Distinct effects of heat shock temperatures on mitotic progression by influencing the spindle assembly checkpoint.

Heat shock is a physiological and environmental stress that leads to the denaturation and inactivation of cellular proteins and is used in hyperthermia cancer therapy. Previously, we revealed that mild heat shock (42 °C) delays the mitotic progression by activating the spindle assembly checkpoint (SAC). However, it is unclear whether SAC activation is maintained at higher temperatures than 42 °C. Here, we demonstrated that a high temperature of 44 °C just before mitotic entry led to a prolonged mitotic delay in the early phase, which was shortened by the SAC inhibitor, AZ3146, indicating SAC activation. Interestingly, mitotic slippage was observed at 44 °C after a prolonged delay but not at 42 °C heat shock. Furthermore, the multinuclear cells were generated by mitotic slippage in 44 °C-treated cells. Immunofluorescence analysis revealed that heat shock at 44 °C reduces the kinetochore localization of MAD2, which is essential for mitotic checkpoint activation, in nocodazole-arrested mitotic cells. These results indicate that 44 °C heat shock causes SAC inactivation even after full activation of SAC and suggest that decreased localization of MAD2 at the kinetochore is involved in heat shock-induced mitotic slippage, resulting in multinucleation. Since mitotic slippage causes drug resistance and chromosomal instability, we propose that there may be a risk of cancer malignancy when the cells are exposed to high temperatures.

The tumor suppressor LATS2 reduces v-Src-induced membrane blebs in a kinase activity-independent manner.

When cells with excess DNA, such as tetraploid cells, undergo cell division, it can contribute to cellular transformation via asymmetrical chromosome segregation-generated genetic diversity. Cell cycle progression of tetraploid cells is suppressed by large tumor suppressor 2 (LATS2) kinase-induced inhibitory phosphorylation of the transcriptional coactivator Yes-associated protein (YAP). We recently reported that the oncogene v-Src induces tetraploidy and promotes cell cycle progression of tetraploid cells by suppressing LATS2 activity. We explore here the mechanism by which v-Src suppresses LATS2 activity and the role of LATS2 in v-Src-expressing cells. LATS2 was directly phosphorylated by v-Src and the proto-oncogene c-Src, resulting in decreased LATS2 kinase activity. This kinase-deficient LATS2 accumulated in a YAP transcriptional activity-dependent manner, and knockdown of either LATS2 or the LATS2-binding partner moesin-ezrin-radixin-like protein (Merlin) accelerated v-Src-induced membrane bleb formation. Upon v-Src expression, the interaction of Merlin with LATS2 was increased possibly due to a decrease in Merlin phosphorylation at Ser518, the dephosphorylation of which is required for the open conformation of Merlin and interaction with LATS2. LATS2 was colocalized with Merlin at the plasma membrane in a manner that depends on the Merlin-binding region of LATS2. The bleb formation in v-Src-expressing and LATS2-knockdown cells was rescued by the reexpression of wild-type or kinase-dead LATS2 but not the LATS2 mutant lacking the Merlin-binding region. These results suggest that the kinase-deficient LATS2 plays a role with Merlin at the plasma membrane in the maintenance of cortical rigidity in v-Src-expressing cells, which may cause tumor suppression.

The tyrosine kinase v-Src modifies cytotoxicities of anticancer drugs targeting cell division.

v-Src oncogene causes cell transformation through its strong tyrosine kinase activity. We have revealed that v-Src-mediated cell transformation occurs at a low frequency and it is attributed to mitotic abnormalities-mediated chromosome instability. v-Src directly phosphorylates Tyr-15 of cyclin-dependent kinase 1 (CDK1), thereby causing mitotic slippage and reduction in Eg5 inhibitor cytotoxicity. However, it is not clear whether v-Src modifies cytotoxicities of the other anticancer drugs targeting cell division. In this study, we found that v-Src restores cancer cell viability reduced by various microtubule-targeting agents (MTAs), although v-Src does not alter cytotoxicity of DNA-damaging anticancer drugs. v-Src causes mitotic slippage of MTAs-treated cells, consequently generating proliferating tetraploid cells. We further demonstrate that v-Src also restores cell viability reduced by a polo-like kinase 1 (PLK1) inhibitor. Interestingly, treatment with Aurora kinase inhibitor strongly induces cell death when cells express v-Src. These results suggest that the v-Src modifies cytotoxicities of anticancer drugs targeting cell division. Highly activated Src-induced resistance to MTAs through mitotic slippage might have a risk to enhance the malignancy of cancer cells through the increase in chromosome instability upon chemotherapy using MTAs.

