B.A., Johns Hopkins University, Bioengineering/Medicine (1980)
M.D., Johns Hopkins School of Medicine (1983)
The goal of this laboratory is to define targets for cancer therapeutics by identifying alterations in signal transduction proteins and then translate these findings into important clinical tools, including one of the first effective peptide vaccines against cancer. The major type of cancer that we study is glioblastoma multiforme, the most common and devastating of the human brain tumors, but this work has also had implications for lung, breast, ovarian and prostate cancers.
Our research direction originated when we first identified a spontaneously occurring mutant EGF receptor in glioblastoma. Known as EGFRvIII (pronounced E-G-F-R-v-three), this molecule represents a deletion of exons 2 through 7 in the extracellular domain of the EGF receptor. This removes 273 amino acids and creates a novel glycine at the fusion junction. We have engaged in both very basic studies on the signal transduction pathways initiated by EGFRvIII, as well as translational work to create diagnostic tools and therapies around EGFRvIII.
Our basic science studies on understanding EGFRvIII signaling have led us to discover the Gab1 docking protein. Orignially discovered as a substrate for the EGF receptor, we and others have found that Gab1 is a substrate for numerous tyrosine kinase receptors, and in turn Gab1 recruits several proteins with SH2 domains. It is now clear that it is a vital link for multiple growth factor and cytokine pathways and has a role in diverse phenotypes such as cell survival, the cellular response to stress, and the cellular entry of parasites.
As another result of our efforts on signaling pathways, we have discovered that glioblastoma tumors preferentially utilize the JNK pathway, unlike many other tumors that use the ERK pathway. More specifically, only one of the 12 known JNK isoforms is highly expressed, JNK2a2. Co-incidentally, we have uncovered that this isoform is constitutively active and that it has a specific activation domain. JNK2a2 also upregulates TGF-a, a ligand for the EGF receptor that is frequently overexpressed in glioblastomas.
Our translational work has encompassed both diagnostics and therapeutics. We have developed antibodies that specifically recognize EGFRvIII and not EGF receptor and have also developed rapid RT-PCR based assays to detect EGFRvIII. Using these tools, we have discovered that expression of EGFRvIII is not limited just to glioblastoma tumors but it is also found in breast, lung, prostate and ovarian tumors.
We were the first to show that a peptide vaccine based on the cancer specific epitope found in EGFRvIII is an effective anti-tumor drug (originally called ALT-110). In animal models, this vaccine can both prevent tumors and induce the regression of existing tumors. We also played a major role in initiating the first clinical trial in humans using this peptide vaccine. Three trials have now been completed with highly interesting results. A Phase II clinical trial at Duke and MD Anderson for glioblastoma patients has shown median survival of 30 months vs. 14 months for conventional therapy in a matched cohort.
An expanded Phase II/III trial for brain tumors using the vaccine (now called CDX-110) is currently enrolling which is sponsored by Celldex Therapeutics. If you have questions about this trial, please visit the NCI trials website at http://clinicaltrials.gov/ct/show/NCT00458601 or www.avantimmune.com.
Disclosure: Dr. Wong is the inventor of EGFRvIII and CDX-110, holds other patents related to EGFRvIII, and owns equity in Celldex.
This is a research study of patients with diffuse intrinsic pontine gliomas. We hope to learn about the safety and efficacy of treating pediatric diffuse intrinsic pontine glioma patients with the EGFRvIII peptide vaccine after conventional radiation.
Stanford is currently not accepting patients for this trial. For more information, please contact Christina Huang, 650-723-0574.
Amplification and rearrangements of the epidermal growth factor receptor (EGFR) gene are frequently found in glioblastoma multiforme (GBM). The most common variant is EGFR variant III (EGFRvIII). Research suggests that EGFRvIII could be a marker for a cancer stem cell or tumor-initiating population. If amplification and rearrangement are early events in tumorigenesis, this implies that they should be preserved throughout the tumor. However, in primary GBM, EGFRvIII expression is focal and sporadic. Unexpectedly, we found EGFR amplification and rearrangement throughout the tumor, including regions with no EGFRvIII expression, suggesting that mechanisms exist to modulate EGFRvIII expression even in the presence of high gene amplification. To study this phenomenon, we characterized three GBM cell lines with endogenous EGFRvIII. EGFRvIII expression was heterogeneous, with both positive and negative populations maintaining the genetic alterations, akin to primary tumors. Furthermore, EGFRvIII defined a hierarchy where EGFRvIII-positive cells gave rise to additional positive and negative cells. Only cells that had recently lost EGFRvIII expression could re-express EGFRvIII, providing an important buffer for maintaining EGFRvIII-positive cell numbers. Epigenetic mechanisms had a role in maintaining heterogeneous EGFRvIII expression. Demethylation induced a 20-60% increase in the percentage of EGFRvIII-positive cells, indicating that some cells could re-express EGFRvIII. Surprisingly, inhibition of histone deacetylation resulted in a 50-80% reduction in EGFRvIII expression. Collectively, this data demonstrates that EGFR amplification and rearrangement are early events in tumorigenesis and EGFRvIII follows a model of hierarchical expression. Furthermore, EGFRvIII expression is restricted by epigenetic mechanisms, suggesting that drugs that modulate the epigenome might be used successfully in glioblastoma tumors.
View details for DOI 10.1038/onc.2012.280
View details for PubMedID 22797070
Gene fusions, like BCR/ABL1 in chronic myelogenous leukemia, have long been recognized in hematologic and mesenchymal malignancies. The recent finding of gene fusions in prostate and lung cancers has motivated the search for pathogenic gene fusions in other malignancies. Here, we developed a "breakpoint analysis" pipeline to discover candidate gene fusions by tell-tale transcript level or genomic DNA copy number transitions occurring within genes. Mining data from 974 diverse cancer samples, we identified 198 candidate fusions involving annotated cancer genes. From these, we validated and further characterized novel gene fusions involving ROS1 tyrosine kinase in angiosarcoma (CEP85L/ROS1), SLC1A2 glutamate transporter in colon cancer (APIP/SLC1A2), RAF1 kinase in pancreatic cancer (ATG7/RAF1) and anaplastic astrocytoma (BCL6/RAF1), EWSR1 in melanoma (EWSR1/CREM), CDK6 kinase in T-cell acute lymphoblastic leukemia (FAM133B/CDK6), and CLTC in breast cancer (CLTC/VMP1). Notably, while these fusions involved known cancer genes, all occurred with novel fusion partners and in previously unreported cancer types. Moreover, several constituted druggable targets (including kinases), with therapeutic implications for their respective malignancies. Lastly, breakpoint analysis identified new cell line models for known rearrangements, including EGFRvIII and FIP1L1/PDGFRA. Taken together, we provide a robust approach for gene fusion discovery, and our results highlight a more widespread role of fusion genes in cancer pathogenesis.
View details for DOI 10.1371/journal.pgen.1003464
View details for PubMedID 23637631
Despite numerous clinical trials over the past 2 decades, the overall survival for children diagnosed with diffuse intrinsic pontine glioma (DIPG) remains 9-10 months. Radiation therapy is the only treatment with proven effect and novel therapies are needed. Epidermal growth factor receptor variant III (EGFRvIII) is the most common variant of the epidermal growth factor receptor and is expressed in many tumor types but is rarely found in normal tissue. A peptide vaccine targeting EGFRvIII is currently undergoing investigation in phase 3 clinical trials for the treatment of newly diagnosed glioblastoma (GBM), the tumor in which this variant receptor was first discovered. In this study, we evaluated EGFRvIII expression in pediatric DIPG samples using immunohistochemistry with a double affinity purified antibody raised against the EGFRvIII peptide. Staining of pediatric DIPG histological samples revealed expression in 4 of 9 cases and the pattern of staining was consistent with what has been seen in EGFRvIII transfected cells as well as GBMs from adult trials. In addition, analysis of tumor samples collected immediately post mortem and of DIPG cells in culture by RT-PCR, western blot analysis, and flow cytometry confirmed EGFRvIII expression. We were therefore able to detect EGFRvIII expression in 6 of 11 DIPG cases. These data suggest that EGFRvIII warrants investigation as a target for these deadly pediatric tumors.
View details for DOI 10.1007/s11060-012-0842-3
View details for Web of Science ID 000305123800007
View details for PubMedID 22382786
The epidermal growth factor receptor (EGFR) is essential to multiple physiological and neoplastic processes via signaling by its tyrosine kinase domain and subsequent activation of transcription factors. EGFR overexpression and alteration, including point mutations and structural variants, contribute to oncogenesis in many tumor types. In this study, we identified an in-frame splice variant of the EGFR called mini-LEEK (mLEEK) that is more broadly expressed than the EGFR and is overexpressed in several cancers. Unlike previously characterized EGFR variants, mLEEK lacks the extracytoplasmic, transmembrane and tyrosine kinase domains. mLEEK localizes in the nucleus and functions as a transcription factor to regulate target genes involved in the cellular response to endoplasmic reticulum (ER) stress, including the master regulator of the unfolded protein response (UPR) pathways, molecular chaperone GRP78/Bip. We demonstrated that mLEEK regulates GRP78 transcription through direct interaction with a cis-regulatory element within the gene promoter. Several UPR pathways were interrogated and mLEEK expression was found to attenuate the induction of all pathways upon ER stress. Conversely, knockdown of mLEEK resulted in caspase-mediated cell death and sensitization to ER stress. These findings indicate that mLEEK levels determine cellular responses to unfavorable conditions that cause ER stress. This information, along with the overexpression of mLEEK in tumors, suggests unique strategies for therapeutic intervention. Furthermore, the identification of mLEEK expands the known mechanisms by which the EGFR gene contributes to oncogenesis and represents the first link between two previously disparate areas in cancer cell biology: EGFR signaling and the UPR.
View details for DOI 10.1038/onc.2011.465
View details for Web of Science ID 000305388400005
View details for PubMedID 21986942
EGFRvIII is a tumor-specific variant of the epidermal growth factor receptor (EGFR). Although EGFRvIII is most commonly found in glioblastoma, its expression in other tumor types remains controversial. In this study, we investigated EGFRvIII expression and amplification in primary breast carcinoma. Our analyses confirmed the presence of EGFRvIII, but in the absence of amplification or rearrangement of the EGFR locus. Nested reverse transcriptase PCR and flow cytometry were used to detect a higher percentage of positive cases. EGFRvIII-positive cells showed increased expression of genes associated with self-renewal and epithelial-mesenchymal transition along with a higher percentage of stem-like cells. EGFRvIII also increased in vitro sphere formation and in vivo tumor formation. Mechanistically, EGFRvIII mediated its effects through the Wnt/?-catenin pathway, leading to increased ?-catenin target gene expression. Inhibition of this pathway reversed the observed effects on cancer stem cell (CSC) phenotypes. Together, our findings show that EGFRvIII is expressed in primary breast tumors and contributes to CSC phenotypes in breast cancer cell lines through the Wnt pathway. These data suggest a novel function for EGFRvIII in breast tumorigenesis.
View details for DOI 10.1158/0008-5472.CAN-11-2656
View details for Web of Science ID 000307346800020
View details for PubMedID 22419663
Glioblastoma multiforme (GBM) is the most common and deadly of the human brain cancers. The EGF receptor is often amplified in GBM and provides a potential therapeutic target. However, targeting the normal receptor is complicated by its nearly ubiquitous and high level of expression in certain tissues. A naturally occurring deletion mutant of the EGF receptor, EGFRvIII, is a constitutively active variant originally identified in a high percentage of brain cancer cases, and more importantly is rarely found in normal tissue. A peptide vaccine, rindopepimut (CDX-110, Celldex Therapeutics), is directed against the novel exon 1-8 junction produced by the EGFRvIII deletion, and it has shown high efficacy in preclinical models. Recent Phase II clinical trials in patients with newly diagnosed GBM have shown EGFRvIII-specific immune responses and significantly increased time to progression and overall survival in those receiving vaccine therapy, as compared with published results for standard of care. Rindopepimut therefore represents a very promising therapy for patients with GBM.
View details for DOI 10.1586/ERV.11.177
View details for Web of Science ID 000300758700009
View details for PubMedID 22309662
Diffuse intrinsic pontine gliomas (DIPGs) are highly aggressive tumors of childhood that are almost universally fatal. Our understanding of this devastating cancer is limited by a dearth of available tissue for study and by the lack of a faithful animal model. Intriguingly, DIPGs are restricted to the ventral pons and occur during a narrow window of middle childhood, suggesting dysregulation of a postnatal neurodevelopmental process. Here, we report the identification of a previously undescribed population of immunophenotypic neural precursor cells in the human and murine brainstem whose temporal and spatial distributions correlate closely with the incidence of DIPG and highlight a candidate cell of origin. Using early postmortem DIPG tumor tissue, we have established in vitro and xenograft models and find that the Hedgehog (Hh) signaling pathway implicated in many developmental and oncogenic processes is active in DIPG tumor cells. Modulation of Hh pathway activity has functional consequences for DIPG self-renewal capacity in neurosphere culture. The Hh pathway also appears to be active in normal ventral pontine precursor-like cells of the mouse, and unregulated pathway activity results in hypertrophy of the ventral pons. Together, these findings provide a foundation for understanding the cellular and molecular origins of DIPG, and suggest that the Hh pathway represents a potential therapeutic target in this devastating pediatric tumor.
