TY - JOUR
T1 - Focal adhesions and associated proteins in medullary thyroid carcinoma cells
AU - Kim, Lawrence T.
AU - Fleming, Jason B.
AU - Lopez-Guzman, Christie
AU - Nwariaku, Fiemu
N1 - Funding Information:
This work was supported by a grant from the Department of Veterans Affairs.
PY - 2003/5/15
Y1 - 2003/5/15
N2 - Background. In medullary thyroid carcinoma (MTC), mutations in the RET protooncogene lead to oncogenic transformation. RET activation in other cell types has been shown to cause phosphorylation of the focal adhesion-associated proteins focal adhesion kinase (FAK), paxillin, and p130CAS. We hypothesized that adhesion-dependent signaling might be deranged in MTC cells. Methods. Indirect immunofluorescence was used to label β1 integrin, FAK, paxillin, and p130CAS. Rhodamine-labeled phalloidin was used to visualize actin microfilaments. Phosphorylated protein was detected by immunoprecipitation followed by Western blotting for phosphotyrosine. MTC cell invasiveness was quantified using a modified Boyden chamber assay. Results. Clustering of β1 integrin, FAK, paxillin, and p130CAS into focal adhesions were not detected in MTC cells under any conditions, although clustering was seen as expected in control HeLa cells. Despite this failure, FAK, paxillin and p130CAS were all found to be phosphorylated. Actin microfilaments were generally not seen although in a few cells, small, poorly formed microfilaments could be detected. MTC cells invaded poorly as compared with highly invasive cell lines. However a clear difference was noted between invasiveness on growth factor depleted Matrigel and regular Matrigel. Conclusions. In MTC cells, focal adhesions are not seen in response to interaction with extracellular matrix. Consistent with this failure, actin microfilaments are absent or poorly formed and invasion is weak. Despite the absence of focal adhesions, focal adhesion proteins remain phosphorylated, even though in normal cells their signaling activity is dependent on focal adhesion formation. Deranged adhesion-dependent signaling may contribute to MTC pathogenesis.
AB - Background. In medullary thyroid carcinoma (MTC), mutations in the RET protooncogene lead to oncogenic transformation. RET activation in other cell types has been shown to cause phosphorylation of the focal adhesion-associated proteins focal adhesion kinase (FAK), paxillin, and p130CAS. We hypothesized that adhesion-dependent signaling might be deranged in MTC cells. Methods. Indirect immunofluorescence was used to label β1 integrin, FAK, paxillin, and p130CAS. Rhodamine-labeled phalloidin was used to visualize actin microfilaments. Phosphorylated protein was detected by immunoprecipitation followed by Western blotting for phosphotyrosine. MTC cell invasiveness was quantified using a modified Boyden chamber assay. Results. Clustering of β1 integrin, FAK, paxillin, and p130CAS into focal adhesions were not detected in MTC cells under any conditions, although clustering was seen as expected in control HeLa cells. Despite this failure, FAK, paxillin and p130CAS were all found to be phosphorylated. Actin microfilaments were generally not seen although in a few cells, small, poorly formed microfilaments could be detected. MTC cells invaded poorly as compared with highly invasive cell lines. However a clear difference was noted between invasiveness on growth factor depleted Matrigel and regular Matrigel. Conclusions. In MTC cells, focal adhesions are not seen in response to interaction with extracellular matrix. Consistent with this failure, actin microfilaments are absent or poorly formed and invasion is weak. Despite the absence of focal adhesions, focal adhesion proteins remain phosphorylated, even though in normal cells their signaling activity is dependent on focal adhesion formation. Deranged adhesion-dependent signaling may contribute to MTC pathogenesis.
KW - Cell adhesion
KW - Focal adhesions
KW - Integrins
KW - Medullary thyroid cancer
KW - Protein tyrosine kinases
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M3 - Article
C2 - 12850460
AN - SCOPUS:0038016865
SN - 0022-4804
VL - 111
SP - 177
EP - 184
JO - Journal of Surgical Research
JF - Journal of Surgical Research
IS - 2
ER -