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The cell death mechanism that prevents aneuploidy caused by a failure of the spindle checkpoint has recently emerged as an important regulatory paradigm. We previously identified a novel type of mitotic cell death, termed caspase-independent mitotic death (CIMD), which is induced during early mitosis by partial BUB1 (a spindle checkpoint protein) depletion and defects in kinetochore–microtubule attachment. In this study, we have shown that survived cells that escape CIMD have abnormal nuclei, and we have determined the molecular mechanism by which BUB1 depletion activates CIMD. BUB3 (a BUB1 interactor and a spindle checkpoint protein) interacts with p73 (a homolog of p53) specifically in cells wherein CIMD occurs. BUB3 that is freed from BUB1 associates with p73 on which Y99 is phosphorylated by c-Abl tyrosine kinase, resulting in the activation of CIMD. These results strongly support the hypothesis that CIMD is the cell death mechanism protecting cells from aneuploidy by inducing the death of cells prone to substantial chromosome missegregation.
Aneuploidy – the presence of an abnormal number of chromosomes – is the primary cause of miscarriages, mental retardation, and congenital abnormalities in humans and is a hallmark of cancer (1). The role of aneuploidy in cancer has been hotly debated for almost a century, and recent evidence has shown a tight link between aneuploidy and tumorigenesis (2, 3). Aneuploidy is frequently caused by chromosome missegregation during mitosis, which results from errors of the mitotic checkpoint, the major cell cycle control mechanism that prevents chromosome missegregation (4, 5).
The spindle checkpoint arrests cell cycle progression prior to anaphase if chromosomes are unattached or incorrectly attached by transiently inhibiting the anaphase-promoting complex (APC/C) (6, 7). Kinetochores that are improperly attached to microtubules send a signal to the spindle checkpoint components – Mad1, Mad2, BUBR1 (Mad3-like, but has a C-terminal kinase domain), Bub1, Bub3, and Mps1 – all originally identified in Saccharomyces cerevisiae (8–10). These components and their functions are highly conserved between yeast and humans (11) and essential for the mitotic checkpoint in human cells as bona fide mitotic checkpoint proteins (12, 13).
Consistent with a link between aneuploidy and cancer, several evidences support the role of the spindle checkpoint in tumorigenesis. For example, mutations in the human homologs of Bub1 (BUB1 and BUBR1) have been found in subtypes of colorectal cancer cells that exhibit chromosome instability (CIN tumor cells) (14). The CIN phenotype has been associated with mutations in spindle checkpoint genes (15–17), decreased levels of spindle checkpoint proteins (18, 19), and loss of spindle checkpoint activity (20, 21). Mad2+/− mice develop lung tumors at high rates after a long latency (22). Bubr1+/− mice and Bub3/Rae1 heterozygotes are prone to tumor development (23, 24). These results strongly suggest a close relationship between altered activity of the spindle checkpoint and tumorigenesis. Also, importantly, many tumor cells have a diminished, but not absent, spindle checkpoint response (12).
When cells cannot satisfy the spindle checkpoint after a long mitotic delay (known as adaptation to D-mitosis), several cell fates can occur: some cells die during mitosis, some exit mitosis as viable cells but die via apoptosis in the G1 phase, and some exit mitosis as viable cells but are tetraploid and reproductively dead (7). Microtubule inhibitors induce mitotic arrest by activating the spindle checkpoint; eventually, these inhibitors cause cytotoxicity. Many reports have described the cytotoxicity of microtubule inhibitors and resultant cell death as either apoptosis in G1 or reproductive death (25). However, questions about cell death during mitosis have remained. Although many studies suggest that cell death occurs during mitosis (26–30), the mechanism remains to be detailed, especially with regard to the relationship between the spindle checkpoint and apoptosis during mitosis.
We have recently identified a novel type of mitotic cell death, which we term caspase independent mitotic death (CIMD) (31, 32). CIMD is a programmed cell death in early mitosis that is induced by defects in kinetochore-microtubule attachment in BUB1-deficient cells. In BUB1-deficient (but not MAD2-deficient) cells, CIMD is induced by conditions that activate the spindle checkpoint (i.e., cold shock or treatment with nocodazole, paclitaxel [Taxol] or 17-allylamino-17-demethoxygeldanamycin [17-AAG]). CIMD depends on p73, a homolog of p53, but not on p53. It also depends on the apoptosis-inducing factor (AIF) and endonuclease G (Endo G), which are effectors of caspase-independent cell death (33). When BUB1 is completely depleted, aneuploidy occurs instead of CIMD. We propose that CIMD is the cell death mechanism that protects cells from aneuploidy by inducing the death of cells prone to substantial chromosome missegregation. In this study, we have determined the molecular mechanism by which CIMD is activated by partial but not complete depletion of BUB1.
