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Donald P Bottaro

Publications and source records attributed to Donald P Bottaro.

18 recordsLinked to original sources
PubMed2006

Beta-catenin signaling: linking renal cell carcinoma and polycystic kidney disease.

Loss of von Hippel-Lindau (VHL) tumor suppressor gene function occurs in familial and most sporadic renal cell carcinoma (RCC), resulting in the aberrant expression of genes that control cell proliferation, invasion and angiogenesis. The molecular mechanisms by which VHL loss leads to tumorigenesis are not yet fully defined. The VHL gene product, pVHL, is part of an E3 ubiquitin ligase complex that targets hypoxia inducible factors for polyubiquitination and proteosomal degradation, implicating hypoxia response genes in RCC oncogenesis. VHL loss also allows robust RCC cell invasiveness and morphogenesis in response to hepatocyte growth factor (HGF), an important regulator of kidney development and renal homeostasis. Recent elucidation of the mechanism by which pVHL represses developmental HGF responses in adult kidney has identified another oncogenically relevant E3 ligase target: beta-catenin. This discovery also further unifies recent insights into the molecular pathogenesis of polycystic kidney disease, where the identification of disease genes has revealed the integration of signaling pathways associated with primary cilia function and the regulation of cell growth and differentiation.

Animals↗
PubMed2006

Utilization of achiral alkenyl amines for the preparation of high affinity Grb2 SH2 domain-binding macrocycles by ring-closing metathesis.

A family of previously reported ring-closing metathesis (RCM)-derived macrocycles that exhibit potent Grb2 SH2 domain-binding affinity is characterized by stereoselectively-introduced upper ring junctions that bear bicyclic aryl substituents. However, the synthetic complexity of these macrocycles presents a potential limit to their therapeutic application. Therefore, the current study was undertaken to simplify these macrocycles through the use of achiral 4-pentenylamides as ring-forming components. A series of macrocycles (5a-f) was prepared bearing both open and cyclic constructs at the upper ring junction. The Grb2 SH2 domain-binding affinities of these macrocycles varied, with higher affinities being obtained with cyclo-substituents. The most potent analogue (5d) contained a cyclohexyl group and exhibited Grb2 SH2 domain-binding affinity (K(D) = 1.3 nM) that was nearly equal to the parent macrocycle (2), which bore a stereoselectively-introduced naphthylmethyl substituent at the upper ring junction (K(D) = 0.9 nM). The results of this study advance design considerations that should facilitate the development of Grb2 SH2 domain-binding antagonists.

Amides↗
PubMed2006

The von Hippel-Lindau tumor suppressor gene product represses oncogenic beta-catenin signaling in renal carcinoma cells.

Loss of von Hippel-Lindau (VHL) tumor suppressor gene function occurs in familial and most sporadic clear cell renal cell carcinoma (RCC), resulting in the aberrant expression of genes that control cell proliferation, invasion, and angiogenesis. The molecular mechanisms by which VHL loss leads to tumorigenesis are not yet fully defined. VHL loss has been shown to allow robust RCC cell motility, invasiveness, and morphogenesis in response to hepatocyte growth factor (HGF) stimulation, processes that are known to contribute to tumor invasiveness and metastatic potential. Among the most likely intracellular mediators of these HGF-driven activities is beta-catenin, a structural link between cadherens and the actin cytoskeleton, as well as a gene transactivator. We show that reconstitution of VHL expression in RCC cells repressed HGF-stimulated beta-catenin tyrosyl phosphorylation, adherens junction disruption, cytoplasmic beta-catenin accumulation, and reporter gene transactivation in RCC cells. Ectopic expression of a ubiquitination-resistant beta-catenin mutant specifically restored HGF-stimulated invasion and morphogenesis in VHL-transfected RCC cells. VHL gene silencing in non-RCC renal epithelial cells phenotypically mimicked VHL loss in RCC, and HGF-driven invasiveness was blocked by the expression of a dominant-negative mutant of Tcf. We conclude that, unlike many other cancers, where HGF pathway activation contributes to malignancy through the acquisition of autocrine signaling, receptor overexpression, or mutation, in RCC cells VHL loss enables HGF-driven oncogenic beta-catenin signaling. These findings identify beta-catenin as a potential target in biomarker and drug development for RCC.

Carcinoma, Renal Cell↗
PubMed2006

c-Met ectodomain shedding rate correlates with malignant potential.

