RESUMO
Circulating tumor cells (CTC) have been studied in various solid tumors but clinical utility of CTC in small cell lung cancer (SCLC) remains unclear. The aim of the CTC-CPC study was to develop an EpCAM-independent CTC isolation method allowing isolation of a broader range of living CTC from SCLC and decipher their genomic and biological characteristics. CTC-CPC is a monocentric prospective non-interventional study including treatment-naïve newly diagnosed SCLC. CD56+ CTC were isolated from whole blood samples, at diagnosis and relapse after first-line treatment and submitted to whole-exome-sequencing (WES). Phenotypic study confirms tumor lineage and tumorigenic properties of isolated cells for the 4 patients analyzed with WES. WES of CD56+ CTC and matched tumor biopsy reveal genomic alteration frequently impaired in SCLC. At diagnosis CD56+ CTC were characterized by a high mutation load, a distinct mutational profile and a unique genomic signature, compared to match tumors biopsies. In addition to classical pathways altered in SCLC, we found new biological processes specifically affected in CD56+ CTC at diagnosis. High numeration of CD56+ CTC (> 7/ml) at diagnosis was associated with ES-SCLC. Comparing CD56+ CTC isolated at diagnosis and relapse, we identify differentially altered oncogenic pathways (e.g. DLL3 or MAPK pathway). We report a versatile method of CD56+ CTC detection in SCLC. Numeration of CD56+ CTC at diagnosis is correlated with disease extension. Isolated CD56+ CTC are tumorigenic and show a distinct mutational profile. We report a minimal gene set as a unique signature of CD56+ CTC and identify new affected biological pathways enriched in EpCAM-independent isolated CTC in SCLC.
Assuntos
Neoplasias Pulmonares , Células Neoplásicas Circulantes , Carcinoma de Pequenas Células do Pulmão , Humanos , Molécula de Adesão da Célula Epitelial , Relevância Clínica , Estudos Prospectivos , Genômica , Carcinogênese , Proteínas de Membrana , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
Recently, HLA-G transgenic mice were shown to exhibit transgene transcription in several extraembryonic tissues. To determine whether HLA-G mRNAs are also expressed in other human tissues, we have undertaken Northern blot and RT-PCR assays using HLA-G locus-specific probe and primers. These studies demonstrate that the HLA-G gene is transcribed in a variety of cells and adult tissues obtained from different individuals (peripheral blood leukocytes, placenta, skin, spleen, thymus, prostate, testicle, ovary, small intestine, colon, heart, brain, lung, liver, and kidney), as well as in fetal tissues (heart, lung, liver, and kidney). The HLA-G mRNA level observed in most tissues is orders of magnitude lower than the level of classic class I genes in the same tissues. RT-PCR studies have demonstrated that alternative splicing of the HLA-G primary transcript is different from tissue to tissue and could be regulated in a tissue-specific fashion. Sequencing of keratinocyte transcripts has confirmed previous observations: (a) three different alternative splicing transcripts are produced (a full-length transcript, an mRNA lacking exon 3, and a transcript devoid of exon 3 and 4) and (b) HLA-G polymorphism is limited in the coding regions. In view of this wide HLA-G tissue distribution, a new hypothesis dealing with possible HLA-G function is proposed.
Assuntos
Antígenos HLA/biossíntese , Antígenos de Histocompatibilidade Classe I/biossíntese , Processamento Alternativo/genética , Sequência de Bases , Northern Blotting , Linhagem Celular , Antígenos HLA/genética , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Queratinócitos/imunologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Transcrição GênicaRESUMO
We previously isolated and characterized a set of overlapping yeast artificial chromosome (YAC) clones spanning 2.4 Mb, including the entire MHC class I region, as a first step towards a detailed genetic analysis. We report here the genomic sequence of the two ends of HLA class I. The centromeric portion of HLA class I, extending from TNF to HLA-C was determined using two different sources of genomic DNA. As a first source, we sequenced cosmids (provided by T. Spies) derived from a total human DNA library which were mapped with conventional restriction digestion and fingerprinting. A second more generalizable approach was used to obtain cosmids for the remainder of the region. The new technology of Multiple-Complete-Digest (MCD) mapping, developed in Maynard Olson's laboratory, was used to map cosmids derived from YACs. This technique involves screening deep cosmid libraries derived from selected YACs and subjecting the cosmids to complete digestion with restriction enzymes, followed by computational assembly into completed maps. This method was also used to obtain material for sequence from three overlapping YACs covering a contiguous region from HLA-G to a point 330 kb telomeric. Among the plethora of new genetic information is a detailed picture of the organization of new multigene families contained within the telomeric end and of members of families spread throughout HLA class I. A clear relationship between the telomeric and centromeric ends of HLA class I has been defined, suggesting that large portions of these regions derived from a common ancestor. Our results demonstrate genomic sequencing to be one of the most effective and efficient means of identifying new genes, yielding information about genomic structure, regulation, and offering new insights into the meaning of physical relationships among functionally interacting genes.
