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Downregulation of FIP200 induces apoptosis of glioblastoma cells and microvascular endothelial cells by enhancing Pyk2 activity.

Wang D, Olman MA, Stewart J, Tipps R, Huang P, Sanders PW, Toline E, Prayson RA, Lee J, Weil RJ, Palmer CA, Gillespie GY, Liu WM, Pieper RO, Guan JL, Gladson CL - PLoS ONE (2011)

Bottom Line: In the human glioblastoma tumor cells, immortalized human astrocytes, and primary human brain MvEC, we found that downregulation of FIP200 increased the activity of Pyk2 without increasing its expression, but did not affect the activity or expression of FAK.Moreover, the pro-apoptotic effect of FIP200 downregulation was inhibited significantly by a TAT-Pyk2-fusion protein containing the Pyk2 autophosphorylation site in these cells.In summary, downregulation of endogenous FIP200 protein in glioblastoma tumor cells, astrocytes, and brain MvECs promotes apoptosis, most likely due to the removal of a direct interaction of FIP200 with Pyk2 that inhibits Pyk2 activation, suggesting that FIP200 expression may be required for the survival of all three cell types found in glioblastoma tumors.

View Article: PubMed Central - PubMed

Affiliation: Division of Neuropathology, Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.

ABSTRACT
The expression of focal adhesion kinase family interacting protein of 200-kDa (FIP200) in normal brain is limited to some neurons and glial cells. On immunohistochemical analysis of biopsies of glioblastoma tumors, we detected FIP200 in the tumor cells, tumor-associated endothelial cells, and occasional glial cells. Human glioblastoma tumor cell lines and immortalized human astrocytes cultured in complete media also expressed FIP200 as did primary human brain microvessel endothelial cells (MvEC), which proliferate in culture and resemble reactive endothelial cells. Downregulation of endogenous expression of FIP200 using small interfering RNA resulted in induction of apoptosis in the human glioblastoma tumor cells, immortalized human astrocytes, and primary human brain MvEC. It has been shown by other investigators using cells from other tissues that FIP200 can interact directly with, and inhibit, proline-rich tyrosine kinase 2 (Pyk2) and focal adhesion kinase (FAK). In the human glioblastoma tumor cells, immortalized human astrocytes, and primary human brain MvEC, we found that downregulation of FIP200 increased the activity of Pyk2 without increasing its expression, but did not affect the activity or expression of FAK. Coimmunoprecipitation and colocalization studies indicated that the endogenous FIP200 was largely associated with Pyk2, rather than FAK, in the glioblastoma tumor cells and brain MvEC. Moreover, the pro-apoptotic effect of FIP200 downregulation was inhibited significantly by a TAT-Pyk2-fusion protein containing the Pyk2 autophosphorylation site in these cells. In summary, downregulation of endogenous FIP200 protein in glioblastoma tumor cells, astrocytes, and brain MvECs promotes apoptosis, most likely due to the removal of a direct interaction of FIP200 with Pyk2 that inhibits Pyk2 activation, suggesting that FIP200 expression may be required for the survival of all three cell types found in glioblastoma tumors.

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Co-Expression of FIP200 and Pyk2 in the cytoplasm of tumor cells and of tumor-associated endothelial cells.Frozen sections of four glioblastoma tumors were reacted with antibodies directed toward vWf (pink), FIP200 (green) and Pyk2 (red), followed by reaction with Alexa 633-, Alexa 563- and Alexa 488-conjugated secondary antibodies, and DAPI staining, as described in the Materials and Methods. Images were viewed and photographed on a Leica DMB microscope at 400× magnification. A1 & A2, triple-labeled tumor-associated endothelial cells; B, double-labeled tumor cells; and C, secondary antibodies alone followed by DAPI staining. Merged images are shown at the reader's far right.
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pone-0019629-g008: Co-Expression of FIP200 and Pyk2 in the cytoplasm of tumor cells and of tumor-associated endothelial cells.Frozen sections of four glioblastoma tumors were reacted with antibodies directed toward vWf (pink), FIP200 (green) and Pyk2 (red), followed by reaction with Alexa 633-, Alexa 563- and Alexa 488-conjugated secondary antibodies, and DAPI staining, as described in the Materials and Methods. Images were viewed and photographed on a Leica DMB microscope at 400× magnification. A1 & A2, triple-labeled tumor-associated endothelial cells; B, double-labeled tumor cells; and C, secondary antibodies alone followed by DAPI staining. Merged images are shown at the reader's far right.

Mentions: Analysis of the cellular localization of these proteins in four frozen glioblastoma biopsy samples by triple-label immunofluorescence indicated cytoplasmic co-expression of FIP200 and Pyk2 in endothelial cells in tumor-associated blood vessels identified by vWf expression (Figure 8A1&2), suggesting that FIP200 and Pyk2 are localized in a manner that is consistent with their potential association. In the tumor cell compartment of all samples, we found cytoplasmic co-expression of FIP200 and Pyk2 in some tumor cells (Figure 8B), also suggesting that FIP200 and Pyk2 are localized in a manner that is consistent with potential association. Nuclear localization of FIP200 also was observed in some tumor cells (data not shown). The cytoplasmic localization of Pyk2 in the tumor-associated endothelial cells and in the tumor cells in frozen glioblastoma biopsies is consistent with the report of another group that examined Pyk2 expression in glioblastoma biopsies [36].


