ABSTRACT
The introduction of new therapies against particular genetic mutations in non-small-cell lung cancer is a promising avenue for improving patient survival, but the target population is small. There is a need to discover new potential actionable genetic lesions, to which end, non-conventional cancer pathways, such as RNA editing, are worth exploring. Herein we show that the adenosine-to-inosine editing enzyme ADAR1 undergoes gene amplification in non-small cancer cell lines and primary tumors in association with higher levels of the corresponding mRNA and protein. From a growth and invasion standpoint, the depletion of ADAR1 expression in amplified cells reduces their tumorigenic potential in cell culture and mouse models, whereas its overexpression has the opposite effects. From a functional perspective, ADAR1 overexpression enhances the editing frequencies of target transcripts such as NEIL1 and miR-381. In the clinical setting, patients with early-stage lung cancer, but harboring ADAR1 gene amplification, have poor outcomes. Overall, our results indicate a role for ADAR1 as a lung cancer oncogene undergoing gene amplification-associated activation that affects downstream RNA editing patterns and patient prognosis.
Subject(s)
Adenosine Deaminase/genetics , Gene Amplification , Lung Neoplasms/etiology , RNA Editing , RNA-Binding Proteins/genetics , Cell Line, Tumor , Humans , Lung Neoplasms/genetics , Oncogenes , Proto-Oncogene Proteins p21(ras)/geneticsABSTRACT
Disruption of the histone modification patterns is one of the most common features of human tumors. However, few genetic alterations in the histone modifier genes have been described in tumorigenesis. Herein we show that the histone methyltransferase SETDB1 undergoes gene amplification in non-small and small lung cancer cell lines and primary tumors. The existence of additional copies of the SETDB1 gene in these transformed cells is associated with higher levels of the corresponding mRNA and protein. From a functional standpoint, the depletion of SETDB1 expression in amplified cells reduces cancer growth in cell culture and nude mice models, whereas its overexpression increases the tumor invasiveness. The increased gene dosage of SETDB1 is also associated with enhanced sensitivity to the growth inhibitory effect mediated by the SETDB1-interfering drug mithramycin. Overall, the findings identify SETDB1 as a bona fide oncogene undergoing gene amplification-associated activation in lung cancer and suggest its potential for new therapeutic strategies.
Subject(s)
Adenocarcinoma/genetics , Carcinoma, Squamous Cell/genetics , Gene Amplification , Lung Neoplasms/genetics , Protein Methyltransferases/genetics , Adenocarcinoma/enzymology , Adenocarcinoma/pathology , Animals , Carcinogenesis/genetics , Carcinoma, Squamous Cell/enzymology , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Gene Dosage , Histone-Lysine N-Methyltransferase , Humans , Lung Neoplasms/enzymology , Lung Neoplasms/pathology , Mice , Mice, Nude , Neoplasm Invasiveness , Neoplasm Transplantation , Protein Methyltransferases/metabolismABSTRACT
VRK2 is a novel Ser-Thr kinase whose VRK2A isoform is located in the endoplasmic reticulum and mitochondrial membranes. We have studied the potential role that VRK2A has in the regulation of mitochondrial-mediated apoptosis. VRK2A can regulate the intrinsic apoptotic pathway in two different ways. The VRK2A protein directly interacts with Bcl-xL, but not with Bcl-2, Bax, Bad, PUMA or Binp-3L. VRK2A does not compete with Bax for interaction with Bcl-xL, and these proteins can form a complex that reduces apoptosis. Thus, high VRK2 levels confer protection against apoptosis. In addition, VRK2 knockdown results in an increased expression of BAX gene expression that is mediated by its proximal promoter, thus VRK2A behaves as a negative regulator of BAX. Low levels of VRK2A causes an increase in mitochondrial Bax protein level, leading to an increase in the release of cytochrome C and caspase activation, detected by PARP processing. VRK2A loss results in an increase in cell death that can be detected by an increase in annexinV+ cells. Low levels of VRK2A increase cell sensitivity to induction of apoptosis by chemotherapeutic drugs like camptothecin or doxorubicin. We conclude that VRK2A protein is a novel modulator of apoptosis.