View details for DOI 10.1073/pnas.1101657108
View details for Web of Science ID 000288450900040
View details for PubMedID 21368213
The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase family and have been implicated in tumorigenesis. One isoform in particular, JNK2?, has been shown to be frequently activated in primary brain tumors, to enhance several tumorigenic phenotypes and to increase tumor formation in mice. As JNK is frequently activated in non-small cell lung carcinoma (NSCLC), we investigated the role of the JNK2? isoform in NSCLC formation by examining its expression in primary tumors and by modulating its expression in cultured cell lines. We discovered that 60% of the tested primary NSCLC tumors had three-fold higher JNK2 protein and two- to three-fold higher JNK2? mRNA expression than normal lung control tissue. To determine the importance of JNK2? in NSCLC progression, we reduced JNK2? expression in multiple NSCLC cell lines using short hairpin RNA. Cell lines deficient in JNK2? had decreased cellular growth and anchorage-independent growth, and the tumors were four-fold smaller in mass. To elucidate the mechanism by which JNK2? induces NSCLC growth, we analyzed the JNK substrate, signal transducer and activator of transcription 3 (STAT3). Our data demonstrates for the first time that JNK2? can regulate the transcriptional activity of STAT3 by phosphorylating the Ser727 residue, thereby regulating the expression of oncogenic genes, such as c-Myc. Furthermore, reintroduction of JNK2?2 or STAT3 restored the tumorigenicity of the NSCLC cells, demonstrating that JNK2? is important for NSCLC progression. Our studies reveal a novel mechanism in which phosphorylation of STAT3 is mediated by a constitutively active JNK2 isoform, JNK2?.
View details for DOI 10.1038/onc.2010.414
View details for Web of Science ID 000286438900011
View details for PubMedID 20871632
Celldex Therapeutics is developing rindopepimut (CDX-110), a 14-mer injectable peptide vaccine for the potential treatment of glioblastoma multiforme (GBM). Rindopepimut specifically targets a novel junctional epitope of the EGFR deletion mutant EGFRvIII, which is a constitutively active receptor that is expressed in approximately 60 to 70% of patients with GBM. EGFRvIII expression is correlated with worse prognosis and reduced overall survival. Importantly, EGFRvIII is not expressed in normal brain tissue, making it an excellent therapeutic target. Preclinical studies demonstrated lasting tumor regression and increased survival times, as well as efficient generation of EGFRvIII-specific humoral and cellular immune responses, in animals expressing EGFRvIII and vaccinated with rindopepimut. Phase I and II clinical trials in patients with GBM demonstrated significantly increased median time to progression and overall survival time in those vaccinated with rindopepimut compared with matched historical controls. Only limited side effects have been observed in patients. Given these results, rindopepimut is an extremely promising therapy for patients with GBM. Phase I and II clinical trials in patients with GBM were ongoing at the time of publication. In the future, it may be beneficial to combine rindopepimut with other treatment modalities to further prolong survival.
View details for Web of Science ID 000285308300013
View details for PubMedID 21154166
EGF receptor variant III (EGFRvIII) is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within the extra-cellular domain of the EGF receptor creates a tumor specific and immunogenic epitope. EGFRvIII expression has been seen in many tumor types including glioblastoma multiforme (GBM), breast adenocarcinoma, non-small cell lung carcinoma, ovarian adenocarcinoma and prostate cancer, but has been rarely observed in normal tissue. Because this variant is tumor specific and highly immunogenic, it can be used for both a diagnostic marker as well as a target for immunotherapy. Unfortunately many of the monoclonal and polyclonal antibodies directed against EGFRvIII have cross reactivity to wild type EGFR or other non-specific proteins. Furthermore, a monoclonal antibody to EGFRvIII is not readily available to the scientific community.In this study, we have developed a recombinant antibody that is specific for EGFRvIII, has little cross reactivity for the wild type receptor, and which can be easily produced. We initially designed a recombinant antibody with two anti-EGFRvIII single chain Fv's linked together and a human IgG1 Fc component. To enhance the specificity of this antibody for EGFRvIII, we mutated tyrosine H59 of the CDRH2 domain and tyrosine H105 of the CDRH3 domain to phenylalanine for both the anti-EGFRvIII sequence inserts. This mutated recombinant antibody, called RAb(DMvIII), specifically detects EGFRvIII expression in EGFRvIII expressing cell lines as well as in EGFRvIII expressing GBM primary tissue by western blot, immunohistochemistry (IHC) and immunofluorescence (IF) and FACS analysis. It does not recognize wild type EGFR in any of these assays. The affinity of this antibody for EGFRvIII peptide is 1.7 × 10? M?¹ as determined by enzyme-linked immunosorbent assay (ELISA).This recombinant antibody thus holds great potential to be used as a research reagent and diagnostic tool in research laboratories and clinics because of its high quality, easy viability and unique versatility. This antibody is also a strong candidate to be investigated for further in vivo therapeutic studies.
View details for DOI 10.1186/1472-6750-10-72
View details for Web of Science ID 000283354200001
View details for PubMedID 20925961
Epidermal growth factor receptor (EGF) receptor gene amplification is commonly seen in cancer and is the target of many therapies. EGF receptor variant III (EGFRvIII) is the most common variant of the EGF receptor and has been detected in a large percentage of patients with glioblastoma multiforme but not in normal brain. Therapies targeting EGFRvIII are currently being investigated in clinical and preclinical trials.A 14-year-old girl who presented with headaches was found to have a pineal parenchymal tumor of intermediate differentiation. We review the histopathological properties that led to the diagnosis of this tumor. EGF receptor gene amplification and EGFRvIII expression have not been analyzed in pineal tumors. We investigated EGF receptor gene status and EGFRvIII expression in this patient's tumor.Tumor tissue was obtained and analyzed with flow cytometry, reverse-transcriptase polymerase chain reaction, and Western blot analysis. EGFRvIII was detected by all 3 methods. The tumor was further analyzed by fluorescence in situ hybridization, which did not reveal EGF receptor gene amplification.This is the first report of EGFRvIII expression in a pineal tumor. It is interesting that this variant is detected in the absence of EGF receptor gene amplification. A larger study evaluating the presence of EGFRvIII in pineal tumors is needed.
View details for DOI 10.1227/01.NEU.0000367726.49003.F1
View details for Web of Science ID 000276970800026
View details for PubMedID 20404701
The c-Jun N-terminal kinases (JNK) are important regulators of cell growth, proliferation, and apoptosis. JNKs are typically activated by a sequence of events that include phosphorylation of its T-P-Y motif by an upstream kinase, followed by homodimerization and translocation to the nucleus. Constitutive activation of JNK has been found in a variety of cancers including non-small cell lung carcinomas, gliomas, and mantle cell lymphoma. In vitro studies show that constitutive activation of JNK induces a transformed phenotype in fibroblasts and enhances tumorigenicity in a variety of cell lines. Interestingly, a subset of JNK isoforms was recently found to autoactivate rendering the proteins constitutively active. These constitutively active JNK proteins were found to play a pivotal role in activating transcription factors that increase cellular growth and tumor formation in mice. In this chapter, we describe techniques and methods that have been successfully used to study the three components of JNK activation. Use of these techniques may lead to a better understanding of the components of JNK pathways and how JNK is activated in cancer cells.
View details for DOI 10.1016/S0076-6879(10)84026-1
View details for Web of Science ID 000284508500026
View details for PubMedID 21036249
Epidermal growth factor variant III (EGFRvIII) is the most common alteration of the epidermal growth factor (EGF) receptor found in human tumors. It is commonly expressed in glioblastoma multiforme (GBM), where it was initially identified. This constitutively active mutant receptor leads to unregulated growth, survival, invasion, and angiogenesis in cells that express it. EGFRvIII results from an in-frame deletion of exons 2 to 7 resulting in the fusion of exon 1 to exon 8 of the EGF receptor gene creating a novel glycine at the junction in the extracellular amino terminal domain. The juxtaposition of ordinarily distant amino acids in combination with the glycine that forms at the junction leads to a novel tumor-specific epitope that would make an ideal tumor-specific target. A peptide derived from the EGFRvIII junction can be used as a vaccine to prevent or induce the regression of tumors. This peptide vaccine has now proceeded to phase 1 and 2 clinical trials where it has been highly successful and is now undergoing investigation in a larger human clinical trial for patients who have newly diagnosed GBM. In this article, the authors discuss the preclinical data that led to the human trials and the exciting preliminary data from the clinical trials.
View details for DOI 10.1016/j.nec.2009.08.004
View details for Web of Science ID 000278059500009
View details for PubMedID 19944969
c-Jun N-terminal kinases (JNKs) are part of the mitogen-activated protein kinase (MAPK) family and are important regulators of cell growth, proliferation, and apoptosis. Typically, a sequential series of events are necessary for MAPK activation: phosphorylation, dimerization, and then subsequent translocation to the nucleus. Interestingly, a constitutively active JNK isoform, JNK2alpha2, possesses the ability to autophosphorylate and has been implicated in several human tumors, including glioblastoma multiforme. Because overexpression of JNK2alpha2 enhances several tumorigenic phenotypes, including cell growth and tumor formation in mice, we studied the mechanisms of JNK2alpha2 autophosphorylation and autoactivation. We find that JNK2alpha2 dimerization in vitro and in vivo occurs independently of its autophosphorylation but is dependent on nine amino acids, known as the alpha-region. Alanine scanning mutagenesis of the alpha-region reveals that five specific mutants (L218A, K220A, G221A, I224A, and F225A) prevent JNK2alpha2 dimerization rendering JNK2alpha2 inactive and incapable of stimulating tumor formation. Previous studies coupled with additional mutagenesis of neighboring isoleucines and leucines (I208A, I214A, I231A, and I238A) suggest that a leucine zipper may play an important role in JNK2alpha2 homodimerization. We also show that a kinase-inactive JNK2alpha2 mutant can interact with and inhibit wild type JNK2alpha2 autophosphorylation, suggesting that JNK2alpha2 undergoes trans-autophosphorylation. Together, our results demonstrate that JNK2alpha2 differs from other MAPK proteins in two major ways; its autoactivation/autophosphorylation is dependent on dimerization, and dimerization most likely precedes autophosphorylation. In addition, we show that dimerization is essential for JNK2alpha2 activity and that prevention of dimerization may decrease JNK2alpha2 induced tumorigenic phenotypes.
View details for DOI 10.1074/jbc.M804970200
View details for Web of Science ID 000261469100050
View details for PubMedID 18940813
The EGF receptor (EGFR) is the first tyrosine kinase receptor ever cloned and remains at the forefront of targeted therapies against cancer. Currently, there are four US FDA-approved drugs and several more in Phase III studies that target the EGFR. These drugs, while resulting in some dramatic remissions, have not resulted in strong nor consistent improvements in survival. EGFR variant III (EGFRvIII) is the most common variant of the EGFR and is present in many different cancer types but not in normal tissue. It results from the fusion of exon 1 to exon 8 of the EGFR gene, which results in a novel glycine at the junction. This mutant receptor is constitutively active in these tumors and can lead directly to cancer phenotypes due to its oncogenic properties. EGFRvIII is an attractive target antigen for cancer immunotherapy because it is not expressed in normal tissue and because cells producing EGFRvIII have an enhanced capacity for dysregulated growth, survival, invasion and angiogenesis. In this review, we will discuss preclinical and clinical data from studies using EGFRvIII as the target antigen for immunotherapy, with a focus on the potential for greatly improved survival for patients diagnosed with glioblastoma multiforme.
View details for DOI 10.1586/14760518.104.22.1687
View details for Web of Science ID 000259334600016
View details for PubMedID 18767947
Vascular endothelial growth factor (VEGF) is involved in the promotion of endothelial cell proliferation, migration, and capillary formation. These activities are mainly mediated by the VEGFR2 receptor tyrosine kinase that upon stimulation, promotes the activation of numerous proteins including phospholipase Cgamma (PLCgamma), phosphatidylinositol 3-kinase (PI3K), Akt, Src, and ERK1/2. However, the VEGFR2-proximal signaling events leading to the activation of these targets remain ill defined. We have identified the Gab1 adapter as a novel tyrosine-phosphorylated protein in VEGF-stimulated cells. In bovine aortic endothelial cells, Gab1 associates with VEGFR2, Grb2, PI3K, SHP2, Shc, and PLCgamma, and its overexpression enhances VEGF-dependent cell migration. Importantly, silencing of Gab1 using small interfering RNAs leads to the impaired activation of PLCgamma, ERK1/2, Src, and Akt; blocks VEGF-induced endothelial cell migration; and perturbs actin reorganization and capillary formation. In addition, co-expression of VEGFR2 with Gab1 mutants unable to bind SHP2 or PI3K in human embryonic kidney 293 cells and bovine aortic endothelial cells mimics the defects observed in Gab1-depleted cells. Our work thus identifies Gab1 as a novel critical regulatory component of endothelial cell migration and capillary formation and reveals its key role in the activation of VEGF-evoked signaling pathways required for angiogenesis.
