RESUMO
Under conditions of environmental stress, prokaryotes and lower eukaryotes such as the yeast Saccharomyces cerevisiae selectively utilize particular subunits of RNA polymerase II (pol II) to alter transcription to patterns favoring survival. In S. cerevisiae, a complex of two such subunits, RPB4 and RPB7, preferentially associates with pol II during stationary phase; of these two subunits, RPB4 is specifically required for survival under nonoptimal growth conditions. Previously, we have shown that RPB7 possesses an evolutionarily conserved human homolog, hsRPB7, which was capable of partially interacting with RPB4 and the yeast transcriptional apparatus. Using this as a probe in a two-hybrid screen, we have now established that hsRPB4 is also conserved in higher eukaryotes. In contrast to hsRPB7, hsRPB4 has diverged so that it no longer interacts with yeast RPB7, although it partially complements rpb4- phenotypes in yeast. However, hsRPB4 associates strongly and specifically with hsRPB7 when expressed in yeast or in mammalian cells and copurifies with intact pol II. hsRPB4 expression in humans parallels that of hsRPB7, supporting the idea that the two proteins may possess associated functions. Structure-function studies of hsRPB4-hsRPB7 are used to establish the interaction interface between the two proteins. This identification completes the set of human homologs for RNA pol II subunits defined in yeast and should provide the basis for subsequent structural and functional characterization of the pol II holoenzyme.
Assuntos
RNA Polimerase II/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Clonagem Molecular , Células HeLa , Humanos , Dados de Sequência Molecular , Mapeamento de Peptídeos , Ligação Proteica , Conformação Proteica , RNA Polimerase II/genéticaRESUMO
Comparative genomic hybridization analysis was used to identify chromosomal imbalances in 20 non-small cell lung carcinoma (NSCLC) biopsies and cell lines. The chromosome arms most often overrepresented were 3q (85%), 5p (70%), 7p (65%), and 8q (65%), which were observed at high copy numbers in many cases. Other common overrepresented sites were 1q, 2p, and 20p. DNA sequence amplification was often observed, with the most frequent site being 3q26 (six cases). Other recurrent sites of amplification included 8q24, 3q13, 3q28-qter, 7q11.2, 8p11-12, 12p12, and 19q13.1-13.2. The most frequent underrepresented segment was 3p21 (50%); other recurrent sites of autosomal loss included 8p21-pter, 15q11.2-13, 5q11.2-15, 9p, 13q12-14, 17p, and 18q21-qter. These regions of copy number decreases are also common sites of allelic loss, further implicating these sites as locations of tumor suppressor genes. Although some of the overrepresented segments harbor known or suspected oncogenes/growth-regulatory genes, we have identified 3q and 5p as new sites that are very frequently overrepresented in NSCLC. These findings could represent entry points for the identification of novel amplified DNA sequences that may contribute to the development or progression of NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , DNA de Neoplasias/metabolismo , Amplificação de Genes , Neoplasias Pulmonares/genética , Biópsia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Cromossomos Humanos Par 3 , Cromossomos Humanos Par 5 , Cromossomos Humanos Par 7 , Cromossomos Humanos Par 8 , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Hibridização de Ácido Nucleico , Análise de Sequência de DNA , Células Tumorais CultivadasRESUMO
Comparative genomic hybridization analysis was performed to identify chromosomal imbalances in 24 human malignant mesothelioma (MM) cell lines derived from untreated primary tumors. Chromosomal losses accounted for the majority of genomic imbalances. The most frequent underrepresented segments were 22q (58%) and 15q1.1-21 (54%); other recurrent sites of chromosomal loss included 1p12-22 (42%), 13q12-14 (42%), 14q24-qter (42%), 6q25-qter (38%), and 9p21 (38%). The most commonly overrepresented segment was 5p (54%). DNA sequence amplification at 3p12-13 was observed in two cases. Whereas some of the regions of copy number decreases (i.e., segments in 1p, 6q, 9p, and 22q) have previously been shown to be common sites of karyotypic and allelic loss in MM, our comparative genomic hybridization analyses identified a new recurrent site of chromosomal loss within 15q in this malignancy. To more precisely map the region of 15q deletion, loss of heterozygosity analyses were performed with a panel of polymorphic microsatellite markers distributed along 15q, which defined a minimal region of chromosomal loss at 15q11.1-15. The identification of frequent losses of a discrete segment in 15q suggests that this region harbors a putative tumor suppressor gene whose loss/inactivation may contribute to the pathogenesis of many MMs.
