduced heat shock factor 1/heat shock element- and NRF2/antioxidant response element-directed luciferase reporter activities in Ras-AI-T2 cells, indicating that NPRL2 depletion led to the suppression of two key cytoprotective processes in oncogenic HRas-transformed cells. Overall, our data suggest that oncogenic HRas-transduced and malignantly transformed cells may depend on NPRL2 for survival and proliferation, and depletion of NPRL2 also induces a stressed state in these cells.

Inhibition of AKT enhances mitotic cell apoptosis induced by arsenic trioxide.

Accumulated evidence has revealed a tight link between arsenic trioxide (ATO)-induced apoptosis and mitotic arrest in cancer cells. AKT, a serine/threonine kinase frequently over-activated in diverse tumors, plays critical roles in stimulating cell cycle progression, abrogating cell cycle checkpoints, suppressing apoptosis, and regulating mitotic spindle assembly. Inhibition of AKT may therefore enhance ATO cytotoxicity and thus its clinical utility. We show that AKT was activated by ATO in HeLa-S3 cells. Inhibition of AKT by inhibitors of the phosphatidyl inositol 3-kinase/AKT pathway significantly enhanced cell sensitivity to ATO by elevating mitotic cell apoptosis. Ectopic expression of the constitutively active AKT1 had no effect on ATO-induced spindle abnormalities but reduced kinetochore localization of BUBR1 and MAD2 and accelerated mitosis exit, prevented mitotic cell apoptosis, and enhanced the formation of micro- or multi-nuclei in ATO-treated cells. These results indicate that AKT1 activation may prevent apoptosis of ATO-arrested mitotic cells by attenuating the function of the spindle checkpoint and therefore allowing the formation of micro- or multi-nuclei in surviving daughter cells. In addition, AKT1 activation upregulated the expression of aurora kinase B (AURKB) and survivin, and depletion of AURKB or survivin reversed the resistance of AKT1-activated cells to ATO-induced apoptosis. Thus, AKT1 activation suppresses ATO-induced mitotic cell apoptosis, despite the presence of numerous spindle abnormalities, probably by upregulating AURKB and survivin and attenuating spindle checkpoint function. Inhibition of AKT therefore effectively sensitizes cancer cells to ATO by enhancing mitotic cell apoptosis.

Establishment of a bioluminescence-based bioassay for the detection of dioxin-like compounds.

Dioxin and dioxin-like compounds are among the most prevalent and toxic environmental pollutants. At present, analytical chemical techniques are considered the gold standard for detection of dioxins. Here, we describe a highly sensitive and cost-effective alternative, based on bioluminescence and bioluminescence resonance energy transfer (BRET). Upon binding to dioxin, aryl hydrocarbon receptor (AHR) dissociates from HSP90 and subsequently translocates to the nucleus, where it interacts with AHR nuclear translocator (ARNT). We generated cell lines that stably co-express a fusion protein of AHR and Renilla luciferase (AHR-RL) and either HSP90 or ARNT tagged with yellow fluorescent protein (HSP90-YFP or ARNT-YFP). The fluorescent signals of YFP are activated by the emission of RL while the interactions between AHR and HSP90 (or ARNT) were monitored. Application of 3-methylcholanthrene, the AHR agonist, enhances BRET signals in cells co-expressing AHR-RL, AIP-His, P23-His and ARNT-YFP (AAPA cells), while suppressing BRET signals in cells co-expressing AHR-RL, AIP-His, P23-His and HSP90-YFP (AAPH cells). In addition, dioxin treatment reduced Renilla luminescence in AAPH cells in a concentration-dependent manner, due to the degradation of AHR. Intriguingly, the detection limit for dioxin in our AHR degradation assay was as low as 10(-17) M. This work highlights the potential of AHR-RL degradation assays to detect dioxin-like pollutants.

Inhibition of acetyl-CoA carboxylase impaired tubulin palmitoylation and induced spindle abnormalities.

