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CXCR4 expression heterogeneity in neuroblastoma cells due to ligand-independent regulation.

Carlisle AJ, Lyttle CA, Carlisle RY, Maris JM - Mol. Cancer (2009)

Bottom Line: Finally, treatment of cells with a proteasome inhibitor resulted in down regulation of CXCR4 surface expression.Taken together, these data show that regulation of CXCR4 surface expression in neuroblastoma cells can occur independently of SDF-1 contribution arguing against an autocrine mechanism.Additionally these data suggest that post-translational modifications of CXCR4, in part through direct ubiquitination, can influence trafficking of CXCR4 to the surface of neuroblastoma cells in a ligand-independent manner.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Oncology, Abramson Research Center, Children's Hospital of Philadelphia, ARC-907A, 3615 Civic Center Blvd, Philadelphia, Pennsylvania 19104-4399, USA. carlisle@email.chop.edu

ABSTRACT

Background: CXCR4, the receptor for the chemokine stromal-derived factor 1 (SDF-1), has been shown to mediate many of the processes essential for cancer progression such as tumor cell proliferation, metastasis, and angiogenesis. To understand the role of CXCR4 in the biology of neuroblastoma, a disease that presents with wide spread metastases in over 50% of patients, we screened ten patient derived-neuroblastoma cell-lines for basal CXCR4 expression and sought to identify characteristics that correlate with tumor cell phenotype.

Results: All cell lines expressed CXCR4 mRNA at variable levels, that correlated well with three distinct classes of CXCR4 surface expression (low, moderate, or high) as defined by flow cytometry. Analysis of the kinetics of CXCR4 surface expression on moderate and high expressing cell lines showed a time-dependent down-regulation of the receptor that directly correlated with cell confluency, and was independent of SDF1. Cell lysates showed the presence of multiple CXCR4 isoforms with three major species of approximately 87, 67 and 55 kDa associating with high surface expression, and two distinct species of 45 and 38 kDa correlating with low to surface expression. Western blot analysis of CXCR4 immunoprecipitates showed that the 87 and 67 kDa forms were ubiquitinated, while the others were not. Finally, treatment of cells with a proteasome inhibitor resulted in down regulation of CXCR4 surface expression.

Conclusions: Taken together, these data show that regulation of CXCR4 surface expression in neuroblastoma cells can occur independently of SDF-1 contribution arguing against an autocrine mechanism. Additionally these data suggest that post-translational modifications of CXCR4, in part through direct ubiquitination, can influence trafficking of CXCR4 to the surface of neuroblastoma cells in a ligand-independent manner.

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Effect of Cell Density on CXCR4 Surface Expression. Individual flasks of culture media were each synchronously seeded with 5 × 106 SK-N-SH cells and grown at 37°C to varying confluency. At each confluency, cells were harvested and processed for cell surface staining of CXCR4. The maximum CXCR4 signal was observed at 50% confluency and set as 100% mean fluorescent intensity. All intensities are represented as % relative values normalized to the maximum intensity. Result represents the mean from three experiments and shows the correlation between cell density and surface expression (Pearson correlation, R2 = 0.9488, P < 0.05).
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Figure 3: Effect of Cell Density on CXCR4 Surface Expression. Individual flasks of culture media were each synchronously seeded with 5 × 106 SK-N-SH cells and grown at 37°C to varying confluency. At each confluency, cells were harvested and processed for cell surface staining of CXCR4. The maximum CXCR4 signal was observed at 50% confluency and set as 100% mean fluorescent intensity. All intensities are represented as % relative values normalized to the maximum intensity. Result represents the mean from three experiments and shows the correlation between cell density and surface expression (Pearson correlation, R2 = 0.9488, P < 0.05).

Mentions: Previous reports have shown that CXCR4 is down-regulated at the neuroblastoma cell surface in response to exogenously added SDF-1 leading to the hypothesis that CXCR4 surface expression is regulated by a negative autocrine feed back loop mediated by an accumulation of SDF-1 secreted from neuroblastoma cells [18]. In an effort to assess the possible effects of endogenously derived SDF-1 accumulated during cell culture on CXCR4 surface expression, cells at different degrees of confluency were measured for CXCR4 at the cell surface using flow cytometry. As shown in fig. 3, increasing confluency resulted in a progressive decrease in CXCR4 at the surface of cells in the higher surface expressing classes, demonstrating a significant correlation between cell density and surface expression of the receptor (Pearson correlation analysis, R2 = 0.9488; P < 0.05); this observation was consistent for all of the moderate to high CXCR4 surface expressing cell lines tested. SH-SY5Y, a sub-clone of SK-N-SH and a low surface expressing cell line showed no difference in CXCR4 surface expression at any confluency; this observation was consistent for all cells in the low CXCR4 surface expressing class. Maximal CXCR4 surface expression was observed for cell cultures less than 50% confluent (data not shown), and upon 100% confluency CXCR4 surface expression was reduced to half maximal levels.


