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. 2014 Jul 12;140(12):2027–2037. doi: 10.1007/s00432-014-1762-8

Bifunctional roles of survivin-ΔEx3 and survivin-2B for susceptibility to apoptosis in endometrial carcinomas

Yuki Tazo 1,2, Atsuko Hara 1, Takashi Onda 2, Makoto Saegusa 1,✉
PMCID: PMC11823821  PMID: 25015769

Abstract

Purpose

Alternative splicing variants of survivin have different biological roles on cell kinetics. Here, we focused on the effects of different variants, including wild type (wt), survivin-ΔEx3, and survivin-2B, on apoptosis and cell proliferation in endometrial carcinomas (Em Cas).

Methods

Expression of survivin-wt, survivin-ΔEx3, and survivin-2B with reference to cell death and proliferation was investigated, using Em Ca cell lines and its clinical tissues.

Results

Ishikawa cells stably overexpressing either survivin-ΔEx3 (Surv-ΔEx3#34) or survivin-2B (Surv-2B#17) demonstrated considerably lower proliferative activity, along with up-regulation of p21waf1. After TNF-α treatment, Surv-ΔEx3#34 cells showed an increase in apoptotic cells, while the effects were relatively minor in Surv-2B#17 cells. In contrast, doxorubicin treatment resulted in increased apoptotic cells in Surv-2B#17 but not Surv-ΔEx3#34 cells, along with decreased expression of bcl-2 relative to bax. Control Ishikawa cells also showed relatively higher endogenous mRNA expression of survivin-ΔEx3 and survivin-2B during treatment of TNF-α and doxorubicin, respectively. In addition, exogenous overexpression of each survivin variant resulted in inhibition of other endogenous isoforms, indicating that the relative proportion may contribute to regulation of the splicing machinery. In clinical samples, level of survivin-ΔEx3 relative to either survivin-wt or survivin-2B was significantly higher in Em Cas than non-neoplastic lesions. Moreover, survivin-ΔEx3 and survivin-wt were positively correlated with apoptosis and cell proliferation, respectively, in Em Cas.

Conclusions

These findings provided evidence that the balance among expression level of survivin variants may contribute to modulation of cell kinetics in Em Ca cells.

Electronic supplementary material

The online version of this article (doi:10.1007/s00432-014-1762-8) contains supplementary material, which is available to authorized users.

Keywords: Survivin, Apoptosis, Alternative splicing, Endometrial carcinoma, Cell proliferation

Introduction

Survivin, a member of the family of inhibitors of apoptosis proteins (IAPs), is a 16.5 kDa protein encoded by a single gene consisting of three introns and four exons (Ambrosini et al. 1997; Li et al. 1998; Li et al. 1999a). The molecular structure is unique among mammalian IAPs, with an NH2-terminal baculovirus IAP repeat (BIR) motif typical of anti-apoptotic proteins and a COOH-terminal amphipathic α-helical coil (Chantalat et al. 2000; Verdecia et al. 2000; Sun et al. 2005). In addition to wild type (wt) survivin transcript, alternative splicing of survivin pre-mRNA produces four other distinct proteins, survivin-2α, survivin-2B, survivin-deleted exon (ΔEx) 3, and survivin-3B (Ambrosini et al. 1997; Zheng et al. 2005; Mahotka et al. 1999; Badran et al. 2004), but little is known about the different biological roles on cell kinetics.

Survivin is functionally involved in both control of cell survival and regulation of mitosis. Survivin-wt is maximally expressed during G2/M phase of cell cycle, directly interacting with chromosomal passenger proteins, such as Aurora-B kinase (Li 2003; Altieri 2004; Wheatley et al. 2001, 2004; Gassmann et al. 2004). In the complexes, survivin-wt can facilitate accurate sister chromatid segregation and stabilization of the microtubules in late mitosis (Yang et al. 2004). Current evidence also indicated that survivin-wt inhibits cell death, functioning upstream of effector caspases by inhibiting caspase 9 (Dohi et al. 2004).

Survivin variants are expressed in many fetal and some proliferating adult tissues, whereas no transcripts are detected in a variety of resting adult tissues (Ambrosini et al. 1997; Li et al. 1999b; Mesri et al. 2001; Komacker et al. 2001). The splicing variants are also overexpressed in almost all types of human malignancies, but the biological significance appears to be different among cancer types (Mahotka et al. 2002a; Krieg et al. 2002; Kappler et al. 2001). Whereas expression of survivin-wt has previously been observed in human endometrial tissues (Fujino et al. 2006; Ai et al. 2006; Chen et al. 2009), there are no data available so far on the role of splicing variants. To clarify their biological functions in endometrial carcinomas (Em Cas), we extensively investigated expression of survivin-wt, survivin-ΔEx3, and survivin-2B with reference to cell death and proliferation.