Assuntos
Genes MHC Classe I/genética , Família Multigênica/genética , Centrômero , Mapeamento Cromossômico/métodos , Cromossomos Artificiais de Levedura , Cromossomos Humanos Par 6/genética , Clonagem Molecular/métodos , Cosmídeos , Antígenos HLA-C/genética , Humanos , Análise de Sequência de DNA , Telômero , Fator de Necrose Tumoral alfa/genéticaRESUMO
The phenomenon of antigenic modulation was studied in the histiocytic lymphoma line U937. A redistribution of cell surface HLA antigen after incubation of U937 cells with the monomorphic anti-HLA class I monoclonal antibody W6/32 was demonstrated by immunofluorescence analysis. As assessed by hybridization of RNA obtained from W6/32-treated U937 cells with a probe corresponding to the alpha 3 domain of HLA Cw3, prolonged W6/32 incubation (24 to 72 hours) induced a decrease in HLA class I transcript abundance. This decrease was about 25% as compared with untreated control cells. These data indicate that W6/32 incubation can induce changes in HLA class I gene expression not only at the antigenic but also at the transcriptional level. Possible implications for the molecular basis of antigenic modulation are discussed.
Assuntos
Anticorpos Monoclonais/imunologia , Genes de Imunoglobulinas , Antígenos HLA/imunologia , Imunoglobulina G/imunologia , Monócitos/imunologia , Transcrição Gênica , Humanos , Linfoma Difuso de Grandes Células B/patologia , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Células Tumorais CultivadasRESUMO
This article presents an overview of the more recent data dealing with the constitutive, transcriptional, and translational expression of classical class Ia and nonclassical HLA-E and -G class Ib products in the different trophoblast cell subpopulations that constitute the maternofetal interface during human pregnancy. Of particular interest is the expression of alternatively spliced HLA-G transcriptional isoforms that may be translated in membrane-bound or soluble protein products. Molecular regulatory mechanisms that may control the differential expression of class Ia and class Ib molecules, according to the cell types, state of differentiation, and stages of gestation are also examined. They may operate at the levels of transcription, translation and/or transport of proteins to the cell surface. Functional significance of the absence of detectable cell surface expression of class Ia molecules in all trophoblast cell subpopulations, and of the presence of membrane-bound HLA-G products in extravillous cytotrophoblast cells is finally questioned.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/imunologia , Genes MHC Classe I/imunologia , Antígenos HLA/biossíntese , Antígenos HLA/genética , Placenta/imunologia , Placenta/metabolismo , Feminino , Humanos , GravidezRESUMO
We report here the genomic sequence of the centromeric portion of HLA class I, extending 424,015 bp from tumor necrosis factor alpha to a newly identified gene approximately 20 kb telomeric of Otf-3. As a source of DNA, we used cosmids centromeric of HLA-B that had been mapped previously with conventional restriction digestion and fingerprinting and previously characterized yeast artificial chromosomes subcloned into cosmids and mapped with multiple complete digest methodologies. The data presented provide a description of the gene content of centromeric HLA class I including new data on intron, promoter and flanking sequences of previously described genes, and a description of putative new genes that remain to be characterized beyond the structural information uncovered. A complete accounting of the repeat structure including abundant di-, tri-, and tetranucleotide microsatellite loci yielded access to precisely localized mapping tools for the major histocompatibility complex. Comparative analysis of a highly polymorphic region between HLA-B and -C was carried out by sequencing over 40 kb of overlapping sequence from two haplotypes. The levels of variation observed were much higher than those seen in other regions of the genome and indeed were higher than those observed between allelic HLA class I loci.