Subject(s)
Apoptosis , Protein Serine-Threonine Kinases/metabolism , bcl-2-Associated X Protein/metabolism , bcl-X Protein/metabolism , Antibiotics, Antineoplastic/toxicity , Apoptosis/drug effects , Camptothecin/toxicity , Caspases/metabolism , Cell Line, Tumor , Cytochromes c/metabolism , Doxorubicin/toxicity , Gene Expression , HEK293 Cells , HeLa Cells , Humans , Mitochondria/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Protein Binding , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , RNA Interference , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , bcl-2-Associated X Protein/geneticsABSTRACT
The vaccinia-related kinases (VRKs) branched off early from the family of casein kinase (CK) I and compose a relatively uncharacterized family of the kinome. The VRKs were discovered due to their close sequence relation to the vaccinia virus B1R serine/threonine kinase. They were first described in phosphorylation of transcription factors that led to the discovery of an autoregulatory mechanism between VRK and the tumor suppressor transcription factor p53. The relevance of VRKs has broadened recently by introduction of its members as essential regulators in cell signaling, nuclear envelope dynamics, chromatin modifications, apoptosis and cellular stress response. Several phosphorylation substrates have been described, as well as the first positive and negative regulators of VRK. We provide an overview of the VRKs across species and discuss the wide diversity of cellular and organismal requirements for this kinase family.
Subject(s)
Protein Serine-Threonine Kinases/physiology , Signal Transduction/physiology , Animals , Humans , PhylogenyABSTRACT
Tetraspanin proteins form signaling complexes between them and with other membrane proteins and modulate cell adhesion and migration properties. The surface expression of several tetraspanin antigens (CD9, CD37, CD53, CD63, and CD81), and their interacting proteins (CD19, CD21, and HLA-DR) were analyzed during normal B-cell maturation and compared to a group of 67 B-cell neoplasias. Three patterns of tetraspanin expression were identified in normal B cells. The first corresponded to bone marrow CD10(+) B-cell precursors (BCP) which showed high expression of CD81 and CD9, low reactivity for CD53 and negativity for CD37. CD10(-) B-lymphocytes showed downregulation of CD9/CD81 and upregulation of CD53/CD37. Plasma cells showed re-expressed CD9 and downregulated CD37. Hierarchical clustering analysis of flow cytometry immunophenotypic data showed a good correlation between the tumor differentiation stage and the pattern of tetraspanin expression, with all analyzed individual samples classified into three major groups, independently of their normal or neoplastic origin. Despite this, neoplastic B-cells frequently showed aberrantly high/low expression of the different markers analyzed. Interestingly, in B-cell chronic lymphocytic leukemia, abnormal expression of CD53 and CD9 were associated with different patterns of disease infiltration, which would support the role of these molecules on modulating adhesion and migration of neoplastic B cells.
Subject(s)
Antigens, CD/analysis , Antigens, Neoplasm/analysis , B-Lymphocytes/pathology , Cell Differentiation , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , B-Lymphocytes/cytology , B-Lymphocytes/physiology , Blood Cells/pathology , Bone Marrow/pathology , Cluster Analysis , Gene Expression Regulation, Neoplastic , Humans , Immunophenotyping , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Lymph Nodes/pathologySubject(s)
Antigens, Neoplasm/biosynthesis , B-Lymphocytes/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/diagnosis , Lymphoproliferative Disorders/diagnosis , Membrane Proteins/biosynthesis , Splenic Neoplasms/diagnosis , Adult , Antigens, Neoplasm/genetics , B-Lymphocytes/pathology , Clone Cells , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/complications , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Lymphoproliferative Disorders/complications , Lymphoproliferative Disorders/genetics , Membrane Proteins/genetics , Splenic Neoplasms/complications , Splenic Neoplasms/geneticsABSTRACT
To determine the types of human papillomaviruses (HPV) in northern Morocco, information which is needed for the design and use of HPV vaccines, we have analysed 129 cervical biopsies from this region. In our study, 91 cases were HPV positive, 45 cases had HPV-16 DNA, and 20 cases had HPV-18 DNA. This distribution of virus type was similar in inflammatory cervical lesions and in invasive cervical carcinomas. In conclusion, the HPV type distribution in Morocco is similar to that in other African Mediterranean countries, where the proportion of HPV-18 cases is significantly higher than in Europe. Determination of virus-type distribution is essential for vaccination programs.