Materials and methods

Plasmids and cell line

Full-length cDNAs for human survivin-wt, survivin-2B, and survivin-ΔEx3 (GeneBank accession number NM001168) were generated by PCR (Mahotka et al. 1999), and subcloned into pEGFP (BD Biosciences Clontech, Mountainview, CA, USA). The promoter sequence between –1,472 and +13 bp relative to the transcription start site (U75285) was also amplified by PCR, using the primers forward: 5′-CTGGGTGAAGGGTATATGAG-3′ and reverse: 5′-TTCAAATCTGGCGGTTAATGG-3′, and subcloned into the pGL3-basic vector (Promega, Madison WI, USA).

Em Ca cell lines, Ishikawa, and Hec251 cells were maintained in Eagle’s MEM with 10 % bovine calf serum. To establish cells stably overexpressing GFP-survivin variants, the expression or empty plasmids were transfected into Ishikawa cells, and stable clones were established, as described previously (Saegusa et al. 2009, 2012a, b).

Antibodies and reagents

Antibodies to survivin-wt (R&D Systems, Minneapolis, MN, USA), survivin-2B (Abcam, Cambridge, MA, USA), survivin-ΔEx3 (Abcam), bcl-2 (Dako, Glostrup, Denmark), bax (BD Transduction Lab, Franklin Lakes, NJ, USA), p21waf1 (Calbiochem, Cambridge, MA, USA), p27kip1 (BD Transduction Lab), Aurora-B kinase (Cell Signaling, Danvers, MA, USA), cleaved caspase 3 (Cell Signaling), cleaved caspase 9 (Cell Signaling), Ki-67 (Dako), γ-tubulin (Sigma-Aldrich Co, St Louis, MO, USA), cyclin A (Novocastra, Newcastle, UK), cyclin E (Dako), and β-actin (Sigma) were employed in this study.

TNF-α, nocodazole, and doxorubicin were purchased from Sigma. Rapamycin and aphidicolin were obtained from Calbiochem.

Transfection

Transfection was carried out using LipofectAMINE PLUS (Invitrogen, Carlsbad, CA, USA), and luciferase activity was assayed, as described previously (Saegusa et al. 2009, 2012a, b).

RT-PCR

Amplification was carried out in the exponential phase to allow comparison among cDNAs synthesized from identical reactions, using specific primers for each survivin variant (Zhu et al. 2004). Efficiency of gene amplification by each specific primer set was also examined using serially diluted cDNA of the three survivin variants. Primers for GAPDH genes were applied as internal control. The intensity of each individual band was measured with ImageJ, NIH software.

Western blot assay

Total cellular proteins and subcellular protein fractions were prepared. Aliquots of 1–10-μg proteins were resolved by SDS-PAGE, transferred to membranes, probed with primary antibodies, and visualized by the ECL detection system (Amersham Pharmacia Biotech, Tokyo, Japan).

Immunofluorescence

After transfection of GFP-survivin cDNAs into Ishikawa cells, they were incubated with anti-γ-tubulin or anti-Aurora-B kinase antibodies. Rhodamine-labeled anti-rabbit IgGs (Molecular Probes, Eugene, OR, USA) were used as secondary antibodies.

TUNEL (TdT-mediated dUTP-biotin nick end labeling) assay

The in situ Cell Death Detection Kit (Roche, Tokyo, Japan) was used for detection of apoptotic cells. At least 700 cells were counted to detect apoptotic cells, and labeling indices (LIs) were then calculated, as described previously (Chaopotong et al. 2012).

Flow cytometry

For cell cycle analysis, cells were fixed using 70 % alcohol and stained with propidium iodide (Sigma). The prepared cells were analyzed by flow cytometry using BD FACS Calibur (BD Biosciences) and CellQuest Pro software (BD Biosciences).

Clinical cases

A total of 94 cases of endometrial carcinomas (Em Cas), including 39 grade (G) 1, 30 G2, and 25 G3 endometrioid type, as well as 21 complex atypical hyperplasia cases, surgically resected at Kitasato University Hospital in the period from 2003 to 2012, were selected from our patient records, according to the criteria of the 2003 World Health Organization classification (Silverberg et al. 2003). Fifty-eight cases were subcategorized as stage I and 36 as stage II to IV, and 60 and 34 demonstrated upper or lower myometrial invasions, while 14 were positive and 80 were negative for nodal metastasis. Thirty specimens of normal endometrial tissues including 10 in the proliferative and 20 in the secretory phase (10 early and 10 late phases) were also investigated. All tissues were routinely fixed in 10 % formalin and processed for embedding in paraffin wax. In addition, 20 cases of normal proliferative endometrium and 24 of Em Cas were snap-frozen for mRNA analysis. Approval for this study was given by the Ethics Committee of the Kitasato University School of Medicine (B13-29).

