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1.
Cell Prolif ; 40(4): 580-94, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17635524

ABSTRACT

OBJECTIVE: Lapatinib (Tykerb, GW572016), a potent inhibitor of the catalytic activities of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) (ErbB2), inhibits population growth of selected EGFR and HER2 overexpressing cell lines. Previous studies with a small number of cell lines suggest a correlation between overexpression of EGFR and/or HER2 and sensitivity to growth inhibition by lapatinib; however, the precise determinants of lapatinib selectivity for tumour and/or other cells remain unclear. MATERIALS AND METHODS: To clarify the determinants of its selectivity in cultured cells, lapatinib-induced cell population growth inhibition and relative EGFR and HER2 protein expression were quantified in 61 different human tumour cell lines from 12 tumour types, two oncogene transformed human cell lines and two normal human cell cultures. Using statistical tools to analyse the data, a model describing the relationship between lapatinib IC(50) (the response variable) and EGFR and HER2 expression and tissue type (explanatory variables) was derived. CONCLUSION: The results suggest that simultaneous consideration of EGFR and HER2 expression, as well as tissue type yields the best determinant of lapatinib selectivity in cultured cells.


Subject(s)
Antineoplastic Agents/pharmacology , ErbB Receptors/metabolism , Protein Kinase Inhibitors/pharmacology , Quinazolines/pharmacology , Receptor, ErbB-2/metabolism , Cell Line , Cell Line, Transformed , Cell Line, Tumor , Humans , Lapatinib , Models, Statistical
2.
Cancer Res ; 61(19): 7196-203, 2001 Oct 01.
Article in English | MEDLINE | ID: mdl-11585755

ABSTRACT

The type I receptor tyrosine kinases constitute a family of transmembrane proteins involved in various aspects of cell growth and survival and have been implicated in the initiation and progression of several types of human malignancies. The best characterized of these proteins are the epidermal growth factor receptor (EGFR) and ErbB-2 (HER-2/neu). We have developed potent quinazoline and pyrido-[3,4-d]-pyrimidine small molecules that are dual inhibitors of ErbB-2 and EGFR. The compounds demonstrate potent in vitro inhibition of the ErbB-2 and EGFR kinase domains with IC(50)s <80 nM. Growth of ErbB-2- and EGFR-expressing tumor cell lines is inhibited at concentrations <0.5 microM. Selectivity for tumor cell growth inhibition versus normal human fibroblast growth inhibition ranges from 10- to >75-fold. Tumor growth in mouse s.c. xenograft models of the BT474 and HN5 cell lines is inhibited in a dose-responsive manner using oral doses of 10 and 30 mg/kg twice per day. In addition, the tested compounds caused a reduction of ErbB-2 and EGFR autophosphorylation in tumor fragments from these xenograft models. These data indicate that these compounds have potential use as therapy in the broad population of cancer patients overexpressing ErbB-2 and/or EGFR.


Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , ErbB Receptors/antagonists & inhibitors , Quinazolines/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Animals , Cell Division/drug effects , Drug Screening Assays, Antitumor , Female , Growth Inhibitors/pharmacology , Humans , Mice , Mice, SCID , Structure-Activity Relationship , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
3.
Protein Expr Purif ; 22(3): 462-6, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11483009

ABSTRACT

The gene mutated in the human disease ataxia telangiectasia (AT), termed ATM, encodes a large protein kinase involved in DNA repair and cell cycle control. Biochemical characterization of ATM function has been somewhat difficult because of its large size (approximately 370 kDa) and relatively low level of expression in several systems. The majority of studies have used immunoprecipitated ATM or purified ATM obtained through relatively complex procedures. Here, we describe an efficient method for the expression and purification of FLAG-epitope-tagged recombinant human ATM protein (F-ATM). This method utilizes the expression of F-ATM in transiently transfected 293T cells followed by anti-FLAG-agarose affinity chromatography. The transfection procedure has been optimized for large (225-cm(2)) culture flasks and F-ATM can be purified to near homogeneity as judged by SDS-PAGE. This procedure yields approximately 1 microg of catalytically active F-ATM protein/225-cm(2) flask that can be used for biochemical studies.