Subject(s)
Papillomaviridae/isolation & purification , Papillomavirus Infections/complications , Tumor Virus Infections/complications , Uterine Cervical Dysplasia/virology , Uterine Cervical Neoplasms/virology , Biopsy , DNA, Viral/chemistry , DNA, Viral/genetics , Female , Humans , Morocco , Papillomaviridae/classification , Papillomaviridae/genetics , Papillomavirus Infections/pathology , Papillomavirus Infections/virology , Polymerase Chain Reaction , Tumor Virus Infections/pathology , Tumor Virus Infections/virology , Uterine Cervical Neoplasms/pathology , Uterine Cervical Dysplasia/pathologyABSTRACT
CD53 is a tetraspanin protein mostly expressed in to the lymphoid-myeloid lineage. We have characterized the human CD53 gene regulatory region. Within the proximal 2 kilobases, and with opposite transcriptional orientation, is located the promoter-enhancer of a second gene, which does not affect CD53. Twenty-four copies of a CA dinucleotide repeat separate these two gene promoters. The proximal enhanceosome of the human CD53 gene is comprised between residues -266 and +84, and can be subdivided into four major subregions, two of them within exon 1. Mutational analysis identified several cooperating sequences. An Sp1 and an ets-1 site, at positions -115 and +62, respectively, are essential for transcriptional competence in all cell lines. Five other regulatory sequences have a dual role, activator or down-regulator, depending on the cell line. At the end of the non-coding exon 1, +64 to +83, there is a second ets-1 regulatory element, which is required for high level of transcription, in cooperation with the Sp1 site, in K562 and Molt-4, but not in Namalwa cells, where it functions as a repressor. This Sp1 site also cooperates with another ets-1/PU.1 site at -172. Different cell types use different regulatory sequences in the enhanceosome for the expression of the same gene.
Subject(s)
Antigens, CD/genetics , Antigens, Differentiation, T-Lymphocyte/genetics , Gene Expression Regulation/genetics , Regulatory Sequences, Nucleic Acid , Base Sequence , Cell Line , Child, Preschool , DNA , Enhancer Elements, Genetic , Humans , Male , Molecular Sequence Data , Mutagenesis , Promoter Regions, Genetic , Tetraspanin 25 , Transcription Factors/metabolismABSTRACT
Synaptosome-associated protein-23 (SNAP23) is a component of the cellular mechanism required for specific membrane fusion and targetting of intracellular vesicles. We have cloned the full-length human cDNA and the SNAP23 gene. The SNAP23 gene has eight exons, with the initiation codon located in exon 2, and maps to the human chromosome 15q21-22 region. The human SNAP23 gene can generate two types of message, the full-length message (SNAP23A) and a shorter message (SNAP23B). The latter is the result of alternative splicing where exon 5 is joined to exon 7 and the skipping of exon 6; it thus lacks a region that is required for non-specific binding to plasma membranes. The two isoforms, expressed as fusion proteins with glutathione-S-transferase, interact in vitro with human syntaxin 6, thus retaining the specific protein interaction required for membrane fusion. Alterations in the SNAP23 gene might be involved in neurological and other diseases with defects in vesicle-membrane fusion processes that map to 15q15-21.