In our previous study, we detected no caspase activity (caspases 1, 3–9) in cells of which BUB1 is partially depleted using small interfering RNA (siRNA) and exposed to 17-AAG or Taxol (32). In addition, pharmacological caspase inhibitors bocaspartyl-(OMe)-fluoromethyl-ketone (BAF) and Z-VAD-FMK (zVAD) did not inhibit DNA fragmentation induced by 17-AAG or Taxol treatment and BUB1 partial depletion using siRNA (32). Therefore, we concluded that CIMD is caspase independent.
To strength our conclusion, we tested caspases 2 and 10–13 and found no caspase activity in cells in which CIMD occurs (Figure S1). We also tested whether CIMD occurs in mouse embryonic fibroblasts deficient in caspase 3 and 7 (34) and found that CIMD occurs in the absence of these caspases (Figure 1A–C). These results strongly suggest that CIMD is independent of caspases.
As we previously reported (32), when BUB1 is partially depleted from HeLa cells by siRNA and cells subsequently treated with 17-AAG or Taxol, substantial synergistic lethality is observed in a colony outgrowth assay (Figure 2A and B). We previously reported that this lethality is due to CIMD. Because the occurrence of DNA fragmentation is dependent on p73 but not on p53 (32), we examined whether depletion of p73 rescues the lethality caused by CIMD in the colony outgrowth assay. Although we could not detect p73 in HeLa cells by immunoblotting (data not shown), the amount of p73α mRNA was significantly suppressed when 2 independent sets of p73 siRNA were used (Figure S2; Table S2). Depletion of p73α rescued the lethality induced by BUB1 partial depletion (Figure S3 [right]) and drug treatment in the colony outgrowth assay, whereas single p73 depletion and drug treatment did not cause significant lethality (Figure 2A and B). These results indicate that p73 depletion rescues the lethality of cells in which CIMD occurs.
We have hypothesized that CIMD is an alternative cell death pathway that protects cells from aneuploidy by killing cells prone to substantial chromosome missegregation (31, 32). If this hypothesis is true, survived cells that escape CIMD by depletion of p73 in the colony outgrowth assay should be aneuploid. There was a significant increase in the number of abnormal nuclei in cells with p73 + BUB1 double siRNA and drug (17-AAG or Taxol) treatment (Figure 2C and D). The resulting abnormal nuclei (i.e., fragmented and aggregated nuclei consisting of more than 3 nuclei-like fragments, micronuclei, chromosome bridges, malformed nuclei, or binuclei; Figure 2C) are similar to those of cells depleted of MAD2 for several cell divisions (35). Some abnormal nuclei types (Types 1, 2, and 3) looked like completely BUB1-depleted cells that show significant reduction in CIMD cells and mitotic index with 17-AAG treatment (Figure S3 [left] and S4) (32).
These data indicate that survived cells that escaped CIMD by the addition of p73 siRNA to BUB1 siRNA have abnormal nuclei, suggesting that CIMD eliminates cells prone to substantial chromosome missegregation. This finding strengths our hypothesis that CIMD is an alternative cell death pathway that protects cells from aneuploidy.
We have proposed a working hypothesis to describe how partial depletion of BUB1 induces CIMD (31). We assume that there exists a mediator protein X that interacts with BUB1. While protein X interacts with BUB1, it cannot associate with p73; when BUB1 is depleted, protein X can bind to p73 to activate its transcriptional activity.