Subject(s)
Protein Serine-Threonine Kinases/genetics , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins , Cell Line , Chromatography, Affinity , DNA-Binding Proteins , Humans , Oligopeptides , Peptides/isolation & purification , Protein Serine-Threonine Kinases/isolation & purification , Protein Serine-Threonine Kinases/metabolism , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Transfection , Tumor Suppressor Proteins
4.
Mol Cancer Ther ; 1(2): 85-94, 2001 Dec.
Article in English | MEDLINE | ID: mdl-12467226

ABSTRACT

The epidermal growth factor receptor (EGFR) and ErbB-2 transmembrane tyrosine kinases are currently being targeted by various mechanisms in the treatment of cancer. GW2016 is a potent inhibitor of the ErbB-2 and EGFR tyrosine kinase domains with IC50 values against purified EGFR and ErbB-2 of 10.2 and 9.8 nM, respectively. This report describes the efficacy in cell growth assays of GW2016 on human tumor cell lines overexpressing either EGFR or ErbB-2: HN5 (head and neck), A-431 (vulva), BT474 (breast), CaLu-3 (lung), and N87 (gastric). Normal human foreskin fibroblasts, nontumorigenic epithelial cells (HB4a), and nonoverexpressing tumor cells (MCF-7 and T47D) were tested as negative controls. After 3 days of compound exposure, average IC50 values for growth inhibition in the EGFR- and ErbB-2-overexpressing tumor cell lines were < 0.16 microM. The average selectivity for the tumor cells versus the human foreskin fibroblast cell line was 100-fold. Inhibition of EGFR and ErbB-2 receptor autophosphorylation and phosphorylation of the downstream modulator, AKT, was verified by Western blot analysis in the BT474 and HN5 cell lines. As a measure of cytotoxicity versus growth arrest, the HN5 and BT474 cells were assessed in an outgrowth assay after a transient exposure to GW2016. The cells were treated for 3 days in five concentrations of GW2016, and cell growth was monitored for an additional 12 days after removal of the compound. In each of these tumor cell lines, concentrations of GW2016 were reached where outgrowth did not occur. Furthermore, growth arrest and cell death were observed in parallel experiments, as determined by bromodeoxyuridine incorporation and propidium iodide staining. GW2016 treatment inhibited tumor xenograft growth of the HN5 and BT474 cells in a dose-responsive manner at 30 and 100 mg/kg orally, twice daily, with complete inhibition of tumor growth at the higher dose. Together, these results indicate that GW2016 achieves excellent potency on tumor cells with selectivity for tumor versus normal cells and suggest that GW2016 has value as a therapy for patients with tumors overexpressing either EGFR or ErbB-2.


Subject(s)
Antineoplastic Agents/pharmacology , Cell Division/drug effects , Enzyme Inhibitors/pharmacology , ErbB Receptors/antagonists & inhibitors , Furans/pharmacology , Neoplasms, Experimental/drug therapy , Quinazolines/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Animals , Apoptosis , Blotting, Western , Cell Cycle/drug effects , Epidermal Growth Factor/pharmacology , ErbB Receptors/metabolism , Female , Fibroblasts/drug effects , Humans , Infant, Newborn , Mice , Mice, Nude , Mice, SCID , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Phosphorylation , Precipitin Tests , Receptor, ErbB-2/metabolism , Signal Transduction/drug effects , Skin/cytology , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism , Xenograft Model Antitumor Assays
5.
Hybridoma ; 19(4): 317-21, 2000 Aug.
Article in English | MEDLINE | ID: mdl-11001404

