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1.
J Transl Sci ; 5(2)2019 Apr.
Article in English | MEDLINE | ID: mdl-30906574

ABSTRACT

INTRODUCTION: Tumors over-expressing the human epithelial receptor 2 (HER2) or exhibiting amplification or mutation of its proto-oncogene have a poorer prognosis. Using trastuzumab and/or other HER2 targeted therapies can increase overall survival in patients with HER2(+) tumors making it critical to accurately identify patients who may benefit. We report on a Phase 0 study of the imaging agent, 111In-CHX-A"-DTPA trastuzumab, in patients with known HER2 status to evaluate its safety and biodistribution and to obtain preliminary data regarding its ability to provide an accurate, whole-body, non-invasive means to determine HER2 status. METHODS: 111In-CHX-A"-DTPA trastuzumab was radiolabeled on-site and slowly infused into 11 patients who underwent single (n=5) or multiple (n=6) ɣ-camera (n=6) and/or SPECT (n=8) imaging sessions. RESULTS: No safety issues were identified. Visual and semi-quantitative imaging data were concordant with tissue HER2 expression profiling in all but 1 patient. The biodistribution showed intense peak liver activity at the initial imaging timepoint (3.3h) and a single-phase clearance fit of the average time-activity curve (TAC) estimated t1/2=46.9h (R2=0.97; 95%CI 41.8 to 53h). This was followed by high gastrointestinal (GI) tract activity peaking by 52h. Linear regression predicted GI clearance by 201.2h (R2 =0.96; 95%CI 188.5 to 216.9h). Blood pool had lower activity with its maximum on the initial images. Non-linear regression fit projected a t1/2=34.2h (R2 =0.96; 95%CI 25.3 to 46.3h). Assuming linear whole-body clearance, linear regression projected complete elimination (x-intercept) at 256.5hr (R2=0.96; 95%CI 186.1 to 489.2h). CONCLUSION: 111In-CHX-A"-DTPA trastuzumab can be safely imaged in humans. The biodistribution allowed for visual and semiquantitative analysis with results concordant with tissue expression profiling in 10 of 11 patients. Advances in Knowledge and Implications for Patient Care Using readily available components and on-site radiolabeling 111In-CHX-A"-DTPA trastuzumab SPECT imaging may provide an economical, non-invasive means to detect HER2 over-expression.

2.
Oncogene ; 25(49): 6497-509, 2006 Oct 19.
Article in English | MEDLINE | ID: mdl-16702950

ABSTRACT

The overexpression or mutation of tyrosine kinases (TKs), such as the epidermal growth factor receptor (EGFR), can lead to the development of cancer. The most common mutation of the EGFR in glioblastomas is the deletion of exons 2-7 known as the EGFRvIII. This mutant receptor cannot bind EGF but, instead, is constitutively active. The Cbl family of ubiquitin ligases (Cbl, Cbl-b, and Cbl-c) targets the activated EGFR for degradation. As the EGFRvIII is transforming, we investigated whether it could be downregulated by the Cbl proteins. The overexpression of all three Cbl proteins resulted in the ubiquitination and degradation of the EGFRvIII. As with the wild-type EGFR, the TK-binding domain and the RING finger of Cbl-b are sufficient for the downregulation of the EGFRvIII. Also, we found that Cbl-b is recruited to the EGFRvIII and inhibits the transformation of NIH 3T3 cells by the EGFRvIII. Mutation of the Cbl-binding site (Y1045F) in the EGFRvIII inhibits its ubiquitination and downregulation by Cbl-b and enhances its ability to transform. Furthermore, the EGFR TK inhibitor, AG 1478, prevents the downregulation of the EGFRvIII by the Cbl proteins and antagonizes the ability of an immunotoxin directed against the EGFRvIII to kill cells expressing this receptor. In conclusion, the EGFRvIII does not transform by escaping regulation by Cbl proteins and this activation-induced downregulation of the EGFRvIII has an important role in mediating the toxicity of anti-EGFRvIII immunotoxins.


Subject(s)
ErbB Receptors/metabolism , Proto-Oncogene Proteins c-cbl/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , CHO Cells , Cell Survival/drug effects , Cell Transformation, Neoplastic , Cells, Cultured , Cricetinae , Down-Regulation , Humans , Mice , NIH 3T3 Cells , Protein Binding , Protein Processing, Post-Translational , Quinazolines , Transfection , Tyrphostins/pharmacology , Ubiquitin/metabolism
3.
Cell Death Differ ; 11(5): 527-41, 2004 May.
Article in English | MEDLINE | ID: mdl-14765134

ABSTRACT

The majority of ovarian cancer cells are resistant to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Subtoxic concentrations of the semisynthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) enhanced TRAIL-mediated apoptosis in ovarian cancer cell lines but not in immortalized nontumorigenic ovarian epithelial cells. The enhancement of TRAIL-mediated apoptosis by 4HPR was not due to changes in the levels of proteins known to modulate TRAIL sensitivity. The combination of 4HPR and TRAIL enhanced cleavage of multiple caspases in the death receptor pathway (including the two initiator caspases, caspase-8 and caspase-9). The 4HPR and TRAIL combination leads to mitochondrial permeability transition, significant increase in cytochrome c release, and increased caspase-9 activation. Caspase-9 may further activate caspase-8, generating an amplification loop. Stable overexpression of Bcl-xL abrogates the interaction between 4HPR and TRAIL at the mitochondrial level by blocking cytochrome c release. As a consequence, a decrease in activation of caspase-9, caspase-8, and TRAIL-mediated apoptosis occurs. These results indicate that the enhancement in TRAIL-mediated apoptosis induced by 4HPR is due to the increase in activation of multiple caspases involving an amplification loop via the mitochondrial-death pathway. These findings offer a promising and novel strategy for the treatment of ovarian cancer.


