J Robert Grammer

Publications of J Robert Grammer

• ABT-510, a modified type 1 repeat peptide of thrombospondin, inhibits malignant glioma growth in vivo by inhibiting angiogenesis.

  Authors: Joshua C Anderson, J Robert Grammer, Wenquan Wang, L Burton Nabors, Jack Henkin, Jerry E Stewart, Candece L Gladson

  Cancer biology & therapy. 04/2007; 6(3):454-62.

  Anti-angiogenic therapies would be particularly beneficial in the treatment of malignant gliomas. Peptides derived from the second type 1 repeat (TSR) of thrombospondin-1 (TSP-1) have been shown toAnti-angiogenic therapies would be particularly beneficial in the treatment of malignant gliomas. Peptides derived from the second type 1 repeat (TSR) of thrombospondin-1 (TSP-1) have been shown to inhibit angiogenesis in non-glioma tumor models and a modified TSR peptide, ABT-510, has now entered into Phase II clinical trials of its efficacy in non-glioma tumors. As microvascular endothelial cells (MvEC) exhibit heterogeneity, we evaluated the ability of the modified TSR peptide (NAcSarGlyValDallolleThrNvalleArgProNHE, ABT-510) to inhibit malignant glioma growth in vivo and to induce apoptosis of brain microvessel endothelial cells (MvEC) propagated in vitro. We found that daily administration of ABT-510 until euthanasia (days 7 to 19), significantly inhibited the growth of human malignant astrocytoma tumors established in the brain of athymic nude mice. The microvessel density was significantly lower and the number of apoptotic MvEC was significantly higher (3-fold) in the tumors of the ABT-510-treated animals. Similar results were found using a model in which the established tumor is an intracerebral malignant glioma propagated in a syngeneic mouse model. ABT-510 treatment of primary human brain MvEC propagated as a monolayer resulted in induction of apoptosis in a dose- and time-dependent manner through a caspase-8-dependent mechanism. It also inhibited tubular morphogenesis of MvEC propagated in collagen gels in a dose- and caspase-8 dependent manner through a mechanism that requires the TSP-1 receptor (CD36) on the MvEC. These findings indicate that ABT-510 should be evaluated as a therapeutic option for patients with malignant glioma.

• A novel technique to quantify glioma tumor invasion using serial microscopy sections.

  Authors: N Shastry Akella, Qiang Ding, Ingrid Menegazzo, Wenquan Wang, G Yancey Gillespie, J Robert Grammer, Candece L Gladson, L Burton Nabors

  Journal of neuroscience methods. 07/2006; 153(2):183-9.

  Here we present a new technique to quantitatively characterize malignant glioma invasion in a syngeneic mouse model. The GL261 mouse malignant glioma cell line was injected intracerebrally into theHere we present a new technique to quantitatively characterize malignant glioma invasion in a syngeneic mouse model. The GL261 mouse malignant glioma cell line was injected intracerebrally into the C57B1/6 black mouse and allowed to propagate for 10 or 17 days, followed by euthanasia of the animal, harvesting of the brain, fixation, and serial sectioning. Histologic examination was performed and the primary tumor mass and discontinuous sites of tumor invasion were traced on digital images of serial microscopy sections, followed by analysis of the invasion characteristics using a custom-written MATLAB program. We found a significant increase in the number of discontinuous tumor invasion sites and in the distance of these sites from the tumor centroid in mice that were euthanized at 17 days post-tumor cell injection, as compared to mice euthanized at 10 days. Furthermore, a scatter plot analyses indicated that the invasion site data could be grouped based on the characteristics of area and distance from the tumor centroid to reveal significant differences between the two experimental groups of mice. This quantitative method will allow a future in vivo analysis of invasion characteristics in glioma cells expressing altered levels or function of invasion genes, and of new therapy targeting invading glioma cells.

• Low-density lipoprotein receptor-related protein contributes to the antiangiogenic activity of thrombospondin-2 in a murine glioma model.

  Authors: Constance Y Fears, J Robert Grammer, Jerry E Stewart, Douglas S Annis, Deane F Mosher, Paul Bornstein, Candece L Gladson

  Cancer research. 11/2005; 65(20):9338-46.