Assuntos
Centrômero , Antígenos de Histocompatibilidade Classe I/genética , Polimorfismo Genético , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Biblioteca Genômica , Haplótipos , Dados de Sequência Molecular , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
Otf-3 (Octamer-binding transcription factor 3) is an octamer binding protein encoded by the murine gene Otf-3. Otf-3 belongs to a multigenic family and maps to the mouse Chromosome 17 between the Q and T regions within the major histocompatibility complex (MHC). We report the mapping of the human homologue: OTF3, to human chromosome 6p21.3-->p22 within or close to the human MHC class I region. Furthermore, one OTF3-like copy (OTF3L) is localized to 12p13.
Assuntos
Cromossomos Humanos Par 12 , Cromossomos Humanos Par 6 , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/genética , Corantes Azur , Bandeamento Cromossômico , Mapeamento Cromossômico/métodos , Genes MHC Classe I , Humanos , Hibridização In Situ , Fator 3 de Transcrição de OctâmeroRESUMO
PROBLEM: We investigated the possible negative regulatory mechanisms that repress classical human leukocyte antigen (HLA) class I gene expression in human spermatozoa and searched for novel testis-specific coding sequences that might be present in MHC class I chromosomal region. METHOD: We performed a comparative DNA methylation analysis of this genomic region in both purified human spermatozoa and mononuclear blood cells from the same donors, using methylation-sensitive restriction enzymes followed by classical or pulsed field gel electrophoresis and hybridization with HLA class I locus-specific probes. RESULTS: Unmethylated CpG sites were detected in the 3' part of HpaII tiny fragments of the HLA-F and HLA-G genes in spermatozoal DNA. In contrast, no difference was observed in the methylation status of the HLA-B, HLA-C, and HLA-E genes between germ and somatic cells. CpG unmethylation events were also detected in several parts of this chromosomal region (outside the known loci) in spermatozoal DNA. CONCLUSIONS: These results suggest that this genomic region undergoes changes in its DNA methylation pattern during the developmental process. We hypothesize that these dynamic changes have functional importance, including a possible transcriptional activity of nonclassical class I genes and/or as yet undescribed testis-specific coding sequences.
Assuntos
DNA/química , DNA/genética , Genes MHC Classe I , Espermatozoides/química , Espermatozoides/imunologia , Sequência de Bases , Mapeamento Cromossômico , Sondas de DNA , Eletroforese em Gel de Campo Pulsado , Antígenos HLA/genética , Humanos , Leucócitos Mononucleares/química , Leucócitos Mononucleares/imunologia , Masculino , Metilação , Hibridização de Ácido NucleicoRESUMO
A testicular form of hormone-sensitive lipase (HSL(tes)), a triacylglycerol lipase, and cholesterol esterase, is expressed in male germ cells. Northern blot analysis showed HSL(tes) mRNA expression in early spermatids. Immunolocalization of the protein in human and rodent seminiferous tubules indicated that the highest level of expression occurred in elongated spermatids. We have previously shown that 0.5 kilobase pairs of the human HSL(tes) promoter directs testis-specific expression of a chloramphenicol acetyltransferase reporter gene in transgenic mice and determined regions binding nuclear proteins expressed in testis but not in liver (Blaise, R., Grober, J., Rouet, P., Tavernier, G., Daegelen, D., and Langin, D. (1999) J. Biol. Chem. 274, 9327-9334). Mutation of a SRY/Sox-binding site in one of the regions did not impair in vivo testis-specific expression of the reporter gene. Further transgenic analyses established that 95 base pairs upstream of the transcription start site were sufficient for correct testis expression. In gel retardation assays using early spermatid nuclear extracts, a germ cell-specific DNA-protein interaction was mapped between -46 and -29 base pairs. The DNA binding nuclear protein showed properties of zinc finger transcription factors. Mutation of the region abolished reporter gene activity in transgenic mice, showing that it is necessary for testis expression of HSL(tes).