Tubulin s-palmitoylation involves the thioesterification of a cysteine residue in tubulin with palmitate. The palmitate moiety is produced by the fatty acid synthesis pathway, which is rate-limited by acetyl-CoA carboxylase (ACC). While it is known that ACC is phosphorylated at serine 79 (pSer79) by AMPK and accumulates at the spindle pole (SP) during mitosis, a functional role for tubulin palmitoylation during mitosis has not been identified. In this study, we found that modulating pSer79-ACC level at the SP using AMPK agonist and inhibitor induced spindle defects. Loss of ACC function induced spindle abnormalities in cell lines and in germ cells of the Drosophila germarium, and palmitic acid (PA) rescued the spindle defects in the cell line treated transiently with the ACC inhibitor, TOFA. Furthermore, inhibition of protein palmitoylating or depalmitoylating enzymes also induced spindle defects. Together, these data suggested that precisely regulated cellular palmitate level and protein palmitoylation may be required for accurate spindle assembly. We then showed that tubulin was largely palmitoylated in interphase cells but less palmitoylated in mitotic cells. TOFA treatment diminished tubulin palmitoylation at doses that disrupt microtubule (MT) instability and cause spindle defects. Moreover, spindle MTs comprised of α-tubulins mutated at the reported palmitoylation site exhibited disrupted dynamic instability. We also found that TOFA enhanced the MT-targeting drug-induced spindle abnormalities and cytotoxicity. Thus, our study reveals that precise regulation of ACC during mitosis impacts tubulin palmitoylation to delicately control MT dynamic instability and spindle assembly, thereby safeguarding nuclear and cell division.

Heat shock factor 1 suppression induces spindle abnormalities and sensitizes cells to antimitotic drugs.

BACKGROUND: Heat shock factor 1 (HSF1) is the master regulator of the heat shock response and supports malignant cell transformation. Recent work has shown that HSF1 can access the promoters of heat shock proteins (HSPs) and allow HSP expression during mitosis. It also acts as a mitotic regulator, controlling chromosome segregation. In this study, we investigated whether the transactivation activity of HSF1 is required for the assembly of mitotic spindles. RESULTS: Our results showed that phosphorylation of HSF1 at serine 326 (S326) and its transactivation activity were increased during mitosis. Inhibition of the transactivation activity of HSF1 by KRIBB11 or CCT251263 during mitosis significantly increased the proportion of mitotic cells with abnormal spindles. It also hampered the reassembly of spindle microtubules after nocodazole treatment and washout by impeding the formation of chromosomal microtubule asters. Depletion of HSF1 led to defects in mitotic spindle assembly, subsequently attenuating cell proliferation and anchorage-independent cell growth (AIG). These HSF1 depletion-induced effects could be rescued by ectopically expressing wild-type HSF1 or a constitutively active mutant (∆202-316, caHSF1) but not the S326A or dominant negative (∆361-529, dnHSF1) mutants. In addition, overexpression of HSP70 partially reduced HSF1 depletion-induced spindle abnormalities. These results indicate that HSF1 may support cell proliferation and AIG by maintaining spindle integrity through its transactivation activity. Furthermore, inhibition of HSF1 transactivation activity by KRIBB11 or CCT251236 can enhance diverse anti-mitosis drug-induced spindle defects and cell death. CONCLUSIONS: The increased transactivation activity of HSF1 during mitosis appears to be required for accurate assembly of mitotic spindles, thereby supporting cell viability and probably AIG. In addition, inhibition of the transactivation activity of HSF1 may enhance the mitotic errors and cell death induced by anti-mitosis drugs.

Mdivi-1 induces spindle abnormalities and augments taxol cytotoxicity in MDA-MB-231 cells.