Subject(s)
Apoptosis/drug effects , Fenretinide/toxicity , Membrane Glycoproteins/metabolism , Mitochondria/metabolism , Retinoid X Receptors/metabolism , Tumor Necrosis Factor-alpha/metabolism , Apoptosis/physiology , Apoptosis Regulatory Proteins , Caspases/metabolism , Cytochromes c/metabolism , DNA Fragmentation/drug effects , Female , Humans , Ovary/metabolism , TNF-Related Apoptosis-Inducing Ligand , Tumor Cells, Cultured
4.
Cancer Res ; 61(12): 4892-900, 2001 Jun 15.
Article in English | MEDLINE | ID: mdl-11406568

ABSTRACT

We investigated whether combined treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and trastuzumab could enhance the specific killing of cells that overexpress the erbB-2 receptor. The combination resulted in an enhancement of TRAIL-mediated apoptosis in all cell lines overexpressing erbB-2 receptor compared with either reagent alone. In contrast, there was no effect in cell lines with low levels of the erb-B2 receptor. Trastuzumab treatment resulted in down-regulation of the erbB-2 receptor in all erbB-2-overexpressing cell lines. Similar enhancement of TRAIL toxicity was observed when the erbB-2 receptor was down-regulated using antisense oligodeoxynucleotides. Down-regulation of the erbB-2 receptor protein by trastuzumab or antisense oligodeoxynucleotides decreased Akt kinase activation but not mitogen-activated protein kinase activation. Down-regulation of Akt kinase activity by a phosphatidylinositol 3'-kinase inhibitor (LY294002) also resulted in enhancement of TRAIL-mediated apoptosis. Expression of a constitutively active form of Akt kinase in an erbB-2-overexpressing cell line completely abrogated the increase in TRAIL-mediated apoptosis by trastuzumab and significantly reduced the biological effect of either reagent alone. Therefore, down-regulation of the erbB-2 receptor by trastuzumab enhances TRAIL-mediated apoptosis by inhibiting Akt kinase activity. These data suggest that the combination of trastuzumab and TRAIL may allow enhanced therapeutic efficacy and specificity in the treatment of erbB-2-overexpressing tumors.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Breast Neoplasms/pathology , Membrane Glycoproteins/pharmacology , Ovarian Neoplasms/pathology , Protein Serine-Threonine Kinases , Receptor, ErbB-2/biosynthesis , Tumor Necrosis Factor-alpha/pharmacology , Antibodies, Monoclonal, Humanized , Apoptosis/physiology , Apoptosis Regulatory Proteins , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Down-Regulation/drug effects , Drug Synergism , Female , Humans , Membrane Glycoproteins/genetics , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Receptor, ErbB-2/genetics , Receptor, ErbB-2/physiology , Recombinant Fusion Proteins/pharmacology , TNF-Related Apoptosis-Inducing Ligand , Trastuzumab , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/genetics
5.
J Biol Chem ; 276(29): 27677-84, 2001 Jul 20.
Article in English | MEDLINE | ID: mdl-11375397

ABSTRACT

Cbl proteins function as ubiquitin protein ligases for the activated epidermal growth factor receptor and, thus, negatively regulate its activity. Here we show that Cbl-b is ubiquitinated and degraded upon activation of the receptor. Epidermal growth factor (EGF)-induced Cbl-b degradation requires intact RING finger and tyrosine kinase binding domains and requires binding of the Cbl-b protein to the activated EGF receptor (EGFR). Degradation of both the EGFR and the Cbl-b protein is blocked by lysosomal and proteasomal inhibitors. Other components of the EGFR-signaling complex (i.e. Grb2 and Shc) are also degraded in an EGF-induced Cbl-b-dependent fashion. Our results suggest that the ubiquitin protein ligase function of Cbl-b is regulated by coordinated degradation of the Cbl-b protein along with its substrate. Furthermore, the data demonstrate that Cbl-b mediates degradation of multiple proteins in the EGFR-signaling complex.


