Stable and Transient Expression of Id1 Induce Tetraploidization and Centrosome/Centriole Amplification
We have reported previously that overexpression of Id1 is common in nasopharyngeal carcinoma (Wang et al., 2002
). The contribution of Id1 to the pathogenesis of nasopharyngeal carcinoma is not fully defined. We first examined the effect of Id1 overexpression on chromosome instability in a telomerase-immortalized nasopharyngeal epithelial cell line (NP460hTert), which has a near-diploid karyotype. Establishment and detailed characterization of NP460hTert has been described previously (Li et al., 2006
Stable expression of Id1 in NP460hTert cells resulted in 30% tetraploids, indicating an effect of Id1 on mitosis (p < 0.0001) (A). Previous studies have indicated that centrosomal defects could be identified at an early premalignant stage of carcinogenesis (Pihan et al., 2003
); centrosome amplification is also strongly associated with rapid onset of aneuploidy (Goepfert et al., 2002
; Pihan et al., 2003
). Furthermore, it has been reported that overexpression of Id1 could induce centrosome overduplication in human foreskin keratinocytes (Hasskarl et al., 2004
). We first sought to examine for the presence of centrosomal aberrations in our immortalized nasopharyngeal epithelial cells stably expressing Id1 (NP460hTert-Id1), by using antibody against γ- tubulin, a major component of centrosomes, (B). Single-nucleated cells with three or more centrosomes as well as binucleated cells with more than four centrosomes are considered abnormal. In NP460hTert-Id1 cells, abnormal numbers of centrosomes was detected in 50% of the cells (n = 200), but <10% in control NP460hTert cells at equivalent population doublings. To further eliminate the possibility that the observed effect of Id1 on centrosome abnormality was the result of selective outgrowth of cell clones with centrosome anomalies, transient expression of Id1 in HeLa cells was performed for examination of centrosome aberration. A flag-tagged Id1 expression vector was used for tracking the Id1-expressing cells. Ectopic expression of Id1 was confirmed by Western blotting analysis (B). Similar to the observation in NP460hTert-Id1 cells, fourfold increase in supernumerary centrosomes was detected in HeLa cells transiently expressing Id1 (B).
Figure 1. Stable expression of Id1 induced tetraploidization. (A) Western blot confirming high level of Id1 expression following NP460 hTert transduced with the Id1 gene. Representative karyotypes of NP460 hTert and NP460 hTert-Id1 performed by conventional cytogenetics. (more ...)
To determine whether Id1 induced extra centrosomes is due to interference on centriole homeostasis, we also performed transient transfection of Id1 into U2OS cells stably transfected with green fluorescent protein (GFP)-tagged centrin to allow direct visualization of individual centriole (C). The effect of Id1 expression in centriole homeostasis in the U2OS cells expressing Id1 was observed at 48 h after transfection. A twofold increase of extra centrioles in single-nucleated (>4) or binucleated cells (flag-positive) cells (>8) (n = 200) (C) was detected. Similar increase in centriole numbers was noted in NP460hTert+Id1 cells by immunofluorescent staining with the centrin antibody (C).
Id1 Disrupts Bipolarity and Induces Multipolar Spindles
Ectopic Id1 expression also induced a high frequency of mitotic spindle defects in cells. Most notable was the increase in the percentage of cells with multipolar and monopolar spindles, revealed by immunofluorescent staining with α-tubulin. Twenty-six percent of the NP460hTert-Id1 cells revealed multipolar mitotic spindles and monopolar spindles (n = 50) (D). Using live cell imaging, multipolar spindles were also observed in 23% of HeLa cells (n = 50) cotransfected with Id1 and GFP-α-tubulin (5:1 ratio), compared with 5% in the control cells (n = 50) (Supplemental Figure 1A). In both cell models, the frequency of mono- and multipolar spindles observed in Id1-overexpressing cells is lower than the frequency of extra centrosomes observed (, B and C), suggesting that some centrosomes in the Id1-overexpressing cells do not possess microtubule-nucleating activity in mitosis (Supplemental Figure 1B).
