The overexpression of SOX2 affects the migration of human teratocarcinoma cell line NT2/D1

The altered expression of the SOX2 transcription factor is associated with oncogenic or tumor suppressor functions in human cancers. This factor regulates the migration and invasion of different cancer cells. In this study we investigated the effect of constitutive SOX2 overexpression on the migration and adhesion capacity of embryonal teratocarcinoma NT2/D1 cells derived from a metastasis of a human testicular germ cell tumor. We detected that increased SOX2 expression changed the speed, mode and path of cell migration, but not the adhesion ability of NT2/D1 cells. Additionally, we demonstrated that SOX2 overexpression increased the expression of the tumor suppressor protein p53 and the HDM2 oncogene. Our results contribute to the better understanding of the effect of SOX2 on the behavior of tumor cells originating from a human testicular germ cell tumor. Considering that NT2/D1 cells resemble cancer stem cells in many features, our results could contribute to the elucidation of the role of SOX2 in cancer stem cells behavior and the process of metastasis.

adhesion; HDM2; migration; p53; SOX2; wound healing

Authorship

DANIJELA DRAKULIC

Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, P.O. BOX 23, 11010 Belgrade, SerbiaUniversity of BelgradeSerbiaBelgrade, SerbiaInstitute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, P.O. BOX 23, 11010 Belgrade, Serbia

JELENA MARJANOVIC VICENTIC

MARIJA SCHWIRTLICH

JELENA TOSIC

Center for Integrative Genomics, University of Lausanne, Génopode, CH-1015 Lausanne, SwitzerlandUniversity of LausanneSwitzerlandLausanne, SwitzerlandCenter for Integrative Genomics, University of Lausanne, Génopode, CH-1015 Lausanne, Switzerland

ALEKSANDAR KRSTIC

Systems Biology Ireland, University College Dublin, Belfield, D4, Dublin, IrelandUniversity College DublinIrelandDublin, IrelandSystems Biology Ireland, University College Dublin, Belfield, D4, Dublin, Ireland

ANDRIJANA KLAJN

MILENA STEVANOVIC

Correspondence to: Milena Stevanovic E-mail: milenastevanovic@imgge.bg.ac.rs

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (5)

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Figure 1
Elevated SOX2 protein expression in SOX2-overexpressing NT2/D1 cell clones. Immunocytochemical analysis of SOX2 expression in NT2/D1 (B), MOCK (E), F5 (H) and G3 (K) cells. Cells were visualized by staining microtubules with α-Tubulin (C, F, I, L). Boxed regions in A-L are enlarged in the same figures. Cell nuclei were co-stained with DAPI (A, D, G, J). Scale bar: 50μm. NT2/ D1 = NT2/D1 cells; MOCK = empty vector-transfected control NT2/D1 cell clone; F5, G3 = SOX2-overexpressing NT2/D1 cell clone.

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Figure 2
Effect of SOX2 overexpression on cell wound recovery. Nearly confluent cell monolayers of NT2/D1, MOCK, F5 and G3 cells were scratched and wound recovery was monitored throughout a 24h time window. Representative phase contrast images of 3 independent wound closure experiments show that F5 (E, F) and G3 (G, H) cells almost closed the gap while control cells did not fill the wounded area in this time window (A - D). Cell migration was monitored with the DM IL LED Inverted Microscope (Leica) using 10x objective. NT2/D1 = NT2/D1 cells; MOCK = empty vector-transfected control NT2/D1 cell clone; F5, G3 = SOX2- overexpressing NT2/D1 cell clone.

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Figure 3
Effect of SOX2 overexpression on NT2/D1 cell migration. Cell motility was monitored using time-lapse microscopy. Panel I. The migration distance of cells from their initial positions was measured after 6h (left) and 12h (right). Panel II. Representative images (A) and graphs (B) comparatively show the mode of cell migration of NT2/D1, F5 and G3 cells 6h after the scratches had been made. Arrows indicate single cells in wounded area. Scale bar: 100 μm. Panel III. 3 representative cell migration trajectories were obtained during 12h (F5, G3) and 20h (NT2/D1) of time-lapse imaging of indicated cells. Arrows indicate exact distance of trajectories (569 μm for cell 3 and 564 μm for cell 8). NT2/D1 = NT2/D1 cells; MOCK = empty vectortransfected control NT2/D1 cell clone; F5, G3 = SOX2-overexpressing NT2/D1 cell clone.

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Figure 4
Adhesion of cells to Matrigel. Cell adhesion was measured using the MTT test. Results are expressed as a fold of adhesion of parental NT2/D1 cells, which was set as 1. Experiments were performed in triplicate and repeated four times. Data of independent experiments are presented in a histogram as the mean ± SD. P ≤ 0.05 were considered as significant. NT2/D1 = NT2/D1 cells; MOCK = empty vectortransfected control NT2/D1 cell clone; F5 and G3 = SOX2- overexpressing NT2/D1 cell clones.