PURPOSE: Many proteins are proteolytically released from the cell surface by a process known as ectodomain shedding. Shedding occurs under normal physiologic conditions and can be increased in certain pathologies. Among the many receptors for which ectodomain shedding has been shown is c-Met, the hepatocyte growth factor (HGF) receptor tyrosine kinase. HGF stimulates mitogenesis, motogenesis, and morphogenesis in a variety of cellular targets during development, homeostasis, and tissue regeneration. Inappropriate HGF signaling resulting in unregulated cell proliferation, motility, and invasion occurs in several human malignancies. This can occur through paracrine signaling, autocrine loop formation, receptor mutation, gene amplification, or gene rearrangement, accompanied frequently with overexpression of ligand and/or receptor proteins. We hypothesized that c-Met overexpression in cancer might result in increased ectodomain shedding, and that its measure could be a useful biomarker of tumor progression. EXPERIMENTAL DESIGN: We developed a sensitive electrochemiluminescent immunoassay to quantitate c-Met protein in cell lysates, culture supernatants, and biological samples. RESULTS: A survey of cultured cell models of oncogenic transformation revealed significant direct correlations (P < 0.001, t test or ANOVA) between malignant potential and the rate of c-Met ectodomain shedding that was independent of steady-state receptor expression level. Moreover, weekly plasma and urine samples from mice harboring s.c. human tumor xenografts (n = 4 per group) displayed soluble human c-Met levels that were measurable before tumors became palpable and that correlated directly with tumor volume (R2 > 0.92, linear regression). CONCLUSIONS: For a variety of human cancers, c-Met ectodomain shedding may provide a reliable and practical indicator of malignant potential and overall tumor burden.

Animals↗
PubMed2006

Targeting the c-Met signaling pathway in cancer.

On binding to the cell surface receptor tyrosine kinase (TK) known as c-Met, hepatocyte growth factor (HGF) stimulates mitogenesis, motogenesis, and morphogenesis in a wide range of cellular targets including, epithelial and endothelial cells, hematopoietic cells, neurons, melanocytes, and hepatocytes. These pleiotropic actions are fundamentally important during development, homeostasis, and tissue regeneration. HGF signaling also contributes to oncogenesis and tumor progression in several human cancers and promotes aggressive cellular invasiveness that is strongly linked to tumor metastasis. Our present understanding of c-Met oncogenic signaling supports at least three avenues of pathway selective anticancer drug development: antagonism of ligand/receptor interaction, inhibition of TK catalytic activity, and blockade of intracellular receptor/effector interactions. Potent and selective preclinical drug candidates have been developed using all three strategies, and human clinical trials in two of the three areas are now under way.

Antineoplastic Agents↗
PubMed2006

Molecular targeting of growth factor receptor-bound 2 (Grb2) as an anti-cancer strategy.

Growth factor receptor-bound 2 (Grb2) is a ubiquitously expressed adapter protein that provides a critical link between cell surface growth factor receptors and the Ras signaling pathway. As such, it has been implicated in the oncogenesis of several important human malignancies. In addition to this function, research over the last decade has revealed other fundamental roles for Grb2 in cell motility and angiogenesis--processes that also contribute to tumor growth, invasiveness and metastasis. This functional profile makes Grb2 a high priority target for anti-cancer drug development. Knowledge of Grb2 protein structure, its component Src homology domains and their respective structure-function relationships has facilitated the rapid development of sophisticated drug candidates that can penetrate cells, bind Grb2 with high affinity and potently antagonize Grb2 signaling. These novel compounds offer considerable promise in our growing arsenal of rationally designed anti-cancer therapeutics.

Animals↗
PubMed2005

Examination of phosphoryl-mimicking functionalities within a macrocyclic Grb2 SH2 domain-binding platform.

Reported herein are the design, synthesis, and Grb2 SH2 domain-binding affinities of several phosphoryl-mimicking groups displayed within the context of a conformationally constrained macrocyclic platform. With use of surface plasmon resonance techniques, single-digit nanomolar affinities were exhibited by phosphonic acid and malonyl-containing diacidic phosphoryl mimetics (for 4h and 4g, K(D) = 1.47 and 3.62 nM, respectively). Analogues containing monoacidic phosphoryl mimetics provided affinities of K(D) = 16-67 nM. Neutral phosphoryl-mimicking groups did not show appreciable binding.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Utilization of a nitrobenzoxadiazole (NBD) fluorophore in the design of a Grb2 SH2 domain-binding peptide mimetic.

Fluorescence labeling has become a general technique for studying the intracellular accumulation and localization of exogenously administered materials. Reported herein is a low nanomolar affinity Grb2 SH2 domain-binding antagonist that utilizes the environmentally-sensitive nitrobenzoxadiazole (NBD) fluorophore as a naphthyl replacement. This novel agent should serve as a useful tool to visualize the actions of this class of Grb2 SH2 domain-binding antagonists in whole cell systems.