View details for DOI 10.1074/jbc.M611327200
View details for Web of Science ID 000245081000003
View details for PubMedID 17178724
c-Jun NH(2)-terminal kinases (JNK) are members of the mitogen-activated protein kinase family and have been implicated in the formation of several human tumors, especially gliomas. We have previously shown that a 55 kDa JNK isoform is constitutively active in 86% of human brain tumors and then showed that it is specifically a JNK2 isoform and likely to be either JNK2alpha2 or JNK2beta2. Notably, we found that only JNK2 isoforms possess intrinsic autophosphorylation activity and that JNK2alpha2 has the strongest activity. In the present study, we have further explored the contribution of JNK2 isoforms to brain tumor formation. Analysis of mRNA expression by reverse transcription-PCR revealed that JNK2alpha2 is expressed in 91% (10 of 11) of glioblastoma tumors, whereas JNK2beta2 is found in only 27% (3 of 11) of tumors. Both JNK2alpha2 and JNK2beta2 mRNAs are expressed in normal brain (3 of 3). Using an antibody specific for JNK2alpha isoforms, we verified that JNK2alpha2 protein is expressed in 88.2% (15 of 17) of glioblastomas, but, interestingly, no JNK2alpha2 protein was found in six normal brain samples. To evaluate biological function, we transfected U87MG cells with green fluorescent protein-tagged versions of JNK1alpha1, JNK2alpha2, and JNK2alpha2APF (a dominant-negative mutant), and derived cell lines with stable expression. Each cell line was evaluated for various tumorigenic variables including cellular growth, soft agar colony formation, and tumor formation in athymic nude mice. In each assay, JNK2alpha2 was found to be the most effective in promoting that phenotype. To identify effectors specifically affected by JNK2alpha2, we analyzed gene expression. Gene profiling showed several genes whose expression was specifically up-regulated by JNK2alpha2 but down-regulated by JNK2alpha2APF, among which eukaryotic translation initiation factor 4E (eIF4E) shows the greatest change. Because AKT acts on eIF4E, we also examined AKT activation. Unexpectedly, we found that JNK2alpha2 could specifically activate AKT. Our data provides evidence that JNK2alpha2 is the major active JNK isoform and is involved in the promotion of proliferation and growth of human glioblastoma tumors through specific activation of AKT and overexpression of eIF4E.
View details for DOI 10.1158/0008-5472.CAN-06-0136
View details for Web of Science ID 000241392700030
View details for PubMedID 17047065
The interactions between cancer cells and the extracellular matrix (ECM) regulate cancer progression. The beta1C and beta1A integrins, two cytoplasmic variants of the beta1 integrin subfamily, are differentially expressed in prostate cancer. Using gene expression analysis, we show here that the beta1C variant, an inhibitor of cell proliferation, which is down-regulated in prostate cancer, up-regulates insulin-like growth factor-II (IGF-II) mRNA and protein levels. In contrast, beta1A does not affect IGF-II levels. We provide evidence that beta1C-mediated up-regulation of IGF-II levels increases adhesion to Laminin-1, a basement membrane protein down-regulated in prostate cancer, and that the beta1C cytoplasmic domain contains the structural motif sufficient to increase cell adhesion to Laminin-1. This autocrine mechanism that locally supports cell adhesion to Laminin-1 via IGF-II is selectively regulated by the beta1 cytoplasmic domain via activation of the growth factor receptor binding protein 2-associated binder-1/SH2-containing protein-tyrosine phosphatase 2/phosphatidylinositol 3-kinase pathway. Thus, the concurrent local loss of beta1C integrin, of its ligand Laminin-1, and of IGF-II in the tumor microenvironment may promote prostate cancer cell invasion and metastasis by reducing cancer cell adhesive properties. It is, therefore, conceivable that reexpression of beta1C will be sufficient to revert a neoplastic phenotype to a nonproliferative and highly adherent normal phenotype.
View details for DOI 10.1158/0008-5472.CAN-05-2588
View details for Web of Science ID 000234529500041
View details for PubMedID 16397247
c-Jun N-terminal kinases (JNKs) are a group of mitogen-activated protein kinase family members that are important in regulating cell growth, proliferation, and apoptosis. Activation of the JNK pathway has been implicated in the formation of several human tumors. We have previously demonstrated that a 55-kDa JNK isoform is constitutively activated in 86% of human brain tumors and more recently demonstrated that this isoform is either JNK2alpha2 or JNK2beta2. Importantly, we have also found that among the 10 known JNK isoforms, the JNK2 isoforms are unique in their ability to autophosphorylate in vitro and in vivo. This does not require the participation of any upstream kinases and also leads to substrate kinase activity in vitro and in vivo. To clarify the mechanism of JNK2alpha2 autoactivation, we have generated a series of chimeric cDNAs joining portions of JNK1alpha2, which does not have detectable autophosphorylation activity, with portions of JNK2alpha2, which has the strongest autophosphorylation activity. Through in vivo and in vitro kinase assays, we were able to define a domain ranging from amino acids 218 to 226 within JNK2alpha2 that is required for its autophosphorylation. Mutation of JNK2alpha2 to its counterpart of JNK1alpha2 in this region abrogated the autophosphorylation activity and c-Jun substrate kinase activity in vivo and in vitro. Notably, switching of JNK1alpha2 to JNK2alpha2 at this 9-amino acid site enabled JNK1alpha2 to gain the autophosphorylation activity in vivo and in vitro. We also found two other functional sites that participate in JNK2alpha2 activity. One site ranging from amino acids 363 to 382 of JNK2alpha2 is required for efficient c-Jun binding in vitro, and a site ranging from amino acids 383 to 424 enhances autophosphorylation intensity, although it is not required for triggering the autophosphorylation in vitro. These findings have uncovered the regions required for JNK2alpha2 autophosphorylation, and this information could be used as potential targets to block JNK2alpha2 activation.
View details for DOI 10.1074/jbc.M412165200
View details for Web of Science ID 000227559600022
View details for PubMedID 15637069
The bacterial surface protein InlB mediates internalization of Listeria monocytogenes into mammalian cells through interaction with the host receptor tyrosine kinase, Met. InlB/Met interaction results in activation of the host phosphoinositide (PI) 3-kinase p85-p110, an event required for bacterial entry. p85-p110 activation coincides with tyrosine phosphorylation of the host adaptor Gab1, and formation of complexes between Gab1 and the p85 regulatory subunit of PI 3-kinase. When phosphorylated in response to agonists, Gab1 is known to recruit several Src-homology 2 (SH2) domain-containing proteins including p85, the tyrosine phosphatase Shp2 and the adaptor CrkII. Here, we demonstrate that Gab1.p85 and Gab1.CrkII complexes promote entry of Listeria. Overexpression of wild-type Gab1 stimulated entry, whereas Gab1 alleles unable to recruit all SH2 proteins known to bind wild-type Gab1 inhibited internalization. Further analysis with Gab1 alleles defective in binding individual effectors suggested that recruitment of p85 and CrkII are critical for entry. Consistent with this data, overexpression of wild-type CrkII stimulated bacterial uptake. Experiments with mutant CrkII alleles indicated that both the first and second SH3 domains of this adaptor participate in entry, with the second domain playing the most critical role. Taken together, these findings demonstrate novel roles for Gab1 and CrkII in Listeria internalization.
View details for DOI 10.1111/j.1462-5822.2004.00475.x
View details for Web of Science ID 000226641500013
View details for PubMedID 15679846
Prostatic carcinogenesis has been associated with alterations in the expression of the androgen receptor (AR) and the epidermal growth factor receptor (WT-EGFR), and over-expression of the constitutively active variant epidermal growth factor receptor (EGFRvIII). Changes in the expression of AR, WT-EGFR and EGFRvIII were evaluated in serial sections from 26 normal and 26 benign hyperplastic and 50 prostate cancer tissues using specific immunostaining techniques. The loss of AR expression in peri-epithelial stroma as prostatic tissues de-differentiated correlated strongly with the depletion of WT-EGFR and with increasing expression of the EGFRvIII in the adjacent epithelium. In contrast, changes in epithelial AR immunopositivity in these tissues correlated weakly with the changes in normal and variant EGFR levels. This is the first report correlating the changes in the expression of these three proteins in archival material from the different human prostatic tissue histotypes. The loss of expression of proteins that contribute to the regulation of prostatic homeostasis (AR and WT-EGFR) correlates strongly with the expression of a constitutively active variant EGF receptor (EGFRvIII) in human prostate cancer. These changes occur at an early stage of neoplastic transformation and may contribute to the progression of the disease to hormone independence.
View details for PubMedID 15819472
Grb2-associated binder 1 (Gab1) is a docking protein that is tyrosine phosphorylated following the activation of multiple cytokine receptors and receptor tyrosine kinases. Its function then is to recruit and activate multiple signaling molecules. In our previous work, we showed that Gab1 enhances cell growth and induces the transformed phenotype in NIH3T3 cells downstream of the epidermal growth factor (EGF) receptor. In this report, we analyze how it produces these effects. Because SHP-2 is the major binding partner of Gab1, we mutated its binding site in the Gab1 cDNA (Gab1/DeltaSHP-2). This construct was stably overexpressed in NIH3T3 cells (3T3-Gab1/DeltaSHP-2) and in the wild-type Gab1 cDNA (3T3-Gab1) or an empty expression vector (3T3-CTR). Our findings show that after EGF stimulation, Gab1/DeltaSHP-2 has a higher level of tyrosine phosphorylation at early time points than Gab1. Gab1/DeltaSHP-2 recruits more phosphatidylinositol 3'-kinase than Gab1 after EGF triggering, which accounts for a higher and more sustained AKT activation in 3T3-Gab1/DeltaSHP-2 cells relative to 3T3-Gab1 fibroblasts. Moreover, 3T3-Gab1/DeltaSHP-2 cells demonstrate a higher level of extracellular-regulated kinase 1 activation at early time points of EGF stimulation. However, there was an unexpected decrease in c-fos promoter induction in 3T3-Gab1/DeltaSHP-2 cells when compared with 3T3-Gab1 cells. Additionally, the 3T3-Gab1/DeltaSHP-2 cells show a reversion of the transformed phenotype, including fewer morphologic changes, an increase in stress fiber cytoskeletal organization, and a decrease in cell proliferation and anchorage independent growth. These results reveal that the Gab1/SHP-2 interaction is essential for cell growth and transformation but that this must occur through a novel pathway that is independent of extracellular-regulated kinase or AKT. On the basis of its role in growth and transformation, the Gab1/SHP-2 interaction may become an attractive target for the pharmacologic intervention of malignant cell growth.
View details for Web of Science ID 000220249100019
View details for PubMedID 15026337
Upon the addition of different growth factors and cytokines, the Gab1 docking protein is tyrosine phosphorylated and in turn activates different signaling pathways. On the basis of the large body of evidence concerning cross talk between the signaling pathways activated by growth factors and oxidative stress, we decided to investigate the role of Gab1 in oxidative injury. We stimulated wild-type mouse embryo fibroblasts (MEF) or MEF with a homozygous deletion of the Gab1 gene (-/- MEF) with H(2)O(2). Our results show that Gab1 is phosphorylated in a dose- and time-dependent manner after H(2)O(2) triggering. Gab1 then recruits molecules such as SHP2, phosphatidylinositol 3-kinase (PI3K), and Shc. Gab1 phosphorylation is sensitive to the Src family kinase inhibitor PP2. Furthermore, we demonstrate that Gab1 is required for H(2)O(2)-induced c-Jun N-terminal kinase (JNK) activation but not for ERK2 or p38 activation. Reconstitution of Gab1 in -/- MEF rescues JNK activation, and we find that this is dependent on the SHP2 binding site in Gab1. Cell viability assays reveal that Gab1 has a dual role in cell survival: a positive one through its interaction with PI3K and a negative one through its interaction with SHP2. This is the first report identifying Gab1 as a component in oxidative stress signaling and one that is required for JNK activation.
