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1.
Oncogene ; 32(7): 903-9, 2013 Feb 14.
Article in English | MEDLINE | ID: mdl-22430205

ABSTRACT

Rac1b, an alternative splice form of Rac1, has been previously shown to be upregulated in colon and breast cancer cells, suggesting an oncogenic role for Rac1b in these cancers. Our analysis of NSCLC tumor and matched normal tissue samples indicates Rac1b is upregulated in a significant fraction of lung tumors in correlation with mutational status of K-ras. To directly assess the oncogenic potential of Rac1b in vivo, we employed a mouse model of lung adenocarcinoma, in which the expression of Rac1b can be conditionally activated specifically in the lung. Although expression of Rac1b alone is insufficient to drive tumor initiation, the expression of Rac1b synergizes with an oncogenic allele of K-ras resulting in increased cellular proliferation and accelerated tumor growth. Finally, we show that in contrast to our previous findings demonstrating a requirement for Rac1 in K-ras-driven cell proliferation, Rac1b is not required in this context. Given the partially overlapping spectrum of downstream effectors regulated by Rac1 and Rac1b, our findings further delineate the signaling pathways downstream of Rac1 that are required for K-ras driven tumorigenesis.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Cell Transformation, Neoplastic/genetics , Lung Neoplasms/genetics , Proto-Oncogene Proteins/physiology , rac1 GTP-Binding Protein/physiology , ras Proteins/physiology , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Animals , Carcinoma, Non-Small-Cell Lung/pathology , Cell Transformation, Neoplastic/pathology , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , Lung Neoplasms/pathology , Mice , Mice, Transgenic , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Isoforms/physiology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras) , Tumor Cells, Cultured , rac1 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/metabolism , ras Proteins/genetics
2.
Article in English | MEDLINE | ID: mdl-16869760

ABSTRACT

Lung cancer is a devastating disease that presents a challenge to basic research to provide new steps toward therapeutic advances. The cell-type-specific responses to oncogenic mutations that initiate and regulate lung cancer remain poorly defined. A better understanding of the relevant signaling pathways and mechanisms that control therapeutic outcome could also provide new insight. Improved conditional mouse models are now available as tools to improve the understanding of the cellular and molecular origins of adenocarcinoma. These models have already proven their utility in proof-of-principle experiments with new technologies including genomics and imaging. Integrated thinking to apply technological advances while using the appropriate mouse model is likely to facilitate discoveries that will significantly improve lung cancer detection and intervention.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/genetics , Animals , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/therapy , Disease Models, Animal , ErbB Receptors/genetics , Genes, p53 , Genes, ras , Genomics , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Lung Neoplasms/therapy , Mice , Mice, Mutant Strains , Mutation , Neoplastic Stem Cells/pathology , Signal Transduction , ras Proteins/metabolism
3.
Mol Cell Biol ; 20(3): 1044-54, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10629061

ABSTRACT

In this study we describe the identification and structure-function analysis of a novel death-associated protein (DAP) kinase-related protein, DRP-1. DRP-1 is a 42-kDa Ca(2+)/calmodulin (CaM)-regulated serine threonine kinase which shows high degree of homology to DAP kinase. The region of homology spans the catalytic domain and the CaM-regulatory region, whereas the remaining C-terminal part of the protein differs completely from DAP kinase and displays no homology to any known protein. The catalytic domain is also homologous to the recently identified ZIP kinase and to a lesser extent to the catalytic domains of DRAK1 and -2. Thus, DAP kinase DRP-1, ZIP kinase, and DRAK1/2 together form a novel subfamily of serine/threonine kinases. DRP-1 is localized to the cytoplasm, as shown by immunostaining and cellular fractionation assays. It binds to CaM, undergoes autophosphorylation, and phosphorylates an exogenous substrate, the myosin light chain, in a Ca(2+)/CaM-dependent manner. The truncated protein, deleted of the CaM-regulatory domain, was converted into a constitutively active kinase. Ectopically expressed DRP-1 induced apoptosis in various types of cells. Cell killing by DRP-1 was dependent on two features: the status of the catalytic activity, and the presence of the C-terminal 40 amino acids shown to be required for self-dimerization of the kinase. Interestingly, further deletion of the CaM-regulatory region could override the indispensable role of the C-terminal tail in apoptosis and generated a "superkiller" mutant. A dominant negative fragment of DAP kinase encompassing the death domain was found to block apoptosis induced by DRP-1. Conversely, a catalytically inactive mutant of DRP-1, which functioned in a dominant negative manner, was significantly less effective in blocking cell death induced by DAP kinase. Possible functional connections between DAP kinase and DRP-1 are discussed.