ABSTRACT

We report on the rapid generation of two monoclonal antibodies, ATM A16.35 and ATM D16.11, that bind to the kinase domain of mutated ataxia telangiectasia (ATM). These antibodies were generated against E. coli-expressed recombinant protein using the RIMMS strategy. We show that ATM A16.35 binds ATM by Western blot analysis, and ATM D16.11 forms immune complexes with native ATM in immunoprecipitations without neutralizing kinase activity.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases/immunology , Animals , Antibodies, Monoclonal/immunology , Antibody Affinity/immunology , Antibody Specificity/immunology , Ataxia Telangiectasia Mutated Proteins , Blotting, Western , Cell Cycle Proteins , Cell Line , DNA-Binding Proteins , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunization , Mice , Peptide Fragments , Precipitin Tests , Recombinant Proteins/immunology , T-Lymphocytes/enzymology , Tumor Suppressor Proteins
7.
Genes Dev ; 12(23): 3686-92, 1998 Dec 01.
Article in English | MEDLINE | ID: mdl-9851975

ABSTRACT

Similarities exist between the progressive cerebellar ataxia in ataxia telangiectasia (AT) patients and a number of neurodegenerative diseases in both mouse and man involving specific mutations in ion channels and/or ion channel activity. These relationships led us to investigate the possibility of defective ion channel activity in AT cells. We examined changes in the membrane potential of AT fibroblasts in response to extracellular cation addition and found that the ability of AT fibroblasts to depolarize in response to increasing concentrations of extracellular K+ is significantly reduced when compared with control fibroblasts. Electrophysiological measurements performed with a number of AT cell lines, as well as two matched sets of primary AT fibroblast cultures, reveal that outward rectifier K+ currents are largely absent in AT fibroblasts in comparison with control cells. These K+ current defects can be corrected in AT fibroblasts transfected with the full-length ATM cDNA. These data implicate, for the first time, a role for ATM in the regulation of K+ channel activity and membrane potential.


Subject(s)
Ataxia Telangiectasia/metabolism , Potassium Channels/metabolism , Potassium/metabolism , Cell Line , Electric Conductivity , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Membrane Potentials
8.
J Med Chem ; 41(11): 1894-908, 1998 May 21.
Article in English | MEDLINE | ID: mdl-9599239

ABSTRACT

The design, synthesis, and evaluation of dipeptide analogues as ligands for the pp60c-src SH2 domain are described. The critical binding interactions between Ac-Tyr-Glu-N(n-C5H11)2 (2) and the protein are established and form the basis for our structure-based drug design efforts. The effects of changes in both the C-terminal (11-27) and N-terminal (51-69) portions of the dipeptide are explored. Analogues with reduced overall charge (92-95) are also investigated. We demonstrate the feasibility of pairing structurally diverse subunits in a modest dipeptide framework with the goal of increasing the druglike attributes without sacrificing binding affinity.


Subject(s)
Dipeptides/pharmacology , Enzyme Inhibitors/pharmacology , Proto-Oncogene Proteins pp60(c-src)/antagonists & inhibitors , src Homology Domains , Crystallography, X-Ray , Dipeptides/chemical synthesis , Dipeptides/chemistry , Dipeptides/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Enzyme-Linked Immunosorbent Assay , Humans , Ligands , Models, Molecular , Molecular Conformation , Proto-Oncogene Proteins pp60(c-src)/metabolism , Structure-Activity Relationship
10.
Bioorg Med Chem Lett ; 8(10): 1189-94, 1998 May 19.
Article in English | MEDLINE | ID: mdl-9871733

ABSTRACT

The X-ray crystal structure of the src SH2 domain revealed the presence of a thiol residue (Cys 188) located proximal to the phosphotyrosine portion of a dipeptide ligand. An aldehyde bearing ligand (1) was designed to position an electrophilic carbonyl group in the vicinity of the thiol. X-ray crystallographic and NMR examination of the complex formed between (1) and the src SH2 domain revealed a hemithioacetal formed by addition of the thiol to the aldehyde group with an additional stabilizing hydrogen bond between the acetal hydroxyl and a backbone carbonyl.