Functional differences between Hsp105/110 family proteins in cell proliferation, cell division, and drug sensitivity.

The mammalian HSP105/110 family consists of four members, including Hsp105 and Apg-1, which function as molecular chaperones. Recently, we reported that Hsp105 knockdown increases sensitivity to the DNA-damaging agent Adriamycin but decreases sensitivity to the microtubule-targeting agent paclitaxel. However, whether the other Hsp105/110 family proteins have the same functional property is unknown. Here, we show that Apg-1 has different roles from Hsp105 in cell proliferation, cell division, and drug sensitivity. We generated the Apg-1-knockdown HeLa S3 cells by lentiviral expression of Apg-1-targeting short hairpin RNA. Knockdown of Apg-1 but not Hsp105 decreased cell proliferation. Apg-1 knockdown increased cell death upon Adriamycin treatment without affecting paclitaxel sensitivity. The cell synchronization experiment suggests that Apg-1 functions in mitotic progression at a different mitotic subphase from Hsp105, which cause difference in paclitaxel sensitivity. Since Apg-1 is overexpressed in certain types of tumors, Apg-1 may become a potential therapeutic target for cancer treatment without causing resistance to the microtubule-targeting agents.

Linderapyrone: A Wnt signal inhibitor isolated from Lindera umbellata.

Linderapyrone, a Wnt signal inhibitor was isolated from the methanolic extract of the stems and twigs of Lindera umbellata together with epi-(-)-linderol A. Linderapyrone inhibited TCF/ß-catenin transcriptional activity that was evaluated using cell-based TOPFlash luciferase assay system. To evaluate the structure-activity relationship and mechanism, we synthesized linderapyrone and its derivatives from piperitone. As the results of further bioassay for synthesized compounds, we found both of pyrone and monoterpene moieties were necessary for inhibitory effect. cDNA microarray analysis in a linderapyrone derivative treated human colorectal cancer cells showed that this compound downregulates Wnt signaling pathway. Moreover, we successes to synthesize the derivative of linderapyrone that has stronger inhibitory effect than linderapyrone and ICG-001 (positive control).

Kinase activity-independent role of EphA2 in the regulation of M-phase progression.

Cell division is a tightly regulated, essential process for cell proliferation. Very recently, we reported that EphA2 is phosphorylated at Ser897, via the Cdk1/MEK/ERK/RSK pathway, during M phase and contributes to proper M-phase progression by maintaining cortical rigidity via the EphA2pSer897/ephexin4/RhoG pathway. Here, we show that EphA2 kinase activity is dispensable for M-phase progression. Although EphA2 knockdown delayed this progression, the delay was rescued by an EphA2 mutant expression with an Asp739 to Asn substitution, as well as by wild-type EphA2. Western blotting analysis confirmed that the Asp739Asn mutant lost its EphA2 kinase activity. Like wild-type EphA2, the Asp739Asn mutant was localized to the plasma membrane irrespective of cell cycle. While RhoG localization to the plasma membrane was decreased in EphA2 knockdown cells, it was rescued by re-expression of wild-type EphA2 but not via the mutant containing the Ser897 to Ala substitution. This confirmed our recent report that phosphorylation at Ser897 is responsible for RhoG localization to the plasma membrane. In agreement with the M-phase progression's rescue effect, the Asp739Asn mutant rescued RhoG localization in EphA2 knockdown cells. These results suggest that EphA2 regulates M-phase progression in a manner independent of its kinase activity.

Combination Treatment of OSI-906 with Aurora B Inhibitor Reduces Cell Viability via Cyclin B1 Degradation-Induced Mitotic Slippage.

