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Fusion of CCL21 non-migratory active breast epithelial and breast cancer cells give rise to CCL21 migratory active tumor hybrid cell lines.

Berndt B, Haverkampf S, Reith G, Keil S, Niggemann B, Zänker KS, Dittmar T - PLoS ONE (2013)

Bottom Line: Cell migration demonstrated that only M13HS hybrid cell lines, but not parental derivatives, responded to CCL21 stimulation with an increased migratory activity.Knockdown of CCR7 expression by siRNA completely abrogated the CCL21 induced migration of hybrid cell lines indicating the necessity of CCL21/CCR7 signaling.Because the CCL21/CCR7 axis has been linked to metastatic spreading of breast cancer to lymph nodes we conclude from our data that cell fusion could be a mechanism explaining the origin of metastatic cancer (hybrid) cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Immunology, Center for Biomedical Education and Research (ZBAF), Witten/Herdecke University, Witten, Germany.

ABSTRACT
The biological phenomenon of cell fusion has been linked to tumor progression because several data provided evidence that fusion of tumor cells and normal cells gave rise to hybrid cell lines exhibiting novel properties, such as increased metastatogenic capacity and an enhanced drug resistance. Here we investigated M13HS hybrid cell lines, derived from spontaneous fusion events between M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics and HS578T-Hyg breast cancer cells, concerning CCL21/CCR7 signaling. Western Blot analysis showed that all cell lines varied in their CCR7 expression levels as well as differed in the induction and kinetics of CCR7 specific signal transduction cascades. Flow cytometry-based calcium measurements revealed that a CCL21 induced calcium influx was solely detected in M13HS hybrid cell lines. Cell migration demonstrated that only M13HS hybrid cell lines, but not parental derivatives, responded to CCL21 stimulation with an increased migratory activity. Knockdown of CCR7 expression by siRNA completely abrogated the CCL21 induced migration of hybrid cell lines indicating the necessity of CCL21/CCR7 signaling. Because the CCL21/CCR7 axis has been linked to metastatic spreading of breast cancer to lymph nodes we conclude from our data that cell fusion could be a mechanism explaining the origin of metastatic cancer (hybrid) cells.

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The CCL21 induced migration of M13HS hybrid cell lines depends on a different signal transduction cascades.The migratory activity was analyzed using the 3D collagen matrix migration assay combined with time-lapse video-microscopy. For a better comparison the locomotory activities of CCL21 and inhibitor stimulated cells were calculated in relation to untreated control cells, which were set to 100%. Cells were treated with 500 ng/ml CCL21, 5 µM U73122, 500 nM Ly294002, and 500 nM PD98059. Shown are the mean±SD of n = 4 independent experiments. (A) HS578T-Hyg breast cancer cells (B) M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics, (C) M13HS-2 hybrid cells, (D) M13HS-8 hybrid cells. Statistical significance was calculated using Student's t-test: “*”  =  statistical significance vs. control: **  =  p<0.01; ***  =  p<0.001. “+”  =  statistical significance vs. CCL21: ++  =  p<0.01; +++  =  p<0.001.
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pone-0063711-g005: The CCL21 induced migration of M13HS hybrid cell lines depends on a different signal transduction cascades.The migratory activity was analyzed using the 3D collagen matrix migration assay combined with time-lapse video-microscopy. For a better comparison the locomotory activities of CCL21 and inhibitor stimulated cells were calculated in relation to untreated control cells, which were set to 100%. Cells were treated with 500 ng/ml CCL21, 5 µM U73122, 500 nM Ly294002, and 500 nM PD98059. Shown are the mean±SD of n = 4 independent experiments. (A) HS578T-Hyg breast cancer cells (B) M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics, (C) M13HS-2 hybrid cells, (D) M13HS-8 hybrid cells. Statistical significance was calculated using Student's t-test: “*”  =  statistical significance vs. control: **  =  p<0.01; ***  =  p<0.001. “+”  =  statistical significance vs. CCL21: ++  =  p<0.01; +++  =  p<0.001.

Mentions: Analysis of HS578T-Hyg cells showed a marked inhibitory effect of U73122 on the cells migratory activity, which was similar to U73122 plus CCL21 treated cells (U73122: 38±7%; p<0.001; U73122 + CCL21: 29±8%; p<0.001; Figure 5A) indicating that the matrix induced migration of HS578T-Hyg breast cancer cells depends on PLC-β/γ signaling. By contrast, treatment of HS578T-Hyg breast cancer cells with the PI3K inhibitor Ly294002 did not alter the cells migratory activity (both control and CCL21 treated cells) (Figure 5A), which is in view with Western Blot studies showing no AKT phosphorylation in CCL21 stimulated HS578T-Hyg cells (Figure 2A). Likewise, inhibition of MAPKp42/44 signaling with PD98059 had no effect on the migration of both control and CCL21 stimulated HS578T-Hyg breast cancer cells albeit cells displayed increased MAPKp42/44 levels upon CCL21 stimulation (Figure 2A). Moreover, because the PI3K inhibitor Ly294002 also blocked the CCL21 induced MAPKp42/44 phosphorylation in HS578T-Hyg cells (Figure 2A), but did not alter the cells migratory behavior these data suggest that MAPKp42/44 signaling is not involved in the migration of HS578T-Hyg breast cancer cells.