Subject(s)
Dipeptides/chemistry , Protein Conformation , Proteins/chemistry , src Homology Domains , Aldehydes , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Cysteine , Dipeptides/chemical synthesis , Hydrogen Bonding , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Phosphotyrosine
11.
Cancer Res ; 57(10): 1962-9, 1997 May 15.
Article in English | MEDLINE | ID: mdl-9157992

ABSTRACT

Previous studies indicated that overexpression of wild-type avian c-src cannot induce neoplastic transformation of NIH 3T3 cells. In this study, we isolated and characterized novel spontaneously derived transforming mutants of avian pp60(c-src) from a Syrian hamster embryo-derived cell line, 10W, transfected with the avian c-src gene. Seventeen independently derived transfected 10W cell clones were injected into athymic nude mice. After a latency period, tumors eventually arose and were established in culture. The tumorigenic phenotype was always accompanied by the presence of the avian c-src DNA and functional expression of pp60(c-src). However, most of the tumor-derived cell lines expressed an electrophoretically altered form of pp60(c-src), suggesting mutations in src. Consistent with this hypothesis, DNAs isolated from the tumor-derived lines, but not the parental 10W cell lines, morphologically transformed NIH 3T3 cells in a focus-forming assay. We characterized pp60(c-src) in detail from three of the tumor-derived lines: 4AT, 4BT, and E2T. Two of these lines contained mutations within the exogenous c-src coding region. Line 4AT has an internal repeat of 29 amino acids immediately following Gln-513, which disrupts the spacing between the end of the kinase domain and Tyr-527, the negative regulatory site in pp60(c-src). Line 4BT has a 5-bp deletion following Phe-520, which results in loss of Tyr-527. However, the DNA sequence of the coding region of pp60(c-src) from a third line, E2T, was completely wild type. Cyanogen bromide cleavage analyses of the altered pp60(c-src) from lines 4AT and 4BT showed that Tyr-527, the site of negative regulation of c-src, is not phosphorylated, but Tyr-416, the site of in vitro autophosphorylation, is phosphorylated. However, in line E2T, Tyr-527 was phosphorylated, and Tyr-416 was phosphorylated to a lesser extent. Additionally, two proteins that indicate activation of src, p85 cortactin and p120(cas), are phosphorylated in at least six of the tumor-derived cell lines, although to a lesser extent in line E2T. These results suggest that dephosphorylation of Tyr-527 and phosphorylation of Tyr-416 correlate with activation of pp60(c-src) in the tumor-derived lines 4AT and 4BT, respectively. However, in line E2T, the high levels of pp60(c-src), in combination with a partial activation of the pp60(c-src) protein as indicated by phosphorylation of Tyr-416, appear to be involved in the neoplastic process, rather than mutation.


Subject(s)
Cell Transformation, Neoplastic/genetics , Mutation , Precancerous Conditions/genetics , Proto-Oncogene Proteins pp60(c-src)/genetics , 3T3 Cells/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cricetinae , DNA/genetics , Mesocricetus , Mice , Molecular Sequence Data , Phenotype , Phosphorylation , Phosphotyrosine/metabolism , Polymerase Chain Reaction , Proto-Oncogene Proteins pp60(c-src)/biosynthesis , Proto-Oncogene Proteins pp60(c-src)/metabolism , Transfection , Tyrosine/metabolism
12.
Cancer Res ; 56(16): 3659-62, 1996 Aug 15.
Article in English | MEDLINE | ID: mdl-8706003

ABSTRACT

The tumor suppressor gene product, p53, appears to be a significant participant in signaling pathways that mediate cellular responses to cytotoxic stresses. In particular, p53 appears to be a critical determinant of whether the cell lives or dies and how it progresses through the cell cycle after the cytotoxic exposure. Many of the molecular details for these signaling pathways remain to be elucidated, and whether all cytotoxic signals utilize the same pathway to increase p53 expression is not clear. Here, we demonstrate the existence of cell types in which the induction of p53 and associated G1 arrest by the antimetabolite, N-(phosphonoacetyl)-L-aspartate (PALA), is defective, whereas p53 induction and G1 arrest induced by ionizing radiation are intact. These observations demonstrate the existence of genetic defects that can alter p53 induction and associated cellular outcomes after some, but not all, cytotoxic insults and suggest distinct pathways of p53 induction by PALA and ionizing radiation.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Aspartic Acid/analogs & derivatives , Gene Expression Regulation , Genes, p53 , Phosphonoacetic Acid/analogs & derivatives , Aspartic Acid/pharmacology , Base Sequence , Cell Cycle/drug effects , Cell Cycle/radiation effects , Cell Line , DNA Damage , Gene Expression Regulation/drug effects , Gene Expression Regulation/radiation effects , Genes, p53/drug effects , Genes, p53/radiation effects , Humans , Molecular Sequence Data , Phosphonoacetic Acid/pharmacology , Tumor Suppressor Protein p53/biosynthesis
13.
Am J Pathol ; 148(5): 1381-5, 1996 May.
Article in English | MEDLINE | ID: mdl-8623910

