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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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Calcium measurements.The diagram shows the mean calcium influx in CCL21 stimulated cells. An increase in cytosolic calcium levels was only observed in M13HS-2 and M13HS-8 hybrid cells. To prove whether the CCL21 induced calcium influx in these cell lines depended on PLC-β/γ activity, appropriate calcium measurements within the presence of 5 µM U73122 were performed. Cells were preincubated for 5 minutes with U73122 prior to analysis. Shown are the mean±SD of n = 3 independent experiments. Statistical significance was calculated using Student's t-test: n.s.  =  not significant; **  =  p<0.01; ***  =  p<0.001.
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pone-0063711-g003: Calcium measurements.The diagram shows the mean calcium influx in CCL21 stimulated cells. An increase in cytosolic calcium levels was only observed in M13HS-2 and M13HS-8 hybrid cells. To prove whether the CCL21 induced calcium influx in these cell lines depended on PLC-β/γ activity, appropriate calcium measurements within the presence of 5 µM U73122 were performed. Cells were preincubated for 5 minutes with U73122 prior to analysis. Shown are the mean±SD of n = 3 independent experiments. Statistical significance was calculated using Student's t-test: n.s.  =  not significant; **  =  p<0.01; ***  =  p<0.001.

Mentions: In addition to Western Blot studies analyzing PI3K/AKT and MAPK signaling we also performed flow cytometry based calcium measurements [23], [43]–[45] to investigate whether the CCL21/CCR7 axis does also active PLC-β/γ signaling. Flow cytometry based calcium measurements revealed no significant CCL21 mediated calcium influx in M13SV1-EGFP-Neo and HS578T-Hyg parental cells (Figure 3). By contrast, both hybrid cell lines showed significantly increased intracellular calcium levels upon CCL21 stimulation indicating activation of PLC-β/γ signaling (M13HS-2 control: 0±8% vs. 500 ng/ml CCL21: 26±9%, p<0.01; M13HS-8 control: 0±5% vs. 500 ng/ml CCL21: 40±6%, p<0.001; Figure 3). This was further supported by addition of the PLC-β/γ inhibitor U73122 that completely abolished the CCL21 mediated calcium influx in M13HS-2 and -8 hybrid cell lines (M13HS-2: 500 ng/ml CCL21: 26±9% vs. 5 µM U73122 + 500 ng/ml CCL21: 0±9%, p<0.001; M13HS-8: 500 ng/ml CCL21: 40±6% vs. 5 µM U73122 + 500 ng/ml CCL21: 3±4%, p<0.001; Figure 3).


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)

Calcium measurements.The diagram shows the mean calcium influx in CCL21 stimulated cells. An increase in cytosolic calcium levels was only observed in M13HS-2 and M13HS-8 hybrid cells. To prove whether the CCL21 induced calcium influx in these cell lines depended on PLC-β/γ activity, appropriate calcium measurements within the presence of 5 µM U73122 were performed. Cells were preincubated for 5 minutes with U73122 prior to analysis. Shown are the mean±SD of n = 3 independent experiments. Statistical significance was calculated using Student's t-test: n.s.  =  not significant; **  =  p<0.01; ***  =  p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646822&req=5

pone-0063711-g003: Calcium measurements.The diagram shows the mean calcium influx in CCL21 stimulated cells. An increase in cytosolic calcium levels was only observed in M13HS-2 and M13HS-8 hybrid cells. To prove whether the CCL21 induced calcium influx in these cell lines depended on PLC-β/γ activity, appropriate calcium measurements within the presence of 5 µM U73122 were performed. Cells were preincubated for 5 minutes with U73122 prior to analysis. Shown are the mean±SD of n = 3 independent experiments. Statistical significance was calculated using Student's t-test: n.s.  =  not significant; **  =  p<0.01; ***  =  p<0.001.
Mentions: In addition to Western Blot studies analyzing PI3K/AKT and MAPK signaling we also performed flow cytometry based calcium measurements [23], [43]–[45] to investigate whether the CCL21/CCR7 axis does also active PLC-β/γ signaling. Flow cytometry based calcium measurements revealed no significant CCL21 mediated calcium influx in M13SV1-EGFP-Neo and HS578T-Hyg parental cells (Figure 3). By contrast, both hybrid cell lines showed significantly increased intracellular calcium levels upon CCL21 stimulation indicating activation of PLC-β/γ signaling (M13HS-2 control: 0±8% vs. 500 ng/ml CCL21: 26±9%, p<0.01; M13HS-8 control: 0±5% vs. 500 ng/ml CCL21: 40±6%, p<0.001; Figure 3). This was further supported by addition of the PLC-β/γ inhibitor U73122 that completely abolished the CCL21 mediated calcium influx in M13HS-2 and -8 hybrid cell lines (M13HS-2: 500 ng/ml CCL21: 26±9% vs. 5 µM U73122 + 500 ng/ml CCL21: 0±9%, p<0.001; M13HS-8: 500 ng/ml CCL21: 40±6% vs. 5 µM U73122 + 500 ng/ml CCL21: 3±4%, p<0.001; Figure 3).

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