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01.12.2014 | Original Article – Cancer Research | Ausgabe 12/2014

Journal of Cancer Research and Clinical Oncology 12/2014

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

Zeitschrift:
Journal of Cancer Research and Clinical Oncology > Ausgabe 12/2014
Autoren:
Yuki Tazo, Atsuko Hara, Takashi Onda, Makoto Saegusa
Wichtige Hinweise

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.

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.

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Zusatzmaterial
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_MOESM1_ESM.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_MOESM2_ESM.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_MOESM3_ESM.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_MOESM4_ESM.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)
432_2014_1762_MOESM5_ESM.tif
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