ABSTRACT

Mutations of the p53 tumor suppressor gene are rarely found in neuroblastoma. Though typically a nuclear protein, a number of tumor cell types have recently been reported to exhibit cytoplasmic p53 immunostaining, and it has been suggested that altered cellular localization is another mechanism of inhibiting p53 function. We examined p53 protein expression, localization, and function in neuroblastoma cell lines with wild-type p53 genes. Basal p53 levels were largely confined to the cytoplasmic compartment in these cells. However, after irradiation, p53 protein levels increased predominately in the nucleus. Transcriptional activity of p53 was intact in these cells because "downstream" proteins, p21WAF1 and MDM2, were induced by irradiation. In contrast to a neuroblastoma cell line harboring a mutant p53 gene, the neuroblastoma cells with wild-type protein were associated with an intact G1 arrest after DNA damage. The induced nuclear protein in these neuroblastoma cells also appeared functional as measured by its capacity to bind to a DNA oligomer containing a p53-consensus sequence. We have concluded that although p53 expression in neuroblastoma cells is primarily localized to the cytosol, ionizing radiation induces a functional p53 protein in the nucleus and that this cytoplasmic sequestration of p53 in human neuroblastoma is not a mechanism of inactivating p53 function.


Subject(s)
Neuroblastoma/chemistry , Neuroblastoma/pathology , Signal Transduction/physiology , Tumor Suppressor Protein p53/analysis , Tumor Suppressor Protein p53/physiology , Blotting, Western , Cell Cycle/radiation effects , Cell Fractionation , Cell Nucleus/chemistry , Cytoplasm/chemistry , DNA Damage , DNA, Neoplasm/analysis , DNA, Neoplasm/genetics , Genes, Tumor Suppressor/genetics , Genes, p53/radiation effects , Humans , Immunohistochemistry , Point Mutation , Polymerase Chain Reaction , Tumor Cells, Cultured , Tumor Suppressor Protein p53/genetics
14.
Oncogene ; 12(1): 169-76, 1996 Jan 04.
Article in English | MEDLINE | ID: mdl-8552388

ABSTRACT

Although experimental studies suggest that aberrant Ras function can promote the malignant progression of human breast epithelial cells, the occurrence of mutated ras genes in breast tumors is infrequent. One possible explanation for this apparent paradox is that aberrant function of the Ras-related protein TC21/R-Ras2, which causes malignant transformation of NIH 3T3 cells via upregulation of the Ras signal transduction pathway, may contribute to breast tumor development in the absence of Ras mutations. To address this possibility, we utilized two complementary approaches. First, we determined that aberrant TC21 function caused transformation of the MCF-10A human breast epithelial cell line. TC21-transformed MCF-10A cells exhibited altered cellular morphology associated with a disruption of cell-cell adherens junctions, formed colonies in soft agar, and showed enhanced motility in vitro. These alterations were similar to, but more dramatic than, those observed with oncogenic Ras-transformed MCF-10A cells. Furthermore, overexpression of normal TC21, but not Ras, also caused transformation of these cells. Second, we observed that TC21 protein expression was greatly elevated in 7 of 9 breast tumor lines when compared to untransformed MCF-10A cells. Taken together, these results support the possibility that overexpression of TC21 may contribute to aberrant growth properties of breast carcinoma cells.