Insulin-like growth factor 1 receptor (IGF1R), a receptor-type tyrosine kinase, transduces signals related to cell proliferation, survival, and differentiation. We recently reported that OSI-906, an IGF1R inhibitor, in combination with the Aurora B inhibitor ZM447439 suppresses cell proliferation. However, the mechanism underlying this suppressive effect is yet to be elucidated. In this study, we examined the effects of combination treatment with OSI-906 and ZM447439 on cell division, so as to understand how cell proliferation was suppressed. Morphological analysis showed that the combination treatment generated enlarged cells with aberrant nuclei, whereas neither OSI-906 nor ZM447439 treatment alone caused this morphological change. Flow cytometry analysis indicated that over-replicated cells were generated by the combination treatment, but not by the lone treatment with either inhibitors. Time-lapse imaging showed mitotic slippage following a severe delay in chromosome alignment and cytokinesis failure with furrow regression. Furthermore, in S-trityl-l-cysteine-treated cells, cyclin B1 was precociously degraded. These results suggest that the combination treatment caused severe defect in the chromosome alignment and spindle assembly checkpoint, which resulted in the generation of over-replicated cells. The generation of over-replicated cells with massive aneuploidy may be the cause of reduction of cell viability and cell death. This study provides new possibilities of cancer chemotherapy.

EphA2 phosphorylation at Ser897 by the Cdk1/MEK/ERK/RSK pathway regulates M-phase progression via maintenance of cortical rigidity.

Successful cell division is accomplished by the proper formation of the mitotic spindle. Here, we show that EphA2 knockdown causes mitotic errors, including a delay in M-phase progression, asymmetric spindle positioning, multipolar spindles, and cell blebs. It has been known that EphA2 is phosphorylated at Tyr588, which is triggered by the ligand binding, and at Ser897 downstream of growth factor signaling. Upon mitotic entry, EphA2 is phosphorylated at Ser897, accompanied by a reduction in Tyr588 phosphorylation. This EphA2 phosphorylation at Ser897 is inhibited by MEK/ERK and 90 kDa ribosomal S6 kinase (RSK) inhibitors and is induced by the introduction of active cyclin-dependent kinase 1 (Cdk1) and cyclin B1. EphA2 knockdown-induced M-phase delay and cell blebs are rescued by wild type EphA2 expression but not by Ser897Ala mutant. The Ras homolog gene family member G (RhoG) guanine nucleotide exchange factor Ephexin4 interacts with EphA2 in a Ser897 phosphorylation-dependent manner, and its knockdown delays M-phase progression and causes RhoG delocalization. RhoG knockdown delays M-phase progression, and EphA2 knockdown-induced M-phase delay is partially rescued by the constitutively active RhoG mutant. These results suggest that, in EphA2-expressing cells, EphA2 phosphorylation at Ser897 participates in proper M-phase progression downstream of the Cdk1/MEK/ERK/RSK pathway because of its role in maintaining cortical rigidity via Ephexin4 and RhoG and thereby regulating mitotic spindle formation.-Kaibori, Y. Saito, Y., Nakayama, Y. EphA2 phosphorylation at Ser897 by the Cdk1/MEK/ERK/RSK pathway regulates M-phase progression via maintenance of cortical rigidity.

Heat shock-induced mitotic arrest requires heat shock protein 105 for the activation of spindle assembly checkpoint.

Heat shock causes proteotoxic stress that induces various cellular responses, including delayed mitotic progression and the generation of an aberrant number of chromosomes. In this study, heat shock delayed the onset of anaphase by increasing the number of misoriented cells, accompanied by the kinetochore localization of budding uninhibited by benzimidazole-related (BubR)1 in a monopolar spindle (Mps)1-dependent manner. The mitotic delay was canceled by knockdown of mitotic arrest defect (Mad)2. Knockdown of heat shock protein (Hsp)105 partially abrogated the mitotic delay with the loss of the kinetochore localization of BubR1 under heat shock conditions and accelerated mitotic progression under nonstressed conditions. Consistent with this result, Hsp105 knockdown increased the number of anaphase cells with lagging chromosomes, through mitotic slippage, and decreased taxol sensitivity more than Mad2 knockdown. Hsp105 was coprecipitated with cell division cycle (Cdc)20 in an Mps1-dependent manner; however, its knockdown did not affect coprecipitation of Mad2 and BubR1 with Cdc20. We propose that heat shock delays the onset of anaphase via the activation of the spindle assembly checkpoint (SAC). Hsp105 prevents abnormal cell division by contributing to SAC activation under heat shock and nonstressed conditions by interacting with Cdc20 but not affecting formation of the mitotic checkpoint complex.-Kakihana, A., Oto, Y., Saito, Y., Nakayama, Y. Heat shock-induced mitotic arrest requires heat shock protein 105 for the activation of spindle assembly checkpoint.