  Host antiangiogenesis factors defend against tumor growth. The matricellular protein, thrombospondin-2 (TSP-2), has been shown to act as an antiangiogenesis factor in a carcinogen-induced model ofHost antiangiogenesis factors defend against tumor growth. The matricellular protein, thrombospondin-2 (TSP-2), has been shown to act as an antiangiogenesis factor in a carcinogen-induced model of skin cancer. Here, using an in vivo malignant glioma model in which the characteristics of the tumors formed after intracerebral implantation of GL261 mouse glioma cells are assessed, we found that tumor growth and microvessel density were significantly enhanced in tumors propagated in TSP-2(-/-) mice. Mechanistically, matrix metalloproteinase (MMP)-2 has been associated with neoangiogenesis and it has been proposed that the levels of available MMP-2 may be down-regulated by formation of a complex with TSP-2 that is internalized by low-density lipoprotein receptor-related protein 1 (LRP1). We found elevated expression of MMP-2 and MMP-9 in tumors propagated in TSP-2(-/-) mice, with a preferential localization in the microvasculature. In wild-type mice, MMP-2 was coexpressed with TSP-2 in the tumor microvasculature. In vitro, addition of recombinant (rec) TSP-2 to mouse brain microvessel endothelial cells reduced MMP-2 levels and invasion through mechanisms that could be inhibited by a competitive inhibitor of ligand binding to LRP1 or by siLRP1. Thus, the antiangiogenic activity of TSP-2 is capable of inhibiting the growth of gliomas in part by reducing the levels of MMP-2 in the tumor microvasculature. This mechanism is mediated by LRP1.

• Lyn kinase activity is the predominant cellular SRC kinase activity in glioblastoma tumor cells.

  Authors: Michelle R Stettner, Wenquan Wang, L Burton Nabors, Suman Bharara, Daniel C Flynn, J Robert Grammer, G Yancey Gillespie, Candece L Gladson

  Cancer research. 07/2005; 65(13):5535-43.

  Cellular Src activity modulates cell migration, proliferation, and differentiation, and recent reports suggest that individual members of the Src family may play specific roles in these processes. AsCellular Src activity modulates cell migration, proliferation, and differentiation, and recent reports suggest that individual members of the Src family may play specific roles in these processes. As we have found that Lyn, but not Fyn, activity promotes migration of glioblastoma cells in response to the cooperative signal generated by platelet-derived growth factor receptor beta and integrin alpha(v)beta3, we compared the activity and expression of Lyn and Fyn in glioblastoma (grade IV) tumor biopsy samples with that in anaplastic astrocytoma (grade III) tumors, nonneoplastic brain, and normal autopsy brain samples. Lyn kinase activity was significantly elevated in glioblastoma tumor samples. Notably, the Lyn kinase activity accounted for >90% of pan-Src kinase activity in glioblastoma samples but only approximately 30% of pan-Src kinase activity in the other groups. The levels of phosphorylation of the autophosphorylation site were consistent with significantly higher Lyn activity in glioblastoma tumor tissue than nonneoplastic brain. Although the normalized levels of Lyn protein and the relative levels of Lyn message were significantly higher in glioblastoma samples than nonneoplastic brain, the normalized levels of Lyn protein did not correlate with Lyn activity in the glioblastoma samples. There was no significant difference in the normalized levels of c-Src and Fyn protein and message in the glioblastoma and nonneoplastic brain. Immunostaining revealed that Lyn is located primarily in the glioblastoma cells in the tumor biopsies. These data indicate that Lyn kinase activity is significantly elevated in glioblastoma tumors and suggest that it is the Lyn activity that promotes the malignant phenotype in these tumors.

• p27Kip1 and cyclin D1 are necessary for focal adhesion kinase regulation of cell cycle progression in glioblastoma cells propagated in vitro and in vivo in the scid mouse brain.

  Authors: Qiang Ding, J Robert Grammer, Mark A Nelson, Jun-Lin Guan, Jerry E Stewart, Candece L Gladson

  The Journal of biological chemistry. 03/2005; 280(8):6802-15.