Subject(s)
Alternative Splicing , Carrier Proteins/genetics , Genes/genetics , Carrier Proteins/metabolism , Chromosome Mapping , Chromosomes, Human, Pair 15/genetics , Cloning, Molecular , Cytoplasmic Vesicles/physiology , DNA/chemistry , DNA/genetics , DNA, Complementary/chemistry , DNA, Complementary/genetics , Exons , HeLa Cells , Humans , In Situ Hybridization, Fluorescence , Introns , Membrane Fusion , Membrane Proteins/metabolism , Molecular Sequence Data , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism , Qa-SNARE Proteins , Qb-SNARE Proteins , Qc-SNARE Proteins , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Analysis, DNASubject(s)
DNA-Binding Proteins/genetics , Genes, Tumor Suppressor , Leucine Zippers/genetics , Lymphoma, Non-Hodgkin/genetics , Protein Isoforms/genetics , Sequence Deletion , Transcription Factors/genetics , Alternative Splicing , Endosomal Sorting Complexes Required for Transport , Gene Expression Regulation, Neoplastic/genetics , Humans , Protein Structure, Tertiary , Structure-Activity RelationshipABSTRACT
To understand the molecular basis of exocytosis in human neutrophils, the role of syntaxin 6 and SNAP-23 in neutrophil degranulation was examined. Human syntaxin 6 was cloned and identified as a 255-amino acid protein with a carboxy-terminal transmembrane region and two coiled-coil domains. Syntaxin 6 was localized mainly in the plasma membrane of human resting neutrophils, whereas SNAP-23 was located primarily in the mobilizable tertiary and specific granules. SNAP-23 was translocated to the cell surface, colocalizing with syntaxin 6, on neutrophil activation. In vitro binding studies established that SNAP-23 binds to syntaxin 6. Coimmunoprecipitation assays indicated that SNAP-23 interacts with syntaxin 6 in vivo, and this interaction was dramatically increased on neutrophil activation. Antibodies against SNAP-23 inhibited Ca(++) and GTP-gamma-S-induced exocytosis of CD67-enriched specific granules, but they hardly affected exocytosis of the CD63-enriched azurophilic granules, when introduced into electropermeabilized neutrophils. Anti-syntaxin 6 antibodies prevented exocytosis of both CD67- and CD63-enriched granules in electropermeabilized neutrophils. These data show that syntaxin 6 and SNAP-23 are involved in human neutrophil exocytosis, demonstrating that vesicle SNAP receptor-target SNAP receptor (v-SNARE- t-SNARE) interactions modulate neutrophil secretion. Syntaxin 6 acts as a target for secretion of specific and azurophilic granules, whereas SNAP-23 mediates specific granule secretion.
Subject(s)
Antigens, Neoplasm , Carrier Proteins/physiology , Cell Adhesion Molecules , Exocytosis , Membrane Proteins/physiology , Neutrophils/physiology , Amino Acid Sequence , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/pharmacology , Antigens, CD/analysis , Calcium/pharmacology , Carrier Proteins/analysis , Carrier Proteins/immunology , Cell Degranulation/physiology , Cell Membrane/chemistry , Cell Membrane Permeability , Cloning, Molecular , Fluorescent Antibody Technique , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Humans , Immunoblotting , Membrane Glycoproteins/analysis , Membrane Proteins/chemistry , Membrane Proteins/genetics , Microscopy, Confocal , Molecular Sequence Data , Neutrophils/chemistry , Neutrophils/ultrastructure , Platelet Membrane Glycoproteins/analysis , Qa-SNARE Proteins , Qb-SNARE Proteins , Qc-SNARE Proteins , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Subcellular Fractions/chemistry , Tetraspanin 30ABSTRACT
The tumour suppressor p53 protein integrates multiple signals regulating cell cycle progression and apoptosis. This regulation is mediated by several kinases that phosphorylate specific residues in the different functional domains of the p53 molecule. The human VRK1 protein is a new kinase related to a poxvirus kinase, and more distantly to the casein kinase 1 family. We have characterized the biochemical properties of human VRK1 from HeLa cells. VRK1 has a strong autophosphorylating activity in several Ser and Thr residues. VRK-1 phosphorylates acidic proteins, such as phosvitin and casein, and basic proteins such as histone 2b and myelin basic protein. Because some transcription factors are regulated by phosphorylation, we tested as substrates the N-transactivation domains of p53 and c-Jun fused to GST. Human c-Jun is not phosphorylated by VRK1. VRK1 phosphorylates murine p53 in threonine 18. This threonine is within the p53 hydrophobic loop (residues 13-23) required for the interaction of p53 with the cleft of its inhibitor mdm-2. The VRK1 C-terminus domain (residues 268-396) that contains a nuclear localization signal targets the protein to the nucleus, as determined by using fusion proteins with the green fluorescent protein. We conclude that VRK1 is an upstream regulator of p53 that belongs to a new signalling pathway.