Involvement of Stat3 phosphorylation in mild heat shock-induced thermotolerance.

Thermotolerance is a phenomenon in which cells become resistant to stress by prior exposure to heat shock, and its development is associated with the induction of heat shock proteins (Hsps), including Hsp70. We previously showed that the expression of Hsp70 is regulated by the cytokine signaling transcription factor Stat3, but the role of Stat3 in thermotolerance is not known. In this study, we examined the possible involvement of Stat3 in the acquisition of thermotolerance. We found that severe heat shock-induced morphological changes and decreases in cell viability, which were suppressed by exposure to non-lethal mild heat shock prior to severe heat shock. This thermotolerance development was accompanied by Stat3 phosphorylation and the induction of Hsps such as Hsp105, Hsp70, and Hsp27. Stat3 phosphorylation and Hsp induction were inhibited by AG490, an inhibitor of JAK tyrosine kinase. Consistent with this, we found that mild heat shock-induced thermotolerance was partially suppressed by AG490 or knockdown of Hsp105. We also found that the Stat3 inhibitor Stattic suppresses the acquisition of thermotolerance by inhibiting the mild heat shock-induced Stat3 phosphorylation and Hsp105 expression. These results suggest that the mild heat shock-dependent stimulation of the JAK-Stat signaling pathway contributes to the development of thermotolerance via the induction of Hsps including Hsp105. This signaling pathway may be a useful target for hyperthermia cancer therapy.

Targeting Insulin-Like Growth Factor 1 Receptor Delays M-Phase Progression and Synergizes with Aurora B Inhibition to Suppress Cell Proliferation.

The insulin-like growth factor 1 receptor (IGF1R) is a receptor-type tyrosine kinase that transduces signals related to cell proliferation, differentiation, and survival. IGF1R expression is often misregulated in tumor cells, but the relevance of this for cancer progression remains unclear. Here, we examined the impact of IGF1R inhibition on cell division. We found that siRNA-mediated knockdown of IGF1R from HeLa S3 cells leads to M-phase delays. Although IGF1R depletion causes partial exclusion of FoxM1 from the nucleus, quantitative real-time PCR revealed that the transcription of M-phase regulators is not affected by decreased levels of IGF1R. Moreover, a similar delay in M phase was observed following 2 h of incubation with the IGF1R inhibitors OSI-906 and NVP-ADW742. These results suggest that the M-phase delay observed in IGF1R-compromised cells is not caused by altered expression of mitotic regulators. Live-cell imaging revealed that both prolonged prometaphase and prolonged metaphase underlie the delay and this can be abrogated by the inhibition of Mps1 with AZ3146, suggesting activation of the Spindle Assembly Checkpoint when IGF1R is inhibited. Furthermore, incubation with the Aurora B inhibitor ZM447439 potentiated the IGF1R inhibitor-induced suppression of cell proliferation, opening up new possibilities for more effective cancer chemotherapy.

The tyrosine kinase v-Src causes mitotic slippage by phosphorylating an inhibitory tyrosine residue of Cdk1.