Assuntos
Cromossomos Humanos Par 15 , Perda de Heterozigosidade , Mesotelioma/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Humanos , Hibridização de Ácido Nucleico , Rad51 Recombinase , Células Tumorais CultivadasRESUMO
Krev-1/rap1A is an evolutionarily conserved Ras-family GTPase whose cellular function remains unclear, but which has been proposed to function as a tumor suppressor gene, and may act as a Ras antagonist. To elucidate Krev-1 activity, we have used LexA-Krev-1 in a two-hybrid screen of a HeLa cell cDNA library. Of the two cDNA classes isolated, one contained a single isolate encoding the known Krev-1 interactor Raf, while the second contained multiple isolates coding for a previously undescribed protein which we have designated Kritl (for Krev Interaction Trapped 1). The full length Krit1 cDNA encodes a protein of 529 amino acids, with an amino-terminal ankyrin repeat domain and a novel carboxy-terminal domain required for association with Krit1. Krit1 interacted strongly with Krev-1 but only weakly with Ras, suggesting it might specifically regulate Krev-1 activities. Krit1 mRNA and protein are expressed endogenously at low levels, with tissue specific variation. Intriguingly, the Krit cDNA has been mapped by FISH to chromosome 7q21-22, a region known to be frequently deleted or amplified in multiple forms of cancer.
Assuntos
Repetição de Anquirina , Cromossomos Humanos Par 7 , Proteínas de Ligação ao GTP/metabolismo , Proteínas Associadas aos Microtúbulos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Regulação da Expressão Gênica , Células HeLa , Humanos , Proteína KRIT1 , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas rap de Ligação ao GTPRESUMO
We isolated mouse cDNA clones containing the entire coding region of the putative oncogene Akt2. Sequence analysis revealed that, like its human homolog, Akt2 encodes a protein-serine/threonine kinase containing a pleckstrin homology domain at its amino terminus. Fluorescence in situ hybridization of the mouse cDNA to rodent metaphase spreads demonstrated that the Akt2 gene maps to mouse chromosome band 7B1 and rat chromosome 1q22. Expression levels of mouse Akt2 mRNA and Akt2 protein varied among tissues, with the highest levels in skeletal muscle. Akt2 expression was low in a multipotent fibroblast cell line, but it was upregulated when these cells were transformed with Myod and induced to differentiate into myocytes. These data demonstrate that Akt2 expression is activated during cellular differentiation and suggest that it functions in the signaling pathways of some adult tissues.
Assuntos
Mapeamento Cromossômico , Proteínas Oncogênicas/genética , Oncogenes , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular Transformada , Clonagem Molecular , DNA Complementar/isolamento & purificação , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Dados de Sequência Molecular , Proteína MyoD/genética , Proteínas Oncogênicas/análise , Proteínas Serina-Treonina Quinases/análise , Proteínas Proto-Oncogênicas c-akt , RatosRESUMO
AKT2 is a serine/threonine kinase implicated in human ovarian and pancreatic cancers. AKT2 is activated by a variety of growth factors and insulin via phosphatidylinositol 3-kinase (PI3K). However, its normal cellular role is not well understood. To gain insight into the function of AKT2, we performed yeast two-hybrid system to screen for interacting proteins. Using this technique, we identified a novel interactor, designated APPL, which contains a pleckstrin homology (PH) domain, a phosphotyrosine binding (PTB) domain and a leucine zipper, classes of motifs defined in signaling molecules as functional interaction domains with specific targets. The PH domain of APPL shows similarity to those found in GTPase-activating proteins such as oligophrenin-1 and Graf, whereas its PTB domain exhibits homology with CED-6, an adaptor protein that promotes engulfment of apoptotic cells, and IB1, a transactivator of the GLUT2 gene. APPL is highly expressed in skeletal muscle, heart, ovary and pancreas, tissues in which AKT2 mRNA is abundant. APPL interacts with the inactive form of AKT2; moreover, APPL binds to the PI3K catalytic subunit, p110alpha. These data suggest that APPL is an adaptor that may tether inactive AKT2 to p110alpha in the cytoplasm and thereby may expedite recruitment of AKT2 and p110alpha to the cell membrane upon mitogenic stimulation. Furthermore, the APPL gene was mapped to human chromosome 3p14.3-p21.1, where deletions and other rearrangements have often been reported in a variety of tumor types. The identification of APPL may facilitate further analysis of the physiological and oncogenic activities of AKT2.