View details for DOI 10.1128/MCB.23.13.4471-4484.2003
View details for Web of Science ID 000183848200006
View details for PubMedID 12808090
With a view to their use in cancer therapy, we have produced rat monoclonal antibodies (MAbs) directed against 5 distinct epitopes (A-E) on the external domain of the wild-type human EGF receptor (EGFR). Here, we have investigated the relative binding and anti-tumour activity of our anti-EGFR MAbs against HC2 20d2/c cells, which have been engineered to overexpress the type-III mutated form of the human EGFR (EGFRvIII). We found that anti-EGFR MAbs that are the most effective antagonists of EGFR ligands (e.g., ICR16, ICR62 and ICR80) also bind to cells that overexpress the EGFRvIII. Although these antibodies are potent inhibitors of the growth of cells which express wild-type EGFR, they did not directly inhibit the growth in vitro of EGFRvIII expressing HC2 20d2/c cells, or the constitutive tyrosine kinase activity of this receptor. However, in the presence of human peripheral blood mononuclear cells (PBMC), the rat IgG2b MAb ICR62 induced strong antibody-dependent cell-mediated cytotoxicity (ADCC) against HC2 20d2/c cells in culture. Interestingly, MAb ICR62 also inhibited very effectively experimental lung metastases of HC2 20d2/c cells in athymic nude mice. Our results suggest that anti-EGFR MAb ICR62, which binds to the EGFRvIII, may have potential in the treatment of tumors which overexpress the EGFRvIII via immunological mechanisms such as ADCC. Since tumours that are EGFRvIII positive may also overexpress the wild-type EGFR, the use of anti-EGFR MAbs that target both wild-type and mutant receptors may have advantages over those that target only1form.
View details for DOI 10.1002/ijc.11055
View details for Web of Science ID 000182359500020
View details for PubMedID 12673691
The c-Jun NH(2)-terminal kinases (JNKs) have a role both in promoting apoptosis and tumorigenesis. The JNKs are encoded by three separate genes (JNK1, 2, and 3), which are spliced alternatively to create 10 JNK isoforms that are either M(r) 55,000 or 46,000 in size. However, the functional significance and distinct role for each splice variant remains unclear. We have noted previously that 86% of primary human glial tumors show activation of almost exclusively the M(r) 55,000 isoforms of JNK. To further study which isoforms are involved, we constructed glutathione S-transferase fusion proteins for all 10 JNK isoforms and examined kinase activity with or without the activating upstream kinase. Surprisingly, five JNK isoforms demonstrate autophosphorylation activity, and in addition, all four JNK2 isoforms (either M(r) 55,000 or 46,000) show a high basal level of substrate kinase activity in the absence of the upstream kinase, especially a M(r) 55,000 JNK2 isoform. Examination revealed autophosphorylation activity at the T-P-Y motif, which is critical for JNK activation, because a mutant lacking the dual phosphorylation sites did not show autophosphorylation or basal kinase activity. Using green fluorescence protein-JNK expression vectors, transient transfection into U87MG cells demonstrates that although the JNK1 isoforms localize predominantly to the cytoplasm, the JNK2 isoforms localize to the nucleus and are phosphorylated, confirming the constitutive activation seen in vitro. We then examined which JNK isoforms are active in glial tumors by performing two-dimensional electrophoresis. This revealed that the M(r) 55,000 isoforms of JNK2 are the principal active JNK isoforms present in tumors. Collectively, these results suggest that these constitutively active JNK isoforms play a significant role in glial tumors. Aside from epidermal growth factor receptor vIII, this is the only other kinase that has been shown to be basally active in glioma. The presence of constitutively active JNK isoforms may have implications for the design of inhibitors of the JNK pathway.
View details for Web of Science ID 000180290500039
View details for PubMedID 12517805
The epidermal growth factor receptor (EGFR) variant type III (variously called EGFRvIII, de2-7 EGFR or deltaEGFR) has an in-frame deletion of the extracellular domain and is found in numerous types of human tumors. Since EGFRvIII has been reported to be tumor-specific and has oncogenic potential, it is being investigated as a potential therapeutic target. Because the cell-specific expression of EGFRvIII in lung has not been well documented, we examined the expression of EGFRvIII in 76 non-small cell lung cancers (NSCLCs) and 10 non-neoplastic lung tissues by immunohistochemistry using a new monoclonal antibody specific for this variant receptor. We found a higher incidence (30 of 76, 39%) of enhanced EGFRvIII expression in NSCLC than previously described. Interestingly, the presence of EGFRvIII was also observed in several normal tissue components of lung (e.g., normal bronchial epithelium). Given the high prevalence of EGFRvIII in NSCLC, a newly developed phospho-specific (activated) EGFR antibody was employed for immunohistochemical analysis that permitted visualization of activated EGFR and/or EGFRvIII in tumors. This study presents evidence, for the first time, that EGFRvIII expressed in human tumors is phosphorylated and hence activated. Our results suggest that the sustained activation of EGFRvIII is implicated in the pathogenesis of NSCLC and thus EGFRvIII is a potential therapeutic target in this challenging disease.
View details for Web of Science ID 000182370000009
View details for PubMedID 12708474
The ERK pathway is typically associated with activation of the EGF receptor and has been shown to play a major role in promoting several tumor phenotypes. An analogous signaling module, the JNK pathway, has not been shown to be consistently activated by the EGF receptor but is instead more uniformly stimulated by cellular stresses and cytokines. The function of the JNK pathway in primary tumors is unclear as it has been implicated in both promoting apoptosis and cell growth in vitro, which may be a reflection of the cell lines chosen. Primary human brain tumors frequently show overexpression of the EGF receptor. To clarify the role of JNK in tumorigenesis, we have investigated the role of JNK in a large panel of primary human brain tumors and tumor derived cell lines. Here we present evidence that JNK has a major role in promoting tumorigenesis both in vivo and in vitro. Western blot analysis demonstrated that 86% (18 of 21) of primary brain tumors showed evidence of JNK activation but only 38% (8 of 21) showed evidence of ERK activation. Kinase assays revealed that 77% of brain tumor cell lines activated JNK in response to EGF (7 of 13) or had high levels of basal activity (3 of 13), whereas none of six normal cell lines analysed, including astrocytes, had these properties. Of several growth factors examined, EGF produced the highest level of JNK induction in tumor cell lines and the duration of activation was greater than that seen for ERK. Expression of a dominant-negative (dn) form of JNK potently inhibited EGF mediated anchorage independent growth and protection from cell death in two glial tumor cell lines. These findings demonstrate that enhanced JNK activation is frequently found in primary brain tumors and that this activation contributes to phenotypes related to transformation.
View details for DOI 10.1038/sj.onc.1205593
View details for Web of Science ID 000176975900002
View details for PubMedID 12140754
Cell surface adhesion molecules are crucial for the development and/or pathogenesis of various diseases including cancer. CD44 has received much interest as a major adhesion molecule that is involved in tumor progression. We have previously demonstrated that the ectodomain of CD44 undergoes proteolytic cleavage by membrane-associated metalloproteases in various tumor cell lines. The remaining membrane-bound CD44 cleavage product can be detected using antibodies against the cytoplasmic domain of CD44 (anti-CD44cyto antibody). However, the cleavage of CD44 in primary human tumors has not been investigated. Using Western blots with anti-CD44cyto antibody to assay human tumor tissues, we show that the CD44 cleavage product can be detected in 58% (42 of 72) of gliomas but not in normal brain. Enhanced CD44 cleavage was also found in 67% (28 of 42) of breast carcinomas, 45% (5 of 11) of non-small cell lung carcinomas, 90% (9 of 10) of colon carcinomas, and 25% (3 of 12) of ovarian carcinomas. Tumors expressing a CD44 splice variant showed a significantly higher incidence of enhanced CD44 cleavage. The wide prevalence of CD44 cleavage suggests that it plays an important role in the pathogenesis of human tumors.
View details for Web of Science ID 000173733500008
View details for PubMedID 11839564
CD44 is a widely distributed cell surface adhesion molecule and is implicated in diverse biological processes. However, the nature of intracellular signaling triggered by CD44 remains to be elucidated. Here, we show that CD44 undergoes sequential proteolytic cleavage in the ectodomain and intracellular domain, resulting in the release of a CD44 intracellular domain (ICD) fragment. Consequently, CD44ICD acts as a signal transduction molecule, where it translocates to the nucleus and activates transcription mediated through the 12-O-tetradecanoylphorbol 13-acetate-responsive element, which is found in numerous genes involved in diverse cellular processes. Expression of an uncleavable CD44 mutant as well as metalloprotease inhibitor treatment blocks CD44-mediated transcriptional activation. In search of the underlying mechanism, we have found that CD44ICD potentiates transactivation mediated by the transcriptional coactivator CBP/p300. Furthermore, we show that cells expressing CD44ICD produce high levels of CD44 messenger RNA, suggesting that the CD44 gene is one of the potential targets for transcriptional activation by CD44ICD. These observations establish a novel CD44 signaling pathway and shed new light on the functional link between proteolytic processing of an adhesion molecule at the cell surface and transcriptional activation in the nucleus.
View details for Web of Science ID 000172459900011
View details for PubMedID 11714729
Storage of unstained paraffin slides may lead to the deterioration of specimens and failure to detect cellular proteins immunohistochemically. Although the implication of age-induced alterations on multicenter immunohistochemical studies would be considerable, they have not been investigated previously. The current study was undertaken to examine the effect of this factor further and to explore new ways of overcoming the resultant shortcomings. The authors now report on the immunodetection of a host of antigens in similarly preserved unstained serial paraffin slides obtained from three centers using a panel of eight antibodies. Staining of recently prepared sections from the authors' centers resulted in similar strong patterns in seven of eight antibodies, with one antibody demonstrating variable immunoreactivity. However, storage of unstained paraffin sections at room temperature resulted in a variable but progressive decrease in expression of several tissue antigens. Although the loss in antigenicity was proportional to the length of storage, the effect was reversible if super antibody concentrations were used. The authors conclude that recently prepared paraffin sections from centers with similar fixation protocols have similar immunoreactivity and are suitable for use in comparative multicenter studies. However, in view of the delays that may attend tissue transportation during these projects, the authors suggest that test systems should be checked for age-induced antigen degradation by incubating sections with higher antibody concentrations.
View details for Web of Science ID 000170625800011
View details for PubMedID 11556755
B cell antigen receptor (BCR) signaling causes tyrosine phosphorylation of the Gab1 docking protein. This allows phosphatidylinositol 3-kinase (PI3K) and the SHP2 tyrosine phosphatase to bind to Gab1. In this report, we tested the hypothesis that Gab1 acts as an amplifier of PI3K- and SHP2-dependent signaling in B lymphocytes. By overexpressing Gab1 in the WEHI-231 B cell line, we found that Gab1 can potentiate BCR-induced phosphorylation of Akt, a PI3K-dependent response. Gab1 expression also increased BCR-induced tyrosine phosphorylation of SHP2 as well as the binding of Grb2 to SHP2. We show that the pleckstrin homology (PH) domain of Gab1 is required for BCR-induced phosphorylation of Gab1 and for Gab1 participation in BCR signaling. Moreover, using confocal microscopy, we show that BCR ligation can induce the translocation of Gab1 from the cytosol to the plasma membrane and that this requires the Gab1 PH domain as well as PI3K activity. These findings are consistent with a model in which the binding of the Gab1 PH domain to PI3K-derived lipids brings Gab1 to the plasma membrane, where it can be tyrosine-phosphorylated and then act as an amplifier of BCR signaling.
View details for Web of Science ID 000168081800110
View details for PubMedID 11278704
The Grb2-associated binder-1 (Gab1) is one of the major adapter molecules downstream of growth factor receptor signaling. Even though insulin causes tyrosine phosphorylation of Gab1, its role in insulin signaling has not been identified yet. We have demonstrated that insulin increased expression of early growth response gene-1 (egr-1), which is one of the most important transcription factors involved in cell proliferation and differentiation. In the present study, the possible role of Gab1 in insulin-induced egr-1 expression was studied using Rat1 fibroblasts expressing human insulin receptors and wildtype Gab1 (HIRc/Gab1(WT)), Gab1 with three tyrosines in the phosphatidylinositol (PI) 3'-kinase binding domain mutated to phenylalanine (HIRc/Gab1(DeltaPI3K)), or histidinol resistance only (HIRc/HIS). Insulin-induced egr-1 expression in HIRc/Gab1(DeltaPI3K) cells was much lower than in the other cells, as determined by Northern blot analysis. These results suggest that Gab1 is involved in the signaling pathway for insulin-induced egr-1 expression through increasing PI3'-kinase activity. The MAP kinase activity increased less with insulin treatment in HIRc/Gab1(DeltaPI3K) cells than in other cells. Inhibition of MAP kinase by the MEK inhibitor completely abolished insulin-induced egr-1 expression. These results suggest that Gab1 increases MAP kinase activity through its PI3'-kinase binding site, which then leads to egr-1 expression. Our results indicate that Gab1 is involved in the control of egr-1 expression regulated by insulin.