The nonreceptor tyrosine kinase v-Src is an oncogene first identified in Rous sarcoma virus. The oncogenic effects of v-Src have been intensively studied; however, its effects on chromosomal integrity are not fully understood. Here, using HeLa S3/v-Src cells having inducible v-Src expression, we found that v-Src causes mitotic slippage in addition to cytokinesis failure, even when the spindle assembly checkpoint is not satisfied because of the presence of microtubule-targeting agents. v-Src's effect on mitotic slippage was also observed in cells after a knockdown of C-terminal Src kinase (Csk), a protein-tyrosine kinase that inhibits Src-family kinases and was partially inhibited by PP2, an Src-family kinase inhibitor. Proteomic analysis and in vitro kinase assay revealed that v-Src phosphorylates cyclin-dependent kinase 1 (Cdk1) at Tyr-15. This phosphorylation attenuated Cdk1 kinase activity, resulting in a decrease in the phosphorylation of Cdk1 substrates. Furthermore, v-Src-induced mitotic slippage reduced the sensitivity of the cells to microtubule-targeting agents, and cells that survived the microtubule-targeting agents exhibited polyploidy. These results suggest that v-Src causes mitotic slippage by attenuating Cdk1 kinase activity via direct phosphorylation of Cdk1 at Tyr-15. On the basis of these findings, we propose a model for v-Src-induced oncogenesis, in which v-Src-promoted mitotic slippage due to Cdk1 phosphorylation generates genetic diversity via abnormal cell division of polyploid cells and also increases the tolerance of cancer cells to microtubule-targeting agents.

Hsp105α suppresses Adriamycin-induced cell death via nuclear localization signal-dependent nuclear accumulation.

Heat shock protein 105 (Hsp105) is a molecular chaperone, and the isoforms Hsp105α and Hsp105ß exhibit distinct functions with different subcellular localizations. Hsp105ß localizes in the nucleus and induces the expression of the major heat shock protein Hsp70, whereas cytoplasmic Hsp105α is less effective in inducing Hsp70 expression. Hsp105 shuttles between the cytoplasm and the nucleus; the subcellular localization is governed by the relative activities of the nuclear localization signal (NLS) and nuclear export signal (NES). Here, we show that nuclear accumulation of Hsp105α but not Hsp105ß is involved in Adriamycin (ADR) sensitivity. Knockdown of Hsp105α induces cell death at low ADR concentration, at which ADR is less effective in inducing cell death in the presence of Hsp105α. Of note, Hsp105 is localized in the nucleus under these conditions, even though Hsp105ß is not expressed, indicating that Hsp105α accumulates in the nucleus in response to ADR treatment. The exogenously expressed Hsp105α but not its NLS mutant localizes in the nucleus of ADR-treated cells. In addition, the expression level of the nuclear export protein chromosomal maintenance 1 (CRM1) was decreased by ADR treatment of cells, and CRM1 knockdown caused nuclear accumulation of Hsp105α both in the presence and absence of ADR. These results indicating that Hsp105α accumulates in the nucleus in a manner dependent on the NLS activity via the suppression of nuclear export. Our findings suggest a role of nuclear Hsp105α in the sensitivity against DNA-damaging agents in tumor cells.

A novel immunofluorescence method to visualize microtubules in the antiparallel overlaps of microtubule-plus ends in the anaphase and telophase midzone.

Cell division, in which duplicated chromosomes are separated into two daughter cells, is the most dynamic event during cell proliferation. Chromosome movement is powered mainly by microtubules, which vary in morphology and are organized into characteristic structures according to mitotic progression. During the later stages of mitosis, antiparallel microtubules form the spindle midzone, and the irregular formation of the midzone often leads to failure of cytokinesis, giving rise to the unequal segregation of chromosomes. However, it is difficult to analyze the morphology of these microtubules because microtubules in the antiparallel overlaps of microtubule-plus ends in the midzone are embedded in highly electron-dense matrices, impeding the access of anti-tubulin antibodies to their epitopes during immunofluorescence staining. Here, we developed a novel method to visualize selectively antiparallel microtubule overlaps in the midzone. When cells are air-dried before fixation, aligned α-tubulin staining is observed and colocalized with PRC1 in the center of the midzone of anaphase and telophase cells, suggesting that antiparallel microtubule overlaps can be visualized by this method. In air-dried cells, mCherry-α-tubulin fluorescence and ß-tubulin staining show almost the same pattern as α-tubulin staining in the midzone, suggesting that the selective visualization of antiparallel microtubule overlaps in air-dried cells is not attributed to an alteration of the antigenicity of α-tubulin. Taxol treatment extends the microtubule filaments of the midzone in air-dried cells, and nocodazole treatment conversely decreases the number of microtubules, suggesting that unstable microtubules are depolymerized during the air-drying method. It is of note that the air-drying method enables the detection of the disruption of the midzone and premature midzone formation upon Aurora B and Plk1 inhibition, respectively. These results suggest that the air-drying method is suitable for visualizing microtubules in the antiparallel overlaps of microtubule-plus ends of the midzone and for detecting their effects on midzone formation.