Adaptor Proteins, Signal Transducing↗
PubMed2004

0.2 T magnetic field inhibits angiogenesis in chick embryo chorioallantoic membrane.

Inhibition of angiogenesis is a major target in the fight against cancer and other diseases. Although the effects of static magnetic fields on cancer development and cell growth have been investigated, effects on angiogenesis have received no attention so far. In this study we report the effects on angiogenesis of exposure to 0.2 T static magnetic field. Angiogenesis was analyzed using the chick embryo chorioallantoic membrane assay. Exposure to 0.2 T static magnetic field was achieved by placing the eggs for 3 hr in the isocentre of the magnet of a sectorial magnetic resonance tomograph used in clinical practice. In sham exposed specimens treated with phosphate buffered saline (negative control), no significant vascular reaction was detectable; 3 hr exposure to 0.2 T static magnetic field did not affect the basal pattern of vascularization or chick embryo viability. Prostaglandin E1 and fetal calf serum elicited a strong angiogenic response in sham exposed eggs. This angiogenic response was significantly inhibited by 3 hr exposure to 0.2 T static magnetic field. These findings point to possible use of static magnetic field in inhibiting angiogenesis; this effect could be exploited for treatment of cancer and other diseases where excessive angiogenesis is involved.

Allantois↗
PubMed2004

Hereditary papillary renal carcinoma type I.

Germline missense mutations in the tyrosine kinase domain of the hepatocyte growth factor/scatter factor (HGF/SF) receptor, c-Met, are thought to be responsible for hereditary papillary renal carcinoma (HPRC) type 1, a form of human kidney cancer. In addition to extensive linkage analysis of HPRC families localizing the HPRC type 1 gene within chromosome 7, the demonstration that individual c-Met mutations reconstituted in cultured cells display enhanced and dysregulated kinase activity, and confer cell transformation and tumorigenicity in mice, solidifies this conclusion. Our prior knowledge of HGF/SF biology and c-Met signaling enabled rapid progress in unraveling the molecular pathogenesis of HPRC type 1, and in laying the framework for the development of novel therapeutics for the treatment of this cancer. At the same time, the study of HPRC type 1 has refined our appreciation of the oncogenic potential of c-Met signaling, and challenges our current understanding of HGF/SF and c-Met function in health and disease.

Carcinoma, Papillary↗
PubMed2004

Inhibition of angiogenesis by growth factor receptor bound protein 2-Src homology 2 domain bound antagonists.

Growth factor receptor bound protein 2 (Grb2) is an intracellular adaptor protein that participates in the signal transduction cascades of several angiogenic factors, including hepatocyte growth factor, basic fibroblast growth factor, and vascular endothelial growth factor. We described previously the potent blockade of hepatocyte growth factor-stimulated cell motility, matrix invasion, and epithelial tubulogenesis by synthetic Grb2-Src homology 2 (SH2) domain binding antagonists. Here, we show that these binding antagonists block basic morphogenetic events required for angiogenesis, including hepatocyte growth factor-, vascular endothelial growth factor-, and basic fibroblast growth factor-stimulated endothelial cell proliferation and migration, as well as phorbol 12-myristate 13-acetate-stimulated endothelial cell migration and matrix invasion. The Grb2-SH2 domain binding antagonists also impair angiogenesis in vitro, as shown by the inhibition of cord formation by macrovascular endothelial cells on Matrigel. We further show that a representative compound inhibits angiogenesis in vivo as measured using a chick chorioallantoic membrane assay. These results suggest that Grb2 is an important mediator of key proangiogenic events, with potential application to pathologic conditions where neovascularization contributes to disease progression. In particular, the well-characterized role of Grb2 in signaling cell cycle progression together with our present findings suggests that Grb2-SH2 domain binding antagonists have the potential to act as anticancer drugs that target both tumor and vascular cell compartments.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Regulation of leukemic cell adhesion, proliferation, and survival by beta-catenin.

In epithelial cells beta-catenin plays a critical role as a component of the cell-cell adhesion apparatus and as a coactivator of the TCF/LEF (T-cell transcription factor/lymphoid enhancer binding factor) family of transcription factors. Deregulation of beta-catenin has been implicated in the malignant transformation of cells of epithelial origin. However, a function for beta-catenin in hematologic malignancies has not been reported. beta-Catenin is not detectable in normal peripheral blood T cells but is expressed in T-acute lymphoblastic leukemia cells and other tumor lines of hematopoietic origin and in primary lymphoid and myeloid leukemia cells. beta-Catenin function was examined in Jurkat T-acute lymphoblastic leukemia cells. Overexpression of dominant-negative beta-catenin or dominant-negative TCF reduced beta-catenin nuclear signaling and inhibited Jurkat proliferation and clonogenicity. Similarly, these constructs inhibited proliferation of K562 and HUT-102 cells. Reduction of beta-catenin expression with beta-catenin antisense down-regulated adhesion of Jurkat cells in response to phytohemagglutinin. Incubation of Jurkat cells with anti-Fas induced caspase-dependent limited proteolysis of beta-catenin N- and C-terminal regions and rapid redistribution of beta-catenin to the detergent-insoluble cytoskeleton, concomitant with a marked decline in nuclear beta-catenin signaling. Fas-mediated apoptosis was potentiated by inhibition of beta-catenin nuclear signaling. The data suggest that beta-catenin can play a significant role in promoting leukemic cell proliferation, adhesion, and survival.