View details for Web of Science ID 000168518800004
View details for PubMedID 11403719
Epithelial morphogenesis is critical during development and wound healing, and alterations in this program contribute to neoplasia. Met, the hepatocyte growth factor (HGF) receptor, promotes a morphogenic program in epithelial cell lines in matrix cultures. Previous studies have identified Gab1, the major phosphorylated protein following Met activation, as important for the morphogenic response. Gab1 is a docking protein that couples the Met receptor with multiple signaling proteins, including phosphatidylinositol-3 kinase, phospholipase Cgamma, the adapter protein Crk, and the tyrosine specific phosphatase SHP-2. HGF induces sustained phosphorylation of Gab1 and sustained activation of extracellular signal-regulated kinase (Erk) in epithelial Madin-Darby canine kidney cells. In contrast, epidermal growth factor fails to promote a morphogenic program and induces transient Gab1 phosphorylation and Erk activation. To elucidate the Gab1-dependent signals required for epithelial morphogenesis, we undertook a structure-function approach and demonstrate that association of Gab1 with the tyrosine phosphatase SHP-2 is required for sustained Erk activation and for epithelial morphogenesis downstream from the Met receptor. Epithelial cells expressing a Gab1 mutant protein unable to recruit SHP-2 elicit a transient activation of Erk in response to HGF. Moreover, SHP-2 catalytic activity is required, since the expression of a catalytically inactive SHP-2 mutant, C/S, abrogates sustained activation of Erk and epithelial morphogenesis by the Met receptor. These data identify SHP-2 as a positive modulator of Erk activity and epithelial morphogenesis downstream from the Met receptor.
View details for Web of Science ID 000090094800020
View details for PubMedID 11046147
Epidermal growth factor receptor vIII (EGFRvIII) is a tumor-specific, ligand-independent, constitutively active variant of the EGFR. Its expression has been detected in gliomas and various other human malignancies. To more fully characterize the function and potential biological role of EGFRvIII in regulating cell proliferation and in tumorigenesis, we transfected EGFRvIII cDNA into a nontumorigenic, interleukin 3 (IL-3)-dependent murine hematopoietic cell line (32D cells). We observed 32D cells expressing high levels of EGFRvIII (32D/EGFRvIII P5) to be capable of abrogating the IL-3-dependent pathway in the absence of ligands. In contrast, the parental cells, 32D/EGFR, 32D/ErbB-4, and 32D/ErbB-2+ErbB-3 cells, all depended on IL-3 or EGF-like ligands for growth. 32D/EGFRvIII P5 cells subjected to long-term culture conditions in the absence of IL-3 revealed further elevation of EGFRvIII expression levels. These results suggested that the IL-3-independent phenotype is mediated by EGFRvIII. The level of expression is a critical driving force for the IL-3-independent phenotype. Dose-response analysis revealed 32D/EGFRvIII cells to require 500-fold higher concentrations (50 ng/ml) of EGF to further stimulate the EGF-mediated proliferation than in the 32D/EGFR cells (100 pg/ml). Similar effects were also observed in beta-cellulin-mediated proliferation. Moreover, 32D cells expressing high levels of EGFRvIII formed large tumors in nude mice, even when no exogenous EGF ligand was administered. In contrast, no tumors grew in mice injected with 32D/EGFR, 32D/ErbB-4, and 32D/ErbB-2+ErbB-3 cells or low-expressing clone 32D/EGFRvIII C2 cells or the parental 32D cells. The changes of the ligand specificity support the notion for an altered conformation of EGFRvIII to reveal an activated ligand-independent oncoprotein with tumorigenic activity analogous to v-erbB. These studies clearly demonstrate that EGFRvIII is capable of transforming a nontumorigenic, IL-3-dependent murine hematopoietic cell line (32D cells) into an IL-3-independent and ligand-independent malignant phenotype in vitro and in vivo. To delineate the biological significance of EGFRvIII in human breast cancer, we expressed EGFRvIII in the MCF-7 human breast cancer cell line. Expression of EGFRvIII in MCF-7 cells produced a constitutively activated EGFRvIII receptor. Expression of EGFRvIII in MCF-7 cells also elevated ErbB-2 phosphorylation, presumably through heterodimerization and cross-talk. These MCF-7/EGFRvIII transfectants exhibited an approximately 3-fold increase in colony formation in 1% serum with no significant effect observed at higher percentages of serum. A similar result was also seen in anchorage-dependent assays. Furthermore, EGFRvIII expression significantly enhanced tumorigenicity of MCF-7 cells in athymic nude mice with P < 0.001. Collectively, these results provide the first evidence that EGFRvIII could play a pivotal role in human breast cancer progression.
View details for Web of Science ID 000087434700055
View details for PubMedID 10850460
Earlier studies have demonstrated an unexplained depletion of the epidermal growth factor receptor (EGFR) protein expression in prostatic cancer. We now attribute this phenomenon to the presence of a variant EGFR (EGFRvIII) that is highly expressed in malignant prostatic neoplasms. In a retrospective study, normal, benign hyperplastic and malignant prostatic tissues were examined at the mRNA and protein levels for the presence of this mutant receptor. The results demonstrated that whilst EGFRvIII was not present in normal prostatic glands, the level of expression of this variant protein increased progressively with the gradual transformation of the tissues to the malignant phenotype. The selective association of high EGFRvIII levels with the cancer phenotype underlines the role that this mutant receptor may maintain in the initiation and progression of malignant prostatic growth, and opens the way for new approaches in the management of this disease including gene therapy.
View details for Web of Science ID 000084335900033
View details for PubMedID 10638988
The Gab1-docking protein has been shown to regulate phosphatidylinositol 3-kinase PI3K activity and potentiate nerve growth factor (NGF)-induced survival in PC12 cells. Here, we investigated the potential of Gab1 to induce neurite outgrowth and DNA synthesis, two other important aspects of NGF-induced neuronal differentiation of PC12 cells and NGF-independent survival. We generated a recombinant adenovirus encoding hemagglutinin (HA)-epitope-tagged Gab1 and expressed this protein in PC12 cells. HA-Gab1 was constitutively tyrosine-phosphorylated in PC12 cells and induced the phosphorylation of Akt/protein kinase B and p44/42 mitogen-activated protein kinase. HA-Gab1-stimulated a 10-fold increase in neurite outgrowth in the absence of NGF and a 5-fold increase in NGF-induced neurite outgrowth. HA-Gab1 also stimulated DNA synthesis and caused NGF-independent survival in PC12 cells. Finally, we found that HA-Gab1-induced neuritogenesis was completely suppressed by pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) activity and 50% suppressed by inhibition of PI3K activity. In contrast, HA-Gab1-stimulated cell survival was efficiently suppressed only by inhibition of both PI3K and MEK activities. These results indicate that Gab1 is capable of mediating differentiation, DNA synthesis, and cell survival and uses both PI3K and MEK signaling pathways to achieve its effects.
View details for Web of Science ID 000084382700068
View details for PubMedID 10601297
Stimulation of the hepatocyte growth factor receptor tyrosine kinase, Met, induces the inherent morphogenic program of epithelial cells. The multisubstrate binding protein Gab1 (Grb2-associated binder-1) is the major phosphorylated protein in epithelial cells following activation of Met. Gab1 contains a pleckstrin homology domain and multiple tyrosine residues that act to couple Met with multiple signaling proteins. Met receptor mutants that are impaired in their association with Gab1 fail to induce a morphogenic program in epithelial cells, which is rescued by overexpression of Gab1. The Gab1 pleckstrin homology domain binds to phosphatidylinositol 3,4, 5-trisphosphate and contains conserved residues, shown from studies of other pleckstrin homology domains to be crucial for phospholipid binding. Mutation of conserved phospholipid binding residues tryptophan 26 and arginine 29, generates Gab1 proteins with decreased phosphatidylinositol 3,4,5-trisphosphate binding, decreased localization at sites of cell-cell contact, and reduced ability to rescue Met-dependent morphogenesis. We conclude that the ability of the Gab1 pleckstrin homology domain to bind phosphatidylinositol 3,4,5-trisphosphate is critical for subcellular localization of Gab1 and for efficient morphogenesis downstream from the Met receptor.
View details for Web of Science ID 000083379400087
View details for PubMedID 10531383
Loss of all or part of one copy of chromosome 17p is very common in ovarian and breast tumors. OVCA1 is a candidate tumor suppressor gene mapping to a highly conserved region on chromosome 17p13.3 that shows frequent loss of heterozygosity in breast and ovarian carcinomas. Western blot analysis of extracts prepared from breast and ovarian carcinomas revealed reduced expression of OVCA1 compared with extracts from normal epithelial cells from these tissues. Subcellular localization studies indicate that OVCA1 is localized to punctate bodies scattered throughout the cell but is primarily clustered around the nucleus. Attempts to create cell lines that stably expressed OVCA1 from the cytomegalovirus promoter were generally unsuccessful in a variety of different cell lines. This reduction of colony formation was quantified in the ovarian cancer cell line A2780, where it was demonstrated that cells transfected with plasmids expressing OVCA1 had a 50-60% reduction in colony number as compared with appropriate controls, and only a few of these clones expressed OVCA1, albeit at low levels. The clones that expressed exogenous OVCA1 were found to have dramatically reduced rates of proliferation. Reduced growth rates correlated with an increased proportion of the cells in the G1 fraction of the cell cycle compared with the parental cell line and decreased levels of cyclin D1. The low levels of cyclin D1 appeared to be caused by an accelerated rate of cyclin D1 degradation. Overexpression of cyclin D1 was able to override OVCA1's suppression of clonal outgrowth. These results suggest that slight alterations in the level of OVCA1, such as would occur after reduction of chromosome 17p13.13 to hemizygosity, may result in cell cycle deregulation and promote tumorigenesis.
View details for Web of Science ID 000083028800036
View details for PubMedID 10519411
Stimulation of the hepatocyte growth factor (HGF) receptor tyrosine kinase, Met, induces mitogenesis, motility, invasion, and branching tubulogenesis of epithelial and endothelial cell lines in culture. We have previously shown that Gab1 is the major phosphorylated protein following stimulation of the Met receptor in epithelial cells that undergo a morphogenic program in response to HGF. Gab1 is a member of the family of IRS-1-like multisubstrate docking proteins and, like IRS-1, contains an amino-terminal pleckstrin homology domain, in addition to multiple tyrosine residues that are potential binding sites for proteins that contain SH2 or PTB domains. Following stimulation of epithelial cells with HGF, Gab1 associates with phosphatidylinositol 3-kinase and the tyrosine phosphatase SHP2. Met receptor mutants that are impaired in their association with Gab1 fail to induce branching tubulogenesis. Overexpression of Gab1 rescues the Met-dependent tubulogenic response in these cell lines. The ability of Gab1 to promote tubulogenesis is dependent on its pleckstrin homology domain. Whereas the wild-type Gab1 protein is localized to areas of cell-cell contact, a Gab1 protein lacking the pleckstrin homology domain is localized predominantly in the cytoplasm. Localization of Gab1 to areas of cell-cell contact is inhibited by LY294002, demonstrating that phosphatidylinositol 3-kinase activity is required. These data show that Gab1 is an important mediator of branching tubulogenesis downstream from the Met receptor and identify phosphatidylinositol 3-kinase and the Gab1 pleckstrin homology domain as crucial for subcellular localization of Gab1 and biological responses.
View details for Web of Science ID 000078697900017
View details for PubMedID 10022866
Gab1 is a member of the docking/scaffolding protein family which includes IRS-1, IRS-2, c-Cbl, p130(cas), and p62(dok). These proteins contain a variety of protein-protein interaction motifs including multiple tyrosine residues that when phosphorylated can act as binding sites for Src homology 2 (SH2) domain-containing signaling proteins. We show in the RAMOS human B cell line that Gab1 is tyrosine-phosphorylated in response to B cell antigen receptor (BCR) engagement. Moreover, tyrosine phosphorylation of Gab1 correlated with the binding of several SH2-containing signaling proteins to Gab1 including Shc, Grb2, phosphatidylinositol 3-kinase, and the SHP-2 tyrosine phosphatase. Far Western analysis showed that the SH2 domains of Shc, SHP-2, and the p85 subunit of phosphatidylinositol 3-kinase could bind directly to tyrosine-phosphorylated Gab1 isolated from activated RAMOS cells. In contrast, the Grb2 SH2 domain did not bind directly to Gab1 but instead to the Shc and SHP-2 associated with Gab1. We also show that Gab1 is present in the membrane-enriched particulate fraction of RAMOS cells and that Gab1/signaling protein complexes are found in this fraction after BCR engagement. Thus, tyrosine-phosphorylated Gab1 may recruit cytosolic signaling proteins to cellular membranes where they can act on membrane-bound targets. This may be a critical step in the activation of multiple BCR signaling pathways.