Subject(s)
Adaptor Proteins, Signal Transducing , Carrier Proteins/physiology , ErbB Receptors/metabolism , Phosphoproteins/physiology , Signal Transduction/physiology , Ubiquitin-Protein Ligases , Carrier Proteins/chemistry , Humans , Hydrolysis , Phosphoproteins/chemistry , Protein Conformation , Proto-Oncogene Proteins c-cbl , Tumor Cells, Cultured , Ubiquitins/metabolism
6.
Gynecol Oncol ; 81(3): 380-90, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11371126

ABSTRACT

OBJECTIVES: The aim of this study was to investigate whether TNF-related apoptosis-inducing ligand (TRAIL) alone or in combination with chemotherapy could induce apoptosis in ovarian cancer cells resistant to chemotherapy. METHODS: Twelve chemoresistant epithelial cancer cell lines were treated with each chemotherapeutic drug alone (cisplatin, doxorubicin, or paclitaxel), TRAIL alone, or the combination. Toxicity was assessed using the MTS assay. To assess whether growth inhibition was due to apoptosis, TUNEL assay, caspase activation (measured by caspase-3 and PARP cleavage), and the sub G0/G1 fraction of cells were measured. Synergism was confirmed by fractional inhibition and dose-effect analysis. Expression of death and decoy receptors was studied by immunoblotting and an RNase protection assay. Statistical comparison of means was performed using Student's t test. RESULTS: The majority of the chemoresistant cells were also resistant to TRAIL alone. In contrast, the combination of TRAIL and chemotherapy resulted in a significant growth inhibition over a wide range of concentrations. This interaction was synergistic by dose-effect analysis. Flow cytometry demonstrated a significant increase in the fraction of apoptotic cells by the combination compared to each reagent alone. A significant enhancement in caspase and PARP cleavage was observed upon treatment with the combination. Finally, no correlation between induction of apoptosis and level of death receptors was found. CONCLUSIONS: The data suggest that almost all the ovarian cancer cells, which are resistant to chemotherapy, are also resistant to TRAIL. The combination of TRAIL and chemotherapy overcomes this resistance in a synergistic fashion by triggering caspase-mediated apoptosis. The combination of TRAIL and chemotherapy could be useful as a therapy for chemoresistant ovarian cancers.


Subject(s)
Antineoplastic Agents/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Membrane Glycoproteins/pharmacology , Ovarian Neoplasms/drug therapy , Tumor Necrosis Factor-alpha/pharmacology , Antineoplastic Agents/administration & dosage , Apoptosis/physiology , Apoptosis Regulatory Proteins , Caspase 3 , Caspases/metabolism , Cell Division/drug effects , Cisplatin/administration & dosage , Cisplatin/pharmacology , Doxorubicin/administration & dosage , Doxorubicin/pharmacology , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Drug Synergism , Female , Humans , Membrane Glycoproteins/administration & dosage , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Paclitaxel/administration & dosage , Paclitaxel/pharmacology , Poly(ADP-ribose) Polymerases/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptors, TNF-Related Apoptosis-Inducing Ligand , Receptors, Tumor Necrosis Factor/biosynthesis , Receptors, Tumor Necrosis Factor/genetics , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/pharmacology , TNF-Related Apoptosis-Inducing Ligand , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/administration & dosage
7.
Breast J ; 7(6): 378-87, 2001.
Article in English | MEDLINE | ID: mdl-11843848

ABSTRACT

The noninvasive technique of nipple aspiration as a potential source of biomarkers of breast cancer risk was evaluated. The feasibility of performing mutagenesis assays, amplifying DNA, and performing protein electrophoresis on nipple aspirate fluid was explored. A tool was developed to measure the level of discomfort, if any, from this procedure. Twenty-five healthy women (20 premenopausal and 5 postmenopausal) were enrolled. Fluid was obtained using a modified breast pump. Premenopausal women were scheduled for four to six weekly aspirations, and postmenopausal women were scheduled for one to two weekly aspirations. Mutagenesis assays were performed using the Salmonella (Ames) assay. DNA amplification of several microsatellite regions was carried out using polymerase chain reaction. Protein was quantified, and two-dimensional protein electrophoresis was performed. Overall, fluid was obtained from 80% of the women, and the level of discomfort was minimal. Acid hydrolysis of one sample resulted in mutagenicity; all six nonhydrolyzed samples were not mutagenic. The ability to amplify DNA ranged from 34% to 96%, depending on length of the microsatellite region examined. The average protein concentration was 71 microg/mL. Two-dimensional protein electrophoresis was successfully performed on samples from two subjects. Nipple aspiration is a simple technique and is easily learned and well tolerated, which yields a reagent useful for a variety of investigations. This technique may facilitate the identification and application of biomarkers for future breast cancer risk assessment and chemopreventive protocols.


Subject(s)
Breast Neoplasms/diagnosis , Cytodiagnosis/methods , DNA/analysis , Exudates and Transudates/cytology , Nipples/metabolism , Suction , Adult , Biomarkers , Electrophoresis, Gel, Two-Dimensional , Exudates and Transudates/metabolism , Feasibility Studies , Female , Humans , Microsatellite Repeats , Middle Aged , Mutagenicity Tests , Pain Measurement , Postmenopause , Premenopause , Proteins/analysis
8.
Mol Cell Biol ; 20(18): 6638-45, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10958661