Subject(s)
Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Proteins , Proto-Oncogene Proteins/metabolism , Threonine/metabolism , Tumor Suppressor Protein p53/metabolism , Viral Proteins/metabolism , 3T3 Cells , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Casein Kinases , Catalytic Domain , Cell Nucleus/metabolism , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins , Mice , Molecular Sequence Data , Nuclear Proteins/classification , Nuclear Proteins/genetics , Phosphorylation , Protein Kinases/classification , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/classification , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins c-mdm2 , Recombinant Fusion Proteins/classification , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Serine/metabolism , Substrate Specificity , Threonine/genetics , Transcription Factors/metabolism , Tumor Suppressor Protein p53/genetics , Viral Proteins/classification , Viral Proteins/geneticsABSTRACT
Detection of human papillomaviruses DNA (HPV) in tumour samples is often determined by a PCR based approach with standard universal consensus oligonucleotides. It is shown that these primers under the same amplification conditions can amplify a human genomic sequence of 1361 nucleotides in oral carcinomas and normal DNA samples. This sequence is detected more easily as the copy number of HPV DNA decreases. Therefore, in tumour samples that have a low copy number of HPV or that are contaminated by normal tissue there is a potential risk of misidentification of the presence of HPV if this observation is not taken into account.
Subject(s)
Carcinoma, Squamous Cell/genetics , DNA, Viral/analysis , Mouth Neoplasms/genetics , Papillomaviridae/genetics , Tumor Virus Infections , Base Sequence , Carcinoma, Squamous Cell/virology , DNA Primers , False Positive Reactions , HeLa Cells , Hematologic Neoplasms/genetics , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Molecular Sequence Data , Mouth Neoplasms/virology , Polymerase Chain Reaction/methodsABSTRACT
In the pathogenesis of cervical carcinoma there are three major components, two of them related to the role of human papillomaviruses (HPV). First, the effect of viral E6 and E7 proteins. Second, the integration of viral DNA in chromosomal regions associated with well known tumour phenotypes. Some of these viral integrations occur recurrently at specific chromosomal locations, such as 8q24 and 12q15, both harbouring HPV18 and HPV16. And third, there are other recurrent genetic alterations not linked to HPV. Recurrent losses of heterozygosity (LOH) have been detected in chromosome regions 3p14-22, 4p16, 5p15, 6p21-22, 11q23, 17p13.3 without effect on p53, 18q12-22 and 19q13, all of them suggesting the alteration of putative tumour suppressor genes not yet identified. Recurrent amplification has been mapped to 3q+ arm, with the common region in 3q24-28 in 90% of invasive carcinomas. The mutator phenotype, microsatellite instability, plays a minor role and is detected in only 7% of cervical carcinomas. The development of cervical carcinoma requires the sequential occurrence and selection of several genetic alterations. The identification of the specific genes involved, and their correlation with specific tumour properties and stages could improve the understanding and perhaps the management of cervical carcinoma.