Assuntos
Proteínas de Transporte/metabolismo , Cromossomos Humanos Par 3/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/genética , Clonagem Molecular , Ativação Enzimática , Feminino , Humanos , Zíper de Leucina , Dados de Sequência Molecular , Neoplasias Ovarianas , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Fosfotirosina/metabolismo , Mapeamento Físico do Cromossomo , Testes de Precipitina , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/análise , RNA Mensageiro/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Células Tumorais Cultivadas , Leveduras/genética , Leveduras/metabolismoRESUMO
We have used a rat model of epithelial ovarian cancer to identify a gene that shows decreased or lost expression in independently transformed rat ovarian surface epithelial cell lines compared to the normal progenitor cells. Hence, we refer to this gene as Lot-1 (Lost on transformation 1, GenBank accession no. U72620). Here, we report the cloning of the likely human homologue and its initial characterization. The deduced amino acid sequences of the cDNAs for rat and human LOT-1 (GenBank accession no. U72621) contain seven zinc finger motifs of the C2H2 type as well as proline and glutamine rich areas. The genes share 76.4% identity at the nucleotide level, 67.7% at the amino acid level and 85.5% within the seven zinc finger motifs. LOT-1 is ubiquitously expressed in normal human tissues but was not expressed in four of 11 (36%) human ovarian cancer cell lines or spontaneously transformed human ovarian surface epithelial cells. The human gene maps to chromosome 6 at band q25. We show that there is a 38% incidence of allelic loss at this chromosomal location in human ovarian cancers. This chromosomal region has also been implicated in the genesis of breast, kidney, and pleural mesothelial cancers. We suggest that this newly identified gene is not only of intrinsic interest as a ubiquitously expressed probable transcription factor but is a plausible candidate for the tumor suppressor gene which likely resides in the region of chromosome 6 defined by band q25.
Assuntos
Proteínas de Ciclo Celular , Cromossomos Humanos Par 6 , Proteínas de Ligação a DNA/genética , Genes Supressores de Tumor , Neoplasias Ovarianas/genética , Fatores de Transcrição , Dedos de Zinco/genética , Sequência de Aminoácidos , Animais , Linhagem Celular Transformada , Deleção Cromossômica , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar , Proteínas de Ligação a DNA/química , Epitélio , Feminino , Glutamina , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Ovário , Reação em Cadeia da Polimerase , Prolina , Ratos , Homologia de Sequência de Aminoácidos , Proteínas Supressoras de TumorRESUMO
Arg and c-Abl represent the mammalian members of the Abelson family of nonreceptor protein-tyrosine kinases. To gain insight into the biological role of Arg we used the two-hybrid approach to identify interacting proteins. Using a C-terminal segment of Arg we identified a novel protein, ArgBP1 (Arg binding protein 1). ArgBP1 contains a C-terminal SH3 domain, several PEST sequences, a serine rich domain and an SH3 binding site. ArgBP1 is ubiquitously expressed as two transcripts of approximately 2.2 kb and approximately 8 kb with highest levels in brain, heart and testis. The association of ArgBP1 with Arg in living cells was confirmed by coimmunoprecipitation in cotransfected COS cells. Analysis of the mechanism of association indicated that the ArgBP1 SH3 domain binds to a C-terminal Arg SH3-binding site, and that an N-terminal ArgBP1 proline-rich sequence binds to the Arg SH3 domain. Immunostaining indicated that the subcellular localization of ArgBP1 is cytoplasmic. The similarity of the ArgBP1 expression pattern and subcellular localization to those of Arg and the potential for a highly specific and potentially strong association mediated by two pairs of SH3 domain/proline-rich motif interactions, suggest that ArgBP1 is likely to be a regulator and/or effector of Arg function.