Assuntos
Regiões Promotoras Genéticas , Espermátides/enzimologia , Esterol Esterase/genética , Testículo/enzimologia , Animais , Sequência Consenso , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Mutação , RNA Mensageiro/biossíntese , Ratos , Espermatozoides/metabolismo , Esterol Esterase/biossíntese , Transcrição GênicaRESUMO
Specific expression of the non classical class I HLA-G gene on trophoblasts, the only fetal tissue in contact with maternal cells which lack MHC class I antigens, may indicate a role of this gene in fetal-maternal tolerance. We recently reported HLA-G transcription in peripheral blood leukocytes. In this work, we have investigated HLA-G transcription in hematopoietic stem cells, in different hematopoietic lineages and in malignant cells by using a RT-PCR technique. PCR amplification with primers specific to the exon 2 and the 3' untranslated region has enabled to detect HLA-G transcription in B and T cell populations. No transcription was found in CD34+ cells, in thymocytes, in polynuclear cells, in monocytes and in natural killer cells. Among the malignancies analyzed, HLA-G is transcribed in 2 of 13 cases of acute leukemia characterized by a monocytic contingent, in 3 of 6 CLL and in all the cases of B-NHL (n = 6). No HLA-G transcription was detected in myeloma (n = 2). The splicing type does not seem to be linked to a lymphocyte subpopulation nor to a malignant proliferation stage. These results suggest that HLA-G is a marker of mature lymphoid cells and may play an immunological function as a peptide presenting molecule. HLA-G transcription in some cases of malignancy might indicate a contribution to the tumoral progression by blocking natural killing reaction.
Assuntos
Antígenos de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Regulação da Expressão Gênica , Antígenos HLA/genética , Neoplasias Hematológicas/imunologia , Hematopoese , Células-Tronco Hematopoéticas/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Subpopulações de Linfócitos/imunologia , Células-Tronco Neoplásicas/imunologia , Transcrição Gênica , Antígenos de Neoplasias/biossíntese , Regulação Leucêmica da Expressão Gênica , Antígenos HLA/biossíntese , Antígenos HLA-G , Neoplasias Hematológicas/genética , Células-Tronco Hematopoéticas/classificação , Células-Tronco Hematopoéticas/metabolismo , Antígenos de Histocompatibilidade Classe I/biossíntese , Humanos , Subpopulações de Linfócitos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Células-Tronco Neoplásicas/metabolismo , Especificidade de Órgãos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Timoma/genética , Timoma/imunologia , Neoplasias do Timo/genética , Neoplasias do Timo/imunologia , Células Tumorais CultivadasRESUMO
Specific expression of the non classical class I HLA-G gene on trophoblasts, the only fetal tissue in contact with maternal cells which lack MHC class I antigens, may indicate a role of this gene in fetal-maternal tolerance. We recently reported HLA-G transcription in peripheral blood leukocytes. In this work, we have investigated HLA-G transcription in hematopoietic stem cells, in different hematopoietic lineages and in malignant cells by using a RT-PCR technique. PCR amplification with primers specific to the exon 2 and the 3' untranslated region has enabled to detect HLA-G transcription in B and T cell populations. No transcription was found in CD34+ cells, in thymocytes, in polynuclear cells, in monocytes and in natural killer cells. Among the malignancies analyzed, HLA-G is transcribed in 2 of 13 cases of acute leukemia characterized by a monocytic contingent, in 3 of 6 CLL and in all the cases of B-NHL (n = 6). No HLA-G transcription was detected in myeloma (n = 2). The splicing type does not seem to be linked to a lymphocyte subpopulation nor to a malignant proliferation stage. These results suggest that HLA-G is a marker of mature lymphoid cells and may play an immunological function as a peptide presenting molecule. HLA-G transcription in some cases of malignancy might indicate a contribution to the tumoral progression by blocking natural killing reaction.
Assuntos
Antígenos HLA/genética , Neoplasias Hematológicas/imunologia , Hematopoese/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos HLA-G , Neoplasias Hematológicas/genética , Hematopoese/genética , Humanos , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Polymorphic as well as HLA-F and -G genes are repressed in the human cell line JAR, derived from a tumor of trophoblast origin. By contrast, the HLA-E gene as well as the non-HLA novel coding-sequence, R1, located 5' to HLA-E, both remain transcriptionally active. We first demonstrated the role of DNA methylation in the repression of class I genes (except HLA-E) in JAR by the use of the 5-Azacytidine demethylating agent. Following treatment, JAR clones reexpressed polymorphic class I transcripts and cell surface alpha chains. Using methylation-sensitive rare cutter enzymes on JAR genomic DNA, followed by classical or pulse field gel electrophoresis and hybridization with HLA locus-specific probes, we found methylated CpG islands in the 5' region of all class I genes, except for HLA-E. These results, establishing an inverse relationship between states of methylation and transcriptional activity within the MHC class I chromosomal region in JAR, and the observations that the HLA-E and R1 genes were ubiquitously expressed, suggest that the HLA-E chromosomal domain might have functional importance including the presence of housekeeping genes.
