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1.
Cancer Res ; 61(21): 7878-81, 2001 Nov 01.
Article in English | MEDLINE | ID: mdl-11691807

ABSTRACT

The mRNA for Rvp.1 (rat ventral prostate) increases in abundance before gland involution after androgen deprivation. Rvp.1 is homologous to CPE-R, the high-affinity intestinal epithelial receptor for Clostridium perfringens enterotoxin (CPE), and is sufficient to mediate CPE binding and trigger subsequent toxin-mediated cytolysis. Rvp.1 (claudin-3) and CPE-R (claudin-4) are members of a larger family of transmembrane tissue-specific claudin proteins that are essential components of intercellular tight junction structures regulating paracellular ion flux. However, claudin-3 and claudin-4 are the only family members capable of mediating CPE binding and cytolysis. The present study was designed to study the expression of claudin-3 and claudin-4 in human prostate tissue as potential targets for CPE toxin-mediated therapy for prostate cancer. On human multiple-tissue Northern blot analysis, mRNAs for both claudin-3 and claudin-4 were expressed at high levels in prostate tissue. In normal prostate tissue, expression of claudin-3 was localized exclusively within acinar epithelial cells by in situ mRNA hybridization. Compared with expression within prostate epithelial cells in surrounding normal glandular tissue, expression of claudin-3 mRNA remained high in the epithelium of prostate adenocarcinoma (10 of 10) and prostatic intraepithelial neoplasia (five of five). Prostate adenocarcinoma cells metastatic to bone were obtained from a patient with disease progression during antiandrogen therapy. These metastatic cells were prostate-specific antigen-positive by immunohistochemical staining and also expressed functional CPE receptors as measured by sensitivity to CPE-induced cell lysis. The persistent high level of claudin-3 expression in prostate adenocarcinoma and functional cytotoxicity of CPE in metastatic androgen-independent prostate adenocarcinoma suggests a new potential therapeutic strategy for prostate cancer.


Subject(s)
Adenocarcinoma/metabolism , Enterotoxins/toxicity , Membrane Proteins/biosynthesis , Prostatic Neoplasms/metabolism , Adenocarcinoma/drug therapy , Adenocarcinoma/secondary , Androgens/physiology , Animals , Bone Marrow Neoplasms/drug therapy , Bone Marrow Neoplasms/metabolism , Bone Marrow Neoplasms/secondary , Chlorocebus aethiops , Claudin-3 , Claudin-4 , Clostridium perfringens/genetics , Clostridium perfringens/metabolism , Enterotoxins/metabolism , Epithelial Cells/metabolism , Epithelial Cells/pathology , Humans , Male , Membrane Proteins/genetics , Neoplasms, Hormone-Dependent/drug therapy , Neoplasms, Hormone-Dependent/metabolism , Prostatic Neoplasms/drug therapy , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Vero Cells
2.
Mamm Genome ; 9(8): 660-5, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9680388

ABSTRACT

Apoptosis in murine myeloid cell lines requires the expression of the Requiem gene, which encodes a putative zinc finger protein. We detected the protein in both cytoplasmic and nuclear subcellular fractions of murine myeloid cells and human K562 leukemia cells, which suggests that the protein might have a function distinct from a transcription factor. This distribution did not alter upon apoptosis induction by IL-3 deprivation. As an approach to investigate its role in development, we determined the spatio-temporal expression pattern in the mouse. Expression was detected in various tissues in earlier gestational age; however, confined to testes, spleen, thymus, and part of the hippocampus in the adult mouse. The expression profile is consistent with a functional role during rapid growth and cell turnover, and in agreement with a regulatory function for hematopoietic cells. The human cDNA clone sequenced showed high homology to its murine counterpart and extended the open reading frame by 20 codons upstream. The gene is located in the proximal region of mouse Chromosome (Chr) 19. In the homologous human region at 11q13, it is located at about 150 kb centromeric from MLK3.