Immunohistochemistry (IHC)

IHC was performed using a combination of microwave oven heating and polymer immunocomplex (Envision, Dako) methods.

For evaluation of IHC findings, nuclear immunopositivity was counted and LIs were then calculated, as described previously (Saegusa et al. 2009, 2012a, b). Scoring of cytoplasmic immunoreactivity was performed on the basis of percentage of immunopositive cells and immunointensity, as described previously (Saegusa et al. 2009, 2012a, b).

Apoptosis and mitosis

Apoptotic cells were identified in HE-stained sections, according to the criteria of Kerr et al. (1994). At least 500 cancer cells per slide in areas remote from necrosis and fibrosis were counted and calculated for apoptotic indices (AIs), as well as mitotic indices (MIs), in a similar manner as analysis of LIs. TUNEL assays were also conducted in 30 samples of Em Cas, and the positive cells were calculated using the same approach.

Statistics

Comparative data were analyzed using the Mann–Whitney U test and Spearman’s correlation coefficient. The cutoff for statistical significance was set as p < 0.05.

Results

Relationship between survivin variants and Em Ca cell kinetics

Because subcellular localization of survivin variants is closely linked to their function (Mahotka et al. 2002b), we first transfected Ishikawa cells with GFP-survivin variants. As shown in Fig. 1a, GFP-survivin-wt and survivin-2B isoforms were predominantly located in cytoplasmic compartment, while GFP-survivin-ΔEx3 showed distinct nuclear localization. Expression of GFP-survivin-wt and GFP-survivin-ΔEx3 at both nuclear and cytoplasmic levels was substantially increased in G2/M-arrested cells induced by nocodazole, as compared to G1- and early S-arrested cells using rapamycin and aphidicolin, respectively. Higher cytoplasmic and lower nuclear expression of GFP-survivin-2B were observed throughout the cell cycle progression, with transient nuclear accumulation at G2/M (Fig. 1b).

Fig. 1.

Fig. 1

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Survivin variants and Em Ca cell kinetics. a Upper after transfection of GFP-survivin variants into Ishikawa cells, mRNA and protein levels were analyzed by RT-PCR (left) and Western blot assays (right GFP-survivin-2B indicated by asterisk), respectively. Lower subcellular localization of GFP-survivin variants (GFP-survivin-ΔEx3 indicated by arrows). b After transfection of GFP-survivin variants, the cells were synchronized in the G1 phase by treatment with rapamycin (50 nM), in the early S phase by aphidicolin (2 μg/ml), or in the G2/M phase by nocodazole (0.25 μg/ml) for 24 h (h). Western blot assays using cytoplasmic (Cyt) and nuclear (Nu) protein fractions. c After transfection of GFP-survivin variants, cells were treated with nocodazole (0.25 μg/ml) for 24 h and stained for Aurora-B kinase. Note the colocalization in metaphase (indicated by arrows), in contrast to cells in prophase (asterisk)

Since survivin-wt acts as a subunit of the chromosome passenger complex (CPC) and as a regulator of microtubule dynamic (Li 2003; Altieri 2004; Wheatley et al. 2001, 2004; Gassmann et al. 2004), we also examined the association with Aurora-B kinase, one of the CPC components. As shown in Fig. 1c, GFP-survivin-ΔEx3 and GFP-survivin-2B were also found to be colocalized with Aurora-B kinase during cell division. Although survivin-2B has been reported to colocalize and interact with γ-tubulin, a major component of the microtubule organization center (Ling et al. 2007), such associations were never observed (Supplementary Figure S1A).

To further examine the association between survivin variants and cell growth, we generated stable cell lines overexpressing either GFP-survivin-ΔEx3 (Surv-ΔEx3#34) or GFP-survivin-2B (Surv-2B#17) in Ishikawa cells. Both stable cells, in particular Surv-ΔEx3#34, showed low proliferation rates in the exponential growth phase (Supplementary Figure S1B). In addition, up-regulation of p21waf1 and bcl-2 was observed in Surv-ΔEx3#34 and Surv-2B#17 cells, respectively (Supplementary Figure S1C).

Pro-apoptotic and anti-apoptotic functions of survivin-ΔEx3 and survivin-2B in Em Ca cells

To examine whether overexpression of survivin variants contributed to alteration in expression of other variants, RT-PCR assay was carried out (Fig. 2a). Efficiency of gene amplification by each specific primer set using serially diluted cDNA appeared to be identical between survivin-ΔEx3 and survivin-2B, in contrast to the relatively higher efficiency with survivin-wt primers (Supplementary Figure S2A). By single transfection of the three survivin cDNAs into Ishikawa cells, overexpression of exogenous survivin-ΔEx3 and survivin-2B resulted in decreases in mRNA expression of other endogenous variants (Fig. 2b), independent of its own promoter status (Supplementary Figure S2B).