CXCR4 expression heterogeneity in neuroblastoma cells due to ligand-independent regulation.

Carlisle AJ, Lyttle CA, Carlisle RY, Maris JM - Mol. Cancer (2009)

Effect of Cell Density on CXCR4 Surface Expression. Individual flasks of culture media were each synchronously seeded with 5 × 106 SK-N-SH cells and grown at 37°C to varying confluency. At each confluency, cells were harvested and processed for cell surface staining of CXCR4. The maximum CXCR4 signal was observed at 50% confluency and set as 100% mean fluorescent intensity. All intensities are represented as % relative values normalized to the maximum intensity. Result represents the mean from three experiments and shows the correlation between cell density and surface expression (Pearson correlation, R2 = 0.9488, P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2807429&req=5

Figure 3: Effect of Cell Density on CXCR4 Surface Expression. Individual flasks of culture media were each synchronously seeded with 5 × 106 SK-N-SH cells and grown at 37°C to varying confluency. At each confluency, cells were harvested and processed for cell surface staining of CXCR4. The maximum CXCR4 signal was observed at 50% confluency and set as 100% mean fluorescent intensity. All intensities are represented as % relative values normalized to the maximum intensity. Result represents the mean from three experiments and shows the correlation between cell density and surface expression (Pearson correlation, R2 = 0.9488, P < 0.05).
Mentions: Previous reports have shown that CXCR4 is down-regulated at the neuroblastoma cell surface in response to exogenously added SDF-1 leading to the hypothesis that CXCR4 surface expression is regulated by a negative autocrine feed back loop mediated by an accumulation of SDF-1 secreted from neuroblastoma cells [18]. In an effort to assess the possible effects of endogenously derived SDF-1 accumulated during cell culture on CXCR4 surface expression, cells at different degrees of confluency were measured for CXCR4 at the cell surface using flow cytometry. As shown in fig. 3, increasing confluency resulted in a progressive decrease in CXCR4 at the surface of cells in the higher surface expressing classes, demonstrating a significant correlation between cell density and surface expression of the receptor (Pearson correlation analysis, R2 = 0.9488; P < 0.05); this observation was consistent for all of the moderate to high CXCR4 surface expressing cell lines tested. SH-SY5Y, a sub-clone of SK-N-SH and a low surface expressing cell line showed no difference in CXCR4 surface expression at any confluency; this observation was consistent for all cells in the low CXCR4 surface expressing class. Maximal CXCR4 surface expression was observed for cell cultures less than 50% confluent (data not shown), and upon 100% confluency CXCR4 surface expression was reduced to half maximal levels.

Bottom Line: Finally, treatment of cells with a proteasome inhibitor resulted in down regulation of CXCR4 surface expression.Taken together, these data show that regulation of CXCR4 surface expression in neuroblastoma cells can occur independently of SDF-1 contribution arguing against an autocrine mechanism.Additionally these data suggest that post-translational modifications of CXCR4, in part through direct ubiquitination, can influence trafficking of CXCR4 to the surface of neuroblastoma cells in a ligand-independent manner.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Oncology, Abramson Research Center, Children's Hospital of Philadelphia, ARC-907A, 3615 Civic Center Blvd, Philadelphia, Pennsylvania 19104-4399, USA. carlisle@email.chop.edu

ABSTRACT

Background: CXCR4, the receptor for the chemokine stromal-derived factor 1 (SDF-1), has been shown to mediate many of the processes essential for cancer progression such as tumor cell proliferation, metastasis, and angiogenesis. To understand the role of CXCR4 in the biology of neuroblastoma, a disease that presents with wide spread metastases in over 50% of patients, we screened ten patient derived-neuroblastoma cell-lines for basal CXCR4 expression and sought to identify characteristics that correlate with tumor cell phenotype.

Results: All cell lines expressed CXCR4 mRNA at variable levels, that correlated well with three distinct classes of CXCR4 surface expression (low, moderate, or high) as defined by flow cytometry. Analysis of the kinetics of CXCR4 surface expression on moderate and high expressing cell lines showed a time-dependent down-regulation of the receptor that directly correlated with cell confluency, and was independent of SDF1. Cell lysates showed the presence of multiple CXCR4 isoforms with three major species of approximately 87, 67 and 55 kDa associating with high surface expression, and two distinct species of 45 and 38 kDa correlating with low to surface expression. Western blot analysis of CXCR4 immunoprecipitates showed that the 87 and 67 kDa forms were ubiquitinated, while the others were not. Finally, treatment of cells with a proteasome inhibitor resulted in down regulation of CXCR4 surface expression.

Conclusions: Taken together, these data show that regulation of CXCR4 surface expression in neuroblastoma cells can occur independently of SDF-1 contribution arguing against an autocrine mechanism. Additionally these data suggest that post-translational modifications of CXCR4, in part through direct ubiquitination, can influence trafficking of CXCR4 to the surface of neuroblastoma cells in a ligand-independent manner.

Show MeSH
Related in: MedlinePlus