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Figure 5
Profile of p53 and HDM2 expression in SOX2-overexpressing NT2/D1 cell clones. Panel I Proteins expression was detected by the Western Blot using p53, HDM2 and GAPDH -specific antibodies. At least three independent experiments were performed and one representative blot was shown. Bands were digitalized, quantified with the ImageJ software and normalized for GAPDH values. The relative p53 and HDM2 expression levels were calculated as a fold expression of parental NT2/D1 cells. A value of 1 was given to the relative p53 and HDM2 expression levels in parental cells. Data were presented as the means ± SD. P ≤ 0.05 were considered as significant. Panel II mRNA expression was detected by semi-quantitative RT-PCR using primers specific for p53, HDM2 and GAPDH. Three independent experiments were performed and one representative image was shown. NT2/D1 = NT2/D1 cells; MOCK = empty vector-transfected control NT2/D1 cell clone; F5 and G3 = SOX2-overexpressing NT2/D1 cell clones.

Figure 1 Elevated SOX2 protein expression in SOX2-overexpressing NT2/D1 cell clones. Immunocytochemical analysis of SOX2 expression in NT2/D1 (B), MOCK (E), F5 (H) and G3 (K) cells. Cells were visualized by staining microtubules with α-Tubulin (C, F, I, L). Boxed regions in A-L are enlarged in the same figures. Cell nuclei were co-stained with DAPI (A, D, G, J). Scale bar: 50μm. NT2/ D1 = NT2/D1 cells; MOCK = empty vector-transfected control NT2/D1 cell clone; F5, G3 = SOX2-overexpressing NT2/D1 cell clone.

Figure 2 Effect of SOX2 overexpression on cell wound recovery. Nearly confluent cell monolayers of NT2/D1, MOCK, F5 and G3 cells were scratched and wound recovery was monitored throughout a 24h time window. Representative phase contrast images of 3 independent wound closure experiments show that F5 (E, F) and G3 (G, H) cells almost closed the gap while control cells did not fill the wounded area in this time window (A - D). Cell migration was monitored with the DM IL LED Inverted Microscope (Leica) using 10x objective. NT2/D1 = NT2/D1 cells; MOCK = empty vector-transfected control NT2/D1 cell clone; F5, G3 = SOX2- overexpressing NT2/D1 cell clone.

Figure 3 Effect of SOX2 overexpression on NT2/D1 cell migration. Cell motility was monitored using time-lapse microscopy. Panel I. The migration distance of cells from their initial positions was measured after 6h (left) and 12h (right). Panel II. Representative images (A) and graphs (B) comparatively show the mode of cell migration of NT2/D1, F5 and G3 cells 6h after the scratches had been made. Arrows indicate single cells in wounded area. Scale bar: 100 μm. Panel III. 3 representative cell migration trajectories were obtained during 12h (F5, G3) and 20h (NT2/D1) of time-lapse imaging of indicated cells. Arrows indicate exact distance of trajectories (569 μm for cell 3 and 564 μm for cell 8). NT2/D1 = NT2/D1 cells; MOCK = empty vectortransfected control NT2/D1 cell clone; F5, G3 = SOX2-overexpressing NT2/D1 cell clone.

Figure 4 Adhesion of cells to Matrigel. Cell adhesion was measured using the MTT test. Results are expressed as a fold of adhesion of parental NT2/D1 cells, which was set as 1. Experiments were performed in triplicate and repeated four times. Data of independent experiments are presented in a histogram as the mean ± SD. P ≤ 0.05 were considered as significant. NT2/D1 = NT2/D1 cells; MOCK = empty vectortransfected control NT2/D1 cell clone; F5 and G3 = SOX2- overexpressing NT2/D1 cell clones.

Figure 5 Profile of p53 and HDM2 expression in SOX2-overexpressing NT2/D1 cell clones. Panel I Proteins expression was detected by the Western Blot using p53, HDM2 and GAPDH -specific antibodies. At least three independent experiments were performed and one representative blot was shown. Bands were digitalized, quantified with the ImageJ software and normalized for GAPDH values. The relative p53 and HDM2 expression levels were calculated as a fold expression of parental NT2/D1 cells. A value of 1 was given to the relative p53 and HDM2 expression levels in parental cells. Data were presented as the means ± SD. P ≤ 0.05 were considered as significant. Panel II mRNA expression was detected by semi-quantitative RT-PCR using primers specific for p53, HDM2 and GAPDH. Three independent experiments were performed and one representative image was shown. NT2/D1 = NT2/D1 cells; MOCK = empty vector-transfected control NT2/D1 cell clone; F5 and G3 = SOX2-overexpressing NT2/D1 cell clones.

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The overexpression of SOX2 affects the migration of human teratocarcinoma cell line NT2/D1

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[1] Correspondence to: Milena Stevanovic E-mail: milenastevanovic@imgge.bg.ac.rs