Fig. 2.

Fig. 2

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Relationship of mRNA expression among survivin variants. a Left conformation of specific primers for each survivin variant. Right schematic representation of primer design for each survivin variant Miura et al. (2012). b Left analysis of endogenous mRNA expression by RT-PCR assay in Ishikawa cells after transfection of GFP-survivin variants (exogenous mRNA products indicated by asterisks). Right relative mRNA levels of endogenous survivin-ΔEx3 (left) and survivin-2B (right) were calculated by normalization to GAPDH using NIH ImageJ program. Expression level in cell with empty vector was set as 1

To examine the functional roles of survivin variants in extrinsic apoptotic pathway, we subjected Surv-ΔEx3#34 and Surv-2B#17 cells to treatment with TNF-α, known as a pro-apoptotic molecule (Kelly et al. 2011; Miura et al. 2012). After the treatment, the amount of apoptotic cells was significantly increased in Surv-ΔEx3#34 cells and decreased in Surv-2B#17 cells (Fig. 3a), along with activation of caspases 3 and 9, independently of the status of bcl-2 and bax (Fig. 3b). Because doxorubicin, a topoisomerase II inhibitor, causes consistent levels of cell damage through the intrinsic apoptotic pathway (Jacob et al. 2012), the treatment was also examined. As shown in Fig. 4a, susceptibility to apoptosis by doxorubicin was significantly higher in Surv-2B#17 cells and lower in Surv-ΔEx3#34 cells with changes in activation of caspases 3 and 9 (Fig. 4b). In addition, expression of bcl-2 relative to bax showed significantly progressive decrease during the treatment in Surv-2B#17 cells, in contrast to Surv-ΔEx3#34 cells (Fig. 4b). Similar findings at both mRNA and protein levels were also observed when cells were treated with higher but not lower doses of both TNF-α and doxorubicin (Supplementary Figure S3), indicating that relatively high concentrations of the reagents may be necessary for activation of both extrinsic and intrinsic apoptotic pathways.

Fig. 3.

Fig. 3

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Functional roles of survivin-ΔEx3 and survivin-2B during TNF-α-mediated cell death. a Left upper after treatment with TNF-α (20 ng/ml), Surv-ΔEx3#34 and Surv-2B#17 cells undergoing apoptosis (indicated by arrows) were detected by TUNEL assay. Original magnification: ×200. Left lower apoptotic indices (AI) following TNF-α (20 ng/ml) treatment shown as mean ± SD. Right upper after treatment with TNF-α (20 ng/ml), Surv-ΔEx3#34 and Surv-2B#17 cells undergoing apoptosis (subG1) were detected by flow cytometry. Right lower percentage of subG1, G1, S, and G2/M fractions following TNF-α treatment. b Upper analysis of protein levels in Surv-ΔEx3#34 and Surv-2B#17 cells after TNF-α (20 ng/ml) treatment by Western blot assay. Lower values of endogenous bcl-2 relative to bax protein were calculated by normalization to β-actin using NIH ImageJ software. Expression levels in absence of TNF-α treatment (0 h) were set as 1

Fig. 4.

Fig. 4

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Functional roles of survivin-ΔEx3 and survivin-2B during doxorubicin-mediated cell death. a Left upper after treatment with doxorubicin (1 μg/ml), Surv-ΔEx3#34 and Surv-2B#17 cells undergoing apoptosis (indicated by arrows) were detected by TUNEL assay. Original magnification: ×200. Left lower apoptotic indices (AI) following doxorubicin (1 μg/ml) treatment shown as mean ± SD. Right upper after treatment with doxorubicin, Surv-ΔEx3#34 and Surv-2B#17 cells undergoing apoptosis (subG1) were detected by flow cytometry. Right lower percentage of subG1, G1, S, and G2/M fractions following doxorubicin treatment. b Upper analysis of protein levels in Surv-ΔEx3#34 and Surv-2B#17 cells after doxorubicin (1 μg/ml) treatment by Western blot assay. Lower values of endogenous bcl-2 relative to bax protein were calculated by normalization to β-actin using NIH ImageJ software. Expression level in absence of doxorubicin treatment (0 h) was set as 1

In addition, we examined whether expression of endogenous survivin variants was altered during apoptosis in Ishikawa and Hec251 cells. In the former, following TNF-α treatment, mRNA levels of survivin-wt and survivin-2B showed significantly time-dependent decrease, resulting in a substantially higher mRNA level of survivin-ΔEx3 relative to survivin-wt or survivin-2B (Fig. 5a). In contrast, considerable decrease in mRNA expression of survivin-ΔEx3 was observed during doxorubicin treatment, leading to significant decrease in mRNA expression of survivin-ΔEx3 relative to either survivin-wt or survivin-2B (Fig. 5b). Similar findings were also observed in the latter, with the exception of some cases (Supplementary Figure S4).