Because BUB3 is one of the proteins that physically interacts with BUB1 (36, 37), we tested BUB3 as a candidate for being protein X and studied p73–BUB3 association by immunoprecipitation while introducing defects in kinetochore–microtubule attachment (KT–MT defects) by drug treatment. Proteins were precipitated using anti-5XHis antibody from HeLa cells cotransfected with 6XHis-tagged BUB3 and p73α expression constructs. 17-AAG- or Taxol-treated cells and cells not treated with drugs were examined to detect this interaction. Interestingly, p73α was found in immunoprecipitates of 6XHis-tagged BUB3 from 17-AAG- or Taxol-treated cells, although the level of 6XHis-tagged BUB3 was below detectable levels in total lysates (Figure 3A [left]; Figure S6). Also, p73α was not precipitated in HeLa cells not treated with drugs (Figure 3A, right). We confirmed the occurrence of DNA fragmentation by performing the TUNEL (TdT-mediated dUTP nick-end labeling) assay under the same conditions as those in which 6XHis-tagged BUB3 and p73α interact (Figure S5; see the next paragraph). To check BUB3–p73 interaction in a reciprocal manner, we also performed an immunoprecipitation analysis using HeLa cells cotransfected with 6XHis-tagged BUB3 and HA-tagged p73α expression constructs and immunoprecipitated HA-tagged p73α using anti-HA antibody and detected 6XHis-tagged BUB3 in the immunoprecipitates (Figure S7). Together, these data strongly suggest that BUB3 interacts with p73α specifically in cells in which CIMD occurs.
If our model is correct, overexpression of protein X should induce CIMD when cells are treated with drugs that generate KT–MT defects. Therefore, we overexpressed BUB3 from the Tet promoter in HeLa Tet-Off cells in the absence of tetracycline/doxycycline (Figure 3B) and treated them with 17-AAG or Taxol. As expected, overexpression of BUB3 induced DNA fragmentation (TUNEL-positive) with 17-AAG or Taxol treatment at early mitosis (Figure 3C and D). There was no caspase activity (caspase 1–10, 13) in TUNEL-positive cells (Figure 3E and data not shown) and caspase inhibitors BAF and zVAD did not inhibit DNA fragmentation induced by 17-AAG and BUB3 overexpression [Figure 3F, see (32) for drug evaluation controls]. Therefore, the mitotic death caused by BUB3 overexpression is caspase independent and thus CIMD.
Because BUB3 overexpression induces CIMD, we next investigated whether BUB3 depletion inhibits CIMD. Although we tried achieving complete depletion of BUB3 by using 11 different sets of siRNA (data not shown), we could deplete only 70% of BUB3 (Figure 3G, top). However, even this partial depletion of BUB3 significantly suppressed CIMD (Figure 3G, bottom). Furthermore, endogenous BUB3 interacted with p73 when BUB1 was partially depleted (Figure S8).
These data indicate that BUB3 is the mediator protein inducing CIMD in response to KT–MT defects.
p73 exists as several C-terminal splice variants (38–47). As it was difficult to clearly detect endogenous levels of individual isoforms, we first depleted all isoforms by siRNA targeting the common 5′-UTR region (Figure 4B) and we confirmed abrogation of CIMD using these siRNA sequences (Figure 4C [second column from the right]; Table S2). Then we expressed each isofom (Figure 4A) to determine the ones that complemented the function of p73 in CIMD. Interestingly, the expression of α, β, γ isoforms but not the ε isoform induced CIMD (Figure 4C).
However, under conditions in which CIMD occurs, BUB3 interacted with α, β, γ, and ε isoforms (Figure 4D), suggesting that binding of p73 isoforms to BUB3 is not sufficient to induce CIMD.
Tomasini et al. described 2 independent promoters that enable expression of full-length, transcriptionally competent proteins (TAp73 isoforms) and amino-truncated transcriptionally incompetent proteins (ΔNp73 isoforms) (48). Furthermore, extensive 3′ splicings generate further isoforms for each TAp73 and ΔNp73 isoform (39, 48). ΔNp73 isoforms, by inhibiting the transactivational activity of both TAp73 and p53, are anti-apoptotic and therefore oncogenic (48). We found that ΔNp73 α, β, and γ suppressed DNA fragmentation in BUB1-depleted cells that were treated with 17-AAG (Figure 4E), indicating that ΔNp73 isoforms inhibit CIMD.
Previous studies report that when p73 is activated in response to DNA damage, p73 Y99 is phosphorylated by c-Abl (human ABL1) tyrosine kinase (49). This phosphorylation is a prerequisite modification for p73 to elicit cell death in fibroblasts (49, 50).
In our study, phosphorylated Y99 was detected with anti-phospho-p73α (Y99)–specific antibody specifically in 6XHis-BUB3 immunoprecipitates (Figure 5A; Figure S6), indicating that Y99-phosphorylated p73 interacts with BUB3 in cells in which CIMD occurs.