Downregulation of FIP200 induces apoptosis of glioblastoma cells and microvascular endothelial cells by enhancing Pyk2 activity.

Wang D, Olman MA, Stewart J, Tipps R, Huang P, Sanders PW, Toline E, Prayson RA, Lee J, Weil RJ, Palmer CA, Gillespie GY, Liu WM, Pieper RO, Guan JL, Gladson CL - PLoS ONE (2011)

Co-Expression of FIP200 and Pyk2 in the cytoplasm of tumor cells and of tumor-associated endothelial cells.Frozen sections of four glioblastoma tumors were reacted with antibodies directed toward vWf (pink), FIP200 (green) and Pyk2 (red), followed by reaction with Alexa 633-, Alexa 563- and Alexa 488-conjugated secondary antibodies, and DAPI staining, as described in the Materials and Methods. Images were viewed and photographed on a Leica DMB microscope at 400× magnification. A1 & A2, triple-labeled tumor-associated endothelial cells; B, double-labeled tumor cells; and C, secondary antibodies alone followed by DAPI staining. Merged images are shown at the reader's far right.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3094350&req=5

pone-0019629-g008: Co-Expression of FIP200 and Pyk2 in the cytoplasm of tumor cells and of tumor-associated endothelial cells.Frozen sections of four glioblastoma tumors were reacted with antibodies directed toward vWf (pink), FIP200 (green) and Pyk2 (red), followed by reaction with Alexa 633-, Alexa 563- and Alexa 488-conjugated secondary antibodies, and DAPI staining, as described in the Materials and Methods. Images were viewed and photographed on a Leica DMB microscope at 400× magnification. A1 & A2, triple-labeled tumor-associated endothelial cells; B, double-labeled tumor cells; and C, secondary antibodies alone followed by DAPI staining. Merged images are shown at the reader's far right.
Mentions: Analysis of the cellular localization of these proteins in four frozen glioblastoma biopsy samples by triple-label immunofluorescence indicated cytoplasmic co-expression of FIP200 and Pyk2 in endothelial cells in tumor-associated blood vessels identified by vWf expression (Figure 8A1&2), suggesting that FIP200 and Pyk2 are localized in a manner that is consistent with their potential association. In the tumor cell compartment of all samples, we found cytoplasmic co-expression of FIP200 and Pyk2 in some tumor cells (Figure 8B), also suggesting that FIP200 and Pyk2 are localized in a manner that is consistent with potential association. Nuclear localization of FIP200 also was observed in some tumor cells (data not shown). The cytoplasmic localization of Pyk2 in the tumor-associated endothelial cells and in the tumor cells in frozen glioblastoma biopsies is consistent with the report of another group that examined Pyk2 expression in glioblastoma biopsies [36].

Bottom Line: In the human glioblastoma tumor cells, immortalized human astrocytes, and primary human brain MvEC, we found that downregulation of FIP200 increased the activity of Pyk2 without increasing its expression, but did not affect the activity or expression of FAK.Moreover, the pro-apoptotic effect of FIP200 downregulation was inhibited significantly by a TAT-Pyk2-fusion protein containing the Pyk2 autophosphorylation site in these cells.In summary, downregulation of endogenous FIP200 protein in glioblastoma tumor cells, astrocytes, and brain MvECs promotes apoptosis, most likely due to the removal of a direct interaction of FIP200 with Pyk2 that inhibits Pyk2 activation, suggesting that FIP200 expression may be required for the survival of all three cell types found in glioblastoma tumors.

View Article: PubMed Central - PubMed

Affiliation: Division of Neuropathology, Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.

ABSTRACT
The expression of focal adhesion kinase family interacting protein of 200-kDa (FIP200) in normal brain is limited to some neurons and glial cells. On immunohistochemical analysis of biopsies of glioblastoma tumors, we detected FIP200 in the tumor cells, tumor-associated endothelial cells, and occasional glial cells. Human glioblastoma tumor cell lines and immortalized human astrocytes cultured in complete media also expressed FIP200 as did primary human brain microvessel endothelial cells (MvEC), which proliferate in culture and resemble reactive endothelial cells. Downregulation of endogenous expression of FIP200 using small interfering RNA resulted in induction of apoptosis in the human glioblastoma tumor cells, immortalized human astrocytes, and primary human brain MvEC. It has been shown by other investigators using cells from other tissues that FIP200 can interact directly with, and inhibit, proline-rich tyrosine kinase 2 (Pyk2) and focal adhesion kinase (FAK). In the human glioblastoma tumor cells, immortalized human astrocytes, and primary human brain MvEC, we found that downregulation of FIP200 increased the activity of Pyk2 without increasing its expression, but did not affect the activity or expression of FAK. Coimmunoprecipitation and colocalization studies indicated that the endogenous FIP200 was largely associated with Pyk2, rather than FAK, in the glioblastoma tumor cells and brain MvEC. Moreover, the pro-apoptotic effect of FIP200 downregulation was inhibited significantly by a TAT-Pyk2-fusion protein containing the Pyk2 autophosphorylation site in these cells. In summary, downregulation of endogenous FIP200 protein in glioblastoma tumor cells, astrocytes, and brain MvECs promotes apoptosis, most likely due to the removal of a direct interaction of FIP200 with Pyk2 that inhibits Pyk2 activation, suggesting that FIP200 expression may be required for the survival of all three cell types found in glioblastoma tumors.

Show MeSH
Related in: MedlinePlus