Requirement of Hsp105 in CoCl2-induced HIF-1α accumulation and transcriptional activation.

The mammalian stress protein Hsp105α protects cells from stress conditions. Several studies have indicated that Hsp105α is overexpressed in many types of solid tumors, which contain hypoxic microenvironments. However, the role of Hsp105α in hypoxic tumors remains largely unknown. We herein demonstrated the involvement of Hsp105α in HIF-1 functions induced by the hypoxia-mimetic agent CoCl2. While Hsp105α is mainly localized in the cytoplasm under normal conditions, a treatment with CoCl2 induces the nuclear localization of Hsp105α, which correlated with HIF-1α expression levels. The overexpression of degradation-resistant HIF-1α enhances the nuclear localization of Hsp105α without the CoCl2 treatment. The CoCl2-dependent transcriptional activation of HIF-1, which is measured using a reporter gene containing a HIF-responsive element, is reduced by the knockdown of Hsp105α. Furthermore, the CoCl2-induced accumulation of HIF-1α is enhanced by heat shock, which results in the nuclear localization of Hsp105, and is suppressed by the knockdown of Hsp105. Hsp105 associates with HIF-1α in CoCl2-treated cells. These results suggest that Hsp105α plays an important role in the functions of HIF-1 under hypoxic conditions, in which Hsp105α enhances the accumulation and transcriptional activity of HIF-1 through the HIF-1α-mediated nuclear localization of Hsp105α.

Inhibitors of the VEGF Receptor Suppress HeLa S3 Cell Proliferation via Misalignment of Chromosomes and Rotation of the Mitotic Spindle, Causing a Delay in M-Phase Progression.

Cell division is the process by which replicated chromosomes are separated into two daughter cells. Although regulation of M phase has been extensively investigated, not all regulating factors have been identified. Over the course of our research, small molecules were screened to identify those that regulate M phase. In the present study, the vascular endothelial growth factor receptor (VEGFR) inhibitors A83-01, SU4312, and Ki8751 were examined to determine their effects on M phase. Treatment of HeLa S3 cells with these inhibitors suppressed cell proliferation in a concentration-dependent manner, and also suppressed Akt phosphorylation at Ser473, a marker of Akt activation. Interestingly, cleaved caspase-3 was detected in Adriamycin-treated cells but not in inhibitor-treated cells, suggesting that these inhibitors do not suppress cell proliferation by causing apoptosis. A cell cycle synchronization experiment showed that these inhibitors delayed M phase progression, whereas immunofluorescence staining and time-lapse imaging revealed that the M phase delay was accompanied by misalignment of chromosomes and rotation of the mitotic spindle. Treatment with the Mps1 inhibitor AZ3146 prevented the SU4312-induced M phase delay. In conclusion, the VEGFR inhibitors investigated here suppress cell proliferation by spindle assembly checkpoint-induced M phase delay, via misalignment of chromosomes and rotation of the mitotic spindle.

ERK Plays a Role in Chromosome Alignment and Participates in M-Phase Progression.

Cell division, a prerequisite for cell proliferation, is a process in which each daughter cell inherits one complete set of chromosomes. The mitotic spindle is a dedicated apparatus for the alignment and segregation of chromosomes. Extracellular signal-regulated kinase (ERK) 1/2 plays crucial roles in cell cycle progression, particularly during M-phase. Although, association with the mitotic spindle has been reported, the precise roles played by ERK in the dynamics of the mitotic spindle and in M-phase progression remain to be elucidated. In this study, we used MEK inhibitors U0126 and GSK1120212 to dissect the roles of ERK in M-phase progression and chromosome alignment. Fluorescence microscopy revealed that ERK is localized to the spindle microtubules in a manner independent of Src kinase, which is one of the kinases upstream of ERK at mitotic entry. ERK inhibition induces an increase in the number of prophase cells and a decrease in the number of anaphase cells. Time-lapse imaging revealed that ERK inhibition perturbs chromosome alignment, thereby preventing cells from entering anaphase. These results suggest that ERK plays a role in M-phase progression by regulating chromosome alignment and demonstrate one of the mechanisms by which the aberration of ERK signaling may produce cancer cells.