To investigate the role of Y99 phosphorylation on p73α in CIMD activation, we constructed an unphosphorylated mutant p73α-Y99A. p73α-Y99A was expressed at the same level as the wild-type control (Figure 5B, top), and it significantly suppressed the CIMD induced by BUB3 overexpression plus 17-AAG or Taxol treatment (Figure 5B, bottom). These data strongly suggest that Y99 phosphorylation on p73α is required for CIMD activation.
Because p73 is phosphorylated at Y99 by c-Abl, we examined whether c-Abl is required for CIMD. c-Abl depletion (Figure 5C, top) significantly suppressed CIMD (Figure 5C, bottom). Also, c-Abl kinase inhibitors imatinib and dasatinib inhibited CIMD (Figure 5D). These results indicate that c-Abl tyrosine kinase is required for CIMD, indicating a novel role of c-Abl in protecting cells from aneuploidy by inducing programmed cell death that occurs during mitosis.
Because BUB1 kinase activity is required to induce CIMD (32), we hypothesized that BUB3 might be phosphorylated by BUB1 to activate p73. First we examined whether BUB1 phosphorylates BUB3 in vitro. We performed BUB1 kinase assay using histone H3 as a positive control (Figure 6A) and found that BUB3 was phosphorylated by BUB1 in vitro (Figure 6A). Mass spectrometry analysis revealed that BUB3 is phosphorylated at serine-19 (S19) by BUB1 in vitro (Figure 6B). To confirm whether this phosphorylation occurs in vivo, we performed 2D-gel electrophoresis and immunoblotting. Alkaline phosphatase treatment of immunopurified BUB3 reduced the intensity of the signal of the dot at the far right of BUB3 (Figure 6C, top [arrow and circle] and middle), indicating that this dot is a phosphorylated form of BUB3. We found that the corresponding dot was absent in an unphosphorylated mutant BUB3-S19A (Figure 6C, bottom), suggesting that S19 is phosphorylated in vivo. Importantly, overexpression of wild-type BUB3 but not the S19A mutant induced CIMD (Figure 6D), indicating that S19 phosphorylation on BUB3 is required for CIMD activation. However, BUB3-S19A mutant protein was able to associate with p73α as well as the wild-type BUB3 protein, indicating that S19 phosphorylation is not necessary for the interaction with p73 (Figure 6E).
The molecular mechanism involved in the activation of CIMD has not been elucidated to date. Here, we show that overexpressed BUB3, a BUB1 interactor, interacts with p73, a structural and functional homolog of the p53 tumor-suppressor protein, specifically in cells in which CIMD occurs. Y99 phosphorylation on p73 by c-Abl tyrosine kinase as well as S19 phosphorylation on BUB3 by BUB1 kinase are required for CIMD activation. These results strengthen the hypothesis that CIMD protects cells from aneuploidy, which leads to tumorigenesis.
To explain how partial BUB1 depletion induces CIMD, our initial hypothesis was that BUB1 phosphorylates p73 to activate it, but binding of BUB1 to p73 silences the transcriptional activity of p73. Therefore, a certain level of BUB1 is needed to phosphorylate p73, but this level should be low enough to release phosphorylated p73 from BUB1 to bind DNA. However, we could not detect the BUB1–p73 interaction by immunoprecipitation under various conditions. Therefore, we proposed another hypothesis that activated BUB1 binds to mediator protein X and phosphorylates it (31). According to this model, overexpression of mediator protein X should induce CIMD. Our results clearly demonstrate that BUB3 interacts with p73α specifically (Figure 3A and and5A,5A, S6–8) and Y99-phosphorylated p73 interacts with BUB3 in cells in which CIMD occurs (Figures 5A; Figure S5 and S6). Therefore, we conclude that BUB3 is the mediator protein X (Figure 7).
A molecular switch should be required to modify BUB3 to activate p73 to induce CIMD conditionally. Because BUB1 kinase activity is required to induce CIMD (32), we tested whether BUB3 is phosphorylated by BUB1. S19 on BUB3 was phosphorylated by BUB1 in vitro (Figure 6A and B) and this site was phosphorylated in vivo (Figure 6C). Overexpression of an unphosphorylated mutant BUB3-S19A protein, but not BUB3, failed to induce CIMD, indicating that S19 phosphorylation is required for CIMD (Figure 6D). However, we found that BUB3-S19A protein was able to bind p73α efficiently (Figure 6E). Therefore, there may be additional modification to regulate the BUB3-p73 association.