Assuntos
Proteínas de Transporte/genética , Sistema Nervoso Central/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Arginina/metabolismo , Sequência de Bases , Proteínas de Transporte/metabolismo , Linhagem Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 2 , Primers do DNA , Humanos , Dados de Sequência Molecular , Ligação Proteica , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Frações Subcelulares , Xenopus/genéticaRESUMO
We report the isolation of a novel human cDNA encoding a putative transmembrane protein, TMC. The predicted protein sequence is highly conserved evolutionarily. The cDNA clone was mapped to human chromosome 11q24-25 by fluorescence in situ hybridization. mRNA expression was observed in all tissues tested with the highest levels in testes and ovary.
Assuntos
Cromossomos Humanos Par 11 , Proteínas de Membrana/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Sequência Conservada , DNA Complementar , Hexosiltransferases , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
In order to identify new transmembrane helix packing motifs in naturally occurring proteins, we have selected transmembrane domains from a library of random Escherichia coli genomic DNA fragments and screened them for homomultimerization via their abilities to dimerize the bacteriophage lambda cI repressor DNA-binding domain. Sequences were isolated using a modified lambda cI headpiece dimerization assay system, which was shown previously to measure transmembrane helix-helix association in the E. coli inner membrane. Screening resulted in the identification of several novel sequences that appear to mediate helix-helix interactions. One sequence, representing the predicted sixth transmembrane domain (TM6) of the E. coli protein YjiO, was chosen for further analysis. Using site-directed mutagenesis and molecular dynamics, a small set of models for YjiO TM6 multimerization interface interactions were generated. This work demonstrates the utility of combining in vivo genetic tools with computational systems for understanding membrane protein structure and assembly.
Assuntos
Membrana Celular/metabolismo , Proteínas de Ligação a DNA , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli , Biblioteca Genômica , Proteínas de Membrana/química , Modelos Moleculares , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Bacteriófago lambda/imunologia , Bacteriófago lambda/fisiologia , Sequência de Bases , Sítios de Ligação , Membrana Celular/química , Clonagem Molecular/métodos , Dimerização , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Genes Bacterianos/genética , Vetores Genéticos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologia , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas Virais , Proteínas Virais Reguladoras e AcessóriasRESUMO
We have identified a repetitive DNA element in the rat genome that we demonstrate to be suitable to detect molecular genetic differences between normal and malignantly transformed rat ovarian surface epithelial cells by genome scanning. With fluorescence in situ hybridization, we show that these elements are widely distributed in the rat genome, and that a member of this family is present within a homogeneously staining chromosomal region of tumorigenic rat ovarian surface epithelial cells. The homogeneously staining chromosomal region infers the presence of an amplified DNA sequence in the tumor cells, and we provide molecular evidence for an amplicon in this DNA by genome scanning using a probe related to these elements. Sequence analysis revealed that these elements have the structural features of retroviral DNA. We show that they are transcriptionally active in the rat ovary, but not in a wide range of other normal rat tissues. In situ hybridization to ovarian tissue sections revealed that the elements are expressed in granulosa and theca interna cells and in the surface epithelial cells adjacent to preovulatory follicles. Based on this property, we refer to these retroviral-like elements as ovary-specific transcribed sequences. Partial sequence analysis of multiple members of this family and the range in transcript sizes strongly suggest that multiple members of this retrovirus-like family are transcriptionally active. We speculate that binding by one or a combination of ovary-specific transcription factors to regulatory sequences within the genomic ovary-specific transcribed sequences units contributes to the tissue specificity of their expression.