Subject(s)
Apoptosis , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Amino Acid Sequence , Apoptosis Regulatory Proteins , Base Sequence , Breast Neoplasms , Calcium-Calmodulin-Dependent Protein Kinases , Catalytic Domain , Cell Line , Cloning, Molecular , Death-Associated Protein Kinases , Female , Gene Library , Humans , Kidney , Molecular Sequence Data , Phylogeny , Protein Biosynthesis , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Spleen/metabolism , Transcription, Genetic , Tumor Cells, Cultured
4.
J Cell Biol ; 146(1): 141-8, 1999 Jul 12.
Article in English | MEDLINE | ID: mdl-10402466

ABSTRACT

Death-associated protein (DAP)-kinase is a calcium/calmodulin regulated serine/threonine kinase that carries ankyrin repeats, a death domain, and is localized to the cytoskeleton. Here, we report that this kinase is involved in tumor necrosis factor (TNF)-alpha and Fas-induced apoptosis. Expression of DAP-kinase antisense RNA protected cells from killing by anti-Fas/APO-1 agonistic antibodies. Deletion of the death domain abrogated the apoptotic functions of the kinase, thus, documenting for the first time the importance of this protein domain. Overexpression of a fragment encompassing the death domain of DAP-kinase acted as a specific dominant negative mutant that protected cells from TNF-alpha, Fas, and FADD/MORT1-induced cell death. DAP-kinase apoptotic function was blocked by bcl-2 as well as by crmA and p35 inhibitors of caspases, but not by the dominant negative mutants of FADD/MORT1 or of caspase 8. Thus, it functions downstream to the receptor complex and upstream to other caspases. The multidomain structure of this serine/threonine kinase, combined with its involvement in cell death induced by several different triggers, place DAP-kinase at one of the central molecular pathways leading to apoptosis.


Subject(s)
Adaptor Proteins, Signal Transducing , Apoptosis , Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Tumor Necrosis Factor-alpha/pharmacology , fas Receptor/physiology , Apoptosis/drug effects , Apoptosis Regulatory Proteins , Blotting, Western , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Carrier Proteins/genetics , Carrier Proteins/physiology , Caspase Inhibitors , Caspases/genetics , Caspases/metabolism , Cell Line , Death-Associated Protein Kinases , Fas-Associated Death Domain Protein , Genes, Dominant/genetics , Humans , Inhibitor of Apoptosis Proteins , Mutation , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/physiology , RNA, Antisense/genetics , RNA, Antisense/physiology , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor/physiology , Serpins/genetics , Serpins/physiology , Transfection , Tumor Cells, Cultured , Viral Proteins/genetics , Viral Proteins/physiology , fas Receptor/genetics
5.
EMBO J ; 18(2): 353-62, 1999 Jan 15.
Article in English | MEDLINE | ID: mdl-9889192