View details for Web of Science ID 000077008100082
View details for PubMedID 9804835
The newly identified insulin receptor (IR) substrate, Gab1 [growth factor receptor bound 2 (Grb2)-associated binder-1] is rapidly phosphorylated on several tyrosine residues by the activated IR. Phosphorylated Gab1 acts as a docking protein for Src homology-2 (SH2) domain-containing proteins. These include the regulatory subunit p85 of phosphatidylinositol 3-kinase and phosphotyrosine phosphatase, SHP-2. In this report, using a modified version of the yeast two-hybrid system, we localized which Gab1 phospho-tyrosine residues are required for its interaction with phosphatidylinositol 3-kinase and with SHP-2. Our results demonstrate that to interact with p85 or SHP-2 SH2 domains, Gab1 must be tyrosine phosphorylated by IR. Further, we found that Gab1 tyrosine 472 is the major site for association with p85, while tyrosines 447 and 589 are participating in this process. Concerning Gab1/SHP-2 interaction, only mutation of tyrosine 627 prevents binding of Gab1 to SHP-2 SH2 domains, suggesting the occurrence of a monovalent binding event. Finally, we examined the role of Gab1 PH (Pleckstrin homology) domain in Gab1/IR interaction and in Gab1 tyrosine phosphorylation by IR. Using the modified two-hybrid system and in vitro experiments, we found that the Gab1 PH domain is not important for IR/ Gab1 interaction and for Gab1 tyrosine phosphorylation. In contrast, in intact mammalian cells, Gab1 PH domain appears to be crucial for its tyrosine phosphorylation and association with SHP-2 after insulin stimulation.
View details for Web of Science ID 000074476700002
View details for PubMedID 9658397
Epidermal growth factor receptor (EGF) variant type III (EGFRvIII) is a constitutively active, naturally occurring mutation of the EGF receptor that is found in many types of human tumors. When overexpressed in NIH3T3 fibroblasts, EGFRvIII induces transformation by enhancing cell growth and reducing apoptosis. Analysis of downstream signaling pathways has revealed that extracellular signal-regulated kinase activity is down-regulated, raising doubt as to the significance of this pathway in promoting transformation. We investigated whether the c-Jun N-terminal kinase (JNK) pathway was affected by EGFRvIII. NIH3T3 cells expressing EGFRvIII exhibited a high basal level of JNK activity, which was not present in cells overexpressing the normal EGF receptor. Treatment of cells overexpressing EGFRvIII with inhibitors of the EGF receptor or phosphatidylinositol 3-kinase resulted in the down-regulation of JNK activity. Furthermore, the down-regulation of JNK activity was associated with a loss of properties related to transformation, and there was no evidence for JNK activity in the promotion of apoptosis in these cells. These findings implicate constitutive activation of the JNK pathway in transformation by EGFRvIII.
View details for Web of Science ID 000071736600045
View details for PubMedID 9446590
Decorin, a small leucine-rich proteoglycan, is capable of suppressing the growth of various tumor cell lines when expressed ectopically. In this report, we investigated the biochemical mechanism by which decorin inhibits cell cycle progression. In A431 squamous carcinoma cells, decorin proteoglycan or protein core induced a marked growth suppression, when either exogenously added or endogenously produced by a transgene. Decorin caused rapid phosphorylation of the EGF receptor and a concurrent activation of mitogen-activated protein (MAP) kinase signal pathway. This led to a protracted induction of endogenous p21, a potent inhibitor of cyclin-dependent kinases, and ultimate cell cycle arrest. Biglycan, a related proteoglycan, had no effect. Moreover, decorin activated the EGF receptor/MAP kinase/ p21 axis in cell lines of various histogenetic backgrounds. These results provide the first evidence that EGF and decorin converge functionally to regulate the cell cycle through activation of a common pathway which ultimately leads to growth suppression.
View details for Web of Science ID 000071668000016
View details for PubMedID 9435313
The most frequently found alteration of the epidermal growth factor receptor (EGFR) in human tumors is a deletion of exons 2-7. This receptor, termed EGFRvIII, can transform NIH 3T3 cells, and the frequent expression of this variant implies that it confers a selective advantage upon tumor cells in vivo. Although EGFRvIII is a constitutively activated tyrosine kinase, there is no increase in Ras.GTP levels and low levels of mitogen-activated protein kinase activity in NIH 3T3 cells expressing this variant. We investigated whether phosphatidylinositol (PI) 3-kinase was an effector in transformation by the EGFRvIII. High levels of PI 3-kinase activity were constitutively present in EGFRvIII-transformed cells and were dependent upon the kinase activity of the receptor. While mitogen-activated protein kinase activity was quickly down-regulated to basal levels after 12 h of continuous EGFR activation, there was a 3-fold increase in PI 3-kinase activity in cells expressing normal EGFR and an 8-fold increase in cells expressing EGFRvIII after 48 h. This increased activity may reflect enhanced binding to EGFRvIII and the presence of novel PI 3-kinase isoforms. Treatment with the PI 3-kinase inhibitors wortmannin and LY294002 blocked both anchorage-independent growth and growth in low serum media and also resulted in morphological reversion of EGFRvIII-transformed cells. These results support an essential role for PI 3-kinase in transformation by this EGFR variant.
View details for Web of Science ID 000071295600031
View details for PubMedID 9417065
Nerve growth factor (NGF) prevents apoptosis through stimulation of the TrkA receptor protein tyrosine kinase. The downstream activation of phosphatidylinositol 3-kinase (PI 3-kinase) is essential for the inhibition of apoptosis, although this enzyme does not bind to and is not directly activated by TrkA. We have found that the addition of NGF to PC-12 cells resulted in the phosphorylation of the Grb2-associated binder-1 (Gab1) docking protein and induced the association of several SH2 domain-containing proteins, including PI 3-kinase. A substantial fraction of the total cellular PI 3-kinase activity was associated with Gab1. PC-12 cells that overexpressed Gab1 show a decreased requirement for the amount of NGF necessary to inhibit apoptosis. The expression of a Gab1 mutant that lacked the binding sites for PI 3-kinase enhanced apoptosis and diminished the protective effect of NGF. Hence, Gab1 has a major role in connecting TrkA with PI 3-kinase activation and for the promotion of cell survival by NGF.
View details for Web of Science ID A1997YF39300033
View details for PubMedID 9356464
Hepatocyte growth factor/scatter factor is a multifunctional factor that induces mitogenesis, motility, invasion, and branching tubulogenesis of several epithelial and endothelial cell lines in culture. The receptor for hepatocyte growth factor has been identified as the Met-tyrosine kinase. Upon stimulation with hepatocyte growth factor, the Met beta subunit becomes highly phosphorylated on tyrosine residues, one of which, tyrosine 1356 within the carboxyl terminus, is crucial for dissociation, motility, and branching tubule formation in Madin-Darby canine kidney epithelial cells. Tyrosine 1356 forms a multisubstrate binding site for the Grb2 and Shc adaptor proteins, the p85 subunit of phosphatidylinositol 3'-kinase, phospholipase Cgamma, and a phosphatase, SHP2. To investigate additional signaling molecules that are activated by the Met receptor, we have identified hepatocyte growth factor-induced phosphoproteins in tubular epithelial cells. We have established that proteins of 100-130 kDa are highly phosphorylated following stimulation of epithelial cells and that one of these is the Grb2-associated binding protein Gab1, a possible insulin receptor substrate-1-like signal transducer. We show that Gab1 is the major substrate for the Met kinase in vitro and in vivo. Association of Gab1 with Met requires a functional Grb2 binding site involving tyrosine 1356 and to a lesser extent tyrosine 1349. Met receptor mutants that fail to induce branching tubulogenesis are impaired in their ability to interact with Gab1, suggesting that Gab1 may play a role in these processes.
View details for Web of Science ID A1997XR22100076
View details for PubMedID 9252406
The Tpr-Met oncoprotein consists of the catalytic kinase domain of the hepatocyte growth factor/scatter factor receptor tyrosine kinase (Met) fused downstream from sequences encoded by the tpr gene. Tpr-Met is a member of a family of tyrosine kinase oncoproteins generated following genomic rearrangement and has constitutive kinase activity. We have previously demonstrated that a single carboxyl-terminal tyrosine residue, Tyr489, is essential for efficient transformation of Fr3T3 fibroblasts by Tpr-Met and forms a multisubstrate binding site for Grb2, phosphatidylinositol 3' kinase, phospholipase Cgamma, SHP2, and an unknown protein of 110 kDa. A mutant Tpr-Met protein that selectively fails to bind Grb2 has reduced transforming activity, implicating pathways downstream of Grb2 in Tpr-Met mediated cell transformation. We show here that the 110-kDa Tpr-Met substrate corresponds to the recently identified Grb2-associated protein, Gab1. Moreover, we show that tyrosine phosphorylation of the Cbl protooncogene product as well as Gab1 required Tyr489 and correlated with the ability of Tpr-Met to associate with Grb2 and to transform cells, providing evidence that pathways downstream of Gab1 and/or Cbl may play a role in Tpr-Met-mediated cell transformation.
View details for Web of Science ID A1997XQ05900079
View details for PubMedID 9242692
The type III EGF receptor (EGFRvIII) is the result of an in-frame deletion from nucleotides 275 to 1075 in the EGF receptor cDNA sequence creating a novel epitope at the fusion junction. This spontaneously occurring alteration is found in a high percentage of primary human brain, breast, lung and ovarian tumors. We have explored whether a peptide derived from the fusion junction could serve as the basis for an antitumor vaccine. Preimmunization of mice with this peptide substantially inhibited tumor formation by cells expressing EGFRvIII. Tumor cell inoculation followed by immunization could also enhance the regression of existing tumors. Antibody production was elicited in animals that was highly specific for the novel epitope and also a CTL response that was mediated by CD8+ T lymphocytes. The alteration present in EGFRvIII could serve as the basis for an antitumor vaccine with potentially wide application in humans.
View details for Web of Science ID A1997WT72300005
View details for PubMedID 9108438
The epidermal growth factor receptor (EGFR) activates formation of the phospholipid signal messenger phosphatidic acid (PA) on ligand binding. We explored the effects of chronic EGF stimulation on cellular PA in NIH3T3 cells expressing intact EGFR a mutant EGFR (EGFRvIII). The presence of EGFRvIII increased PA levels to twice those induced by chronic EGFR activation. Fatty acid methyl ester analysis revealed a marked increase in oleic acid containing PA. No apparent increase in phospholipase D (PLD) activity was detected, and diacylglycerol (DAG) kinase assays demonstrated a marked preference for dioleoyl DAG in the presence of activated EGFR or EGFRvIII. Levels of PA which were lower than would be predicted by DAG kinase activation are explained by increased phosphatidate phosphohydrolase activity. Specific inhibitors of EGFR kinase and DAG kinase suppressed DAG kinase activation and PA production by EGFRvIII. EGFR kinase activation by chronic exposure to ligand or by deletional mutation stimulates formation of a specific form of signalling PA.
View details for Web of Science ID A1997WN21300023
View details for PubMedID 9125112
Mutation of the autophosphorylation sites of receptor protein-tyrosine kinases alters ligand dependent internalization and down-regulation, indicating a critical role for these sites in receptor processing. Currently, no differences in receptor processing based on an individual autophosphorylation site have been defined. By using a glutathione S-transferase fusion protein containing the src homology 2 domains of phospholipase C-gamma1 to specifically recognize tyrosine 992 on the EGF receptor (Tyr(P)992), we have found differences in this subpopulation of receptors. Following EGF stimulation, the number of Tyr(P)992 receptors increased 2-fold over receptors identified by an antibody that recognizes activated EGF receptors (alpha-Act. EGFR) in A431 cells. Confocal fluorescence microscopy showed that Tyr(P)992 receptors underwent endocytosis at a slower rate and did not rapidly concentrate in juxtanuclear bodies. Tyr(P)992 receptors were associated with more SOS, Ras-GTPase activating protein, phosphatidylinositol 3-kinase, and SHPTP2/syp, but less Grb2, than receptors in the general population, and these receptors were more heavily phosphorylated than the general population of active receptors. These findings suggest that autophosphorylation status is relevant to the endocytosis, degradation, and effector molecule interaction of individual EGF receptors. Further investigations based on phosphorylation status should provide new insights into how receptor protein-tyrosine kinase signaling is regulated.
View details for Web of Science ID A1997WH01900033
View details for PubMedID 9020117
SH2/SH3 adaptor proteins are essential components of the signal transduction pathways initiated by tyrosine kinases. Nck is a ubiquitously expressed adaptor protein whose function has been enigmatic. We performed confocal microscopy to localize Nck in NIH3T3 and A431 cells. Surprisingly, Nck was identified in the nucleus as well as the cytoplasm with no visible change in localization due to PDGF or EGF stimulation. Western blot analysis of nuclear and cytosolic fractions confirmed that there was no translocation in response to growth factor and that tyrosine phosphorylation was specific to only cytosolic Nck. Far Western blot analysis with either Nck, the SH2 domain, or the SH3 domains revealed differential binding in nuclear and cytosolic lysates, indicating specific binding partners for each subcellular location. The major target of c-Src during mitosis is SAM68, a RNA-binding protein ordinarily localized to the nucleus. SAM68 was identified as a nuclear specific binding partner of Nck in both nonmitotic and mitotic cells. Several tyrosine kinases can be found in the nucleus but their signal transduction remains undefined. The discovery of an adaptor protein in the nucleus suggests there are signal transduction mechanisms within the nucleus that recapitulate those found in the cytoplasm.