Fig. 5.

Fig. 5

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Changes in mRNA expression of endogenous survivin variants during TNF-α and doxorubicin treatments. a, b Left analysis of endogenous survivin mRNA expression in Ishikawa cells after treatment with TNF-α (20 ng/ml) or doxorubicin (1 μg/ml) by RT-PCR assay. Middle relative mRNA levels of endogenous survivin variants were calculated by normalization to GAPDH using NIH ImageJ software. Expression level in absence of TNF-α or doxorubicin treatment (0 h) was set as 1. Right relative mRNA expression among survivin variants after treatment with TNF-α or doxorubicin for the time shown

Expression of survivin variants in normal and malignant endometrium

Nuclear immunostainings for survivin-wt and survivin-ΔEx3 were mainly observed in glandular components, while survivin-2B but not survivin-wt showed cytoplasmic immunoreactivity (Fig. 6a). Nuclear LI values for survivin-wt showed significantly progressive decrease from proliferative to late secretory phase. Nuclear survivin-ΔEx3 LIs appeared to be decreased in early secretory phase as compared to proliferative and late secretory endometrium, while IHC scores for cytoplasmic survivin-2B did not alter during the menstrual cycle. RT-PCR assay also demonstrated that relative mRNA levels for survivin-wt and survivin-2B were significantly higher than those of survivin-ΔEx3 in 20 proliferative endometrial samples (Fig. 6b).

Fig. 6.

Fig. 6

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IHC analyses of survivin-wt, survivin-ΔEx3, and survivin-2B in serial sections of normal endometrial tissues. a Left HE and IHC analysis for survivin variants in the proliferative (upper panels) and late secretory (lower panels) stages. Note the sporadic immunopositivity (as indicated by arrows and magnified in the insets) for survivin-2B in the proliferative and for survivin-ΔEx3 in the secretory phase. Original magnification, ×200 and ×400 (inset). b Upper labeling indices of nuclear (N) survivin-wt and survivin-ΔEx3 and IHC scores of survivin-2B in proliferative (Pro), and early (SE) and late secretory (SL) stages. Lower left example of survivin variants (mRNA) by RT-PCR assay in normal proliferative (P) endometrial samples. Lower right the relative mRNA levels were calculated by normalization to GAPDH using NIH ImageJ program. The data are shown as mean ± SD

The subcellular immunolocalizations in Em Cas were similar to those in normal endometrium (Fig. 7a). Nuclear LI values for survivin-wt showed significantly progressive increase from normal to G1 to G3 Em Cas. Survivin-ΔEx3 LIs were also significantly increased in Em Cas as compared to non-neoplastic samples, but the LI values were not different among G1, G2, and G3 tumors. No difference in IHC scores for survivin-2B was observed among non-neoplastic and malignant endometrial lesions.

Fig. 7.

Fig. 7

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IHC findings in serial sections of Em Cas. a Upper HE and IHC analysis for survivin variants (immunopositive cells indicated by arrows are magnified in the inset) in G1 (upper) and G3 (lower) Em Cas. Original magnification, ×100 and ×400 (inset). Lower nuclear (N) LI values for survivin-wt and survivin-ΔEx3, and IHC scores for survivin-2B in normal proliferative (P) and complex atypical hyperplastic (H) endometrium and G1, G2, and G3 Em Cas. b Upper relative values among LIs of nuclear survivin-wt and survivin-ΔEx3 and IHC scores of survivin-2B. c Left example of survivin variants (mRNA) by RT-PCR assay in Em Ca tissues. Right the relative mRNA levels were calculated by normalization to GAPDH using NIH ImageJ program. The data are shown as mean ± SD. d Indices of apoptosis and mitosis, and labeling indices of Ki-67. The data are shown as mean ± SD

Expression of survivin-ΔEx3 relative to either survivin-wt or survivin-2B was significantly higher in Em Cas as compared to non-neoplastic lesions, in contrast to significantly lower survivin-2B expression relative to survivin-wt (Fig. 7b). RT-PCR assay also revealed that survivin-wt and survivin-ΔEx3, but not survivin-2B, were the predominant variant isoforms in 25 cases of Em Cas (Fig. 7c).

Apoptotic cells were readily detected in HE-stained sections, on the basis of characteristic features, the AIs being positively correlated with those detected by TUNEL assay (Supplementary Figure S5A). Average AI, MI, and Ki-67 LI values also demonstrated significantly progressive increases from normal to G3 Em Ca samples (Fig. 7d).