Fusion of CCL21 non-migratory active breast epithelial and breast cancer cells give rise to CCL21 migratory active tumor hybrid cell lines.

Berndt B, Haverkampf S, Reith G, Keil S, Niggemann B, Zänker KS, Dittmar T - PLoS ONE (2013)

The CCL21 induced migration of M13HS hybrid cell lines depends on a different signal transduction cascades.The migratory activity was analyzed using the 3D collagen matrix migration assay combined with time-lapse video-microscopy. For a better comparison the locomotory activities of CCL21 and inhibitor stimulated cells were calculated in relation to untreated control cells, which were set to 100%. Cells were treated with 500 ng/ml CCL21, 5 µM U73122, 500 nM Ly294002, and 500 nM PD98059. Shown are the mean±SD of n = 4 independent experiments. (A) HS578T-Hyg breast cancer cells (B) M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics, (C) M13HS-2 hybrid cells, (D) M13HS-8 hybrid cells. Statistical significance was calculated using Student's t-test: “*”  =  statistical significance vs. control: **  =  p<0.01; ***  =  p<0.001. “+”  =  statistical significance vs. CCL21: ++  =  p<0.01; +++  =  p<0.001.
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pone-0063711-g005: The CCL21 induced migration of M13HS hybrid cell lines depends on a different signal transduction cascades.The migratory activity was analyzed using the 3D collagen matrix migration assay combined with time-lapse video-microscopy. For a better comparison the locomotory activities of CCL21 and inhibitor stimulated cells were calculated in relation to untreated control cells, which were set to 100%. Cells were treated with 500 ng/ml CCL21, 5 µM U73122, 500 nM Ly294002, and 500 nM PD98059. Shown are the mean±SD of n = 4 independent experiments. (A) HS578T-Hyg breast cancer cells (B) M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics, (C) M13HS-2 hybrid cells, (D) M13HS-8 hybrid cells. Statistical significance was calculated using Student's t-test: “*”  =  statistical significance vs. control: **  =  p<0.01; ***  =  p<0.001. “+”  =  statistical significance vs. CCL21: ++  =  p<0.01; +++  =  p<0.001.
Mentions: Analysis of HS578T-Hyg cells showed a marked inhibitory effect of U73122 on the cells migratory activity, which was similar to U73122 plus CCL21 treated cells (U73122: 38±7%; p<0.001; U73122 + CCL21: 29±8%; p<0.001; Figure 5A) indicating that the matrix induced migration of HS578T-Hyg breast cancer cells depends on PLC-β/γ signaling. By contrast, treatment of HS578T-Hyg breast cancer cells with the PI3K inhibitor Ly294002 did not alter the cells migratory activity (both control and CCL21 treated cells) (Figure 5A), which is in view with Western Blot studies showing no AKT phosphorylation in CCL21 stimulated HS578T-Hyg cells (Figure 2A). Likewise, inhibition of MAPKp42/44 signaling with PD98059 had no effect on the migration of both control and CCL21 stimulated HS578T-Hyg breast cancer cells albeit cells displayed increased MAPKp42/44 levels upon CCL21 stimulation (Figure 2A). Moreover, because the PI3K inhibitor Ly294002 also blocked the CCL21 induced MAPKp42/44 phosphorylation in HS578T-Hyg cells (Figure 2A), but did not alter the cells migratory behavior these data suggest that MAPKp42/44 signaling is not involved in the migration of HS578T-Hyg breast cancer cells.

Bottom Line: Cell migration demonstrated that only M13HS hybrid cell lines, but not parental derivatives, responded to CCL21 stimulation with an increased migratory activity.Knockdown of CCR7 expression by siRNA completely abrogated the CCL21 induced migration of hybrid cell lines indicating the necessity of CCL21/CCR7 signaling.Because the CCL21/CCR7 axis has been linked to metastatic spreading of breast cancer to lymph nodes we conclude from our data that cell fusion could be a mechanism explaining the origin of metastatic cancer (hybrid) cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Immunology, Center for Biomedical Education and Research (ZBAF), Witten/Herdecke University, Witten, Germany.

ABSTRACT
The biological phenomenon of cell fusion has been linked to tumor progression because several data provided evidence that fusion of tumor cells and normal cells gave rise to hybrid cell lines exhibiting novel properties, such as increased metastatogenic capacity and an enhanced drug resistance. Here we investigated M13HS hybrid cell lines, derived from spontaneous fusion events between M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics and HS578T-Hyg breast cancer cells, concerning CCL21/CCR7 signaling. Western Blot analysis showed that all cell lines varied in their CCR7 expression levels as well as differed in the induction and kinetics of CCR7 specific signal transduction cascades. Flow cytometry-based calcium measurements revealed that a CCL21 induced calcium influx was solely detected in M13HS hybrid cell lines. Cell migration demonstrated that only M13HS hybrid cell lines, but not parental derivatives, responded to CCL21 stimulation with an increased migratory activity. Knockdown of CCR7 expression by siRNA completely abrogated the CCL21 induced migration of hybrid cell lines indicating the necessity of CCL21/CCR7 signaling. Because the CCL21/CCR7 axis has been linked to metastatic spreading of breast cancer to lymph nodes we conclude from our data that cell fusion could be a mechanism explaining the origin of metastatic cancer (hybrid) cells.

Show MeSH
Related in: MedlinePlus