ABSTRACT

A novel approach to the isolation of positive mediators of programmed cell death, based on random inactivation of genes by expression of anti sense RNAs, was employed to identify mediators of interferon-gamma-induced apoptosis. One of the several genes identified is DAP3, which codes for a 46 kDa protein with a potential nucleotide-binding motif. Structure-function studies of the protein indicate that the intact full-length protein is required for its ability to induce apoptosis when overexpressed. The N-terminal 230 amino acids, on the other hand, act in a dominant-negative fashion. Both of these functions are dependent on the integrity of the nucleotide binding motif. Expression of anti-sense DAP3 RNA and of the dominant interfering form of DAP3 both protected cells from apoptosis induced by activation of Fas and tumor necrosis factor alpha (TNF-alpha) receptors. Thus, DAP3 is implicated as a positive mediator of these death-inducing stimuli. It functions downstream of the receptor signaling complex and its death promoting effects depend on caspase activity. In the nematode Caenorhabditis elegans, a potential homolog of DAP3 showing 35% identity and 64% similarity to the human protein was isolated. Overexpression of the nematode DAP3 cDNA in mammalian cells induced cell death, indicating that the protein is conserved at the functional level as well as the structural level.


Subject(s)
Apoptosis/physiology , Proteins/chemistry , Proteins/physiology , Tumor Necrosis Factor-alpha/physiology , fas Receptor/physiology , Amino Acid Sequence , Animals , Apoptosis Regulatory Proteins , Caenorhabditis elegans/cytology , Caenorhabditis elegans/genetics , Caenorhabditis elegans/physiology , Caspases/physiology , Evolution, Molecular , Gene Expression , HeLa Cells , Humans , Molecular Sequence Data , Mutation , Proteins/genetics , RNA, Antisense/genetics , RNA, Antisense/pharmacology , RNA-Binding Proteins , Ribosomal Proteins , Sequence Homology , Signal Transduction , Species Specificity , Transfection
6.
Mol Med Today ; 4(6): 268-74, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9679246

ABSTRACT

Aberrations of apoptosis are implicated in many diseases, including cancer, autoimmune disease, cardiovascular disease and neurodegeneration. The cell's apoptotic machinery is, therefore, an important potential target for the development of new therapies. Our laboratory has used a strategy called technical knockout (TKO) to identify novel genes involved in apoptosis. TKO is based on random inactivation of gene expression with antisense cDNA libraries, followed by selection of those cells that survive in the continuous presence of an apoptotic stimulus. Using this approach, we have isolated five novel genes, including a serine/threonine kinase, a nucleotide-binding protein and a homologue of the p220 translation initiation factor. Expression of one of these genes (DAP kinase) is lost in some cancers, and this loss appears to increase the metastatic potential of some tumours.


Subject(s)
Apoptosis/genetics , Apoptosis/physiology , Genes, Tumor Suppressor/physiology , Proteins/genetics , Proteins/physiology , Animals , Apoptosis Regulatory Proteins , Humans , Proteins/chemistry , RNA-Binding Proteins , Ribosomal Proteins , Structure-Activity Relationship
7.
Cancer Lett ; 126(1): 33-41, 1998 Apr 10.
Article in English | MEDLINE | ID: mdl-9563646

ABSTRACT

In addition to its structural role, beta-catenin has recently been identified as an oncogene, while its homologue gamma-catenin (plakoglobin) seems to suppress tumorigenicity. Twenty-five epithelial tumor cell lines were screened; 18 expressed both beta- and gamma-catenin, two expressed neither protein, four showed beta- but not gamma-catenin expression, while only one cell line showed gamma- but not beta-catenin expression. As per literature search, the cell line expressing gamma- but not beta-catenin appeared to be unique. This cell line, SKBR-3, is a human breast cancer cell line which does not express beta-catenin or E-cadherin protein. There is, however, expression of beta-catenin, but not E-cadherin, mRNA. In order to determine the mechanism for this unique expression pattern, SKBR-3 cells were transfected with E-cadherin which resulted in expression of beta-catenin protein. Immunofluorescent staining of the E-cadherin transfected SKBR-3 cells revealed beta-catenin in the adherens junctions while transfection with just an epitope tagged (VSV) beta-catenin showed expression only in the nucleus. Double transfection with E-cadherin and VSV beta-catenin showed the beta-catenin in the adherens junction of the E-cadherin transfected cells. These results indicate that the mechanism for the lack of beta-catenin expression in the SKBR-3 cell line is possibly post-translational degradation and that when E-cadherin is transfected into these cells, the beta-catenin is stabilized in the adherens junction and not degraded. This cell line should be of interest to those studying the role of the homologues, beta- and gamma-catenin, in cancer pathogenesis.