As shown in Table 1, there were no correlations among nuclear LIs of survivin-wt and survivin-ΔEx3, and survivin-2B scores. Survivin-ΔEx3 LIs were positively correlated with AI values, while survivin-wt LIs were positively associated with both Ki-67 LIs and MIs. In addition, expression of survivin-wt, survivin-ΔEx3, and survivin-2B was not associated with several clinicopathological markers investigated (Supplementary Figure S5B).

Table 1.

Correlations among survivin variants and cell kinetic markers in endometrial carcinomas

Surv-wild Surv-ΔE3 Surv-2B AI Ki-67 LI
ρ (p) ρ (p) ρ (p) ρ (p) ρ (p)
Surv-ΔE3 0.07 * * * *
(0.45)
Surv-2B −0.09 0.31 * * *
(0.6) (0.08)
AI 0.22 0.67 0.2 * *
(0.014) (<0.0001) (0.25)
Ki-67 LI 0.87 −0.03 −0.29 0.11 *
(<0.0001) (0.73) (0.1) (0.06)
MI 0.61 0.26 −0.13 0.44 0.59
(<0.0001) (0.003) (0.45) (<0.0001) (<0.0001)
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ρ Spearman’s correlation coefficient, Surv survivin, AI apoptotic index, MI mitotic index, LI labeling index

Discussion

Evidence has been provided that exogenous overexpression of GFP-survivin-wt resulted in a predominantly cytoplasmic localization in Ishikawa cells, while the immunoreactivity was mainly detected in nuclear compartment in clinical samples. Given our results showing substantial increase in expression of survivin-wt in G2/M-arrested cells, one possible explanation for the discrepant results may be due to the relatively lower level of endogenous survivin-wt in cytoplasmic component, with the exception of G2/M phase. In fact, survivin-wt is subsequently down-regulated at G1 through the combined action of G1 transcriptional repressor and ubiquitin–proteasome pathway (Zhao et al. 2000).

Conflicting descriptions on regulation of survivin-wt have been reported in human normal endometrium (Konno et al. 2000; Nabilsi et al. 2010). In our study, nuclear survivin-wt expression was significantly higher in the proliferative as compared to late secretory phase and was positively correlated with cell proliferation (data not shown). The cyclic expression therefore appeared to be simply due to changes in cell proliferative activity during menstrual cycle.

Survivin-wt is capable of binding to the multifunctional mitotic regulator Aurora-B kinase, directing it to the kinetochores of metaphase chromosomes (Chen et al. 2003). In this study, survivin-ΔEx3 and survivin-2B were also colocalized with Aurora-B kinase in metaphase. Together with the results demonstrating up-regulation of both variants in G2/M-arrested cells, it was expected that the overexpression may promote cell proliferation through modulation of cell division. However, the two stable cell lines demonstrated lower proliferative activity, along with up-regulation of p21waf1. In addition, there were no associations between expression of the two variants and cell proliferation in Em Cas tissues. These observations therefore suggested that their overexpression may have a minor role in control of cell proliferation. In fact, survivin-ΔEx3 and survivin-2B could not rescue cell proliferation inhibited by siRNA-mediated survivin-wt depletion (Noton et al. 2006).

The extrinsic apoptotic pathway is initiated by activation of cell death receptors, such as TNF-α receptor (Kelly et al. 2011; Miura et al. 2012). In this study, overexpression of survivin-ΔEx3 caused an enhancement of TNF-α-mediated apoptosis, while survivin-2B inhibited this phenomenon. In addition, expression level of bcl-2 and bax did not alter during the process, allowing us to speculate that the TNF-α-mediated apoptosis may occur through mitochondrial-independent pathway in Ishikawa cells, since changes in expression of bcl-2 and bax are essential for the induction (Ling et al. 2007). Given the findings showing relatively higher endogenous mRNA expression of survivin-ΔEx3 relative to either survivin-wt or survivin-2B during the treatment, it is possible that survivin-ΔEx3 may have a potential dominant negative effect on function of the other variants in the extrinsic pathway. Interestingly, survivin variants can interact either directly or indirectly among themselves, and the heterodimers are located at new cellular regions, which may give them the capacity to develop new functions (Noton et al. 2006; Caldas et al. 2005).

Toxic insults such as radiation and chemotherapy cause activation of the intrinsic apoptotic pathway, leading to changes in mitochondrial permeability (Kelly et al. 2011; Miura et al. 2012). As shown by our results, forced expression of survivin-ΔEx3 was able to inhibit doxorubicin-mediated apoptosis, while overexpression of survivin-2B promoted the process, along with changes in expression level of bcl-2 relative to bax. Given that survivin-ΔEx3 is the only variant that has a mitochondrial localization signal and a BH2 domain, the latter of which is required for binding to bcl-2 (Caldas et al. 2005), it appeared that this variant may serve as an inhibitor for mitochondrial-dependent apoptosis through preserved bcl-2 expression. In fact, our results indicated that endogenous mRNA expression level of survivin-ΔEx3 was significantly decreased during doxorubicin treatment, in line with another study (Zhu et al. 2004).