Taxol is a first-line chemotherapeutic for numerous cancers, including the highly refractory triple-negative breast cancer (TNBC). However, it is often associated with toxic side effects and chemoresistance in breast cancer patients, which greatly limits the clinical utility of the drug. Hence, compounds that act in concert with taxol to promote cytotoxicity may be useful to improve the efficacy of taxol-based chemotherapy. In this study, we demonstrated that mdivi-1, a putative inhibitor of mitochondrial fission protein Drp1, enhances the anticancer effects of taxol and overcomes taxol resistance in a TNBC cell line (MDA-MB-231). Not only did mdivi-1 induce mitotic spindle abnormalities and mitotic arrest when used alone, but it also enhanced taxol-induced antimitotic effects when applied in combination. In addition, mdivi-1 induced pronounced spindle abnormalities and cytotoxicity in a taxol-resistant cell line, indicating that it can overcome taxol resistance. Notably, the antimitotic effects of mdivi-1 were not accompanied by prominent morphological or functional alterations in mitochondria and were Drp1-independent. Instead, mdivi-1 exhibited affinity to tubulin at µM level, inhibited tubulin polymerization, and immediately disrupted spindle assembly when cells entered mitosis. Together, our results show that mdivi-1 associates with tubulin and impedes tubulin polymerization, actions which may underlie its antimitotic activity and its ability to enhance taxol cytotoxicity and overcome taxol resistance in MDA-MB-231 cells. Furthermore, our data imply a possibility that mdivi-1 could be useful to improve the therapeutic efficacy of taxol in breast cancer.

Antitumor Effects of the Novel Quinazolinone Holu-12: Induction of Mitotic Arrest and Apoptosis in Human Oral Squamous Cell Carcinoma CAL27 Cells.

BACKGROUND/AIM: Quinazolinone is a privileged chemical structure employed for targeting various types of cancer. This study aimed to demonstrate the antitumor activity of synthesized 6,7-disubstituted-2-(3-fluorophenyl) quinazolines (HoLu-11 to HoLu-14). MATERIALS AND METHODS: The cytotoxicity was assessed by the sulforhodamine B (SRB) assay. The cell cycle was examined by flow cytometry. The expression levels of cell cycle- and apoptosis-related proteins were estimated by western blotting. A xenograft animal model was used to explore the antitumor effects of HoLu-12. RESULTS: Among four synthetic quinazolinone derivatives, HoLu-12 significantly reduced the viability of oral squamous cell carcinoma (OSCC) cells. HoLu-12 induced G2/M arrest and increased the expression of cyclin B, histone H3 (Ser10) phosphorylation, and cleaved PARP, indicating that HoLu-12 could induce mitotic arrest and then apoptosis. Moreover, the combination of HoLu-12 and 5-fluorouracil (5-FU) displayed synergistic toxic effect on OSCC cells. HoLu-12 significantly inhibited tumor growth in vivo. CONCLUSION: HoLu-12 induces mitotic arrest and leads to apoptosis of OSCC cells. Furthermore, HoLu-12 alone or in combination with 5-FU is a potential therapeutic agent for OSCC.

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.

HSP70 regulates Eg5 distribution within the mitotic spindle and modulates the cytotoxicity of Eg5 inhibitors.