  We have reported previously that the expression of focal adhesion kinase (FAK) is elevated in glioblastomas and that expression of FAK promotes the proliferation of glioblastoma cells propagated inWe have reported previously that the expression of focal adhesion kinase (FAK) is elevated in glioblastomas and that expression of FAK promotes the proliferation of glioblastoma cells propagated in either soft agar or in the C.B.17 severe combined immunodeficiency (scid) mouse brain. We therefore determined the effect of FAK on cell cycle progression in these cells. We found that overexpression of wild-type FAK promoted exit from G(1) in monolayer cultures of glioblastoma cells, enhanced the expression of cyclins D1 and E while reducing the expression of p27(Kip1) and p21(Waf1), and enhanced the kinase activity of the cyclin D1-cyclin-dependent kinase-4 (cdk4) complex. Transfection of the monolayers with a FAK molecule in which the autophosphorylation site is mutated (FAK397F) inhibited exit from G(1) and reduced the expression of cyclins D1 and E while enhancing the expression of p27(Kip1) and p21(Waf1). Small interfering RNA (siRNA)-mediated down-regulation of cyclin D1 inhibited the enhancement of cell cycle progression observed on expression of wild-type FAK, whereas siRNA-mediated down-regulation of cyclin E had no effect. siRNA-mediated down-regulation of p27(Kip1) overcame the inhibition of cell cycle progression observed on expression of FAK397F, whereas down-regulation of p21(Waf1) had no effect. These results were confirmed in vivo in the scid mouse brain xenograft model in which propagation of glioblastoma cells expressing FAK397F resulted in a 50% inhibition of tumor growth and inhibited exit from G(1). Taken together, our results indicate that FAK promotes proliferation of glioblastoma cells by enhancing exit from G(1) through a mechanism that involves cyclin D1 and p27(Kip1).

• Focal adhesion kinase is expressed in the angiogenic blood vessels of malignant astrocytic tumors in vivo and promotes capillary tube formation of brain microvascular endothelial cells.

  Authors: Henry Haskell, Meera Natarajan, Timothy P Hecker, Qiang Ding, Jerry Stewart, J Robert Grammer, Candece L Gladson

  Clinical cancer research : an official journal of the American Association for Cancer Research. 07/2003; 9(6):2157-65.

  PURPOSE: Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that has been shown to promote proliferation, migration, and invasion of several cell types in vitro, and we have shown recentlyPURPOSE: Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that has been shown to promote proliferation, migration, and invasion of several cell types in vitro, and we have shown recently that FAK promotes proliferation of malignant astrocytoma cells in vivo. To determine the role of FAK in angiogenesis in malignant astrocytic tumors, we investigated the expression and function of FAK in brain endothelial cells. EXPERIMENTAL DESIGN: We characterized the expression of FAK and activated FAK in endothelial cells by immunohistochemistry. We also determined the function of FAK in brain microvascular endothelial cells by transfecting these cells with a dominant interfering form of FAK [FAK-related nonkinase (FRNK)] or a mutant FRNK (Leu-1034 to Ser) and assessed the effect on capillary tube formation and cell migration. RESULTS: We found that FAK was expressed in the endothelial cells of grade III (4 of 9 samples) and IV (9 of 10 samples) astrocytoma biopsies but not in the endothelial cells of normal brain (0 of 9 samples) and not in grade I (0 of 5 samples) or II (0 of 4 samples) astrocytoma biopsies. Furthermore, we found that both FAK and activated FAK were expressed in the endothelial cells in malignant astrocytoma tumors propagated intracerebrally in the severe combined immunodeficient mouse brain. As expected, immunofluorescence analysis showed FRNK protein to localize to focal adhesions, whereas mutant FRNK protein did not. FRNK-transfected endothelial cells showed a 55% reduction in branched tube formation and a 40% reduction in tube length when propagated in three-dimensional collagen gels, compared with cells transfected with the mutant FRNK construct. Furthermore, FRNK-transfected cells showed a 35-50% reduction in haptotactic migration toward fibronectin and collagen, compared with mutant FRNK-transfected cells. CONCLUSIONS: These data suggest that FAK promotes angiogenesis and that this occurs, at least in part, through the promotion of endothelial cell migration.

• Promotion of malignant astrocytoma cell migration by osteopontin expressed in the normal brain: differences in integrin signaling during cell adhesion to osteopontin versus vitronectin.