View details for Web of Science ID A1997WC81800010
View details for PubMedID 9010224
An amino-truncated variant form of the epidermal growth factor receptor (EGFRvIII) has been identified in human brain, breast, lung and ovarian tumors. We have found that overexpression of this mutant EGF receptor in NIH3T3 cells results in transformation as a result of the activation of the receptor kinase via ligand-independent dimerization. Transformation was correlated with tyrosine phosphorylation of only a subset of the proteins observed in cells overexpressing the normal EGF receptor. This suggested that further studies on cells expressing the EGFRvIII might provide insights into the pathways most relevant to transformation. In clones expressing high levels of mutant EGF receptor, the levels of both Grb2 and SHC were decreased. Despite this decrease, much of the endogenous Grb2 immunoprecipitated with EGFRvIII. Interestingly, no increase in ras-GTP loading was found in clones expressing the EGFRvIII and MAP kinase assays indicated only a small increase in activity. These results indicate that high-level expression of the EGFRvIII induces down-regulation of the ras-MAP kinase pathway and that other components involved in EGF receptor signal transduction may play a greater role in neoplastic transformation by the EGFRvIII.
View details for Web of Science ID A1996UX31900010
View details for PubMedID 8700557
A second tumor suppressor locus on 17p that is distinct from TP53 has been identified in brain, breast, lung, and ovarian tumors. Using allelic loss mapping and positional cloning methods, we have recently identified two novel genes, which we refer to as OVCA1 and OVCA2, that map to 17p13.3. The two genes are ubiquitously expressed and encode proteins of 443 and 227 amino acids, respectively, with no known functional motifs. Sequence comparison of OVCA1 and OVCA2 revealed extensive sequence identity and similarity to hypothetical proteins from Saccharomyces cerevisiae, Caenorhabditis elegans, and Rattus species. Northern blot analysis reveals that OVCA1 and OVCA2 mRNA were expressed in normal surface epithelial cells of the ovary, but the level of this transcript is significantly reduced or is undetectable in 92% (11/12) of the ovarian tumors and tumor cell lines analyzed. The location, high degree of amino acid conservation, and reduced expression in ovarian tumors and tumor cell lines suggest that decreased expression of these two genes contributes to ovarian tumorigenesis and should be considered candidate tumor suppressor genes.
View details for Web of Science ID A1996UG75600010
View details for PubMedID 8616839
The protein Grb2 plays a central role in signalling by receptor protein-tyrosine kinases, where its SH2 domain binds to the receptor and its two SH3 domains link to effectors. One target effector is Sos, so Grb2 links receptor protein-tyrosine kinases with the Ras signalling pathway. The SH3 domains can also couple to other signalling proteins, including Vav, c-Abl and dynamin. We have identified several bands in glial and medulloblastoma tumours that are recognized by Grb2 but these did not correspond to any known protein. Here we use recombinant Grb2 to isolate a complementary DNA called Gab1 (for Grb2-associated binder-1). Gab1 shares amino-acid homology and several structural features with IRS-1 (insulin-receptor substrate-1; refs 6,7), is a substrate of the EGF and insulin receptors, and can act as a docking protein for several SH2-containing proteins. Over-expression of Gab1 enhances cell growth and results in transformation. We conclude that Gab1 is a new protein in EGF and insulin receptor signalling which could integrate signals from different systems.
View details for Web of Science ID A1996TU69300056
View details for PubMedID 8596638
A paradigm has been established whereby mutant tyrosine kinase receptors such as the v-erbB and v-fms gene products function as oncoproteins in the absence of ligand. A spontaneously occurring deletional mutant of the human epidermal growth factor receptor (EGFR-vIII) has been isolated from astrocytic neoplasms and transforms NIH3T3 cells in the absence of ligand. The EGFRvIII is constitutively complexed with the majority of cellular GRB2, suggesting a link to the Ras-Mitogen-activated protein (MAP) kinase pathway (D. Moscatello, R. B. Montgomery, P. Sundareshan, H. McDanel, M. Y. Wong, and A. J. Wong, submitted for publication). In this report, we document that expression of EGFRvIII in fibroblasts is associated with downstream activation of mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (MEK) and modest activation of p42 and p44 MAP kinases. The presence of EGFRvIII suppresses activation of p42 and p44 MAP kinases by phorbol 12-myristate 13-acetate (PMA) and serum; however, MEK activation by PMA is not suppressed by EGFRvIII. Basal and PMA-stimulated MAP kinase activity in EGFRvIII-transfected cells is augmented by the tyrosine phosphatase inhibitor sodium vanadate. EGFR-vIII is capable of transducing downstream signals through MAP kinase as evidenced by activation of cytoplasmic phospholipase A2 at levels similar to that induced by intact EGFR. Our results suggest that EGFR-vIII constitutively activates downstream signal transduction through MAP kinase, and this chronic stimulation of the MAP kinase pathway may represent one means by which mutant EGFR transduces an oncogenic signal.
View details for Web of Science ID A1995TL67500054
View details for PubMedID 8530489
The epidermal growth factor receptor has received much interest as a target for various antineoplastic agents, but a complication is that many normal tissues also express this receptor. We have previously identified in human glial tumors an 801-bp in-frame deletion within the epidermal growth factor receptor gene that created a novel epitope at the junction. By using Western blot assays with a mutant-specific antibody as a rapid and sensitive means for detecting this alteration in primary human tumors, it was found that 57% (26 of 46) of high-grade and 86% (6 of 7) of low-grade glial tumors, but not normal brain, express this protein. This altered receptor was also present in 66% (4 of 6) of pediatric gliomas and 86% (6 of 7) of medulloblastomas, 78% (21 of 27) of breast carcinomas, and 73% (24 of 32) of ovarian carcinomas. The fact that this receptor is frequently found in tumors but not in normal tissue makes it an attractive candidate for various antitumor strategies.
View details for Web of Science ID A1995TG21100012
View details for PubMedID 7585629
Amplification and rearrangement of the epidermal growth factor receptor (EGFR) gene are characteristics of many types of tumors. One class of EGFR mutations, EGFRvIII, is characterized by an in-frame deletion resulting in a truncated external domain of the receptor. EGFRvIII was first identified in a subset of gliomas and has since been found in some non-small cell lung carcinomas and breast carcinomas. mAbs specific for this variant form of EGFR but unreactive with the wild-type EGFR have been reported from our laboratory. This study further characterizes three of these antibodies. We determined, via radiolabeling techniques and immunofluorescence microscopy, that, after cell binding in vitro, the anti-EGFRvIII-specific mAbs internalize at 37 degrees C. Furthermore, subsequent to internalization, the antibodies were processed intracellularly, presumably by lysosomal degradation. We also examined the use of an alternative radiolabeling procedure that uses nonmetabolizable radio-iodinated tyramine cellobiose. Our results show that the tyramine cellobiose labeling method allows for greater tumor cell retention of radiolabel in vitro (76% for tyramine cellobiose and 27% for Iodo-Gen after 24 h). Paired-label biodistribution studies in athymic mice indicate that anti-EGFRvIII mAb L8A4 localizes specifically to EGFRvIII-expressing tumor xenografts with a maximum of 34.3 +/- 7.6% injected dose/g when labeled using tyramine cellobiose compared with a maximum of 14.9 +/- 4.3% injected dose/g using Iodo-Gen; similar results were obtained with mAb H10. These results suggest that the anti-EGFRvIII mAbs may serve as potential carriers for radioconjugate- and immunotoxin-based therapies for tumors expressing EGFRvIII.
View details for Web of Science ID A1995RX11400032
View details for PubMedID 7671250
Despite molecular biological advances in understanding human cancers, translation into therapy has been less forthcoming; targeting neoplastic cells still requires that tumor-specific markers, preferably those on the cell surface, be identified. The epidermal growth factor receptor (EGFR) exists in a deletion-mutant form, EGFRvIII, which has been identified by genetic and immunological means in a subset of gliomas and non-small cell lung carcinomas. Specific polyvalent antisera to the extracellular portion of the variant were readily induced, but immunization using a synthetic linear peptide representing the unique EGFRvIII primary sequence has been unsuccessful in mice or macaques. We report here five specific monoclonal antibodies (mAbs) developed through long-term immunization protocols using the EGFRvIII-specific synthetic peptide and the intact variant in different formats that maintained secondary and tertiary conformation. These mAbs identify the EGFRvIII on the cell surface with relatively high affinity (KA range, 0.13 to 2.5 x 10(9) M-1) by live cell Scatchard analysis. These mAbs are specific for EGFRvIII as determined by RIA, ELISA, Western blot, analytical flow cytometry, autophosphorylation, and immunohistochemistry. Isolating specific mAbs enabled us to analyze normal and neoplastic human tissue and establish that EGFRvIII is truly tumor specific for subsets of breast carcinomas and for previously reported non-small cell lung carcinomas and gliomas. Also, this receptor is not expressed by any normal human tissues thus far examined, including elements of the peripheral, central nervous, and lymphoid systems. With mAbs, we identified a higher incidence of EGFRvIII positivity in gliomas than previously described and identified an EGFRvIII-positive subset of breast tumors; also, we observed that the EGFRvIII epitope is not expressed in normal tissues, and we demonstrated the localizing and therapeutic potential of the mAbs for tumors expressing this epitope. Our observations strongly warrant development of this mAb-antigen system as therapy for breast, lung, and central nervous system tumors.
View details for Web of Science ID A1995RH80200034
View details for PubMedID 7606735
A polymorphism at codon 36 in exon 4 of the p53 gene was identified by single strand conformation polymorphism (SSCP) analysis and direct sequencing of genomic DNA PCR products. The polymorphic allele, present in the heterozygous state in genomic DNAs of four of 100 individuals (4%), changes the codon 36 CCG to CCA, eliminates a FinI restriction site and creates a BccI site. Including this polymorphism there are four known polymorphisms in the p53 coding sequence.
View details for Web of Science ID A1994MW24700041
View details for PubMedID 8302598
Cancer biologists now have a unified concept to guide their search for specific genetic abnormalities and new therapeutic approaches: The genes and proteins that participate in the conversion of normal into malignant cells are also involved in the key process that converts extracellular signals into intracellular events that culminate in division and growth.
View details for Web of Science ID A1993LM16800018
View details for PubMedID 8325909
The development of novel immunotherapy strategies for non-small cell lung cancer (NSCLC) will be facilitated by the identification of tumor-specific targets. Although the epidermal growth factor receptor (EGFR) is overexpressed in many cases of NSCLC, its wide distribution in normal tissue may limit its suitability as an immunotherapeutic target. However, mutations within the EGFR that are unique to malignancies may provide specific targets for immunotherapeutic intervention. For example, one mutant form, the type III deletion mutant of the EGFR, that has been identified in glioblastomas contains a novel peptide sequence in its extracellular domain which is detectable by anti-peptide antisera. In this study, the prevalence of this type of mutation of the EGFR in NSCLC was determined. Thirty-two frozen sections of primary NSCLC were examined by immunocytochemistry to determine the presence of native and mutated EGFR. Native EGFR was overexpressed in 12 of the 32 sections and a diffuse cellular distribution of the EGFR type III deletion mutation was identified in five (16%) of the specimens (2 of 13 squamous, 2 of 2 mixed, 0 of 10 adenocarcinoma, and 1 of 7 undifferentiated). This mutated EGFR was not detected in sections of normal breast, lung, skin, ovary, colon, kidney, endometrium, and placenta. The type III EGFR deletion mutant, expressed in some cases of NSCLC, may be a molecularly defined, tumor-specific antigen in lung cancer.
View details for Web of Science ID A1993LM67700001
View details for PubMedID 8391918
The epidermal growth factor receptor (EGFR) gene is amplified in 40% of malignant gliomas, and the amplified genes are frequently rearranged. We have characterized the genetic alterations associated with these rearrangements in five malignant gliomas. In one tumor the rearrangement resulted in the deletion of most of the extracytoplasmic domain of the receptor, resulting in a hybrid mRNA between new sequences and the truncated EGFR sequence. The predicted amino acid sequence of the protein from this tumor was remarkably similar to that described for several viral erbB oncogenes. Four other tumors were noted to have internal deletions of the EGFR gene. These rearrangements brought about in-frame deletions affecting either of two cysteine-rich domains in the extracytoplasmic portion of the molecule. The clonal nature of these alterations, and the fact that identical alterations were seen in more than one tumor, suggests a role for these mutant receptor proteins in tumorigenesis. Further, these studies document the existence of tumor-specific cell surface molecules resulting from somatic mutation.
View details for Web of Science ID A1992HL81600094
View details for PubMedID 1557402
Malignant human glioma D-298 MG amplifies a rearranged epidermal growth factor receptor (EGFR) gene (c-erbB proto-oncogene), resulting in an in-frame deletion of 83 amino acids in domain IV of the extracellular domain of the EGFR. EGF and transforming growth factor-a (TGF-a) bound to the mutant EGFR with high affinity and enhanced the intrinsic mutant EGFR kinase activity. The mutant EGFR was capable of transducing EGF-stimulated glioma cell proliferation and invasiveness in an in vitro three-dimensional spheroid model. The deletion-mutant EGFR in D-298 MG is capable of being activated by growth factor; this suggests that overexpression of this mutant EGFR protein rather than structural alteration may be the more significant biologic event.