Subject(s)
Breast Neoplasms/etiology , Membrane Proteins/physiology , Monomeric GTP-Binding Proteins , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Division , Cell Movement , Cell Transformation, Neoplastic , Female , Genes, ras , Humans , Tumor Cells, Cultured
15.
Hybridoma ; 14(4): 355-9, 1995 Aug.
Article in English | MEDLINE | ID: mdl-8522347

ABSTRACT

The production of two different murine monoclonal antibodies to human Gadd45, a protein that is induced in response to DNA damage, is reported. Antibodies were generated in a SJL mouse using a recombinant form of the human Gadd45 protein. Monoclonal antibody 4TCYA1, which recognizes the denatured form of human Gadd45 in Western blots, was selected based upon the recognition of Gadd45 induced by functional p53 in the human myeloid leukemia cell line, ML-1. A second monoclonal antibody, designated 30T.14, immunoprecipitates native human Gadd45 in lysates produced from RKO cells, a colorectal carcinoma cell line that expresses relatively high basal levels of Gadd45, as well as from cell lysates made from ML-1 cells after exposure to ionizing irradiation (IR). Since 4TCYA1 fails to immunoprecipitate Gadd45, and 30T.14 fails to bind to IR-induced Gadd45 in immunoblotting, these two monoclonal antibodies probably recognize different epitopes.


Subject(s)
Antibodies, Monoclonal/biosynthesis , DNA Damage , Proteins/immunology , Recombinant Proteins/immunology , Animals , Antibodies, Monoclonal/chemistry , Blotting, Western , Cell Division/genetics , DNA Damage/genetics , Female , Humans , Hybridomas/metabolism , Intracellular Signaling Peptides and Proteins , Mice , Mice, Inbred Strains , Proteins/metabolism , Recombinant Proteins/biosynthesis , Tumor Cells, Cultured , GADD45 Proteins
16.
Genes Dev ; 9(5): 600-11, 1995 Mar 01.
Article in English | MEDLINE | ID: mdl-7698649

ABSTRACT

Irradiation of mammalian cells can cause cell cycle perturbations and apoptotic cell death. We have investigated the modulation of these physiologic end points by growth factor stimulation: irradiation of a murine hematopoietic cell line in the presence of interlekin-3 (IL-3) induces G1 arrest, and irradiation in the absence of IL-3 results in rapid apoptotic cell death. Both of these end points are dependent on p53. Transient removal of IL-3 at the time of irradiation results in decreased clonogenic survival of irradiated cells. The removal of IL-3 results in a failure of the irradiated cells to arrest at the G1 checkpoint, despite induction of p53 and p21WAF1/CIP1, and then the cells enter S-phase where they undergo apoptosis. There are no cytokine-related changes in Bcl-2, Bax, or Bcl-x protein levels that could account for the modulation of G1 arrest versus apoptosis by growth factors. In contrast, rapid p53-independent alterations of basal levels of gadd45 and p21WAF1/CIP1 expression are linked to IL-3 withdrawal, suggesting a potential mechanism for this modulation. Constitutive activation of cytokine-like pathways with induced expression of v-Src or activated c-Raf inhibits the radiation-induced apoptosis and the alterations in p21WAF1/CIP1 and gadd45 expression. These observations suggest additional molecular mechanisms that can contribute to the development of radioresistance and resistance to apoptosis during tumorigenesis and provide an explanation for the observed lack of p53 mutations in some tumor types. In addition, these data suggest that oncogenic changes occurring during multistep tumorigenesis could be classified as those that either enhance or decrease apoptosis tendencies.