ABSTRACT

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) (Apo2 ligand [Apo2L]) is a member of the TNF superfamily and has been shown to have selective antitumor activity. Although it is known that TRAIL (Apo2L) induces apoptosis and activates NF-kappaB and Jun N-terminal kinase (JNK) through receptors such as TRAIL-R1 (DR4) and TRAIL-R2 (DR5), the components of its signaling cascade have not been well defined. In this report, we demonstrated that the death domain kinase RIP is essential for TRAIL-induced IkappaB kinase (IKK) and JNK activation. We found that ectopic expression of the dominant negative mutant RIP, RIP(559-671), blocks TRAIL-induced IKK and JNK activation. In the RIP null fibroblasts, TRAIL failed to activate IKK and only partially activated JNK. The endogenous RIP protein was detected by immunoprecipitation in the TRAIL-R1 complex after TRAIL treatment. More importantly, we found that RIP is not involved in TRAIL-induced apoptosis. In addition, we also demonstrated that the TNF receptor-associated factor 2 (TRAF2) plays little role in TRAIL-induced IKK activation although it is required for TRAIL-mediated JNK activation. These results indicated that the death domain kinase RIP, a key factor in TNF signaling, also plays a pivotal role in TRAIL-induced IKK and JNK activation.


Subject(s)
Apoptosis , Membrane Glycoproteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Proteins/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , Apoptosis Regulatory Proteins , Cell Line , Enzyme Activation , Fibroblasts/cytology , HeLa Cells , Humans , I-kappa B Kinase , JNK Mitogen-Activated Protein Kinases , Ligands , Membrane Glycoproteins/genetics , Mice , Mutagenesis , Protein Serine-Threonine Kinases/genetics , Proteins/genetics , Proteins/physiology , Receptor-Interacting Protein Serine-Threonine Kinases , Receptors, TNF-Related Apoptosis-Inducing Ligand , Receptors, Tumor Necrosis Factor/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction , TNF Receptor-Associated Factor 2 , TNF-Related Apoptosis-Inducing Ligand , Tumor Necrosis Factor-alpha/genetics
9.
Nature ; 403(6766): 211-6, 2000 Jan 13.
Article in English | MEDLINE | ID: mdl-10646608

ABSTRACT

The signalling thresholds of antigen receptors and co-stimulatory receptors determine immunity or tolerance to self molecules. Changes in co-stimulatory pathways can lead to enhanced activation of lymphocytes and autoimmunity, or the induction of clonal anergy. The molecular mechanisms that maintain immunotolerance in vivo and integrate co-stimulatory signals with antigen receptor signals in T and B lymphocytes are poorly understood. Members of the Cbl/Sli family of molecular adaptors function downstream from growth factor and antigen receptors. Here we show that gene-targeted mice lacking the adaptor Cbl-b develop spontaneous autoimmunity characterized by auto-antibody production, infiltration of activated T and B lymphocytes into multiple organs, and parenchymal damage. Resting cbl-b(-/-) lymphocytes hyperproliferate upon antigen receptor stimulation, and cbl-b(-/-) T cells display specific hyperproduction of the T-cell growth factor interleukin-2, but not interferon-gamma or tumour necrosis factor-alpha. Mutation of Cbl-b uncouples T-cell proliferation, interleukin-2 production and phosphorylation of the GDP/GTP exchange factor Vav1 from the requirement for CD28 co-stimulation. Cbl-b is thus a key regulator of activation thresholds in mature lymphocytes and immunological tolerance and autoimmunity.


Subject(s)
Adaptor Proteins, Signal Transducing , Carrier Proteins/physiology , Lymphocyte Activation , Phosphoproteins/physiology , T-Lymphocytes/immunology , Ubiquitin-Protein Ligases , Animals , Antigens, CD/biosynthesis , Autoantibodies/biosynthesis , Autoimmunity/genetics , B-Lymphocytes/immunology , Carrier Proteins/genetics , Carrier Proteins/metabolism , Female , Gene Targeting , Lymph Nodes/immunology , Lymph Nodes/pathology , Male , Mice , Mice, Inbred C57BL , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphorylation , Proto-Oncogene Proteins c-cbl , Receptors, Antigen, T-Cell/immunology , Self Tolerance , Spleen/immunology , Spleen/pathology , Tyrosine/metabolism
10.
Cell Signal ; 12(11-12): 769-79, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11152963

ABSTRACT

Expression of the src homology 3 (SH3)-encoding, expressed in tumorigenic astrocytes (SETA) gene is associated with astrocyte transformation in culture and tumors in the adult brain. SETA binds to the apoptosis regulator apoptosis-linked gene 2 (ALG-2) interacting protein 1 (AIP1), and modulates apoptosis in astrocytes. The predicted protein structure of SETA revealed two SH3 domains, while related proteins were reported to have three. Here we report the identification of an additional SH3 domain N-terminal to the previously identified SETA sequence. Yeast two-hybrid screening of a p53(-/-) astrocyte cDNA library with this SH3 domain identified a novel gene, SETA binding protein 1 (SB1), with 55% amino acid identity to the renal tumor antigen, NY-REN-45. In vitro confrontation and co-immunoprecipitation experiments confirmed the binding of SB1 to SETA. Evidence that SETA binds to the CD2 protein, the proto-oncogene c-Cbl, and the signal transduction molecule Grb2, and can dimerize via its C-terminal coiled coil (CC) domain is also presented.