View details for Web of Science ID A1991GB27300090
View details for PubMedID 1678600
Both permanent cultured cell lines and athymic mouse xenografts were established from two human glioblastomas. Biopsies from D-245 MG and D-270 MG contained amplified and rearranged epidermal growth factor receptor (EGFR) genes. Although the gene amplification and rearrangement seen originally was maintained in the xenografts, cultured cell lines established from these biopsies lost the amplified rearranged genes in vitro. Analysis of these cell lines and 11 additional permanent human glioma cell lines with normal EGFR gene copy number showed from 2.7 x 10(3) to 4.1 x 10(5) high affinity EGFRs/cell by radioreceptor assay. The RNase A protection assay showed minimal differences in the quantity of EGFR mRNA among the 13 glioma lines, while the D-245 MG and D-270 MG xenografts expressed approximately 10-20 times as much EGFR mRNA as the corresponding cell lines. Immunoprecipitation of EGFR from these lines, including D-245 MG and D-270 MG, demonstrated only the intact Mr 170,000 Da form, while truncated Mr 145,000 Da and 100,000 Da EGFR proteins were immunoprecipitated from the D-270 MG and D-245 MG xenografts, respectively. These studies demonstrate that gliomas with amplification of the EGFR gene are capable of establishing in culture but that the amplified rearranged genes are not maintained. Possible explanations are that the abnormal genes are lost during serial passage or that the cells with amplified rearranged genes only represent a minor subpopulation of cells, which are unable to grow in culture. In either case, these observations suggest that high expression and structural abnormalities of EGFR proteins generated by amplification and rearrangement of the EGFR gene provide a growth advantage for gliomas in vivo but not in vitro.
View details for Web of Science ID A1990EM16500051
View details for PubMedID 2253244
We have investigated human gliomas that amplify and rearrange the epidermal growth factor receptor gene, with generation of an in-frame deletion mutation of 802 nucleotides in the external domain. This in-frame deletion mutation generates a local amino acid sequence at the fusion junction of what normally were distant polypeptide sequences in the intact epidermal growth factor receptor. This 14-amino acid peptide was chemically synthesized, coupled to keyhole limpet hemocyanin, and used as an immunogen in rabbits. The elicited antibody reacted specifically with the fusion peptide in ELISA. The anti-fusion junction peptide antibody was purified by passage of the antiserum over a peptide affinity column with acidic elution. The purified antibody selectively bound the glioma deletion mutant as compared to the intact epidermal growth factor receptor as assessed by immunocytochemistry, immunofluorescence, immunoprecipitation with gel electrophoresis, and binding experiments using radioiodinated antibody. These data indicate that it is feasible to generate site-specific anti-peptide antibodies that are highly selective for mutant proteins in human tumors. The anti-peptide antibody described here, and other mutation site-specific antibodies, should be ideal candidates for tumor immunoimaging and immunotherapy.
View details for Web of Science ID A1990DG09200039
View details for PubMedID 1693434
Previous characterization of GLI, a gene found to be amplified and expressed in a subset of human brain tumors, revealed the presence of five tandem zinc fingers related to those of Krüppel (Kr), a Drosophila segmentation gene of the gap class. We have used the GLI cDNA as a molecular probe to isolate related sequences from the human genome. Partial characterization of six related loci, including sequence determination, expression studies, and chromosome localization, revealed that each locus could encode a separate finger protein. The predicted proteins all had similar H-C links, i.e., a conserved stretch of 9 amino acids connecting the C-terminal histidine of one finger to the N-terminal cysteine of the next. On the basis of amino acid sequence and intron-exon organization, the genes could be placed into one of two subgroups: the GLI subgroup (with the consensus finger amino acid sequence [Y/F]XCX3GCX3[F/Y]X5LX2HX3-4H[T/S]GEKP) or the Kr subgroup (with the consensus finger amino acid sequence [Y/F]XCX2CX3FX5LX2HXRXHTGEKP). Unlike GLI or Kr, most of the newly isolated genes were expressed in many adult tissues. The predicted proteins probably control the expression of other genes and, by analogy with Kr and GLI, may be important in human development, tissue-specific differentiation, or neoplasia.
View details for Web of Science ID A1988P431500014
View details for PubMedID 2850480
Gene amplification occurs in 45-50% of malignant human gliomas (MHG). In the present study, 64 genetically characterized gliomas were evaluated to determine if tumors with amplification of the epidermal growth factor receptor (EGFR), N-myc, c-myc, or gli genes had distinctive histopathologic features. There was no significant difference in age (p = 0.10) or gender (p = 0.78) between patients whose tumors contained amplified genes and those whose tumors did not exhibit this characteristic. Although the patients with amplified genes in their tumors survived slightly longer than patients whose tumors had no detectable gene amplification, these differences were not statistically significant (p = 0.21). The 28 tumors with amplification included 24/48 (50%) glioblastoma multiforme, 2/6 (33%) anaplastic astrocytomas and 2/5 (40%) gliosarcomas. No amplification was seen in one oligodendroglioma, three anaplastic mixed gliomas or one giant cell glioblastoma multiforme. Necrosis and endothelial proliferation were equally prevalent among tumors with and without amplification. Comparison of tumors with gene amplification and tumors without this characteristic revealed similar distributions of most morphologic cells types. Although prominent perivascular lymphocytic infiltrates were more frequent in tumors without amplification, this association was of borderline significance statistically. In situ hybridization of tumors with amplification using an EGFR mRNA probe showed intense labeling of different neoplastic cell types including fibrillary and protoplasmic astrocytes, gemistocytes, anaplastic cells, and multinucleated giant cells. Non-neoplastic cells such as hyperplastic endothelium within the tumors did not express detectable EGFR mRNA. These studies demonstrate that (a) cells with quite different morphology within the same tumor can contain the same genetic alteration; (b) tumors of identical histological appearance may have arisen and evolved by different molecular mechanisms; and (c) molecular analyses are necessary to evaluate gene amplification in MHG since this characteristic cannot be accurately predicted by the morphologic or clinical criteria used in this study.
View details for Web of Science ID A1988N498700001
View details for PubMedID 3367154
Xenografts from eight malignant human gliomas were established in athymic mice and were used to study amplification and expression of the epidermal growth factor receptor (EGFR) gene. Tissue identity between biopsy and xenografts was confirmed by karyotypic profiles, which showed that each glioma xenograft retained structural abnormalities, including double minute chromosomes, present in the parent glioma. EGFR gene amplification was found in six of the eight glioma biopsies and their corresponding xenografts. Expression of the EGFR gene was measured by Scatchard analysis, affinity reactions, immunoprecipitations, Western immunoblots, and immunocytochemistry; significant expression of the EGFR gene was only detectable in xenografts with EGFR gene amplification. Moreover, five of the six xenografts with EGFR gene amplification demonstrated structural alterations of the EGFR gene, which was associated with low-molecular-weight EGFR proteins. These xenografts represent an excellent tissue source and in vivo model system for characterizing the epidermal growth factor receptor in malignant human gliomas.
View details for Web of Science ID A1988M876900042
View details for PubMedID 3258189
Biopsies of 33 malignant human gliomas were karyotyped and evaluated for amplification (more than eight gene copies per cell) of the epidermal growth factor receptor (EGFR), N-myc, c-myc, and gli genes by Southern blot analysis. Fifteen of 33 tumors showed amplification of EGFR, none had amplified c-myc, one tumor had amplified N-myc, and one had amplification of gli. Thirteen of the 16 (81%) evaluable tumors with gene amplification contained double minutes (DM), and only four of 16 (25%) tumors without demonstrable amplification contained these structures. Polysomy for chromosome #7, in contrast, occurred in 58% of tumors with EGFR amplification and 53% of tumors without amplification of the gene. Structural abnormalities of 7p occurred in two tumors with EGFR amplification and in one tumor without amplification of this gene. These studies suggest that DM are the usual locus for amplified genes (usually EGFR) in human glioma biopsies, but that structural abnormalities of 7p may be associated with EGFR amplification in a small proportion of these tumors. The presence of polysomy 7, however, probably is unrelated to amplification of the EGFR gene.
View details for Web of Science ID A1987K678300021
View details for PubMedID 3478127
Primary malignant gliomas from 63 patients were analyzed to determine the relationship between amplification of the gene encoding the epidermal growth factor receptor (EGFR) and expression of the corresponding mRNA. Twenty-four tumors were found to have amplified the EGFR gene and amplification of other genes occurred in three additional tumors. Hybridization with synthetic RNA probes was used to quantitate mRNA levels in situ. All 24 tumors with amplification of the EGFR gene had high levels of expression of this gene, while none of the 39 tumors without amplification had increased levels. This shows that, in human gliomas, large increases in the expression of the EGFR gene are invariably associated with alterations in gene structure.
View details for Web of Science ID A1987K314300061
View details for PubMedID 3477813
A gene, termed gli, was identified that is amplified more than 50-fold in a malignant glioma. The gene is expressed at high levels in the original tumor and its derived cell line and is located at chromosome 12 position (q13 to q14.3). The gli gene is a member of a select group of cellular genes that are genetically altered in primary human tumors.
View details for Web of Science ID A1987G661400029
View details for PubMedID 3563490
The relationship of the copy numbers of the c-myc and N-myc oncogenes to tumor formation and progression was studied in small cell carcinoma of the lung. When 96 neoplastic lesions from 45 patients were examined, these lesions could be grouped into three categories: high copy (tumors with greater than 3 copies of the N-myc or c-myc gene per haploid genome), middle copy (1.5 to 3 copies per genome), and normal copy. Fourteen of the patients had middle copy tumors, but this was almost always a result of chromosome duplication rather than the amplification of a small genetic locus. In contrast, five patients had high copy tumors, with the increased copy number in each case due to gene amplification. The amplification did not occur in a heterogeneous fashion within individual patients, since all metastatic lesions from patients with high copy lung tumors were also high copy, while none of 41 metastatic lesions from the other patients were high copy. These data suggest that gene amplification is an important step in neoplastic growth in a subset of patients with small cell carcinoma of the lung and that this genetic event occurs relatively early (before metastasis) in this subset.
View details for Web of Science ID A1986D234300039
View details for PubMedID 3014659
Fibrinogen fragment D, which is heterogeneous, has several important biological functions. Human fibrinogen fragments D94 (molecular weight, 94,000), D78 (78,000), and E (52,000) were purified. Fragments D78 and D94 but not purified fibrinogen or fragment E specifically caused disorganization of bovine aortic endothelial cells cultured as monolayers. Within 2 hours of exposure to pathophysiological concentrations of fragment D, the confluent endothelial cells retracted from each other and projected pseudopodia. These disturbed cells subsequently became rounded and detached from the substrate. The actin present in stress fibers in stationary monolayer cells was diffusely redistributed in cells with fragment D-induced alterations in morphology. This effect was not observed in monolayers of kidney epithelial cells. The results demonstrate a specific effect of fibrinogen fragment D on the disorganization of cultured vascular endothelial cell monolayers and suggest that fragment D plays a role in the pathogenesis of syndromes with vascular endothelial damage.
View details for Web of Science ID A1985ADG0600031
View details for PubMedID 4038818
We have purified myosin from human erythrocytes using methods similar to that for other cytoplasmic myosins with a yield of about 500 micrograms/100 ml of packed cells. It consists of a 200-kDa heavy chain and light chains of 26- and 19.5 kDa and therefore differs from the isozyme in platelets which has light chains of 20- and 15 kDa. At low ionic strength, the myosin forms short bipolar filaments like those of platelet myosin. Eight of eight monoclonal antibodies to platelet myosin also bind to erythrocyte myosin. Like most myosins, it has a high ATPase activity in the presence of Ca2+ or EDTA, but is inhibited by Mg2+. Myosin light-chain kinase transfers 1 phosphate from ATP to the 20-kDa light chain, and this stimulates the actin-activated ATPase. Thus, myosin may play a role in shape changes in the erythrocytes.
View details for Web of Science ID A1985TZ26100003
View details for PubMedID 3155518
Fluorescence microscopy with 7-nitrobenz-2-oxa-3-diazole phallacidin was used to survey vertebrate tissues for actin filament bundles comparable to the stress fibers of cultured cells. Such bundles were found only in vascular endothelial cells. Like the stress fibers of cultured cells, these actin filament bundles were stained in a punctate pattern by fluorescent antibodies to both alpha-actinin and myosin. The stress fibers were oriented parallel to the direction of blood flow and were prominent in endothelial cells from regions exposed to high-velocity flow, such as the left ventricle, aortic valve, and aorta. Actin bundles may help the endothelial cell to withstand hemodynamic stress.
View details for Web of Science ID A1983QB45400041
View details for PubMedID 6681677
View details for PubMedID 11696458
View details for PubMedID 11550299