Subject(s)
Apoptosis/drug effects , Cell Division/drug effects , Gene Expression Regulation/drug effects , Interleukin-3/pharmacology , Nuclear Proteins , Repressor Proteins , Tumor Suppressor Protein p53/physiology , Animals , Apoptosis/radiation effects , Bone Marrow Cells , Cell Cycle/drug effects , Cell Cycle/radiation effects , Cell Division/radiation effects , Cell Line , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/genetics , Gamma Rays , Gene Expression Regulation/radiation effects , Genes, p53/genetics , Intracellular Signaling Peptides and Proteins , Mice , Oncogene Protein pp60(v-src)/genetics , Oncogene Protein pp60(v-src)/physiology , Oncogene Proteins, Viral/genetics , Proteins/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2 , RNA, Messenger/biosynthesis , Tumor Suppressor Protein p53/biosynthesis , Tumor Suppressor Protein p53/genetics , GADD45 Proteins
17.
J Biol Chem ; 269(51): 32672-7, 1994 Dec 23.
Article in English | MEDLINE | ID: mdl-7798274

ABSTRACT

GADD45 (growth arrest and DNA damage) is a DNA-damage-inducible gene regulated in part by the tumor suppressor p53. A role in negative growth control has recently been suggested based on significant (more than 75%) reduction of colony formation following over expression of Gadd45. To better understand the role of Gadd45, we have developed specific rabbit and murine antibodies raised against the human recombinant protein. Using these antibodies, we have found that in ML-1 cells Gadd45 is predominantly a nuclear protein. MyD118, a protein induced by terminal differentiation sharing 57% homology with Gadd45, does not cross-react with any of the antibodies produced. As expected, the induction of Gadd45 protein by ionizing radiation (IR) was also found to be dependent on a wild type p53 phenotype. Interestingly, WI-L2-NS, a human lymphoid cell line, showed very high basal levels of Gadd45 mRNA and protein in addition to a high constitutive level of a mutated p53 protein. In this cell line, the high levels of GADD45 did not inhibit cellular growth in spite of the fact that no mutations were found in GADD45 sequence. These results indicate that some cell line(s) can tolerate high levels of Gadd45 and abrogate its growth suppression function.


Subject(s)
Gene Expression Regulation , Genes, p53 , Proteins/metabolism , Animals , Baculoviridae/genetics , Base Sequence , Cell Line , Cloning, Molecular , DNA Damage , DNA Primers , Gene Expression Regulation/radiation effects , Humans , Intracellular Signaling Peptides and Proteins , Molecular Sequence Data , Protein Biosynthesis , Proteins/genetics , RNA, Messenger/metabolism , Radiation, Ionizing , Spodoptera , Tumor Cells, Cultured , GADD45 Proteins
18.
Science ; 266(5189): 1376-80, 1994 Nov 25.
Article in English | MEDLINE | ID: mdl-7973727

ABSTRACT

GADD45 is a ubiquitously expressed mammalian gene that is induced by DNA damage and certain other stresses. Like another p53-regulated gene, p21WAF1/CIP1, whose product binds to cyclin-dependent kinases (Cdk's) and proliferating cell nuclear antigen (PCNA), GADD45 has been associated with growth suppression. Gadd45 was found to bind to PCNA, a normal component of Cdk complexes and a protein involved in DNA replication and repair. Gadd45 stimulated DNA excision repair in vitro and inhibited entry of cells into S phase. These results establish GADD45 as a link between the p53-dependent cell cycle checkpoint and DNA repair.


Subject(s)
DNA Repair , Genes, p53 , Proliferating Cell Nuclear Antigen/metabolism , Proteins/metabolism , S Phase/drug effects , Cell Division/drug effects , Cell Line , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/metabolism , DNA/biosynthesis , DNA Damage , Humans , Intracellular Signaling Peptides and Proteins , Proteins/pharmacology , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Transfection , Tumor Cells, Cultured , GADD45 Proteins
19.
Laryngoscope ; 104(11 Pt 1): 1337-47, 1994 Nov.
Article in English | MEDLINE | ID: mdl-7968162