Subject(s)
Adaptor Proteins, Signal Transducing , Carrier Proteins/metabolism , Neoplasm Proteins/chemistry , Neoplasm Proteins/metabolism , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Ubiquitin-Protein Ligases , src Homology Domains , Amino Acid Sequence , Animals , Antigens, Neoplasm/chemistry , Astrocytes/metabolism , CD2 Antigens/metabolism , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cells, Cultured , Dimerization , Exons/genetics , GRB2 Adaptor Protein , Gene Deletion , Gene Library , Mice , Molecular Sequence Data , Neoplasm Proteins/genetics , Nerve Tissue Proteins/genetics , Precipitin Tests , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Tertiary , Proteins/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-cbl , Rats , Sequence Alignment , Tumor Cells, Cultured , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/physiology , Two-Hybrid System Techniques
11.
Breast Cancer Res Treat ; 64(2): 211-9, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11194457

ABSTRACT

Most breast cancer cell lines are resistant to TNF-related apoptosis inducing ligand (TRAIL) induced apoptosis. In sensitive breast cancer cell lines TRAIL rapidly induces the cleavage and activation of caspases leading to the subsequent cleavage of downstream caspase substrates. In contrast, there is no caspase activation in the resistant cell lines. The transcription factor NF-KB can inhibit apoptosis induced by a variety of stimuli including activation of death receptors. We investigated whether NF-kappaB contributes to the resistance of breast cancer cells to TRAIL induced apoptosis. All of the resistant breast cancer cell lines expressed NF-kappaB and had detectable NF-kappaB activity in nuclear extracts prior to treatment with TRAIL. Upon TRAIL treatment, a significant increase in NF-kappaB activity was seen in most of the cell lines. To directly test if NF-kappaB activity contributes to the resistance of these cell lines to TRAIL, we transiently transfected the resistant cell lines with an inhibitor of NF-kappaB (IkappaBdeltaN) and measured TRAIL induced apoptosis in control and transfected cells. All of the resistant cell lines tested showed an increase in TRAIL induced apoptosis when transfected with the IKBdeltaN. These results demonstrate that TRAIL resistant breast cancer cells fail to rapidly activate the apoptotic machinery but they do activate NF-kappaB. Inhibition of NF-kappaB activity increases the sensitivity to TRAIL mediated apoptosis in resistant cells. These results suggest that agents which inhibit NF-kappaB should increase the clinical efficacy of TRAIL in breast cancer cells.


Subject(s)
Breast Neoplasms/pathology , Membrane Glycoproteins/pharmacology , NF-kappa B/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Apoptosis , Apoptosis Regulatory Proteins , Drug Interactions , Female , Humans , TNF-Related Apoptosis-Inducing Ligand , Transfection , Tumor Cells, Cultured/drug effects
12.
Mol Cell Biol Res Commun ; 1(3): 216-20, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10425229

ABSTRACT

The retinoblastoma tumor suppressor protein (RB) has been shown to play a role in regulating the eukaryotic cell cycle, promoting cellular differentiation, and modulating programmed cell death. Although regulation of RB tumor suppressor activity is mediated by reversible phosphorylation, an additional posttranslational modification involves the cleavage of 42 residues from the carboxy terminus of RB during the onset of drug-induced or receptor-mediated apoptosis. We now demonstrate that a recombinant p100cl RB species localizes to the nucleus where it may retain wildtype "pocket" protein binding activity. In addition, using immunocytochemistry, we show that cleavage of the endogenous RB protein occurs in vivo in human cells and that p100cl is predominantly retained within the nuclear compartment of cells during early apoptosis. We also show that the carboxy-terminal cleavage of RB is detected immediately following caspase-3 and PARP cleavage during FAS-mediated apoptosis of MCF10 cells. These findings suggest that this cleavage event may be a component of a downstream cascade during programmed cell death.


Subject(s)
Apoptosis , Cell Nucleus/pathology , Retinoblastoma Protein/metabolism , Cell Compartmentation , Cell Nucleus/metabolism , Humans , Peptide Fragments/metabolism , Recombinant Proteins/metabolism , Tumor Cells, Cultured
13.
Mol Cell Biol Res Commun ; 2(2): 111-8, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10542134

ABSTRACT

Studies in C. elegans and Drosophila melanogaster suggest that cbl proteins are inhibitors of epidermal growth factor receptor (EGFR) function. Here we describe that overexpression of cbl-b, a homologue of the c-cbl protooncogene, inhibits EGFR-induced apoptosis in MDA-MB-468 breast cancer cells. Overexpression of cbl-b results in a shortened duration of EGFR activation upon EGF stimulation. This is demonstrated by decreased amounts of phosphorylated EGFR as well as by inhibition of multiple downstream signaling pathways. The inhibition of signaling by cbl-b results from increased ubiquitination and degradation of the activated EGFR. The inhibitory effects of cbl-b overexpression on apoptosis and on EGFR signaling are reversed by blocking proteosomal degradation of the EGFR. These data demonstrate that the mechanism by which cbl-b inhibits EGFR-induced apoptosis is by activation-dependent degradation of the EGFR. They imply that this mechanism may be a general one whereby cbl proteins regulate intracellular signaling.