Id1 Induced Binucleation May Arise as a Consequence of Defects in Cytokinesis
Quantitative analysis of the NP460hTert-Id1 cells revealed that around one third of the Id1-overexpressing cells that carried extra centrosomes were binucleated (D). Conversely, binucleated cells were not common in NP460hTert-Id1 cells with normal number of centrosomes. This indicates that the extra centrosomes in the binucleated cells may result from cytokinesis defect.
Binucleated cells could be generated by cytokinesis failure or cell–cell fusion (Nigg et al., 2002
). To corroborate the interpretation that high level of Id1 may affect cytokinesis, time-lapse microscopy was performed in Id1-expressing HeLa cells and control cells. After cotransfecting the Id1 with the DsRed expression plasmids (at a 5:1 ratio) for 36 h, cells were followed by time-lapse microscopy for 16 h. Pictures were taken every 5 min for a 16-h duration. Mitotic cells were analyzed from metaphase cells when the condensed chromosomes lined up on the equatorial plane, to the exit of mitosis after abscission of the cleavage furrow. Id1-expressing cells exhibited a defect in mitotic exit. A prolonged transition time from anaphase to abscission of furrow was detected in Id1-expressing cells (37 5.6 min, n = 44), compared with (20 2.5 min; n = 46) in control cells (p < 0.0001) (Supplemental Figure 1C). These observations suggest that Id1 may interfere with the cytokinesis process leading to binucleation and tetraploidization.
Id1 Perturbs Organization of Interphase Microtubules and Integrity of Mitotic Spindles
In addition to abnormal number of spindle poles, we also detected diminished GFP intensity that represents poorly formed mitotic spindles in 12% (n = 50) of the Id1-transfected HeLa cells (Supplemental Figure 1D), compared with 2% in the control cells. This would imply that overexpression of Id1 also has pronounced effect on microtubule organization.
The ability of Id1 to perturb interphase microtubules has not been reported previously. Failure of microtubule polymerization was detected in 45% of the NP460hTert-Id1 interphase cells (Supplemental Figure 2A) compared with 3% in control cells (n = 200). The effect of Id1 overexpression on microtubule polymerization was examined by live cell imaging in HeLa cells cotransfected with Id1 and GFP-tagged α-tubulin expression plasmids (5:1 ratio). Twenty-six percent of HeLa cells transiently expressing Id1 resulted in loss of microtubule integrity (Supplemental Figure 2A).
Id1 Up-Regulates Aurora A Expression
We next sought to identify molecular event(s) responsible for the generation of the abnormal phenotypes induced by Id1. Considering the role of several mitotic kinases in the centrosome cycle, many of the Id1-induced phenotypes (tetraploidization, extra centrosomes, binucleation, monopolar/multipolar mitotic spindles) are similar to the phenotypes associated with Aurora A overexpression (Meraldi et al., 2002
). We further examined whether Id1 could up-regulate Aurora A expression to induce these abnormal phenotypes. Western blotting analysis of NP460hTert-Id1 cells revealed up-regulation of Aurora A expression (A). We then examined the ability of Id1 to up-regulate Aurora A expression by transient transfection of Id1 into HeLa cells. Up-regulation of Aurora A by transient Id1 expression could also be demonstrated in HeLa cells in a dose-dependent manner (B).
Figure 2. Aurora A mediates the mitotic defects in Id1-overexpressing cells. (A) High expression level of Aurora A in stable and transient Id1 overexpressing cells. Detection of Aurora A expression level by Western blotting in NP460hTert cells transduced with the (more ...)