Subject(s)
Papillomaviridae/genetics , Uterine Cervical Neoplasms/genetics , Chromosome Mapping , Chromosomes, Human , Female , Humans , Loss of Heterozygosity , Papillomaviridae/isolation & purification , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/virology , Virus IntegrationABSTRACT
The 3p14.2 chromosome region, which contains the FHIT gene and the FRA3B fragile site, is frequently altered in carcinomas. We analyzed the expression of the FHIT gene in 21 Burkitt's lymphoma cell lines and normal lymphoid populations. Seventeen (80%) of these cell lines had a common aberrant FHIT transcript as well as the normal transcript. Exon 2 was often aberrantly spliced to several coding exons, skipping exons 3 and 4, which overlap FRA3B. Other aberrant transcripts lacked exons 4-7 or exons 5-8. Exon 5, which has the initiation codon, was the most commonly affected. In two cell lines, Raji and KK124, there were aberrant transcripts retaining only the coding exons, which were able to make a normal protein, as demonstrated by in vitro transcription-translation analysis. In these aberrant messages, the additional deletion of 11 nucleotides at the beginning of exon 10 resulted in loss of translation. The cell line Ramos did not have a normal transcript. Some transcripts had common insertions of unknown origin that replaced coding exons, mainly exons 6 and 7. None of these aberrant messages coded for a protein, whether normal or aberrant. Within an individual cell line, aberrant messages appeared to result from sequential splicing reactions of a transcriptional unit derived from one allele. There was no correlation between aberrant FHIT transcription and the type of Burkitt's lymphoma regarding chromosomal translocation or presence of Epstein-Barr virus. In normal tonsils, spleen, and peripheral blood lymphocytes, aberrant transcripts were not detected and might represent a very minor subpopulation if detectable.
Subject(s)
Acid Anhydride Hydrolases , Burkitt Lymphoma/genetics , Neoplasm Proteins , Proteins/genetics , RNA Splicing/genetics , RNA, Messenger/analysis , Alleles , Base Sequence , Cell Line , Chromosome Fragile Sites , Chromosome Fragility/genetics , Chromosomes, Human, Pair 3/genetics , Codon/genetics , DNA Mutational Analysis , Exons/genetics , Gene Expression Regulation, Neoplastic , Humans , Mutation , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Neoplasm/analysis , RNA, Neoplasm/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Deletion/genetics , Transcription, Genetic , Tumor Cells, CulturedABSTRACT
The tumor susceptibility gene, TSG101, has been identified as a candidate tumor suppressor gene. We have examined the expression of TSG101 in Burkitt lymphoma cell lines. Several aberrant messages were detected in all cell lines. Aberrant splice donor sites are located within exon 1 at positions 132, 154, 172 and 284. Splice acceptors are located at positions 847 and 1054 within exon 5. The aberrant messages are coexpressed with a normal message and could be the result of additional splicing reactions of the mature message that behaves as an intermediate. The normal message codes for 46 kDa protein (TSG101A). One aberrant message joins in frame nucleotides 283-1055 and codes for a protein isoform of 17 kDa (TSG101B), as demonstrated by in vitro translation assays. The TSG101B isoform lacks the leucine zipper near the C-terminus, a transcriptional repressor domain, and retains most of the N-terminal region which has homology to E2 ubiquitin regulatory enzymes and the CROC-1 transcriptional regulator. The TSG101B isoform was detected in sixteen out of twenty-two (72%) BL cell lines, but not in normal lymphoid populations. The presence of two TSG101 isoforms with different dimerization potential opens up a new level of regulation of the TSG101 proteins possibly affecting cell cycle regulation.