Subject(s)
Adaptor Proteins, Signal Transducing , Apoptosis/physiology , Carrier Proteins/metabolism , ErbB Receptors/physiology , Phosphoproteins/metabolism , Proto-Oncogene Proteins/metabolism , Ubiquitin-Protein Ligases , Ubiquitins/metabolism , Acetylcysteine/analogs & derivatives , Acetylcysteine/pharmacology , Apoptosis/drug effects , Breast Neoplasms , Carrier Proteins/genetics , Cysteine Endopeptidases/drug effects , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Epidermal Growth Factor/pharmacology , ErbB Receptors/drug effects , Humans , Mitogen-Activated Protein Kinase Kinases/drug effects , Mitogen-Activated Protein Kinase Kinases/metabolism , Multienzyme Complexes/drug effects , Multienzyme Complexes/metabolism , Phosphoproteins/genetics , Proteasome Endopeptidase Complex , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-cbl , Signal Transduction , Tumor Cells, Cultured
14.
Oncogene ; 18(22): 3365-75, 1999 Jun 03.
Article in English | MEDLINE | ID: mdl-10362357

ABSTRACT

We have cloned a new human gene, cbl-3, which encodes a protein with marked homology to the cbl family of proteins. The predicted protein encoded by this gene retains the conserved phosphotyrosine binding domain (PTB) in the N-terminal and the zinc finger but is significantly shorter (MW 52.5 kDa) than the other mammalian cbl proteins. The protein lacks the extensive proline rich domain and leucine zipper seen in c-cbl and cbl-b and structurally most resembles the C. elegans and Drosophila cbl proteins. The gene is ubiquitously expressed with highest expression in the aerodigestive tract, prostate, adrenal gland, and salivary gland. The protein is phosphorylated and recruited to the EGFR upon EGF stimulation and inhibits EGF stimulated MAP kinase activation. In comparison to the other mammalian cbl proteins (e.g. cbl-b), cbl-3 interacts with a restricted range of proteins containing Src Homology 3 regions. An alternatively spliced form of the cbl-3 protein was also identified which deletes a critical region of the PTB domain and which does not interact with the EGFR nor inhibit EGF stimulated MAP kinase activation. These data demonstrate that cbl-3, a novel mammalian cbl protein, is a regulator of EGFR mediated signal transduction.


Subject(s)
Adaptor Proteins, Signal Transducing , Chromosomes, Human, Pair 19 , Drosophila Proteins , Proteins/genetics , Proteins/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Ubiquitin-Protein Ligases , Amino Acid Sequence , Base Sequence , Binding Sites , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Carrier Proteins/genetics , Chromosome Mapping , Cloning, Molecular , Enzyme Activation , ErbB Receptors/metabolism , Gene Expression Regulation , Humans , Molecular Sequence Data , Phosphoproteins/genetics , Phosphorylation , Proto-Oncogene Proteins c-cbl , Sequence Homology, Amino Acid , Signal Transduction , src Homology Domains
15.
Oncogene ; 18(10): 1855-66, 1999 Mar 11.
Article in English | MEDLINE | ID: mdl-10086340

ABSTRACT

The role of cbl-b in signaling by the epidermal growth factor receptor (EGFR) was studied and compared with c-cbl. We demonstrate in vivo, that cbl-b, like c-cbl, is phosphorylated and recruited to the EGFR upon EGF stimulation and both cbl proteins can bind to the Grb2 adaptor protein. To investigate the functional role of cbl proteins in EGFR signaling, we transfected cbl-b or c-cbl into 32D cells overexpressing the EGFR (32D/EGFR). This cell line is absolutely dependent on exogenous IL-3 or EGF for sustained growth. 32D/EGFR cells overexpressing cbl-b showed markedly inhibited growth in EGF compared to c-cbl transfectants and vector controls. This growth inhibition by cbl-b was the result of a dramatic increase in the number of cells undergoing apoptosis. Consistent with this finding, cbl-b overexpression markedly decreased the amplitude and duration of AKT activation upon EGF stimulation compared to either vector controls or c-cbl overexpressing cells. In addition, the duration of EGF mediated MAP kinase and Jun kinase activation in cells overexpressing cbl-b is shortened. These data demonstrate that cbl-b inhibits EGF-induced cell growth and that cbl-b and c-cbl have distinct roles in EGF mediated signaling.


Subject(s)
ErbB Receptors/metabolism , Proto-Oncogene Proteins/metabolism , Signal Transduction , Ubiquitin-Protein Ligases , Animals , Cell Line, Transformed , Epidermal Growth Factor/metabolism , Epidermal Growth Factor/pharmacology , Humans , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-cbl , Rabbits
16.
Cancer Res ; 59(3): 734-41, 1999 Feb 01.
Article in English | MEDLINE | ID: mdl-9973225