Id1 Depletion Down-Regulates Endogenous Aurora a Level and Suppresses Centrosome Amplification/Microtubule Perturbation in Cancer Cells
To examine whether Aurora A overexpression may mediate the abnormal mitotic phenotypes observed in Id1-overexpressing cells, we performed knockdown experiments using shRNAs. An undifferentiated nasopharyngeal cancer cell line, HONE1, was chosen for this part of the knockdown study, due to its endogenous high levels of Aurora A and Id1 as well as the presence of supernumerary centrosomes (55%). Transfection of HONE1 cells with a previously characterized Id1-antisense expression vector (pcDNA3-Id1AS) (Ling et al., 2003
) resulted in a marked reduction in Id1 level (Supplemental Figure 2B). Id1 depletion in HONE1 cells also resulted in a decrease in the endogenous Aurora A expression level (Supplemental Figure 2B), suggesting that overexpression of Aurora A is dependent on Id1. In parallel, the percentage of HONE1 cells with extra centrosomes was reduced by fourfold and twofold, after suppression of Id1 or Aurora A, respectively (Supplemental Figure 2C), indicating that overexpression of Id1 plays an important role in mediating supernumerary centrosomes in HONE1 cells. We also reported previously that U2OS cells also have endogenous supernumerary centrioles (Gromley et al., 2003
) as well as high level of Id1. The number of supernumerary centrioles in U2OS cells stably expressing GFP-centrin was suppressed by 2- to 3.5-fold, when transiently transfected with shAurA alone, Id1-AS alone (B). This set of knockdown experiments indicates the involvement of Aurora A in mediating centrosome and centriole amplification in Id1-overexpressing cells. When Id1 and shAurA plasmids were cotransfected into U2OS cells, the extra centriole phenotype was partially suppressed (B), indicating that Id1 is mediating some other events involved in centrosome regulation.
Gene silencing studies, in HONE1 cells, were performed to examine the role of Id1 and Aurora A in association with the microtubule phenotype (Supplemental Figure 2D), which was revealed by transiently expressing GFP-tagged α-tubulin, Id1 depletion suppressed the disturbed microtubule organization in HONE1 cells by fourfold (Supplemental Figure 2D), suggesting that high level of Id1 expression is responsible for the abnormal microtubule phenotype observed. The defective microtubules of HONE1 cells could be suppressed, but to a lesser extent, when Aurora A expression was silenced (Supplemental Figure 2D). This set of data suggests that Id1-induced microtubule aberration might be mediated through other factor in addition to Aurora A.
The above-mentioned findings were confirmed in a telomerase immortalized RPEhTert epithelial cells stably expressing GFP-α-tubulin, previously used in our microtubule studies (Rosa et al., 2006
; Man et al., 2007
). Exogenous expression of Id1 in RPEhTert cells also induced abnormal interphase microtubules and monopolar mitotic spindles (C), compared with the network-like microtubules in the control cells. Number of cells with perturbed microtubules/spindles were suppressed by 50% when Aurora A expression was depleted by shRNA in Id1-overexpressing cells (C). To ensure these phenotypic changes were not artifacts of DNA transfection or cytotoxic effects, cells scoring for abnormal microtubule structures were checked under phase-contrast microscopy for the presence of intact membrane structure (C). In addition, cells were subsequently fixed and immunostained with DM1α-tubulin to verify the distorted microtubules (data not shown). Similar to the centrosome scenario, partial rescue of the microtubule phenotypes by Aurora A in both cell models overexpressing Id1 suggests that other events mediated by Id1 may be responsible for the microtubule effect.
Mechanisms Involved in the Up-Regulation of Aurora A by Id1
Because up-regulation of Aurora A seems to play a role in mediating the abnormal phenotypes, we carried a series of experiments to examine some of the plausible mechanisms involved.
Induction of Aurora A by Id1 Is Not Related to Cell Cycle
The expression of Aurora A peaks at G2/M because of the enrichment of this protein either to the centrosomes (Hirota et al., 2003
) or to the microtubule compartments (Lee et al., 2001
; Kinoshita et al., 2005
). Aurora A is rapidly degraded after anaphase, and it could be detected, weakly, after G1 phase of the next cell cycle (Castro et al., 2002b
; Taguchi et al., 2002
). We examined whether the induction of Aurora A expression by Id1 may be a result of cells accumulating at G2/M phase of the cell cycle. FACS analysis did not reveal significance accumulation of cells at G2/M in Id1-overexpressing HeLa cells (A). Furthermore, elevated Aurora A expression could be detected in Id-1–expressing HeLa cells synchronized at various phases of the cell cycle, and the efficacy of successful synchronization was verified by FACS analysis (B).