Assuntos
Transformação Celular Neoplásica , DNA Viral/análise , Neoplasias Ovarianas/etiologia , Ovário/virologia , Retroviridae/genética , Animais , Sequência de Bases , Feminino , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Ratos , Sequências Repetitivas de Ácido NucleicoRESUMO
Murine MyD88, an RNA with homology both to the interleukin-1 receptor signaling domain and to 'death-domains', is rapidly upregulated during differentiation of the myeloleukemic cell line M1. We have cloned the human homologue of murine MyD88 and re-evaluated the murine sequence. The open reading frame for both species encodes a 296 amino acid protein, which for murine MyD88 is 53 amino acids longer than originally published. Human MyD88 cDNA is encoded by 5 exons, and maps to chromosome 3p21.3-p22 by fluorescence in situ hybridization (FISH). Overexpression of the death domain region leads to transcriptional activation of the IL-8 promoter.
Assuntos
Antígenos de Diferenciação , Proteínas/genética , Receptores Imunológicos , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Mapeamento Cromossômico , Cromossomos Humanos Par 3 , DNA Complementar/genética , Éxons , Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Interleucina-8/genética , Íntrons , Camundongos , Dados de Sequência Molecular , Fator 88 de Diferenciação Mieloide , Oligonucleotídeos Antissenso , Regiões Promotoras Genéticas , Proteínas/química , RNA Mensageiro/genética , Receptores de Interleucina-1/química , Receptores de Interleucina-1/genética , TransfecçãoRESUMO
OBJECTIVE: To isolate a portion of the amplicon inferred to be present in a malignant rat tumor cell line, NuTu 26, by the presence of a homogeneously staining chromosomal region (hsr) and identify genes embedded within it. METHODS: Genome scanning was used to identify an EcoRI fragment (8.6 kbp) within the amplified region of the NuTu 26 genome using a recently identified rat repetitive sequence, OST17 as a probe. The 8.6 kbp amplified fragment was sequenced and used as starting material to obtain additional sequence information by screening a P1 clone-derived DNA library to identify any genes likely embedded in the amplicon. Use of the microdissected hsr as a probe for fluorescence in situ hybridization (FISH) and application of Southern, Northern, and Western blot analysis confirmed the amplification of this region in the NuTu 26 genome. RESULTS: The cathepsin B gene was within the amplicon of the hsr-containing marker chromosome of NuTu 26. FISH analysis and chromosomal banding further revealed that the marker chromosome was a derivative of chromosomes 4 and 15, i.e., der(15)t(4;15). CONCLUSION: Cathepsin B gene amplification may contribute to some aspect of the biology of ovarian cancer. This concept is strengthened by the finding that the gene is overexpressed frequently in independently transformed rat ovarian surface epithelial cells.
Assuntos
Catepsina B/genética , Amplificação de Genes , Neoplasias Ovarianas/genética , Animais , Southern Blotting , Linhagem Celular Transformada , DNA/análise , DNA/metabolismo , Desoxirribonuclease EcoRI/metabolismo , Epitélio , Feminino , Marcadores Genéticos , Hibridização in Situ Fluorescente , Reação em Cadeia da Polimerase , Ratos , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Células Tumorais CultivadasRESUMO
The MEF2 family of transcription factors control the expression of muscle-specific and mitogen-induced genes. Here we describe the isolation and structure of the human MEF2A gene. The protein coding region of MEF2A is divided by 10 introns. The 3' untranslated region (UTR) is 3.7 kb in length, and it contains a region that is highly homologous with a portion of the 3' UTR of Xenopus MEF2A. A partially processed pseudogene (MEF2AP) corresponding to MEF2A was also isolated and characterized. Human MEF2A was mapped by fluorescence in situ hybridization to chromosome 15q26, and MEF2AP was mapped to chromosome 1q24 --> q25.
Assuntos
Cromossomos Humanos Par 15 , Cromossomos Humanos Par 1 , Proteínas de Ligação a DNA/genética , Pseudogenes , Fatores de Transcrição/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Primers do DNA , Humanos , Proteínas de Domínio MADS , Fatores de Transcrição MEF2 , Dados de Sequência Molecular , Fatores de Regulação Miogênica , Homologia de Sequência do Ácido Nucleico , XenopusRESUMO
We previously reported the cloning of a murine cDNA encoding the protein-serine/threonine kinase Akt2, and we used this clone to map the Akt2 gene to mouse chromosome (MMU) 7B1 by fluorescence in situ hybridization. We now have cloned and partially sequenced a mouse Akt2 pseudogene. An analysis of two sets of multilocus crosses revealed that the Akt2 gene is closely linked to the Cyp2a locus in proximal MMU7. The Akt2 pseudogene was mapped to proximal MMU11 by both multilocus mapping and fluorescence in situ hybridization.