Apoptosis↗
PubMed2002

Mitogenic synergy through multilevel convergence of hepatocyte growth factor and interleukin-4 signaling pathways.

Hepatocyte growth factor (HGF) regulates various physiological and developmental processes in concert with other growth factors, cytokines and hormones. We examined interactions between cell signaling events elicited by HGF and the cytokine interleukin (IL)-4, in the IL-3-dependent murine myeloid cell line 32D transfected with the human HGF receptor, c-Met. HGF was a potent mitogen in these cells, and prevented apoptosis in response to IL-3 withdrawal. IL-4 showed modest anti-apoptotic activity, but no significant mitogenic activity. IL-4 synergistically enhanced HGF-stimulated DNA synthesis, whereas only additive prevention of apoptosis was observed. IL-4 did not enhance HGF-dependent tyrosine phosphorylation of c-Met or Shc. In contrast, HGF-stimulated activation of MAP kinases was enhanced by IL-4, suggesting that the IL-4 and HGF signaling pathways converge upstream of these events. Although phosphatidylinositol 3-kinase (PI3K) inhibitors diminished HGF-induced mitogenesis, anti-apoptosis, and MAP kinase activation, IL-4 enhanced HGF signaling persisted even in the presence of these inhibitors. IL-4 enhancement of HGF signaling was partially blocked in 32D/c-Met cells treated with inhibitors of MEK1 or c-Src kinases, completely blocked by expression of a catalytically inactive mutant of Janus kinase 3 (Jak3), and increased in 32D/c-Met cells overexpressing STAT6. Our results suggest that the IL-4 and HGF pathways converge at multiple levels, and that IL-4-dependent Jak3 and STAT6 activities modulate signaling events independent of PI3K to enhance HGF-dependent mitogenesis in myeloid cells, and possibly other common cellular targets.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Direct application of keratinocyte growth factor, basic fibroblast growth factor and transforming growth factor-alpha during healing of tympanic membrane perforation in glucocorticoid-treated rats.

Peptide growth factors and cytokines modulate both normal and impaired wound healing. Topical application of growth factors in the form of ear drops may counteract impairment of wound healing in the tympanic membrane (TM). We applied keratinocyte growth factor (KGF), transforming growth factor (TGF)-alpha or basic fibroblast growth factor (bFGF) to the perforated TMs of rats in which healing impairment had been caused by systemic administration of a glucocorticoid. Histologic studies of the injured TM, including anti-5-bromo-2'-deoxyuridine immunohistochemistry, were performed on the third day after wounding. In the control ear, epidermal migration was markedly inhibited by glucocorticoid treatment and no hyperplasia was observed in any layer at the perforation edge. TMs treated with KGF showed marked hyperplasia in the epithelial layer at the perforation edge. In the bFGF- and TGF-alpha-treated groups, hyperplasia was observed in the epithelial and intermediate layers of the TM near the malleus handle and annulus, while no hyperplasia was seen in any layer at the perforation edge. Only KGF, therefore, improved epidermal migration in the TM, while all the growth factors tested induced hyperplasia in the TM.

Animals↗
PubMed2002

Molecular signaling in bioengineered tissue microenvironments.

Biological tissues and organs consist of specialized living cells arrayed within a complex structural and functional framework known generally as the extracellular matrix (ECM). The great diversity observed in the morphology and composition of the ECM contributes enormously to the properties and function of each organ and tissue. For example, the ECM contributes to the rigidity and tensile strength of bone, the resilience of cartilage, the flexibility and hydrostatic strength of blood vessels, and the elasticity of skin. The ECM is also important during growth, development, and wound repair: its own dynamic composition acts as a reservoir for soluble signaling molecules and mediates signals from other sources to migrating, proliferating, and differentiating cells. Artificial three-dimensional substitutes for ECM, called tissue scaffolds, may consist of natural or synthetic polymers or a combination of both. Scaffolds have been used successfully alone and in combination with cells and soluble factors to induce tissue formation or promote tissue repair. Appropriate numbers of properly functioning living cells are central to many tissue-engineering strategies, and significant efforts have been made to identify and propagate pluripotent stem cells and lineage-restricted progenitor cells. The study of these and other living cells in artificial microenvironments, in turn, has led to the identification of signaling events important for their controlled proliferation, proper differentiation, and optimal function.

Animals↗