ABSTRACT

Mutational activation and overexpression of the family of ras proto-oncogenes have been associated with many human tumors. The role of mutations of H-ras, K-ras, and N-ras, as well as expression of the respective protein products (p21s) in normal mucosa, dysplastic mucosa, and squamous cell carcinomas (SCCs) of the head and neck has not been fully described. In our study, 51 tumors (40 paraffin embedded and 11 fresh frozen) were examined to determine if mutational activation of ras is an important molecular event in head and neck SCC. Analyses of codons 12, 13, and 61 of H-ras, K-ras, and N-ras revealed no mutations, suggesting that mutational activation of ras is not important in the majority of head and neck SCCs. Immunocytochemistry (ICC) was used to define the expression of H-ras, K-ras, and N-ras in normal mucosa, dysplastic mucosa, and SCC of the head and neck and to determine if expression of ras family members correlated with early or late events in the development of SCC. Expression of p21N-ras in nine samples of histologically normal head and neck mucosa revealed moderate staining in the basal proliferative layers with progressively less staining as cells matured. The most superficial layers of normal mucosa failed to express p21N-ras. A low level of p21H-ras was expressed in all layers of normal mucosa while K-ras was not expressed. ICC of SCC tumor sections revealed cytoplasmic expression of N-ras in nine of nine tumors, H-ras in five of nine tumors, and K-ras in one of nine tumors. Expression of H-ras, K-ras, and N-ras in head and neck SCC was not related to histologic differentiation or TNM staging; however, p21N-ras was overexpressed in seven of nine tumors. Furthermore, the pattern of N-ras expression in dysplastic lesions revealed expression in all layers of the mucosa in contrast to normal mucosa, which expresses p21N-ras primarily in the basal proliferative layer. The change in p21N-ras expression pattern in dysplastic mucosa and its overexpression in the majority of tumors suggest that loss of control of N-ras expression may be an early step in carcinogenesis of head and neck SCC.


Subject(s)
Carcinoma, Squamous Cell/genetics , Codon/genetics , Gene Expression Regulation, Neoplastic , Genes, ras/genetics , Head and Neck Neoplasms/genetics , Mutation/genetics , Oncogene Protein p21(ras)/genetics , Adult , Aged , Aged, 80 and over , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/secondary , DNA, Neoplasm/genetics , Exons/genetics , Fibrosarcoma/genetics , Fibrosarcoma/metabolism , Gene Amplification , Head and Neck Neoplasms/metabolism , Humans , Lymphatic Metastasis/genetics , Middle Aged , Oligonucleotides/genetics , Oncogene Protein p21(ras)/metabolism
20.
Proc Natl Acad Sci U S A ; 91(1): 83-7, 1994 Jan 04.
Article in English | MEDLINE | ID: mdl-7506422

ABSTRACT

The phosphotyrosine residues of receptor tyrosine kinases serve as unique binding sites for proteins involved in intracellular signaling, which contain SRC homology 2 (SH2) domains. Since overexpression or activation of the pp60c-src kinase has been reported in a number of human tumors, including primary human breast carcinomas, we examined the interactions of the SH2 and SH3 domains of human SRC with target proteins in human carcinoma cell lines. Glutathione S-transferase fusion proteins containing either the SH2, SH3, or the entire SH3/SH2 region of human SRC were used to affinity purify tyrosine-phosphorylated proteins from human breast carcinoma cell lines. We show here that in human breast carcinoma cell lines, the SRC SH2 domain binds to activated epidermal growth factor receptor (EGFR) and p185HER2/neu. SRC SH2 binding to EGFR was also observed in a nontumorigenic cell line after hormone stimulation. Endogenous pp60c-src was found to tightly associate with tyrosine-phosphorylated EGFR. Association of the SRC SH2 with the EGFR was blocked by tyrosyl phosphopeptides containing the sequences surrounding tyrosine-530, the regulatory site in the SRC C terminus, or sequences surrounding the major sites of autophosphorylation in the EGFR. These results raise the possibility that association of pp60c-src with these receptor tyrosine kinases is an integral part of the signaling events mediated by these receptors and may contribute to malignant transformation.


Subject(s)
Breast Neoplasms/metabolism , ErbB Receptors/metabolism , Proto-Oncogene Proteins pp60(c-src)/metabolism , Proto-Oncogene Proteins/metabolism , Amino Acid Sequence , Binding, Competitive , Humans , In Vitro Techniques , Molecular Sequence Data , Phosphopeptides/metabolism , Protein Binding , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, ErbB-2 , Signal Transduction , Tumor Cells, Cultured
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