Subject(s)
Burkitt Lymphoma/genetics , DNA-Binding Proteins/biosynthesis , Leucine Zippers/genetics , Neoplasm Proteins/biosynthesis , Protein Isoforms/biosynthesis , RNA Splicing , Transcription Factors/biosynthesis , Amino Acid Sequence , Burkitt Lymphoma/metabolism , Burkitt Lymphoma/pathology , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Endosomal Sorting Complexes Required for Transport , Exons/genetics , Gene Expression Regulation, Neoplastic , Humans , Molecular Sequence Data , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Protein Biosynthesis , Protein Isoforms/chemistry , Protein Isoforms/genetics , RNA, Messenger/biosynthesis , RNA, Neoplasm/biosynthesis , Sequence Alignment , Sequence Homology, Amino Acid , Transcription Factors/chemistry , Transcription Factors/genetics , Tumor Cells, CulturedABSTRACT
Syntaxins are major components of vesicle trafficking and their pattern of expression depends on the cell type. Using reverse transcriptase-polymerase chain reaction (RT-PCR), cloning, and sequencing techniques, we have found that human neutrophils and neutrophil-differentiated HL-60 cells co-express syntaxins 1A, 3, 4, 5, 6, 7, 9, 11, and 16. These genes are also expressed in human peripheral blood lymphocytes and SH-SY5Y neuroblastoma cells, which, unlike neutrophils, also expressed syntaxin 10. We have identified two isoforms of syntaxin 3. Syntaxin 3A, similar to the previously reported syntaxin 3, and the novel isoform syntaxin 3B, which is identical to syntaxin 3A but lacks 37 amino acid residues at the carboxy-terminal region. Syntaxin 1 was mainly located to neutrophil granule membranes by confocal microscopy and by immunoblotting of subcellular fractions. These data indicate that syntaxin 1 cannot be considered specific to neural tissues. The level of expression of syntaxins 3, 4, 6, and 11 was increased during neutrophil differentiation of HL-60 cells, whereas that of syntaxins 1A, 5, 9, and 16 was unchanged. Syntaxin 7 was not expressed in undifferentiated HL-60 cells, but its expression was induced on neutrophil differentiation. The expression of several syntaxin genes in human neutrophils could be related to the high secretory capacity of these cells as well as to the presence of different cytoplasmic granules with distinct exocytic capabilities.
Subject(s)
Antigens, Surface/genetics , Gene Expression Regulation , Nerve Tissue Proteins/genetics , Neutrophils/physiology , Amino Acid Sequence , Antigens, Surface/biosynthesis , Cell Differentiation/genetics , HL-60 Cells , Humans , Molecular Sequence Data , Nerve Tissue Proteins/biosynthesis , Neutrophils/cytology , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Sequence Alignment , Syntaxin 1ABSTRACT
Tetraspanin antigens are implicated in the prognosis of different types of tumours. In this study we determine by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) the level of 13 tetraspan messages in 21 Burkitt lymphoma (BL) cell lines. All tumour cell lines have a common pattern of tetraspanin gene expression. There are five antigens which are detected in 90% of cell lines at high levels, CD53, CD81, CD63, SAS and CD82. Another two, CD9 and CD37, were detected in 60% of cell lines, and have a very variable level of expression. The remaining antigens, A15, CoO29, KRAG, L6, TI-1 and il-TMP, are expressed at low levels in very few cell lines without any specific pattern. The level of gene expression corresponds with the level of cell surface antigen determined by flow cytometry. The average number of tetraspan proteins expressed per cell line is six. These proteins may form subunits of an oligomeric structure with 24 transmembrane domains. There are no major differences in tetraspan expression pattern among sporadic or endemic tumours, type of translocation or Epstein-Barr virus status, suggesting the original cell of these tumours is the same, probably a late pre-B cell, at the CD9 to CD37 transition point. Tetraspanin gene expression is consistent with BL being a single entity, despite variations in other parameters.
Subject(s)
Antigens, Neoplasm/genetics , Burkitt Lymphoma/genetics , Membrane Glycoproteins , Membrane Proteins/genetics , Antigens, CD/analysis , Gene Expression , Glycoproteins/analysis , Humans , Polymerase Chain Reaction , Tetraspanin 29 , Tetraspanins , Translocation, Genetic , Tumor Cells, CulturedABSTRACT
Genital carcinomas are associated with human papillomaviruses, and the viral DNA is frequently integrated in the host cell genome. Recurrent chromosomal alterations are genetic markers for specific tumor phenotypes. To demonstrate that papillomavirus DNA integration is indeed a recurrent chromosomal aberration, we mapped two independent papillomavirus integration sites in the human 12q14-15 region, one containing HPV16 DNA and the other HPV18 DNA. The two HPV integration sites map approximately 10 kbp from each other within the cosmid LLNL12NCO1-196E1 clone. The integration site corresponding to HPV16 DNA in SK-v cells is proximal to the 5' end of a DNA segment known to be rearranged by integration of HPV18 DNA in another cervical carcinoma cell line, SW756. Both integrations are located in the PAL2 locus within the uterine leiomyoma cluster region of translocation.