Yeast Two-Hybrid and One-Hybrid Screenings Identify Regulators of hsp70 Gene Expression.

The mammalian stress protein Hsp105ß, which is specifically expressed during mild heat shock and localizes to the nucleus, induces the major stress protein Hsp70. In the present study, we performed yeast two-hybrid and one-hybrid screenings to identify the regulators of Hsp105ß-mediated hsp70 gene expression. Six and two proteins were detected as Hsp105ß- and hsp70 promoter-binding proteins, respectively. A luciferase reporter gene assay revealed that hsp70 promoter activation is enhanced by the transcriptional co-activator AF9 and splicing mediator SNRPE, but suppressed by the coiled-coil domain-containing protein CCDC127. Of these proteins, the knockdown of SNRPE suppressed the expression of Hsp70 irrespective of the presence of Hsp105ß, indicating that SNRPE essentially functions as a transcriptional activator of hsp70 gene expression. The overexpression of HSP70 in tumor cells has been associated with cell survival and drug resistance. We here identified novel regulators of Hsp70 expression in stress signaling and also provided important insights into Hsp70-targeted anti-cancer therapy. J. Cell. Biochem. 117: 2109-2117, 2016. © 2016 Wiley Periodicals, Inc.

v-Src Causes Chromosome Bridges in a Caffeine-Sensitive Manner by Generating DNA Damage.

An increase in Src activity is commonly observed in epithelial cancers. Aberrant activation of the kinase activity is associated with malignant progression. However, the mechanisms that underlie the Src-induced malignant progression of cancer are not completely understood. We show here that v-Src, an oncogene that was first identified from a Rous sarcoma virus and a mutant variant of c-Src, leads to an increase in the number of anaphase and telophase cells having chromosome bridges. v-Src increases the number of γH2AX foci, and this increase is inhibited by treatment with PP2, a Src kinase inhibitor. v-Src induces the phosphorylation of KAP1 at Ser824, Chk2 at Thr68, and Chk1 at Ser345, suggesting the activation of the ATM/ATR pathway. Caffeine decreases the number of cells having chromosome bridges at a concentration incapable of inhibiting Chk1 phosphorylation at Ser345. These results suggest that v-Src induces chromosome bridges via generation of DNA damage and the subsequent DNA damage response, possibly by homologous recombination. A chromosome bridge gives rise to the accumulation of DNA damage directly through chromosome breakage and indirectly through cytokinesis failure-induced multinucleation. We propose that v-Src-induced chromosome bridge formation is one of the causes of the v-Src-induced malignant progression of cancer cells.

Nmi interacts with Hsp105ß and enhances the Hsp105ß-mediated Hsp70 expression.

The mammalian stress protein Hsp105α is expressed constitutively and is further induced under stress conditions, whereas the alternative spliced form, Hsp105ß is only expressed during mild heat shock. We previously reported that Hsp105α is localized mainly in the cytoplasm, whereas Hsp105ß is localized in the nucleus. Consistent with the different localization of these proteins, Hsp105ß but not Hsp105α induces the expression of the major stress protein Hsp70. We here identified N-myc and Stat interactor (Nmi), as an Hsp105ß-binding protein by yeast two-hybrid screening. Immunoprecipitation and pull-down assay showed that Nmi interacts with Hsp105ß in vivo and in vitro. Luciferase reporter gene assay and Western blotting showed that Nmi enhanced both the Hsp105ß-induced phosphorylation of Stat3 and the Hsp105ß-induced activation of the hsp70 promoter in a manner that is dependent on the Stat3-binding site, which results in an increase in Hsp70 protein levels. Most importantly, mild heat shock-induced Hsp70 expression, which is dependent on Hsp105ß, is suppressed by knockdown of endogenous Nmi. These results suggest that Nmi has a role as a positive regulator of Hsp105ß-mediated hsp70 gene expression along the Stat3 signaling pathway.