ABSTRACT

Expression and function of the TRAIL apoptotic pathway was investigated in normal and malignant breast epithelial cells. Glutathione-S-transferase (GST)-TRAIL extracellular domain fusion proteins were produced to analyze TRAIL-induced apoptosis. Only GST-TRAIL constructs containing regions homologous to the Fas self-association and ligand binding domains could induce apoptosis. GST-TRAIL induced significant (>90%) apoptosis in just one of eight normal and one of eight malignant breast cell lines. All other lines were relatively resistant to TRAIL-induced apoptosis. Activating TRAIL receptors DR4 and DR5 were expressed in all normal and malignant breast cell lines. The inhibitory receptor TRID was highly expressed in one of four normal and two of seven malignant breast cell lines. DR4, DR5, or TRID expression did not correlate with sensitivity to TRAIL-induced apoptosis. Incubation of cell lines with doxorubicin or 5-fluorouracil significantly augmented TRAIL-induced apoptosis in most breast cell lines. By fractional inhibition analysis, the toxicity of the combination of TRAIL and doxorubicin or 5-fluorouracil was synergistic compared with either agent alone. In contrast, melphalan and paclitaxel augmented TRAIL-induced apoptosis in few cell lines, and methotrexate did not augment it in any cell line. Augmentation of TRAIL-induced apoptosis by doxorubicin or 5-fluorouracil was mediated through caspase activation. This was evidenced by the fact that chemotherapy agents that synergized with TRAIL (e.g., doxorubicin) themselves caused cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP), and their toxicity was blocked by the caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2 (ZVAD-fmk). The combination of TRAIL and doxorubicin caused significantly greater caspase-3 and PARP cleavage, and the combined toxicity also was inhibited by ZVAD-fmk. In contrast, chemotherapy agents that did not augment TRAIL-induced apoptosis (e.g., methotrexate) caused minimal caspase-3 and PARP cleavage by themselves, and their toxicity was not inhibited by ZVAD-fmk. These drugs also did not increase caspase-3 or PARP cleavage when combined with TRAIL. In summary, few breast cell lines are sensitive to TRAIL-induced apoptosis, and no difference in sensitivity is found between normal and malignant cell lines. Treatment with chemotherapy provides an approach to sensitize breast cancer cells to TRAIL-induced apoptosis.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Apoptosis/physiology , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Membrane Glycoproteins/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Antineoplastic Agents/administration & dosage , Apoptosis Regulatory Proteins , Caspase 3 , Caspases/metabolism , Doxorubicin/administration & dosage , Doxorubicin/pharmacology , Enzyme Activation , Fluorouracil/administration & dosage , Fluorouracil/pharmacology , Glutathione Transferase/pharmacology , Glutathione Transferase/physiology , Humans , Melphalan/administration & dosage , Melphalan/pharmacology , Membrane Glycoproteins/administration & dosage , Membrane Glycoproteins/physiology , Paclitaxel/administration & dosage , Paclitaxel/pharmacology , Recombinant Fusion Proteins/pharmacology , TNF-Related Apoptosis-Inducing Ligand , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/administration & dosage , Tumor Necrosis Factor-alpha/physiology
17.
Oncogene ; 18(5): 1147-56, 1999 Feb 04.
Article in English | MEDLINE | ID: mdl-10022120

ABSTRACT

Cbl-b, a mammalian homolog of Cbl, consists of an N-terminal region (Cbl-b-N) highly homologous to oncogenic v-Cbl, a Ring finger, and a C-terminal region containing multiple proline-rich stretches and potential tyrosine phosphorylation sites. In the present study, we demonstrate that upon engagement of the T cell receptor (TCR), endogenous Cbl-b becomes rapidly tyrosine-phosphorylated. In heterogeneous COS-1 cells, Cbl-b was phosphorylated on tyrosine residues by both Syk- (Syk/Zap-70) and Src- (Fyn/Lck) family kinases, with Syk kinase inducing the most prominent effect. Syk associates and phosphorylates Cbl-b in Jurkat T cells. A Tyr-316 Cbl-binding site in Syk was required for the association with and for the maximal tyrosine phosphorylation of Cbl-b. Mutation at a loss-of-function site (Gly-298) in Cbl-b-N disrupts its interaction with Syk. Cbl-b constitutively binds Grb2 and becomes associated with Crk-L upon TCR stimulation. The Grb2- and the Crk-L-binding regions were mapped to the C-terminus of Cbl-b. The Crk-L-binding sites were further determined to be Y655DVP and Y709KIP, with the latter being the primary binding site. Taken together, these results implicate that Cbl-b is involved in TCR-mediated intracellular signaling pathways.


Subject(s)
Adaptor Proteins, Signal Transducing , CD3 Complex/metabolism , Proto-Oncogene Proteins/metabolism , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/metabolism , Ubiquitin-Protein Ligases , Binding Sites , Humans , Jurkat Cells , Lymphocyte Activation , Nuclear Proteins/metabolism , Phosphorylation , Proline , Protein Binding , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-cbl , Signal Transduction , Tyrosine , ZAP-70 Protein-Tyrosine Kinase , src-Family Kinases/metabolism
18.
Mol Cell ; 4(6): 1029-40, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10635327

ABSTRACT

Receptor desensitization is accomplished by accelerated endocytosis and degradation of ligand-receptor complexes. An in vitro reconstituted system indicates that Cbl adaptor proteins directly control downregulation of the receptor for the epidermal growth factor (EGFR) by recruiting ubiquitin-activating and -conjugating enzymes. We infer a sequential process initiated by autophosphorylation of EGFR at a previously identified lysosome-targeting motif that subsequently recruits Cbl. This is followed by tyrosine phosphorylation of c-Cbl at a site flanking its RING finger, which enables receptor ubiquitination and degradation. Whereas all three members of the Cbl family can enhance ubiquitination, two oncogenic Cbl variants, whose RING fingers are defective and phosphorylation sites are missing, are unable to desensitize EGFR. Our study identifies Cbl proteins as components of the ubiquitin ligation machinery and implies that they similarly suppress many other signaling pathways.