Assuntos
Coriocarcinoma/genética , Genes MHC Classe I , Antígenos HLA/genética , Antígenos de Histocompatibilidade Classe I/genética , Azacitidina/farmacologia , Sequência de Bases , Eletroforese em Gel de Campo Pulsado , Regulação da Expressão Gênica , Genes , Técnicas In Vitro , Metilação , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Polimorfismo Genético , Polimorfismo de Fragmento de Restrição , RNA Mensageiro/genética , Mapeamento por Restrição , Transcrição Gênica , Células Tumorais Cultivadas , Antígenos HLA-ERESUMO
In human spermatogenic cells, in contrast to somatic cells, expression of major histocompatibility complex (MHC) class I molecules is undetectable. This lack of expression may contribute to the absence of female immune reaction against spermatozoa and may be necessary for gamete fusion. Among the molecular repressor mechanisms that may be used at the DNA level, we investigated 5' CpG methylation of the different class Ia and class Ib loci in meiotic pachytene spermatocytes and postmeiotic round spermatids, which had been purified from human testes by centrifugal elutriation. These results were compared with those obtained with mature spermatozoa and peripheral blood mononuclear cells. Using methylation-sensitive restriction enzymes and DNA locus-specific probes, we found that HLA-A, HLA-B/C, and HLA-E loci were similarly unmethylated in the germ and somatic cells tested, whereas HLA-F and HLA-G were even less methylated in the former cells. Together with the observation that spermatozoon DNA contains class I genes that are transfectable and able to direct transcription and protein synthesis in murine L cells, these data suggest that HLA class I genes are in an active conformation in male germ cells. We indeed found that both spermatocytes and spermatids contained low levels of class Ia and class Ib mRNA. Using reverse transcriptase-polymerase chain reaction, followed by DNA sequencing, we also detected three HLA-G transcriptional isoforms, resulting from alternative splicings, which suggested that this class Ib gene may have a potential function in these germ cells. Although intracellular expression of beta2-microglobulin (the light chain that associates with HLA class I heavy chains) was found in spermatocytes but not in round spermatids, no membrane-bound nor intracellular translated HLA class I heavy chain was detected in either germ cell type, when monomorphic anti-HLA class I monoclonal antibodies were used. Thus, lack of expression of HLA class I proteins in the male germ line is likely to involve post-transcriptional mechanisms of regulation.
Assuntos
Expressão Gênica , Genes MHC Classe I/genética , Meiose , Espermatozoides/imunologia , Processamento Alternativo , Animais , Sequência de Bases , Metilação de DNA , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Conformação de Ácido Nucleico , RNA Mensageiro/análise , Espermátides/imunologia , Espermatogênese , Transfecção , Células Tumorais CultivadasRESUMO
Of the various molecular regulatory mechanisms that may be used by human trophoblast cells to down-regulate expression of HLA class I genes, we chose to investigate the methylation of DNA, generally associated with inhibition of transcription. We analyzed the methylation status of different HLA class I loci in villous and extravillous cytotrophoblast cells and in vitro-differentiated syncytiotrophoblast, purified from human term placenta, as well as in the human trophoblast-derived JAR and JEG-3 cell lines. We then compared methylation status and transcriptional activity. An inverse relationship was established between JAR and JEG-3: HLA-A, -B, and -G are methylated and repressed in JAR, whereas in JEG-3, HLA-A is methylated and repressed but HLA-B and -G are partially methylated and transcribed. HLA-E is unmethylated and transcribed in both cell lines. Apart from HLA-E, which is always unmethylated and transcribed, no such relationship exists for the other class I loci in trophoblast cells. Whereas nonclassical HLA-G and classical HLA-A and -B class I genes are undermethylated in both cytotrophoblast and syncytiotrophoblast, they are clearly transcribed in the former but minimally transcribed in the latter subpopulation. Thus, the down-regulation of class I gene expression in the in vitro-differentiated syncytiotrophoblast is unlikely to be caused by DNA methylation. Furthermore, there is no detectable expression of any class I molecule at the cell surface of either trophoblast cell subpopulation, suggesting a negative control on translation and/or on the secretory pathway to the plasma membrane.