The online version of this article (https://doi.org/10.1186/s12885-017-3932-y) contains supplementary material, which is available to authorized users.
Survivin, belonging to the inhibitor of apoptosis (IAP) gene family, is abundantly expressed in tumors. It has been hypothesized that Survivin facilitates carcinogenesis by inhibition of apoptosis resulting in improved survival of tumorigenic progeny. Additionally, Survivin plays an essential role during mitosis. Together with its molecular partners Aurora B, Borealin and inner centromere protein it secures bipolar chromosome segregation. However, whether increased Survivin levels contribute to progression of tumors by inducing chromosomal instability remains unclear.
We overexpressed Survivin in U251-MG, SVGp12, U87-MG, HCT116 and p53-deficient U87-MGshp53 and HCT116p53−/− cells. The resulting phenotype was investigated by FACS-assisted cell cycle analysis, Western Blot analysis, confocal laser scan microscopy, proliferation assays, spectral karyotyping and in a U251-MG xenograft model using immune-deficient mice.
Overexpression of Survivin affected cells with knockdown of p53, cells harboring mutant p53 and SV40 large T antigen, respectively, resulting in the increase of cell fractions harboring 4n and >4n DNA contents. Increased γH2AX levels, indicative of DNA damage were monitored in all Survivin-transduced cell lines, but only in p53 wild type cells this was accompanied by an attenuated S-phase entry and activation of p21waf/cip. Overexpression of Survivin caused a DNA damage response characterized by increased appearance pDNA-PKcs foci in cell nuclei and elevated levels of pATM S1981 and pCHK2 T68. Additionally, evolving structural chromosomal aberrations in U251-MG cells transduced with Survivin indicated a DNA-repair by non-homologous end joining recombination. Subcutaneous transplantation of U251-MG cells overexpressing Survivin and mycN instead of mycN oncogene alone generated tumors with shortened latency and decreased apoptosis. Subsequent SKY-analysis of Survivin/mycN-tumors revealed an increase in structural chromosomal aberrations in cells when compared to mycN-tumors.
Our data suggest that increased Survivin levels promote adaptive evolution of tumors through combining induction of genetic heterogeneity with inhibition of apoptosis.
Additional file 1: Figure S1. Indirect immunofluorescence analyses demonstrating typical localization of endogenous and ectopic Survivin at kinetochores. Depicted are representative images of SVGp12 cells in metaphase after transduction of control plasmid (first panel) or after transduction with Survivin vector (second panel). Arrowhead depicts an additional metaphase plate in the Survivin-transduced cell. From left to right: DAPI (DNA), FITC (Survivin), WGA-TexasRed (membrane) and merge. (TIFF 1364 kb)
Additional file 2: Figure S2. a: Proliferation index of the U251-MG and SVGp12 cells transduced with Survivin and empty vector controls. Depicted are mean values ± SD. **p < 0.01. b: BrdU-incorporation in U251-MG and SVGp12 cell fractions with DNA content >4n. Depicted are mean values ± SD. **p < 0.01. All data were collected 72 h after transduction of cells. (TIFF 117 kb)
Additional file 3: Figure S3. Western blot analysis of HCT116 and HCT116p53−/− cell lysates after transduction of Survivin and control vectors. Membranes were probed with anti-p53 (53 kDa), anti-p21waf/cip (21 kDa), anti-p53(S15) (53 kDa) and anti-γH2AX (16 kDa) antibodies. Membranes were re-probed with α-actin (42 kDa) to confirm equal loading. After densitometric analysis the relative expression levels of proteins in Survivin-transduced cells (fold increase) were compared to controls. (TIFF 243 kb)
Additional file 4: Figure S4. Indirect immunofluorescence analyses images of Survivin- and mock-transduced cells stained with a monoclonal antibody specific for phosphoDNA-PKcs. Nuclei were counterstained with DAPI. a: Representative images of SVGp12 cells. The Survivin-transduced cell contains multiple containing phosphoDNAPKcs foci b: Representative images of transduced U251-MG cells. Note the Survivin-transduced multinuclear U251-MG cell containing numerous phosphoDNAPKcs foci in the nuclei. Magnification bars: 10 μm. Data were collected 72 h after transduction of cells. (TIFF 2132 kb)
Additional file 5: Figure S5. SKY-Analysis showing chromosomal instability (increased numerical and structural chromosomal aberrations) in sorted tumor Survivin-overexpressing U251-MG cells compared to sorted mock-control cells. Representative karyograms of mock-control (upper figure) and Survivin-overexpressing cells (lower figure) with white arrows indicating clonal aberrations already present in the parental cell line. Survivin-overexpressing U251-MG show additional non-clonal structural changes indicated by purple arrows. (TIFF 1830 kb)
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