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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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Quantitative Analysis of SDF-1 Secretion by Neuroblastoma Cells. Conditioned media from several neuroblastoma cell lines was collected at maximum confluency. Conditioned media was concentrated from 12 ml to 0.5 ml by filter centrifugation using membranes with 3000 molecular weight cut-offs. Quantitative Analysis of SDF-1 was performed using an enzyme linked immunoadsorbent assay (ELISA). Result represents the mean of three experiments for representatives from each surface expressing class compared to media background levels (*P < 0.005 and **P > 0.05, unpaired t-test).Concentrations are expressed in pg/ml.
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Figure 5: Quantitative Analysis of SDF-1 Secretion by Neuroblastoma Cells. Conditioned media from several neuroblastoma cell lines was collected at maximum confluency. Conditioned media was concentrated from 12 ml to 0.5 ml by filter centrifugation using membranes with 3000 molecular weight cut-offs. Quantitative Analysis of SDF-1 was performed using an enzyme linked immunoadsorbent assay (ELISA). Result represents the mean of three experiments for representatives from each surface expressing class compared to media background levels (*P < 0.005 and **P > 0.05, unpaired t-test).Concentrations are expressed in pg/ml.

Mentions: Autocrine downregulation of CXCR4 by endogenously derived SDF-1 in the extracellular medium has previously been suggested to explain the low levels of CXCR4 seen on some neuroblastoma cell lines [18]. In an effort to test this theory and more accurately assess basal levels of CXCR4 surface expression an attempt was made to block release of any endogenous SDF-1 and prevent receptor down-regulation by treating cells with Brefeldin A, an inhibitor of vesicular protein secretion. As shown in fig. 4, Brefeldin A treatment did not result in a recovery of CXCR4 on the surface of the low expressing cell line SH-SY5Y, this observation was true for the other low expressing cell lines as well (data not shown). In addition, NGP and SK-N-SH, moderate and high surface expressing cell lines respectively, both showed significant decreases in surface levels as a consequence of Brefeldin A treatment (paired t test, P < 0.005); this observation was also made for the other moderate and high expressing cell lines from our panel (data not shown). In light of the absence of any upregulation of CXCR4 on the surface of Brefeldin A treated cells we decided to determine the presence and abundance of any endogenously derived SDF-1 in the extracellular environment of neuroblastoma cell lines used by us. Quantitative ELISA was performed on conditioned medium collected from cells at 100% confluency. As shown in fig. 5, SDF-1 levels observed in representatives from the high (SK-N-SH or LAN-5), or the low (SH-SY5Y) surface expressing classes were either at, or below background levels of the chemokine measured in culture media (8 pg/ml), and found not to be significant (P > 0.05, One-way ANOVA). The concentration of extracellular SDF-1 for the moderate surface expressing cell line NGP, calculated at 184 pg/ml, was significantly higher than background levels of SDF-1 (P > 0.005, One-way ANOVA); however this level was determined to be below levels used to activate CXCR4 and deemed physiologically irrelevant [18]. With the exception of NGP, SDF-1 levels were observed to be below background levels for all the other cell lines examined regardless of expression class (data not shown). In an attempt to clarify the status of endogenously expressed SDF-1 in neuroblastoma cells localization of the chemokine was assessed using indirect fluorescent immunostaining. SDF-1 was present in both the high surface expressing cell line (SK-N-SH) and its low surface expressing sub-clone (SH-SY5Y) (Figs. 6A and 6B), and appeared to localize predominately at the inner periphery of the plasma membrane. Staining in SK-N-SH cells appearing more focal and polarized with respect to CXCR4, while SH-SY5Y cells showed a more diffuse staining pattern; there was little to no co-localization of SDF-1 and CXCR4 in either cell line. The staining pattern for SK-N-SH was observed for all medium and high CXCR4 surface expressing cell lines in our panel while the pattern for SH-SY5Y was observed for all the low surface expressing lines (Data not shown). Transcriptional expression of SDF-1 was determined in all cell lines examined to confirm their ability to endogenously synthesis the chemokine (Data not shown).