Assuntos
Mapeamento Cromossômico , Proteínas Oncogênicas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas , Pseudogenes , Animais , Clonagem Molecular , Cruzamentos Genéticos , DNA Complementar/genética , Biblioteca Gênica , Genes , Ligação Genética , Células Híbridas , Hibridização in Situ Fluorescente , Camundongos , Muridae/genética , Proteínas Proto-Oncogênicas c-aktRESUMO
We used comparative genomic hybridization (CGH) to identify recurrent chromosomal imbalances in tumor DNA from 25 malignant ovarian carcinomas and two ovarian tumors of low malignant potential (LMP). Many of the carcinoma specimens displayed numerous imbalances. The most common sites of copy number increases, in order of frequency, were 8q24.1, 20q13.2-qter, 3q26.3-qter, 1q32, 20p, 9p21-pter, and 12p. DNA amplification was identified in 12 carcinomas (48%). The most frequent sites of amplification were 8q24.1-24.2, 3q26.3, and 20q13.2-qter. Other recurrent sites of amplification included 7q36, 17q25, and 19q13.1-13.2. The most frequent sites of copy number decreases were 5q21, 9q, 17p, 17q12-21, 4q26-31, 16q, and 22q. Underrepresentation of 17p was observed in six of 16 stage III/IV tumors, but in none of seven stage I/II tumors, suggesting that this change may be a late event associated with the transition of ovarian carcinomas to a more metastatic disease. Overrepresentation of 3q26.3-qter, 5p14-pter, 8q24.1, 9p21-pter, 20p, and 20q13.2-qter and underrepresentation of 4q26-31 and 17q12-21 also tended to be more common in advanced-stage tumors. All ten grade 3 tumors had copy number increases involving 8q24.1, compared to only three of nine grade 2 tumors. Overrepresentation of 3q26.3-qter and 20q13.2-qter was also observed at a higher frequency in high-grade tumors. One of the two LMP tumors displayed chromosomal alterations, which consisted of overrepresentation of 5p and 9p only. Taken collectively, these findings and data from other CGH studies of ovarian cancers define a set of small chromosome segments that are consistently over- or underrepresented and, thus, highlight sites of putative oncogenes and tumor suppressor genes that contribute to the pathogenesis of these highly malignant neoplasms.
Assuntos
Aberrações Cromossômicas/genética , Amplificação de Genes , Dosagem de Genes , Neoplasias Ovarianas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Mapeamento Cromossômico , Cromossomos Humanos Par 20/genética , Cromossomos Humanos Par 3/genética , Cromossomos Humanos Par 8/genética , DNA de Neoplasias/isolamento & purificação , Feminino , Humanos , Pessoa de Meia-Idade , Hibridização de Ácido NucleicoRESUMO
The roles that human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV) play in the genesis of acquired immune deficiency syndrome (AIDS)-related lymphomas are not understood. A human B cell line (B-HIV), developed to study AIDS-related lymphomagenesis, contains EBV and HIV genomes and is malignantly transformed. This line was produced by exposing B cells from an EBV-seropositive donor to HIV. To investigate the number of independent transformation events that took place at the time of HIV infection, we sought to determine how many transformed lineages are present in this cell line. B-HIV was found to have multiple different sites of HIV integration (> or = 25) as determined by fluorescence in situ hybridization. As a control, we analyzed a clonal cell line of HIV-infected human T cells, 8E5, and found HIV sequences located exclusively at band q22 in chromosome 13. We conclude that B-HIV is polyclonal, and viral sequences are located at multiple variable chromosomal sites in different B-HIV cells.