Figure 3. Id1 up-regulates Aurora A expression throughout the cell cycle. (A) FACS analysis showing the cell cycle distribution of the HeLa cells transfected with the vector control or the Id1 expression plasmid. (B) Aurora A expression levels were detected in (more ...)
The results in B were further confirmed by bivariate antigen flow analyses (C). Double immunofluorescent staining, by using Aurora A antibody and PI staining of cellular DNA content, was performed in HeLa cells expressing Id1 or the vector control. Isotypic control was performed to reveal antibody background (data not shown). Each spot represents an individual cell with a specific Aurora A level (y-axis) and DNA content (x-axis). Identical reference lines were drawn in corresponding panels to assist comparison of expression levels. Consistent with the results from B, Aurora A expression was lower at G1/S phase but higher at G2/M phase in the cells transfected with control vector (C). Id1-expressing cells revealed a substantial increase of Aurora A level, revealed by the FITC intensity above the identical reference lines, at all phases of the cell cycle. The mean FITC intensities of both G1 and S phase populations were at least four- to sixfold higher (p < 0.005) than the mean FITC intensities in the corresponding controls (C). The mean FITC intensity of the G2/M phase population was two- to threefold higher (p < 0.005) than the intensity of the control (C). Here, we show that the increased Aurora A level by Id1 is not a consequence of redistribution of cells to the G2/M phase of the cell cycle.
Gene Amplification Is Not Involved in the Elevated Aurora A Level by Id1
Our previous studies had identified amplification of 20q13.2 as a nonrandom event during immortalization in human ovarian epithelial cells, in which numerous copies of Aurora A
were mapped to homogeneous staining regions (hsr) on chromosome 20q13 (Chung et al., 2005
). In breast and ovarian cancer cell lines, a 2.5- to 8-fold amplification of Aurora A accounted for its high expression (Zhou et al., 1998
). The possibility that Aurora A
gene was amplified in NP460 hTert-Id1 cells were examined using the PAC clone (RP5843L14), which mapped to the chromosomal locus of Aurora A
. Another PAC clone (RP5-1107C24) juxtaposed to RP5843L14 was cohybridized to metaphases as internal control. Two normal signals from each PAC clone (RP5-843L14 and RP5-1107C24) were revealed in NP460hTert (n = 200) (A). NP460 hTert-Id1 cells did not reveal any multiple tandem gene copies mapped on 20q13.2 (A). Two extra signals detected in NP460 hTert-Id1 cells (A) were due to the induction of tetraploid progeny by Id1. The third extra signal was arisen from the immortalization process (Li et al., 2006
). Our data suggest that Aurora A
amplification is an unlikely mechanism for the elevated Aurora A level detected in NP460 hTert-Id1.
Figure 4. Mechanism(s) involved in Aurora A up-regulation by Id1. (A) Overexpression of Aurora A in NP460 hTert-Id1 cells is not a consequence of gene amplification. FISH analyses of NP460 hTert and NP460 hTert-Id1 cells by using the PAC clone (RP5-843L14), which (more ...)
Elevated Aurora A Expression in Id1-expressing Cells Only Partially Resulted from Transcriptional Activation of the Promoter of Aurora A
We then examined whether Id1 may result in transcriptional up-regulation of Aurora A promoter activity by luciferase reporter assay. HeLa cells were cotransfected with various 5′-truncated Aurora A promoter luciferase constructs (pGL1486, pGL189, pGL124, and pGL75) and the Id1 expression plasmid (or the pGL basic vector). A 1.5- to 2-fold increase in Aurora A promoter activities was observed in Id1-transfected cells, the activity being highest between −189 and −124 of the core promoter (B). Our results suggest that Id1 may regulate the transcriptional activity of Aurora A at its core promoter between the −124 to −189 from the transcription start site. This weak increase in promoter activation (do not exceed 2-fold) in Id1-expressing cells indicates that transcriptional activation of Aurora A by Id1 may not be the mechanism involved in its up-regulation.