Subject(s)
Chromosome Mapping , Chromosomes, Human, Pair 11 , DNA-Binding Proteins/genetics , Gene Expression Regulation, Developmental , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Centromere , Crosses, Genetic , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/chemistry , Embryonic and Fetal Development , Female , Genetic Markers , Humans , In Situ Hybridization, Fluorescence , Leukemia , Male , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Muridae , Organ Specificity , Pregnancy , Transcription Factors , Tumor Cells, Cultured , Zinc Fingers
3.
Blood ; 85(3): 804-11, 1995 Feb 01.
Article in English | MEDLINE | ID: mdl-7833480

ABSTRACT

Studies of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in a cell-free system showed that the low molecular-weight guanosine triphosphatase (GTPase) Rac was required, and that Rap1a may participate in activation of the catalytic complex. Full-length posttranslationally modified Rac2 was active, whereas only the 1-166 truncated form of Rap1a was functional in the cell-free system, and thus, clarification of the function of Rap1a and Rac2 in intact human phagocytes is needed to provide further insight into their roles as signal transducers from plasma membrane receptors. In the present studies, oligonucleotide-directed mutagenesis was used to introduce a series of mutations into human rap1a or rac2 in the mammalian expression vector pSR alpha neo. HL60 cells transfected with wild-type or mutated rac2 or rap1a cDNA constructs and control HL60 cells transfected with the pSR alpha neo vector containing no inserted cDNA were selected in G418-containing media, then subclones were isolated. Compared with the parent HL60 cells, each of the stable transfected cell lines differentiated similarly into neutrophil-like cells and expressed comparable levels of NADPH oxidase components p47-phox, p67-phox and gp91-phox. The differentiated vector control cell line produced O2. in response to receptor stimulation at rates that were not significantly different from parent HL60 cells. O2-. production by differentiated cell lines expressing mutated N17 Rap1a or N17 Rac2 dominant-negative proteins was inhibited, whereas O2-. production by the subline overexpressing wild-type Rap1a was increased by fourfold. O2-. production by the differentiated cell line expressing GTPase-defective V12 Rap1a was also significantly inhibited, a finding that is consistent with a requirement for cycling between guanosine diphosphate- and GTP-bound forms of Rap1a for continuous NADPH oxidase activation in intact neutrophils. A model is proposed in which Rac2 mediates assembly of the p47 and p67 oxidase components on the cytosolic face of the plasma membrane via cytoskeletal reorganization, whereas Rap1a functions downstream as the final activation switch involving direct physical interaction with the transmembrane flavocytochrome component of the NADPH oxidase.


Subject(s)
GTP Phosphohydrolases/metabolism , GTP-Binding Proteins/metabolism , NADH, NADPH Oxidoreductases/metabolism , Superoxides/metabolism , Animals , Base Sequence , Cell Differentiation , Cell Line , Codon/genetics , Dimethyl Sulfoxide/pharmacology , Enzyme Activation , Genetic Vectors , Humans , Leukemia, Promyelocytic, Acute , Mammals , Molecular Sequence Data , Mutagenesis, Site-Directed , NADPH Oxidases , Oligodeoxyribonucleotides , Plasmids , Recombinant Proteins/metabolism , Transcription Factors/metabolism , Transfection , Tumor Cells, Cultured , rac GTP-Binding Proteins , rap GTP-Binding Proteins
4.
J Biol Chem ; 269(47): 29515-9, 1994 Nov 25.
Article in English | MEDLINE | ID: mdl-7961935

ABSTRACT

To identify genes mediating programmed cell death triggered by interleukin 3 (IL-3)-deprivation of myeloid cells, the IL-3-dependent murine myeloid cell line FDCP-1 was used to screen a mammalian cell expression library for cDNAs that would promote survival following withdrawal of IL-3. A unique 892-base pair cDNA was cloned that prevented the programmed cell death response following IL-3 deprivation by causing antisense suppression of an endogenous 2.4-kilobase (kb) mRNA. A 2.3-kb cDNA containing the identical 892-base pair over-lapping sequence was cloned that encoded a deduced 371-amino acid protein containing a single Kruppel-type zinc finger and a cluster of 4 cysteine/histidine-rich repeats resembling atypical zinc fingers. The 2.4-kb mRNA was found to be ubiquitously expressed in murine tissues and its abundance in FDCP-1 cells was not altered in response to IL-3 deprivation. Since expression of this 2.4-kb mRNA was a prerequisite for the apoptosis response following IL-3 deprivation, the gene encoding it was named requiem. Requiem is likely to encode a transcription factor required for the apoptosis response following survival factor withdrawal from myeloid cells.