Assuntos
Linfócitos B/virologia , Transformação Celular Viral , Infecções por HIV/virologia , HIV , Herpesvirus Humano 4 , Linfoma Relacionado a AIDS/virologia , Linfócitos B/patologia , Linhagem Celular , Células Clonais/patologia , DNA Viral/análise , HIV/genética , Infecções por HIV/patologia , Humanos , Hibridização in Situ Fluorescente , Linfoma Relacionado a AIDS/patologia , Dados de Sequência MolecularRESUMO
Comparative genomic hybridization (CGH) was used in a retrospective analysis of chromosomal imbalances in frozen primary tumor specimens from 14 endometrial carcinoma patients. Chromosome changes were detected in nine cases (64%), and tumor stage and grade tended to parallel the degree of genomic imbalances. Gain of the entire long arm of chromosome 1 was observed in six cases (43%), three of which displayed only this chromosome change. Other common sites of copy number increases included 8q21-->qter (4 cases), 10p15 (4 cases), 10q11-->q24 (3 cases), and 13q21-->qter (3 cases, each with stage III disease). Two of the tumors with gains of chromosome 10 involved the whole chromosome, and this was the sole abnormality in one case. DNA amplification at 5p14-->p15 was identified in one specimen, a stage III tumor having numerous imbalances. DNA microsatellite analysis revealed multiple replication errors (RER), indicative of the RER+ phenotype, in four of 13 (31%) cases evaluated. The RER+ phenotype was observed in four of six stage la tumors but in none of seven stage Ib or stage III tumors. Multiple genomic imbalances detected by CGH were not observed in RER+ tumors but were detected in five of nine tumors without the RER+ phenotype. These investigations demonstrate the feasibility of CGH for the retrospective assessment of chromosomal changes in endometrial carcinoma specimens. Moreover, these data suggest that the etiologies in tumors with and without the RER+ phenotype may differ.
Assuntos
DNA de Neoplasias/genética , Neoplasias do Endométrio/genética , Idoso , Aberrações Cromossômicas , Transtornos Cromossômicos , Feminino , Fluorescência , Heterozigoto , Humanos , Pessoa de Meia-Idade , Hibridização de Ácido Nucleico , Fenótipo , Estudos RetrospectivosRESUMO
Mybl2 encodes a transcription factor that is thought to play an important role in cell cycle progression. Here we report the chromosomal localization of Mybl2 in mouse and human. Using mouse Mybl2 cDNA clones as probes, we assigned Mybl2 in an interspecific backcross panel to distal Chromosome 2. Using human cDNA probes in combination with FISH analysis, we localized MYBL2 to chromosome 20q13.1, a region that is commonly deleted in myeloid disorders. Both chromosomal regions are highly homologous, and the map positions, therefore, confirm each other. However, our findings are in contrast to a previous report by Barletta et al. (Cancer Res. 51:3821-3824, 1991) that placed the MYBL2 gene on human chromosome Xq13.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 12 , Proteínas Oncogênicas de Retroviridae/genética , Fatores de Transcrição/genética , Animais , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas v-mybRESUMO
We previously demonstrated that the putative oncogene AKT2 is amplified and overexpressed in some human ovarian carcinomas. We have now identified amplification of AKT2 in approximately 10% of pancreatic carcinomas (2 of 18 cell lines and 1 of 10 primary tumor specimens). The two cell lines with altered AKT2 (PANC1 and ASPC1) exhibited 30-fold and 50-fold amplification of AKT2, respectively, and highly elevated levels of AKT2 RNA and protein. PANC1 cells were transfected with antisense AKT2, and several clones were established after G418 selection. The expression of AKT2 protein in these clones was greatly decreased by the antisense RNA. Furthermore, tumorigenicity in nude mice was markedly reduced in PANC1 cells expressing antisense AKT2 RNA. To examine further whether overexpression of AKT2 plays a significant role in pancreatic tumorigenesis, PANC1 cells and ASPC1 cells, as well as pancreatic carcinoma cells that do not overexpress AKT2 (COLO 357), were transfected with antisense AKT2, and their growth and invasiveness were characterized by a rat tracheal xenotransplant assay. ASPC1 and PANC1 cells expressing antisense AKT2 RNA remained confined to the tracheal lumen, whereas the respective parental cells invaded the tracheal wall. In contrast, no difference was seen in the growth pattern between parental and antisense-treated COLO 357 cells. These data suggest that overexpression of AKT2 contributes to the malignant phenotype of a subset of human ductal pancreatic cancers.