Id1 Affects Aurora A Degradation
Aurora A is rapidly degraded at mitotic exit by the proteasomal proteolysis pathway (Littlepage and Ruderman, 2002
). We examined whether Id1 overexpression may affect this process. The endogenous turnover rate of Aurora A was detected in HeLa cells by treatment with cycloheximide, a protein biosynthesis inhibitor in eukaryotic cells. The endogenous Aurora A in HeLa cells has a rapid turnover rate with a half-life of ~2 h (C). In line with other studies, we confirmed that degradation of Aurora A in HeLa cells could be prevented by proteasomal inhibition by using MG132 (inhibitor of the proteasomal degradation pathway) (C). Furthermore, Aurora A could not be degraded in Id1-overexpressing cells, even in the presence of cycloheximide, indicating that Id1 overexpression resulted in stabilization of Aurora A (D).
Aurora A and Id1 Are Not Binding Partners
Aurora A is a known substrate of the anaphase-promoting complex. This protein contains the D-box and the A-box sequences known to be required for the recognition and destruction by the activated APC/CCdh1
(Pfleger et al., 2001
; Castro et al., 2002a
; Crane et al., 2004
) at mitotic exit and during the following G1 phase. To examine whether the resistance of Aurora A to proteasomal degradation by Id1 overexpression could be a result of direct interaction between Id1 and Aurora A, we performed coimmunoprecipitation studies. HeLa cells were transfected with a wild-type Id1 flag-tagged expression vector. Coimmunoprecipitation studies using the flag and Aurora A antibodies as baits failed to reveal any interaction between Id1 and Aurora A (A).
Figure 5. Id1 affects Aurora A degradation. (A) Id1 and Aurora A are not interacting proteins. HeLa cells were transfected with flag-tagged Id1 expression plasmid or the control vector. Cellular extracts were sequentially immunoprecipitated with either rabbit immunoglobulin (more ...)
Binding of Id1 to the Anaphase-promoting Complex Coactivator (cdh1) Affects the Stabilization of Aurora A
targets Aurora A for the ubiquitin proteasomal-dependent degradation. The enzymatic E3 ubiquitin ligase activity of APC/C requires Cdh1 as one of the coactivators at late mitosis (Pfleger et al., 2001
; Littlepage and Ruderman, 2002
; Castro et al., 2002b
; Crane et al., 2004
). Cdh1 can recognize APC/C substrates by interacting with specific recognition elements (D-box) in these substrates (Kraft et al., 2005
). It has been reported previously that Aurora A could be immunoprecipitated with Cdh1 (Crane et al., 2004
). Stability of Aurora A could be associated with depletion of Cdh1 expression in somatic, cells whereas restoration of Cdh1 rapidly induces fall in Aurora A level (Taguchi et al., 2002
; Crane et al., 2004
). We then explored whether Id1 associates with Cdh1 and competes for the binding of cdh1 to Aurora A. Our coimmunoprecipitation studies revealed that Id1 and cdh1 are indeed interacting partners (B). Excessive Id1–cdh1 interaction could inhibit the association between Aurora A and Cdh1 (B). Hence, decreased recognition or binding of Cdh1 with Aurora A may result in stabilization of Aurora A.
Id1ΔC Fails to Induce Cdh1/Aurora A Dissociation and the D-Box Motif in the C-Terminal of Id1 Is Involved in Its Binding to Cdh1
We next investigated on the functional domain(s) of Id1 responsible for the dissociation of Cdh1 and Aurora A. Immunoprecipitation studies were performed on cells expressing the wild-type Id1 and other Id1 mutants. Decreased Cdh1 and Aurora A association was clearly observed in cells expressing the wild-type Id1 (C). Inhibition of Cdh1 and Aurora A association was also observed in cells expressing the Id1 mutant carrying a specific mutation at residue 98 of the second helix. Deletion of the C-terminal 44 amino acids resulted in a full recovery of Cdh1/Aurora A association; indicating that the C terminus of Id1 comprises a motif that may be crucial for Cdh1/Aurora A recognition (C). It was also noted that deletion of the N-terminal 65 amino acids resulted in weak recovery of Cdh1/Aurora A association; suggesting the possible involvement of the Id1 N terminus in Cdh1/Aurora A association.