Subject(s)
Apoptosis/genetics , Hematopoietic Stem Cells/cytology , Zinc Fingers/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Division , Cells, Cultured , DNA, Complementary , Interleukin-3/physiology , Mice , Molecular Sequence Data , Sequence Alignment , Transfection
5.
J Biol Chem ; 266(21): 13964-70, 1991 Jul 25.
Article in English | MEDLINE | ID: mdl-1906890

ABSTRACT

Activation of the membrane-associated NADPH oxidase in intact human neutrophils requires a receptor-associated heterotrimeric GTP-binding protein that is sensitive to pertussis toxin. Activation of this NADPH oxidase by arachidonate in a cell-free system requires an additional downstream pertussis toxin-insensitive G protein (Gabig, T. G., English, D., Akard, L. P., and Schell, M. J. (1987) (J. Biol. Chem. 262, 1685-1690) that is located in the cytosolic fraction of unstimulated cells (Gabig, T. G., Eklund, E. A., Potter, G. B., and Dykes, J. R. (1990) J. Immunol. 145, 945-951). In the present study, immunodepletion of G proteins from the cytosolic fraction of unstimulated neutrophils resulted in a loss of the ability to activate NADPH oxidase in the membrane fraction. The activity in immunodepleted cytosol was fully reconstituted by a partially purified fraction from neutrophil cytosol that contained a 21-kDa GTP-binding protein. Purified human recombinant Krev-1 p21 also completely reconstituted immunodepleted cytosol whereas recombinant human H-ras p21 or yeast RAS GTP-binding proteins had no reconstitutive activity. Rabbit antisera raised against a synthetic peptide corresponding to the effector region of Krev-1 (amino acids 31-43) completely inhibited cell-free NADPH oxidase activation, and this inhibition was blocked by the synthetic 31-43 peptide. An inhibitory monoclonal antibody specific for ras p21 amino acids 60-77 (Y13-259) had no effect on cell-free NADPH oxidase activation. Activation of the NADPH oxidase in intact neutrophils by stimulation with phorbol myristate acetate caused a marked increase in the amount of membrane-associated antigen recognized by 151 antiserum on Western blot. Thus a G protein in the cytosol of unstimulated neutrophils antigenically and functionally related to Krev-1 may be the downstream effector G protein for NADPH oxidase activation. This system represents a unique model to study molecular interactions of a ras-like G protein.


Subject(s)
GTP-Binding Proteins/physiology , NADH, NADPH Oxidoreductases/metabolism , Neutrophils/enzymology , Cell-Free System , Cytosol/metabolism , Enzyme Activation , GTP-Binding Proteins/isolation & purification , Humans , In Vitro Techniques , NADPH Oxidases , Neutrophils/chemistry , Oligonucleotides/chemistry , Polymerase Chain Reaction , Recombinant Proteins/metabolism , Signal Transduction , Structure-Activity Relationship , rap GTP-Binding Proteins
6.
Leukemia ; 3(9): 662-8, 1989 Sep.
Article in English | MEDLINE | ID: mdl-2668657

ABSTRACT

The functional role of a mutant RAS gene in immortal myeloid cell proliferation was examined in a fastidious interleukin-3 (IL-3) dependent cell line (NFS/N1.H7) formed by forced proliferation in IL-3 of marrow cells of the NFS/N mouse. The NFS/N1.H7 cell line was strictly dependent upon IL-3 for growth, and the cell line could be activated by phorbol esters (PMA) to augment IL-3 dependent proliferation, but when pKC was downregulated, diminished IL-3 proliferative response resulted. Transfection (electroporation) of the T24 RAS-containing vector pAL8 to NFS/N1.H7 led to clones (H7 NeoRas.F3, H7 NeoRas.E2) that had incorporated the entire 6.6 Kb human mutant H-RAS genome. The mutant RAS-containing clones demonstrated greater proliferation than parent cells or cells containing a control (neo-resistance) vector over a range of suboptimal IL-3 does and in optimal IL-3 concentrations had a faster doubling rate than parent cells. The clone H7 NeoRas.F3 was studied biochemically, and found to constitutively form 3-fold more 3H-diacylglycerol than the parent cell line upon exposure to 3H-glycerol. PMA could partially repair the proliferative defect of NFS/N1.H7 compared to the RAS-expressor. These studies affirm a secondary, accelerating role for a mutant RAS gene product acting through pKC to promote clonal expansion of immortal myeloid cells stimulated by IL-3.


Subject(s)
Bone Marrow Cells , Caenorhabditis elegans Proteins , Cell Division/drug effects , Genes, ras , Interleukin-3/pharmacology , Protein Kinase C/physiology , Animals , Carrier Proteins , Cell Line , Diglycerides/physiology , Enzyme Activation , Mice , Mutation , Receptors, Drug/metabolism , Transfection
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