Several studies have suggested specific roles for particular p73 isoforms (38, 42, 48, 51, 52). p73 exists as several C-terminal splice variants (38–47). Tomasini et al. described 2 independent promoters that enable expression of full-length, transcriptionally competent proteins (TAp73 isoforms) and amino-truncated transcriptionally incompetent proteins (ΔNp73 isoforms) (48). Furthermore, extensive 3′ splicings generate further isoforms for each TAp73 and ΔNp73 isoform (39, 48). Tomasini et al. reported that because TAp73 isoforms can transactivate similar proapoptotic genes as p53, they can act as tumor suppressors; on the other hand, ΔNp73 isoforms, by inhibiting the transactivational activity of both TAp73 and p53, are anti-apoptotic and therefore oncogenic (48). The first study of p73-deficient mice (Trp73−/−) by Yang et al. showed that they have neurological, pheromonal, and inflammatory defects but lack spontaneous tumors (53). However, Flores et al. reported that p73+/− mice showed spontaneous tumors, and 6 of 10 p73−/− mice showed spontaneous lung adenocarcinoma (54), which is not consistent with the findings of Yang et al. A recent study also showed that mice specifically lacking TAp73 isoforms exhibit spontaneous and carcinogen-induced tumors, infertility, and aging, as well as hippocampal dysgenesis (52). Interestingly, cells from TAp73−/− mice exhibit genomic instability associated with enhanced aneuploidy, which may explain the increased incidence of spontaneous tumors in these mutants. Therefore, TAp73 isoforms have a tumor-suppressive role and Trp73 may be involved in maintaining genomic stability.
We used siRNA sequences targeting the p73α isoform and confirmed the reduction of p73α by RT-PCR (Figure S2; Figure 4B). Because it was difficult to confirm the specific depletion of each isoform, we expressed each isoform when all isoforms were depleted (Figure 4A and B). Interestingly, overexpression of α, β, γ but not the ε isoform was sufficient to induce CIMD (Figure 4C). However, α, β, γ, and ε isoforms interacted with BUB3 (Figure 4D). The ε isoform bound to BUB3 but could not induce CIMD, suggesting that specific regions of α, β, and γ isoforms are important for CIMD activation. Further analysis is required to determine these specific domains and their functions. We also found that ΔNp73 isoforms inhibit TAp73 forms in CIMD activation (Figure 4E) (40), raising the possibility that CIMD does not occur in cancer cells expressing ΔNp73, such as neuroblastoma cells, which exhibit aneuploidy (55, 56).
We found that under the conditions in which CIMD occurs, p73 interacts with BUB3 but not BUB1. However, very recently, Vernole et al. have reported that p73α binds to BUB1 and BUB3 in colchicine-treated cells (57), and Tomasini et al. found that p73α interacts with BUBR1 and a C-terminal fragment of BUB1 interacts with p73α in nocodazole-treated cells (58). We do not know the reason for this disparity, but the fact that they used the conditions under which CIMD occurred on our studies could be an important factor. For example, both studies overexpressed these proteins to study interactions in the presence of microtubule inhibitors (57, 58), and we know that overexpression of BUB3 causes CIMD when cells are treated with microtubule inhibitors. Tomasini et al. also report a putative function of p73 in the spindle checkpoint activity; however, the premature mitotic exit in nocodazole-treated TAp73−/− cells they observed was minor compared with that seen in authentic spindle checkpoint mutant/depleted cells (52). This minor defect can be explained by loss of CIMD in p73 knocked down cells (Figure 2). Further analysis is required to clarify these issues.
Defects in the spindle checkpoint can promote aneuploidy, and an altered checkpoint is common in tumor cells (3, 12). Various mutations have been identified in spindle checkpoint components (3, 12), but no correlation has been established between the mutations and protein function. Some studies have found that levels of checkpoint components are different in tumor and normal cells (59–62), but there is no consensus on whether levels of specific proteins increase or decrease in tumor cells and how altered expressions affect spindle checkpoint function. Our results and previous studies suggest that the expression and balance in levels of BUB1 and BUB3 are important to induce CIMD (32, 63). Thus, it would be interesting to detect the balances of these proteins in cancer cells.