targets mitotic proteins for ubiquitin-mediated degradation mostly through the D-box recognition sequence/motif (RxxLxxxN) (Zachariae, 2004
), and it has been reported that the Id2 protein contains this recognition sequence for interacting with the APC/C (Lasorella et al., 2006
). Apparently, this motif is also present in Id1 and Id4. We then focused on the C terminus (residues 126-133) of Id1 (in which the D box is located), and we examined whether this motif is essential for the interaction between Id1 and Cdh1. Three flag-tagged Id1 mutants with mutations at specific residues within the destruction box (mDB126
, and mDB133
) were generated, and their binding abilities with Cdh1 were examined (D). We showed that these DB mutants, as well as Id1ΔC-flag, are less effective to precipitate Cdh1 than Id1wt-flag, indicating that the destruction box recognition motif (residues 126-133) is required for Id1–Cdh1 association. Results from , B–D, suggest that Id1 promotes binding with Cdh1 at the destruction box and that it does inhibit Cdh1 to bind to its substrate, Aurora A.
If Id1 inhibits APC/CCdh1, it should thereby accumulate Aurora A at late M phase and at G1. According to the results from D, one would expect that expression of wild-type Id1, Id1-ΔN, and Id1-mHLH, but not the Id1-ΔC (which lacks the D box) could stabilize Aurora A level. Indeed, Aurora A level was shown to accumulate in cells expressing wild-type Id1, compared with cells expressing the vector control (A, , B and D, D, and E). Aurora A level was seen accumulated in cells expressing the Id1-ΔN as expected, but at a relatively lower level compared with wild-type Id1. However, our results showed that Aurora A level could still be detected in cells expressing the Id1-ΔC mutant (E), albeit at a lower level compared with wild-type Id1; furthermore, Aurora A expression was not detected in cells expressing the Id1-mHLH mutant. These unexpected results indicate that other molecular events or motifs at the N terminus/HLH domain may possibly affect Aurora A stabilization (see Discussion). The D-box motif at the C terminus of Id1 that binds to Cdh1 may not be the sole mechanism in modulating Aurora A stabilization in cells.
Stabilization of Other Cdh1 Substrates Because of Excessive Id1–cdh1 Association
If Id1–Cdh1 association is involved in stabilization of Aurora A, one would also expect accumulations of other Cdh1 substrates to occur in Id1-overexpressing cells. We next examined the levels of selected Cdh1 substrates (Aurora B, Cyclin B1, Plk1, and Survivin) in Id1-overexpressing cells synchronized at either G1/S or G2/M phases of the cell cycle. Similar to the Aurora A scenario, stabilization of Aurora B, Plk1, Cyclin B1, and Survivin was detected in G1 and M phase of the Id1-overexpressing cells (A).
Figure 6. Stabilization of other Cdh1 substrates due to excessive Id1–cdh1 association. (A) Levels of selected cdh1 substrates in Id1-overexpressing cells were examined. Cells were transfected with either the control vector or the Id1 wild-type expression (more ...)
Immunoprecipitation studies were then performed (B) to examine the degree of binding between Cdh1 and the selected substrates. In agreement with our hypothesis, decreased association of Cdh1 to these substrates was generally observed in cells overexpressing wild-type-Id1 (B), compared with the vector controls. Id1ΔC mutant, which lacks the D box, failed to block the interactions between Cdh1 and its substrates, whereas wild-type Id1, Id1-ΔN and Id1-mHLH mutants retain their abilities to block the associations of Cdh1 to these Cdh1-specific substrates (C). Although the general trend is clear, still it is unclear why the degree of inhibition of cdh1–substrate bindings varies in cells transfected with the mutants. That is, whereas the level of Survivin associated with Cdh1 is reduced with the Id1-ΔN mutant more effectively than the wild-type Id1, the level of Plk1 associated with Cdh1 is reduced the same as the wild-type Id1. The N terminus and the HLH domain of Id1 may also participate in regulating the recognition and binding of Cdh1 to these targets. The complexities of the molecular events involved warrant further experiments. Nonetheless, our results suggest that the destruction box motif within the Id1 C terminus is involved in inhibiting Cdh1 to interact with Aurora A and other specific substrates (C).