A rapidly growing body of evidence indicates that survivin-ΔEx3 and survivin-2B may play an opposing role in tumor progression in a variety of human malignancies (Li 2005). According to our results, although expression of survivin variants did not show any associations with several clinicopathological factors in Em Cas, survivin-wt and survivin-ΔEx3 showed a significant correlation with cell proliferation and apoptotic values, respectively. Together with the results demonstrating significantly lower level of survivin-2B mRNA expression in Em Ca tissues, it is likely that survivin-wt and survivin-ΔEx3 may be key factors for regulation of the balance between proliferation and cell death in Em Cas.

Another finding of interest in this study was that overexpression of survivin variants resulted in an inhibition of other endogenous variant isoforms in Ishikawa cells. In Em Cas, protein expression of survivin-ΔEx3 relative to survivin-2B was significantly higher in G1 tumors than non-neoplastic lesions, in line with the results of changes in the mRNA expression values. It is therefore suggested that the relative proportion of each variant may contribute to regulation of the alternative splicing machinery.

Finally, our observations suggest a model for functional roles of survivin-ΔEx3 and survivin-2B in Em Ca cells (Fig. 8), although the possibility of cell-type-specific effects cannot be entirely. Survivin-ΔEx3 and survivin-2B may act as natural antagonists of the function of each variant, leading to promotion or inhibition of the extrinsic and intrinsic apoptotic pathways in Em Ca cells.

Fig. 8.

Fig. 8

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Schematic representation for function of survivin variants in apoptotic pathways in Em Cas

Electronic supplementary material

Below is the link to the electronic supplementary material.

432_2014_1762_MOESM1_ESM.tif (4.2MB, tif)

Supplementary Figure S1. (A) After transfection of GFP-survivin-wt (upper), GFP-survivin-ΔEx3 (middle), and GFP-survivin-2B (lower), Ishikawa cells were treated with nocodazole (0.25 μg/ml) for 24 h and stained for γ-tubulin (indicated by arrows). (B) Upper: Surv-2B#17 and Surv-ΔEx3#34 cells were seeded at low density and monitored for growth. Cell numbers are presented as mean ± SD. P0, P3, P5, and P7: 0, 3, 5, and 7 days after cell passage. Lower: Ishikawa cells stably overexpressing GFP-survivin-ΔEx3 (left: Surv-ΔEx3#34 cells. Nuclear staining is indicated by arrows) and GFP-survivin-2B (right: Surv-2B#17 cells). (B) Western blot analyses of the stably transfected cells at different days of cell growth. (TIFF 4297 kb)

432_2014_1762_MOESM2_ESM.tif (2MB, tif)

Supplementary Figure S2. (A) Efficiency of gene amplification by specific primer sets for each survivin variant. (B) Ishikawa cells were transfected with survivin reporter constructs, together with expression plasmids of survivin-wt, survivin-ΔEx3, and survivin-2B. (TIFF 2058 kb)

432_2014_1762_MOESM3_ESM.tif (3MB, tif)

Supplementary Figure S3. (A) Left: analysis of protein levels in Surv-ΔEx3#34 and Surv-2B#17 cells after treatment with TNF-α (10 and 40 ng/ml) by Western blot assay. Middle: values of endogenous bcl-2 relative to bax protein were calculated by normalization to β-actin using NIH ImageJ software. Expression level in absence of TNF-α treatment (0 ng/ml) was set as 1. Right: analysis of endogenous survivin mRNA expression in Ishikawa cells after treatment with TNF-α (10 and 40 ng/ml). (B) Left: analysis of protein levels in Surv-ΔEx3#34 and Surv-2B#17 cells after treatment with doxorubicin (0.5 and 2 μg/ml) by Western blot assay. Middle: values of endogenous bcl-2 relative to bax protein were calculated by normalization to β-actin using NIH ImageJ software. Expression level in absence of doxorubicin treatment (0 μg/ml) was set as 1. Right: analysis of endogenous survivin mRNA expression in Ishikawa cells after treatment with doxorubicin (0.5 and 2 μg/ml) by RT-PCR assay. (TIFF 3048 kb)

432_2014_1762_MOESM4_ESM.tif (1.9MB, tif)

Supplementary Figure S4. (A, B) Left: analysis of endogenous survivin mRNA expression in Hec251 cells after treatment with TNF-α (20 ng/ml) or doxorubicin (1 μg/ml) by RT-PCR assay. Middle: relative mRNA levels of endogenous survivin variants were calculated by normalization to GAPDH using NIH ImageJ software. Expression level in absence of TNF-α or doxorubicin treatment (0 h) was set as 1. Right: relative mRNA expression among survivin variants after treatment with TNF-α or doxorubicin for the time shown. (TIFF 1987 kb)