The tyrosine kinase c-Abl is the cellular protooncogenic counterpart of the oncogenic Bcr-Abl (64, 65), which is the fusion gene product of the Philadelphia (Ph) chromosome generated from a reciprocal translocation between chromosome 9 containing the tyrosine kinase c-Abl and chromosome 22 with the BCR gene (t[9;22]) (66, 67). Bcr-Abl tyrosine kinase activity is essential to induce in vitro cellular transformation (68) and in vivo leukemogenesis (69, 70). Deregulated activity of BCR-ABL tyrosine kinase is responsible for the development of 90% of chronic myelogenous leukemia cases and 5–15% of acute B-cell lymphoblastic leukemias (71). c-Abl is also activated by certain DNA-damaging agents (72) and can induce programmed cell death (apoptosis) by p53-dependent and p53-independent mechanisms (73). p73 is regulated by tyrosine kinase c-Abl in the apoptotic response to DNA damage (49). In our study, Y99-phosphorylated p73 interacted with BUB3 in cells in which CIMD occurs (Figure 5A; Figure S5 and S6), expression of the p73α-Y99A mutant significantly suppressed CIMD induced by BUB3 overexpression plus drug treatment (Figure 5B), and c-Abl tyrosine kinase and its activity were required for CIMD (Figure 5C and D). These data support that c-Abl has an important role in inducing programmed cell death during mitosis.
Jeganathan et al. generated a series of mutant mice with a gradient of reduced Bub1 expression, using wild-type, hypomorphic, and knockout alleles (63) and found that Bub1 hypomorphic but not Bub1 haploinsufficient mice are highly susceptible to spontaneous tumors.
We speculate that the loss of generation of spontaneous tumors in Bub1 haploinsufficient mice may be because of CIMD. This hypothesis should be tested by using mouse embryonic fibroblast (MEF) cells derived from Bub1 mutant mice (63, 74) and determining whether CIMD occurs. If spontaneous tumorigenesis in Bub1+/− mice is suppressed by CIMD, then reduction or deletion of p73 in Bub1+/− mice should increase spontaneous tumorigenesis. These genetic analyses will determine the in vivo function of CIMD. CIMD has also been found to be dependent on AIF and Endo G, which are effectors of caspase-independent cell death (33), and EndoG and Aif double-knockout mice can be used to test whether CIMD prevents tumorigenesis. Physiological studies on CIMD in mouse models can help determine the in vivo function of CIMD.
Supplemental Table S1 lists the antibodies, Supplemental Table S2 the siRNA/shRNA sequences, Supplemental Table S3 the plasmid vectors, Supplemental Table S4 the drugs, Supplemental Table S5 the cell lines, and Supplemental Table S6 the DNA oligonucleotide primer sequences used in this study. Custom-made siRNA and DNA oligonucleotide primers were synthesized by the Hartwell Center for Bioinformatics and Biotechnology, St. Jude Children’s Research Hospital.
HeLa cells or HeLa Tet-Off cells (Clontech. Mountain View, CA) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (BioWhittaker, Wokingham, UK) with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA) and 1mM penicillin-streptomycin. caspase 3−/−/caspase 7−/− MEF cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (BioWhittaker) with 10% FBS (Invitrogen), 2mM L-glutamine (Invitrogen), 0.1mM MEM non-essential amino acids (Invitrogen, Carlsbad, CA), 55μM β-mercaptoethanol (Invitrogen), and 10 μg/mL gentamicin (Invitrogen). Cells were grown at 37°C in 5% CO2 in a humidified incubator. Cells were transfected with annealed double-stranded siRNA or mammalian expression plasmids by using Lipofectamine 2000 (Invitrogen), Lipofectamine LTX (Invitrogen), or Fugene 6 (Roche). HeLa Tet-Off cells were transiently transfected with pTRM4 overexpression vector whose transcription was regulated by the TRE promoter and cultured in the absence of tetracycline/doxycycline.
The colony outgrowth assay was performed as described previously (75–77) but with a minor modification. HeLa cells were transfected with siRNAs by using Lipofectamine 2000. Twenty-four hours after transfection, cells were incubated with 100 nM 17-AAG or 1.5 nM Taxol for 2 days, and the drug was removed by washing while retaining the mitotic cells. Cells including mitotic cells that were recovered from the supernatant, n = 500 or 2000, were spread in 1 well of a 6-well cluster (Corning Costar, Acton, MA) and incubated for approximately 20 days or 12–14 days, respectively, to allow colony formation. Colonies stained with Giemsa solution (HEMA-QUIK stain solution, Fisher Scientific, Hampton, NH) were counted. The viability (%) was normalized; the percentage of surviving colonies of untreated cells transfected with control luciferase (Luc) siRNA was arbitrarily set to 100.