Subject(s)
Cell Line , Cytoskeletal Proteins/metabolism , Trans-Activators , Cell Adhesion Molecules/metabolism , Cell Nucleus/metabolism , Desmoplakins , Epithelium/metabolism , Humans , Transfection , Tumor Cells, Cultured , beta Catenin , gamma Catenin
8.
Oncogene ; 15(4): 403-7, 1997 Jul 24.
Article in English | MEDLINE | ID: mdl-9242376

ABSTRACT

DAP-kinase is a novel calmodulin dependent serine/threonine kinase that carries ankyrin repeats and the death domain. It was recently isolated, by a functional selection approach of gene cloning, as a positive mediator of programmed cell death. In this study the expression of DAP-kinase was examined in the cell lines derived from various human neoplasms. DAP-kinase mRNA and protein expression were below the limit of detection in eight out of ten neoplastic derived B-cell lines. In six out of 14 examined bladder carcinoma, in three out of five renal cell carcinoma, and in four out of ten tested breast carcinoma cell lines, the DAP-kinase protein levels were below detection limits or lower than 1% compared to the positive cell lines. Interestingly, DAP-kinase expression could be restored in some of the negative bladder carcinoma and B-cell lines by treatment of cells with 5'-azadeoxycytidine that causes DNA demethylation. The high frequency of loss of DAP-kinase expression in human tumor cell lines, and the occasional involvement of methylation in this process raise the possibility that this novel mediator of cell death may function as a tumor suppressor gene.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/physiology , Genes, Tumor Suppressor , Lymphoma, B-Cell/enzymology , Neoplasms/enzymology , Apoptosis Regulatory Proteins , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Death-Associated Protein Kinases , Humans , RNA, Messenger/analysis , Tumor Cells, Cultured
10.
J Biol Chem ; 270(46): 27932-6, 1995 Nov 17.
Article in English | MEDLINE | ID: mdl-7499268

ABSTRACT

Interaction of certain cytokines with their corresponding cell-surface receptors induces programmed cell death. Interferon-gamma induces in HeLa cells a type of cell death with features characteristic of programmed cell death. Here, we report the isolation of a novel gene, DAP3 (death-associated protein-3), involved in mediating interferon-gamma-induced cell death. The rescue of this gene was performed by a functional selection approach of gene cloning that is based on transfection with an antisense cDNA expression library. The antisense RNA-mediated inactivation of the DAP3 gene protected the cells from interferon-gamma-induced cell death. This property endowed the cells expressing it with a growth advantage in an environment restrictive due to the continuous presence of interferon-gamma and thus provided the basis of its selection. The gene is transcribed into a single 1.7-kilobase mRNA, which is ubiquitously expressed in different tissues and codes for a 46-kDa protein carrying a potential P-loop motif. Ectopic expression of DAP3 in HeLa cells was not compatible with cell growth, resulting in a 16-fold reduction in the number of drug-resistant stable clones. The data presented suggest that DAP3 is a positive mediator of cell death induced by interferon-gamma.


Subject(s)
Cell Death/physiology , Gene Expression , Interferon-gamma/pharmacology , Protein Biosynthesis , Amino Acid Sequence , Animals , Antibodies , Apoptosis Regulatory Proteins , Base Sequence , Cell Death/drug effects , Cell Division , Cloning, Molecular , DNA, Antisense , DNA, Complementary , Gene Library , HeLa Cells , Humans , Molecular Sequence Data , Proteins/genetics , Proteins/isolation & purification , RNA, Antisense/metabolism , RNA, Messenger/biosynthesis , RNA-Binding Proteins , Rabbits/immunology , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Reticulocytes/metabolism , Ribosomal Proteins , Transfection
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