The heat shock protein 70 (HSP70) is a conserved molecular chaperone and proteostasis regulator that protects cells from pharmacological stress and promotes drug resistance in cancer cells. In this study, we found that HSP70 may promote resistance to anticancer drugs that target the mitotic kinesin, Eg5, which is essential for assembly and maintenance of the mitotic spindle and cell proliferation. Our data show that loss of HSP70 activity enhances Eg5 inhibitor-induced cytotoxicity and spindle abnormalities. Furthermore, HSP70 colocalizes with Eg5 in the mitotic spindle, and inhibition of HSP70 disrupts this colocalization. Inhibition or depletion of HSP70 also causes Eg5 to accumulate at the spindle pole, altering microtubule dynamics and leading to chromosome misalignment. Using ground state depletion microscopy followed by individual molecule return (GSDIM), we found that HSP70 inhibition reduces the size of Eg5 ensembles and prevents their localization to the inter-polar region of the spindle. In addition, bis(maleimido)hexane-mediated protein-protein crosslinking and proximity ligation assays revealed that HSP70 inhibition deregulates the interaction between Eg5 tetramers and TPX2 at the spindle pole, leading to their accumulation in high-molecular-weight complexes. Finally, we showed that the passive substrate-binding activity of HSP70 is required for appropriate Eg5 distribution and function. Together, our results show that HSP70 substrate-binding activity may regulate proper assembly of Eg5 ensembles and Eg5-TPX2 complexes to modulate mitotic distribution/function of Eg5. Thus, HSP70 inhibition may sensitize cancer cells to Eg5 inhibitor-induced cytotoxicity.

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.

HSP70 is required for the proper assembly of pericentriolar material and function of mitotic centrosomes.

BACKGROUND: At the onset of mitosis, the centrosome expands and matures, acquiring enhanced activities for microtubule nucleation and assembly of a functional bipolar mitotic spindle. However, the mechanisms that regulate centrosome expansion and maturation are largely unknown. Previously, we demonstrated in an immortalized human cell line CGL2 and cancer cell line HeLa that the inducible form of heat shock protein 70 (HSP70) accumulates at the mitotic centrosome and is required for centrosome maturation and bipolar spindle assembly. RESULTS: In this study, we further show that HSP70 accumulated at the spindle pole in a PLK1-dependent manner. HSP70 colocalized with pericentrin (PCNT), CEP215 and γ-tubulin at the spindle pole and was required for the 3D assembly of these three proteins, which supports mitotic centrosome function. Loss of HSP70 disrupted mitotic centrosome structure, reduced pericentriolar material recruitment and induced fragmentation of spindle poles. In addition, HSP70 was necessary for the interaction between PCNT and CEP215 and also facilitated PLK1 accumulation and function at the spindle pole. Furthermore, we found that HSP70 chaperone activity is required for PCNT accumulation at the mitotic centrosome and assembly of mitotic spindles. CONCLUSION: Our current results demonstrate that HSP70 is required for the accurate assembly of the pericentriolar material and proper functioning of mitotic centrosomes.

Phosphatidylinositol-5-phosphate 4-kinase gamma accumulates at the spindle pole and prevents microtubule depolymerization.

BACKGROUND: A previous screen of a human kinase and phosphatase shRNA library to select genes that mediate arsenite induction of spindle abnormalities resulted in the identification of phosphatidylinositol-5-phosphate 4-kinase type-2 gamma (PIP4KIIγ), a phosphatidylinositol 4,5-bisphosphate (PIP2)-synthesizing enzyme. In this study, we explored how PIP4KIIγ regulates the assembly of mitotic spindles. RESULTS: PIP4KIIγ accumulates at the spindle pole before anaphase, and is required for the assembly of functional bipolar spindles. Depletion of PIP4KIIγ enhanced the spindle pole accumulation of mitotic centromere-associated kinesin (MCAK), a microtubule (MT)-depolymerizing kinesin, and resulted in a less stable spindle pole-associated MT. Depletion of MCAK can ameliorate PIP4KIIγ depletion-induced spindle abnormalities. In addition, PIP2 binds to polo-like kinase (PLK1) and reduces PLK1-mediated phosphorylation of MCAK. These results indicate that PIP4KIIγ and PIP2 may negatively regulate the MT depolymerization activity of MCAK by reducing PLK1-mediated phosphorylation of MCAK. Consequently, depletion of PLK1 has been shown to counteract the PIP4KIIγ depletion-induced instability of spindle pole-associated MT and cell resistance to arsenite. CONCLUSIONS: Our current results imply that PIP4KIIγ may restrain MT depolymerization at the spindle pole through attenuating PLK1-mediated activation of MCAK before anaphase onset.