Immunoblotting was performed as previously described (78, 79). Alternatively, the Odyssey Infrared Imaging System (LI-COR Biosciences) was used, especially for coimmunoblotting. Cells were added to lysis buffer A (80), and the mixture was frozen in liquid nitrogen, thawed, and sonicated. Before electrophoresis, cell lysates were mixed with an equal volume of 2X SDS sample buffer.
Indirect immunofluorescent staining was performed by using previously described methods (81, 82), but with the following modifications. HeLa cells were grown for 48 h on coverslip slides after siRNA and/or overexpression plasmid vector transfection (seeding approximately 1.8 × 105 cells, and cells were grown for 18 h before the transfection). Asynchronous populations of HeLa cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C for 30 min and then treated with 0.5% Triton X-100 in KB (10 mM Tris HCl [pH 7.5], 150 mM NaCl, 0.5% bovine serum albumin) at room temperature for 30 min. Cells were then incubated with a specific primary antibody for 1 h at 37°C. After the cells were washed once with KB, they were incubated with the fluorescent secondary antibodies fluorescein isothiocyanate–conjugated AffiniPure IgG, Texas Red–conjugated AffiniPure IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), Alexa Fluor 488 (Molecular Probes) or coincubated with the TUNEL reaction mixture (see TUNEL assay) for 1 h at 37°C. Slides were washed once with KB and then incubated in KB containing 0.1 μg/mL DAPI (Sigma, St. Louis, MO). Cells were observed through a Leica DM IRE2 motorized fluorescence microscope equipped with an HCX PL APO 63× oil immersion lens (Leica), an Leica EL6000 compact light source (Leica), and an ORCA-ER high-resolution digital charge-coupled device (CCD) camera (Hamamatsu, Hamamatsu City, Japan). Image acquisition and processing were performed by using Openlab version 5 Scientific Imaging Software (Improvision, Lexington, MA).
Forty-eight hours after siRNA and/or overexpression plasmid vector transfection, HeLa cells were incubated with 500 nM 17-AAG (A.G. Scientific, San Diego, CA) for 24 h. Cells were fixed with 4% paraformaldehyde in PBS (pH 7.4), and the TUNEL assay was performed by using an in situ cell death detection system that contained tetra-methyl-rhodamine (TMR) red (Roche).
HeLa cells were transfected with siRNA, and 48 h later they were incubated in 500 nM 17-AAG for 24 h. The fluorochrome inhibitor of caspases (FLICA) assay was performed by using the carboxyfluorescein FLICA (Poly-Caspases FLICA [SR-VAD-FMK], Caspase 2 FLICA [FAM-VDVAD-FMK], Caspase 10 FLICA [FAM-AEVD-FMK], or Caspase 13 FLICA [FAM-LEED-FMK]) apoptosis detection system (Immunochemistry Technologies, LLC, Bloomington, MN).
We used a previously described method for the immunoprecipitation assay (83), but with the following modifications. Extracts of HeLa cells cultured in 100-mm dishes were obtained by lysing cells in cold buffer B1 (40 mM Tris-HCl [pH 7.5], 300 mM NaCl, 0.5% Triton X-100, and 1 tablet of EDTA-free protease inhibitor cocktail [Roche] per 10 mL) or buffer B2 (50 mM HEPES-KOH [pH 7.2], 120 mM NaCl, 0.1% NP-40, 1 mM EDTA, 60 mM β-glycerolphosphate, 0.1 mM NaF, 0.1 mM Na3VO4, 1 mM PMSF, and 1 tablet of EDTA-free protease inhibitor cocktail [Roche] per 10 mL), and the mixture was frozen in liquid nitrogen, thawed, and sonicated on ice. Approximately 1–4 mg of the protein lysates were used per immunoprecipitation experiment. After centrifuging (15000 g for 20 min at 4°C) the lysates, 6XHis-tagged protein was precipitated by using 25 μL of Ni-NTA Agarose (QIAGEN) at 4°C for 2 h to overnight. To precipitate HA-tagged protein, 4 μg of anti-HA antibody (Roche) was preincubated with 25 μL of protein A-Sepharose (Amersham Biosciences, Piscataway, NJ) at 4°C for 1 h to overnight, and then the HA-tagged protein in the supernatant was precipitated with anti-HA antibody-linked protein A Sepharose at 4°C for 2 h to overnight. Flag-tagged protein was precipitated by using 25 μL of ANTI-FLAG M2 Affinity Gel (SIGMA) at 4°C for 2 h to overnight. myc-tagged protein was precipitated by using 25 μL of c-Myc Monoclonal Ab-Agarose Beads (Clontech) at 4°C for 2 h to overnight. Immunoprecipitates were washed 6 times with buffer B1 or B2, and the precipitated protein was eluted in buffer C (50 mM NaH2PO4 [pH 8.0], 300 mM NaCl, 250 mM imidazole) or in SDS sample buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 0.1% bromophenol blue, and 10% glycerol).