Subject(s)
Caenorhabditis elegans Proteins , Epidermal Growth Factor/physiology , ErbB Receptors/physiology , Helminth Proteins/physiology , Ligases/physiology , Proto-Oncogene Proteins/physiology , Signal Transduction , Cell-Free System , Enzyme Activation , Phosphorylation , Proto-Oncogene Proteins c-cbl , Tyrosine , Ubiquitin-Activating Enzymes , Ubiquitin-Protein Ligases
19.
J Biol Chem ; 272(15): 9979-85, 1997 Apr 11.
Article in English | MEDLINE | ID: mdl-9092538

ABSTRACT

Stimulation of the T cell antigen receptor (TCR).CD3 complex induces rapid tyrosine phosphorylation of Cbl, a protooncogene product which has been implicated in intracellular signaling pathways via its interaction with several signaling molecules. We found recently that Cbl associates directly with a member of the 14-3-3 protein family (14-3-3tau) in T cells and that the association is increased as a consequence of anti-CD3-mediated T cell activation. We report here that phorbol 12-myristate 13-acetate stimulation of T cells also enhanced the interaction between Cbl and two 14-3-3 isoforms (tau and zeta). Tyrosine phosphorylation of Cbl was not sufficient or required for this increased interaction. Thus, cotransfection of COS cells with Cbl plus Lck and/or Syk family protein-tyrosine kinases caused a marked increase in the phosphotyrosine content of Cbl without a concomitant enhancement of its association with 14-3-3. Phorbol 12-myristate 13-acetate stimulation induced serine phosphorylation of Cbl, and dephosphorylation of immunoprecipitated Cbl by a Ser/Thr phosphatase disrupted its interaction with 14-3-3. By using successive carboxyl-terminal deletion mutants of Cbl, the 14-3-3-binding domain was mapped to a serine-rich 30-amino acid region (residues 615-644) of Cbl. Mutation of serine residues in this region further defined a binding motif distinct from the consensus sequence RSXSXP, which was recently identified as a 14-3-3-binding motif. These results suggest that TCR stimulation induces both tyrosine and serine phosphorylation of Cbl. These phosphorylation events allow Cbl to recruit distinct signaling elements that participate in TCR-mediated signal transduction pathways.


Subject(s)
Enzyme Inhibitors/metabolism , Phorbol Esters/pharmacology , Protein-Tyrosine Kinases/metabolism , Proteins/metabolism , Retroviridae Proteins, Oncogenic/metabolism , Serine/pharmacology , T-Lymphocytes/metabolism , Tyrosine 3-Monooxygenase , 14-3-3 Proteins , Amino Acid Sequence , Animals , Binding Sites , COS Cells , Carcinogens/pharmacology , Enzyme Activation , Humans , Jurkat Cells , Mice , Molecular Sequence Data , Oncogene Protein v-cbl , Peptide Mapping , Phosphorylation , Protein Conformation , Rabbits , Receptor-CD3 Complex, Antigen, T-Cell/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Tyrosine/metabolism
20.
Oncogene ; 14(10): 1243-8, 1997 Mar 13.
Article in English | MEDLINE | ID: mdl-9121775

ABSTRACT

Dephosphorylation of the RB protein has been reported to be associated with apoptosis. In contrast, we show that treatment of HL60 cells with etoposide or cytosine arabinoside or treatment of breast epithelial cells with alpha-FAS is associated with the cleavage of a 5 kDa fragment from the C-terminus of RB, resulting in a truncated product that we have designated as p100cl. This cleavage event coincides with the activation of cysteine proteases at the onset of apoptosis, is blocked by the addition of iodoacetamide to cells prior to the onset of apoptosis, and results in the expression of faster migrating protein species which can mimic dephosphorylated RB. The free 5 kDa fragment is detected only during apoptosis, predicts a cleavage site that we have mapped to a unique CPP32-like recognition sequence which is present at the C-terminus of all reported RB homologues, and results in a truncated RB protein with enhanced E2F binding affinity. While the causality for this cleavage event in the apoptotic process is still under investigation, our findings suggest distinct post-translational pathways for the RB product between cells examined during growth arrest (p105 hypophosphorylated RB) or apoptosis (p100cl).


Subject(s)
Apoptosis/physiology , Carrier Proteins , Cell Cycle Proteins , DNA-Binding Proteins , Peptide Fragments/metabolism , Retinoblastoma Protein/metabolism , Transcription Factors/metabolism , Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Cysteine Endopeptidases/metabolism , Cytarabine/pharmacology , E2F Transcription Factors , Etoposide/pharmacology , Female , HL-60 Cells/drug effects , HL-60 Cells/metabolism , Humans , Phosphorylation , Retinoblastoma-Binding Protein 1 , Transcription Factor DP1 , fas Receptor/physiology
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