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

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

Quantitative Analysis of SDF-1 Secretion by Neuroblastoma Cells. Conditioned media from several neuroblastoma cell lines was collected at maximum confluency. Conditioned media was concentrated from 12 ml to 0.5 ml by filter centrifugation using membranes with 3000 molecular weight cut-offs. Quantitative Analysis of SDF-1 was performed using an enzyme linked immunoadsorbent assay (ELISA). Result represents the mean of three experiments for representatives from each surface expressing class compared to media background levels (*P < 0.005 and **P > 0.05, unpaired t-test).Concentrations are expressed in pg/ml.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Quantitative Analysis of SDF-1 Secretion by Neuroblastoma Cells. Conditioned media from several neuroblastoma cell lines was collected at maximum confluency. Conditioned media was concentrated from 12 ml to 0.5 ml by filter centrifugation using membranes with 3000 molecular weight cut-offs. Quantitative Analysis of SDF-1 was performed using an enzyme linked immunoadsorbent assay (ELISA). Result represents the mean of three experiments for representatives from each surface expressing class compared to media background levels (*P < 0.005 and **P > 0.05, unpaired t-test).Concentrations are expressed in pg/ml.
Mentions: Autocrine downregulation of CXCR4 by endogenously derived SDF-1 in the extracellular medium has previously been suggested to explain the low levels of CXCR4 seen on some neuroblastoma cell lines [18]. In an effort to test this theory and more accurately assess basal levels of CXCR4 surface expression an attempt was made to block release of any endogenous SDF-1 and prevent receptor down-regulation by treating cells with Brefeldin A, an inhibitor of vesicular protein secretion. As shown in fig. 4, Brefeldin A treatment did not result in a recovery of CXCR4 on the surface of the low expressing cell line SH-SY5Y, this observation was true for the other low expressing cell lines as well (data not shown). In addition, NGP and SK-N-SH, moderate and high surface expressing cell lines respectively, both showed significant decreases in surface levels as a consequence of Brefeldin A treatment (paired t test, P < 0.005); this observation was also made for the other moderate and high expressing cell lines from our panel (data not shown). In light of the absence of any upregulation of CXCR4 on the surface of Brefeldin A treated cells we decided to determine the presence and abundance of any endogenously derived SDF-1 in the extracellular environment of neuroblastoma cell lines used by us. Quantitative ELISA was performed on conditioned medium collected from cells at 100% confluency. As shown in fig. 5, SDF-1 levels observed in representatives from the high (SK-N-SH or LAN-5), or the low (SH-SY5Y) surface expressing classes were either at, or below background levels of the chemokine measured in culture media (8 pg/ml), and found not to be significant (P > 0.05, One-way ANOVA). The concentration of extracellular SDF-1 for the moderate surface expressing cell line NGP, calculated at 184 pg/ml, was significantly higher than background levels of SDF-1 (P > 0.005, One-way ANOVA); however this level was determined to be below levels used to activate CXCR4 and deemed physiologically irrelevant [18]. With the exception of NGP, SDF-1 levels were observed to be below background levels for all the other cell lines examined regardless of expression class (data not shown). In an attempt to clarify the status of endogenously expressed SDF-1 in neuroblastoma cells localization of the chemokine was assessed using indirect fluorescent immunostaining. SDF-1 was present in both the high surface expressing cell line (SK-N-SH) and its low surface expressing sub-clone (SH-SY5Y) (Figs. 6A and 6B), and appeared to localize predominately at the inner periphery of the plasma membrane. Staining in SK-N-SH cells appearing more focal and polarized with respect to CXCR4, while SH-SY5Y cells showed a more diffuse staining pattern; there was little to no co-localization of SDF-1 and CXCR4 in either cell line. The staining pattern for SK-N-SH was observed for all medium and high CXCR4 surface expressing cell lines in our panel while the pattern for SH-SY5Y was observed for all the low surface expressing lines (Data not shown). Transcriptional expression of SDF-1 was determined in all cell lines examined to confirm their ability to endogenously synthesis the chemokine (Data not shown).

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