Total RNA from HeLa cells was extracted by using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. To examine the siRNA silencing activity, cDNA was synthesized from RNA (500 ng) and DNA amplification was performed by using the One Step RT-PCR kit (QIAGEN). For amplification of gene-specific fragments, the RT-PCR conditions were as follows: (reverse transcription 50°C for 30 min, 30 cycles 94°C for 60 s, 50°C for 60 s, and 72°C for 60 s, final extension 72°C for 10 min)
6XHis-human BUB3 or 6XHis-human BUB1 was expressed in E. coli or sf9 cells, respectively, purified with Ni-NTA agarose (QIAGEN) and dialyzed with dialysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.1 % Triton X-100, 1 mM DTT). Histone H3/H4 (US Biological) or 6XHis-human BUB3 was incubated in the reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 10 μM ATP) with or without 6XHis-human BUB1 at 30°C for 1 h in the presence of 2 μCi of [γ-32P]-ATP. PhosphorImager (Molecular Dynamics, Sunnyvale, CA) was used to detect phosphorylation.
The BUB3 region from the gel was reduced by 10mM dithiothreitol, alkylated with 25mM iodoacetamide and, digested with 0.2 μg trypsin. Mass spectrometric analysis was performed on an LTQ Linear Ion Trap Mass Spectrometer from Thermo Electron (San Jose, CA) using electrospray ionization (ESI). The tryptic peptides were introduced into the instrument by gradient elution (by increasing the concentration of acetonitrile), using reverse phase (C18) ultra-high pressure liquid chromatography on the nanoAcquity (Waters, MA). Waters BEHC18 with an I.D. of 75 μm and particle size of 1.7 μm was used. Data on peptide mass (MS) spectra followed by fragmentation of the 10 most intense ions in the MS were acquired. Database searches were performed on these MS/MS data, using the Mascot search engine on the Swissprot database. The parameters used in the search were 1.5 Da and 0.7 Da for precursor and fragment ion mass tolerances, respectively, and static modification of carbamidomethyl on C. Oxidation of M and phosphorylation on STY were used as variable modifications. Phosphopeptides identified from the search were manually confirmed.
We applied a method for the two-dimensional gel electrophoresis which was previously described (84), but with the following modifications. Flag-tagged BUB3 was immunoprecipitated from HeLa cell lysates by using an anti-Flag antibody. Isoelectric focusing was performed with 17-cm immobilized pH 3–10 gradient strips (Bio-Rad) as per manufacturer’s instructions. Gel electrophoresis was performed in a Bio-Rad PROTEAN Plus Dodeca cell. After two-dimensional gel electrophoresis, proteins were transferred on to a Immobilon-FL polyvinylidene difluoride membrane (Millipore, Danvers, MA) and immunoblotted by using anti-Flag antibodies.
We thank D. R. Green, Y. Fujii and P. Bansal for their helpful comments; P. Houghton and H. Cam for stimulating conversation and advice; R. Abdulle, P. Sprouse, S. Watanabe, Y. Sakuraba, Y. Fujii, G. Stocco., G. Neale, A. Inoue, R. Chitta, X. Ding and V. Pagala for their technical assistance; W.G. Kaelin, Jr. and P. Houghton for their generous gifts of reagents; and V. Shanker for editing this manuscript. This work was supported by the Cancer Center Support Grant CA21765 from the National Cancer Institute, by NIH grant GM68418, by American Cancer Society Research Grant RSG-07-144-01-CCG and by the American Lebanese Syrian Associated Charities (ALSAC).
The authors declare that they have no conflict of interest.
Supplemental data including supplemental experimental procedures, 8 figures, 6 tables, and supplemental references are available with this article online at http://JJJJJJ/XXXXX