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.

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.

Induction of C-anaphase and diplochromosome through dysregulation of spindle assembly checkpoint by sodium arsenite in human fibroblasts.

Cytogenetic alterations induced by arsenite are associated with its carcinogenic activity. Cytogenetic analysis revealed first that arsenite induced c-anaphases in a time- and dose-dependent manner in human fibroblasts (HFW). With additional incubation of arsenite-arrested mitotic cells in drug-free medium for 0-48 h, approximately 35% exited from mitosis without cell division. This was confirmed by the appearance of tetraploid metaphase, mainly diplochromosomes, in the subsequent cell division. Treatment of HFW cells with both nocodazole, a known agent of microtubular depolymerization, and Taxol, which induces tubulin polymerization and inhibits disassembly of microtubules, resulted in remarkable mitotic arrest but induced only negligible c-anaphase, tetraploidy, and diplochromosomes. Staurosporine, a kinase inhibitor that could effectively reduce arsenite-induced c-anaphase, could also decrease the development of diplochromosomes in the subsequent cell division cycle. These results imply that arsenite-induced c-anaphases mainly exited from mitosis without cell division and became tetraploid in the subsequent cell cycle. Antitubulin immunofluorescent staining confirmed no formation of bipolar spindles in nocodazole-arrested mitotic HFW cells, whereas in arsenite-arrested mitotic cells bipolar spindles were present but distorted in appearance and apparently dysfunctional. Mitotic arrest deficient 2 (Mad2) signal was, as expected, clearly visible at centromeres of nocodazole-arrested mitotic cells. However, the Mad2 signal at centrosomes became insignificant in either arsenite-arrested or nocodazole/arsenite-arrested mitotic cells. In addition, the association of Mad2 with the APC/C(cdc20) complex and the accumulation of Pds1, an anaphase inhibitor, were remarkably reduced in arsenite-arrested mitotic cells as compared with nocodazole-arrested mitotic cells. These results support the observation that nocodazole can inhibit spindle formation and, hence, activate spindle assembly checkpoint to arrest cells at metaphase. In contrast, the dysfunctional bipolar spindles in arsenite-arrested mitotic cells could not effectively activate spindle assembly checkpoint and, hence, resulted in formation of c-anaphase and diplochromosomes in the subsequent cell division.

Derivatives of 6-cinnamamido-quinoline-4-carboxamide impair lysosome function and induce apoptosis.

Autophagy is a lysosomal degradative process that protects cancer cells from multiple types of stress. In this study, we synthesized a series of derivatives of 6-cinnamamido-quinoline-4-carboxamide (CiQ), and investigated their effects on the proliferation and autophagy of cancer cells in vitro. These derivatives effectively inhibited the proliferation of a broad spectrum of cancer cell lines. Further study revealed that CiQ derivatives may induce autophagy and result in disruption of autophagy propagation. Consequently, these derivatives triggered massive apoptosis, as evidenced by caspase-9 activation and PARP cleavage. Blockage of autophagy by depletion of autophagy related gene ATG5 or BECN1 considerably alleviated CiQ-induced cell death, indicating that autophagy may mediate CiQ-induced cell death. Furthermore, treatment with CiQ derivatives increased lysosome membrane permeability (LMP) and enhanced accumulation of ubiquitinated proteins, which collectively indicate impaired lysosome function. In addition, treatment of cells with CiQ derivatives activated extracellular signal-regulated kinase (ERK); abrogation of ERK activation, either by treating cells with U0126, an inhibitor of mitogen-activated protein/ERK kinase 1 (MEK1), or by ectopically overexpressing a dominant-negative MEK1, significantly reduced CiQ derivative-induced LMP, LC3 and p62 accumulation, and cytotoxicity. These results indicate that CiQ derivatives activate ERK and disrupt lysosome function, thereby altering autophagic flux and resulting in apoptotic cell death.