432_2014_1762_MOESM5_ESM.tif (2.9MB, tif)

Supplementary Figure S5. (A) Left: detection of apoptotic cells (indicated by arrows) in hematoxylin and eosin (HE) sections by histological analysis and TUNEL assay. Original magnification: x400. Right: relationship between HE section and TUNEL assay in detection of apoptotic cells. N, number of cases (B) Relationship between expression of survivin variants and clinicopathological factors. The data are shown as mean ± SD. U, upper myometrial invasion; L, lower myometrial invasion; P, positive for nodal metastasis; N, negative for nodal metastasis (TIFF 3010 kb)

Acknowledgments

We would like to thank Miki Hashimura and Tomoko Tsuruta for their technical assistance. This study was supported by a grant from JSPS KAKENHI Grand Number 23590415.

Conflict of interest

Potential conflicts do not exist.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

432_2014_1762_MOESM1_ESM.tif (4.2MB, tif)

Supplementary Figure S1. (A) After transfection of GFP-survivin-wt (upper), GFP-survivin-ΔEx3 (middle), and GFP-survivin-2B (lower), Ishikawa cells were treated with nocodazole (0.25 μg/ml) for 24 h and stained for γ-tubulin (indicated by arrows). (B) Upper: Surv-2B#17 and Surv-ΔEx3#34 cells were seeded at low density and monitored for growth. Cell numbers are presented as mean ± SD. P0, P3, P5, and P7: 0, 3, 5, and 7 days after cell passage. Lower: Ishikawa cells stably overexpressing GFP-survivin-ΔEx3 (left: Surv-ΔEx3#34 cells. Nuclear staining is indicated by arrows) and GFP-survivin-2B (right: Surv-2B#17 cells). (B) Western blot analyses of the stably transfected cells at different days of cell growth. (TIFF 4297 kb)

432_2014_1762_MOESM2_ESM.tif (2MB, tif)

Supplementary Figure S2. (A) Efficiency of gene amplification by specific primer sets for each survivin variant. (B) Ishikawa cells were transfected with survivin reporter constructs, together with expression plasmids of survivin-wt, survivin-ΔEx3, and survivin-2B. (TIFF 2058 kb)

432_2014_1762_MOESM3_ESM.tif (3MB, tif)

Supplementary Figure S3. (A) Left: analysis of protein levels in Surv-ΔEx3#34 and Surv-2B#17 cells after treatment with TNF-α (10 and 40 ng/ml) by Western blot assay. Middle: values of endogenous bcl-2 relative to bax protein were calculated by normalization to β-actin using NIH ImageJ software. Expression level in absence of TNF-α treatment (0 ng/ml) was set as 1. Right: analysis of endogenous survivin mRNA expression in Ishikawa cells after treatment with TNF-α (10 and 40 ng/ml). (B) Left: analysis of protein levels in Surv-ΔEx3#34 and Surv-2B#17 cells after treatment with doxorubicin (0.5 and 2 μg/ml) by Western blot assay. Middle: values of endogenous bcl-2 relative to bax protein were calculated by normalization to β-actin using NIH ImageJ software. Expression level in absence of doxorubicin treatment (0 μg/ml) was set as 1. Right: analysis of endogenous survivin mRNA expression in Ishikawa cells after treatment with doxorubicin (0.5 and 2 μg/ml) by RT-PCR assay. (TIFF 3048 kb)

432_2014_1762_MOESM4_ESM.tif (1.9MB, tif)

Supplementary Figure S4. (A, B) Left: analysis of endogenous survivin mRNA expression in Hec251 cells after treatment with TNF-α (20 ng/ml) or doxorubicin (1 μg/ml) by RT-PCR assay. Middle: relative mRNA levels of endogenous survivin variants were calculated by normalization to GAPDH using NIH ImageJ software. Expression level in absence of TNF-α or doxorubicin treatment (0 h) was set as 1. Right: relative mRNA expression among survivin variants after treatment with TNF-α or doxorubicin for the time shown. (TIFF 1987 kb)

432_2014_1762_MOESM5_ESM.tif (2.9MB, tif)

Supplementary Figure S5. (A) Left: detection of apoptotic cells (indicated by arrows) in hematoxylin and eosin (HE) sections by histological analysis and TUNEL assay. Original magnification: x400. Right: relationship between HE section and TUNEL assay in detection of apoptotic cells. N, number of cases (B) Relationship between expression of survivin variants and clinicopathological factors. The data are shown as mean ± SD. U, upper myometrial invasion; L, lower myometrial invasion; P, positive for nodal metastasis; N, negative for nodal metastasis (TIFF 3010 kb)

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