  Authors: Qiang Ding, Jerry Stewart, Charles W Prince, Pi-Ling Chang, Mohit Trikha, Xiaosi Han, J Robert Grammer, Candece L Gladson

  Cancer research. 10/2002; 62(18):5336-43.

  The extracellular matrix of the normal adult brain lacks expression of most of the adhesive glycoproteins that are known to promote cell attachment, and it has been thought that the malignantThe extracellular matrix of the normal adult brain lacks expression of most of the adhesive glycoproteins that are known to promote cell attachment, and it has been thought that the malignant invasion of astrocytoma tumor is mediated primarily by remodeling of the matrix by the tumor cells. It has been reported, however, that normal brain neuropil does contain a protein(s) that promotes cell attachment. Therefore, we explored the possibility that the cell attachment protein, osteopontin, is expressed in the normal human brain. Here, we report that osteopontin is expressed in the cortical gray and white matter of normal adult brain, with the levels of osteopontin expression being equivalent to those in malignant astrocytic tumor biopsies as assessed by Western blot analysis. Immunoblotting identified osteopontin polypeptides with relative molecular weights of 60- and 65-kDa in normal brain white matter and in astrocytic tumors, with an additional 70-kDa polypeptide being identified in normal cortical gray matter and in some astrocytic tumors. Recombinant osteopontin was found to promote attachment of U-251MG human malignant astrocytoma cells in a process that was inhibited by anti-integrin monoclonal antibodies anti-alphavbeta3 (75%), anti-alphavbeta5 (80%), and anti-alpha5 (40%). On attachment, integrins alphavbeta5 and alphavbeta3 localized to focal adhesions, and there was an alteration in cell morphology with the formation of lamellae-like processes. The attachment was associated with activation of Rac in a slow and prolonged fashion and rapid activation of Rho. Similarly, integrins alphavbeta5 and alphavbeta3 localized to focal adhesions on attachment of the U-251MG cells to vitronectin, but on this substrate, the cells assumed a spread and flat morphology, and there was rapid activation of both Rac and Rho. Extracts of normal brain white matter were capable of promoting haptotactic migration, and this response was inhibitable by monoclonal antibodies anti-alphavbeta3 and anti-alpha5. Depletion of the osteopontin in these extracts abrogated the haptotactic response significantly (50%). These data indicate that the cell attachment protein, osteopontin, is expressed in the normal adult brain and that it has the potential to promote malignant astrocytoma cell invasion.

• Focal adhesion kinase enhances signaling through the Shc/extracellular signal-regulated kinase pathway in anaplastic astrocytoma tumor biopsy samples.

  Authors: Timothy P Hecker, J Robert Grammer, G Yancey Gillespie, Jerry Stewart, Candece L Gladson

  Cancer research. 06/2002; 62(9):2699-707.

  Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that on activation generates signals that can modulate crucial cell functions, including cell proliferation, migration, and survival. InFocal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that on activation generates signals that can modulate crucial cell functions, including cell proliferation, migration, and survival. In vitro, overexpression of FAK has been shown to promote cell proliferation by signaling through the Ras/mitogen-activated protein kinase cascade in several cell types. We have shown previously that overexpression of exogenous FAK lacking alternative splicing in malignant astrocytoma clones injected intracerebrally into SCID mouse brains promotes tumor cell proliferation. Here, we show that in anaplastic astrocytoma biopsy samples, FAK is expressed as an unspliced variant and migrates with a faster mobility similar to that observed in embryonic brain. Compared with nonneoplastic adult brain biopsies, the levels of FAK protein are elevated as are its levels of activation as assessed by autophosphorylation and overall tyrosine phosphorylation. The activity of Src kinase in these tumors is also elevated, as well as the activity of Src kinase associated with FAK; the latter may result in enhanced Src kinase phosphorylation of FAK. Phosphorylated Shc is associated with FAK in the anaplastic astrocytoma biopsy samples and in astrocytoma cells overexpressing FAK in vitro but not in nonneoplastic brain biopsy samples. Elevated extracellular signal-regulated kinase-2 activation and elevated expression of cyclins D and E are also found in anaplastic astrocytoma biopsy samples. These data provide evidence that the increased FAK activity in these tumors contributes to phosphorylation of Shc and likely to the promotion of Ras activity, extracellular signal-regulated kinase-2 activation, and cell proliferation in vivo.