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1.
Oral Oncol ; 39(3): 248-58, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12618197

RESUMO

The near completion of the human genome project and the recent development of novel, highly sensitive high-throughput techniques have now afforded the unique opportunity to perform a comprehensive molecular characterization of normal, precancerous, and malignant cells, including those derived from squamous carcinomas of the head and neck (HNSCC). As part of these efforts, representative cDNA libraries from patient sets, comprising of normal and malignant squamous epithelium, were generated and contributed to the Head and Neck Cancer Genome Anatomy Project (HN-CGAP). Initial analysis of the sequence information indicated the existence of many novel genes in these libraries [Oral Oncol 36 (2000) 474]. In this study, we surveyed the available sequence information using bioinformatic tools and identified a number of known genes that were differentially expressed in normal and malignant epithelium. Furthermore, this effort resulted in the identification of 168 novel genes. Comparison of these clones to the human genome identified clusters in loci that were not previously recognized as being altered in HNSCC. To begin addressing which of these novel genes are frequently expressed in HNSCC, their DNA was used to construct an oral-cancer-specific microarray, which was used to hybridize alpha-(33)P dCTP labeled cDNA derived from five HNSCC patient sets. Initial assessment demonstrated 10 clones to be highly expressed (>2-fold) in the normal squamous epithelium, while 14 were highly represented in the malignant counterpart, in three of the five patient sets, thus suggesting that a subset of these newly discovered transcripts might be highly expressed in this tumor type. These efforts, together with other multi-institutional genomic and proteomic initiatives are expected to contribute to the complete understanding of the molecular pathogenesis of HNSCCs, thus helping to identify new markers for the early detection of preneoplastic lesions and novel targets for pharmacological intervention in this disease.


Assuntos
Carcinoma de Células Escamosas/genética , Perfilação da Expressão Gênica/métodos , Neoplasias de Cabeça e Pescoço/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Idoso , DNA Complementar/genética , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Biblioteca Gênica , Genoma , Humanos , Masculino , Análise de Sequência de DNA
2.
Proteomics ; 1(10): 1271-8, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11721638

RESUMO

Critical changes in protein expression that enable tumors to initiate and progress originate in the local tissue microenvironment, and there are increasing indications that these microenvironmental alterations in protein expression play critical roles in shaping and directing this process. As a model to better understand how patterns of protein expression shape the tissue microenvironment, we analyzed protein expression in tissue derived from squamous cell carcinoma of the oral cavity through an antibody microarray approach for high-throughput proteomic analysis. Utilizing laser capture microdissection to procure total protein from specific microscopic cellular populations, we demonstrate that quantitative, and potentially qualitative, differences in expression patterns of multiple proteins within epithelial cells reproducibly correlate with oral cavity tumor progression. Furthermore, differential expression of multiple proteins was also found in stromal cells surrounding and adjacent to regions of diseased epithelium that directly correlated with tumor progression of the epithelium. Most of the proteins identified in both cell types are involved in signal transduction pathways, thus we hypothesize that extensive molecular communication involving complex cellular signaling between epithelium and stroma play a key role in driving oral cavity cancer progression.


Assuntos
Anticorpos Antineoplásicos/imunologia , Antígenos de Neoplasias/imunologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Proteoma/metabolismo , Antígenos de Neoplasias/metabolismo , Western Blotting , Dissecação , Eletroforese em Gel de Poliacrilamida , Humanos , Lasers , Neoplasias Bucais/imunologia , Neoplasias Bucais/metabolismo , Neoplasias de Células Escamosas/imunologia , Neoplasias de Células Escamosas/metabolismo
3.
Cancer J ; 7(1): 32-9, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11269646

RESUMO

Tissue microdissection is a laboratory method that is used to procure specific cells or cell populations from a histology slide under direct microscopic visualization. The recovered cells can be studied with a variety of DNA, messenger RNA, and protein analysis methods, including new high-throughput gene expression and proteomics technologies. This approach is permitting investigators to comprehensivelyexamine the molecular anatomy of cells in tissue sections forthe first time. This article reviews the development and evolution of tissue microdissection techniques, summarizes examples of research studies, and discusses related challenges that the research community must address. Additional information and complete laboratory protocols are available on a website at http://cgap-mf.nih.gov/.


Assuntos
DNA de Neoplasias/análise , Neoplasias/patologia , RNA Neoplásico/análise , Eletroforese em Gel Bidimensional , Biblioteca Gênica , Genes Supressores de Tumor/genética , Humanos , Terapia a Laser/métodos , Masculino , Monitorização Intraoperatória/métodos , Neoplasia Endócrina Múltipla Tipo 1/genética , Neoplasia Endócrina Múltipla Tipo 1/patologia , Neoplasias/genética , Neoplasias/cirurgia , Neoplasias da Próstata/patologia , RNA Mensageiro/análise , Análise de Sequência de DNA , Proteína Supressora de Tumor p53/análise
5.
Oral Oncol ; 36(5): 474-83, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10964057

RESUMO

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer among men in the developed world affecting the oral cavity, salivary glands, larynx and pharynx. Utilizing tissue from patients with HNSCC, we sought to systematically identify and catalog genes expressed in HNSCC progression. Here, we demonstrate the successful use of laser capture microdissection for procuring pure populations of cells from patient tissue sets comprised of oral squamous cell carcinomas (OSCCs) and matching normal tissue. From the estimated 5000 cells procured for each sample, we were able to extract total RNA (14.7-18.6 ng) of sufficient quality to transcribe GAPDH by reverse transcriptase-polymerase chain reaction (RT-PCR). The RNA was used for the synthesis of blunt-ended, double-strand complementary DNAs (cDNAs) by oligo (dT)-mediated reverse transcription, followed by addition of linkers. Primers specific for these linkers with uracil deglycosylase-compatible ends were used to amplify these cDNAs by PCR and the product was subcloned into the pAMP10 cloning vector. Ninety-six clones from each of six libraries were randomly sequenced and results indicated that 76-96% of the inserts represent either anonymous expressed sequence tags (ESTs) (25-48%), known genes (9-29%) or novel sequences (27-51%), respectively, with very little redundancy. These results demonstrate that high quality, representative cDNA libraries can be generated from microdissected OSCC tissue. Furthermore, these finding suggest the existence of at least 132 novel genes expressed in our cDNA libraries, which may have a role in the pathogenesis of HNSCC, and may represent novel markers for early detection as well as targets for pharmacological intervention in this disease.


Assuntos
Carcinoma de Células Escamosas/genética , DNA Complementar/genética , Dissecação/métodos , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Lasers , Neoplasias Bucais/genética , Idoso , Biópsia/métodos , Carcinoma de Células Escamosas/patologia , Estudos de Casos e Controles , Dissecação/instrumentação , Humanos , Masculino , Pessoa de Meia-Idade , Mucosa Bucal/patologia , Neoplasias Bucais/patologia , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa
6.
Mol Carcinog ; 28(1): 12-22, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10820484

RESUMO

A cDNA microarray comprising 5184 different cDNAs spotted onto nylon membrane filters was developed for prostate gene expression studies. The clones used for arraying were identified by cluster analysis of > 35 000 prostate cDNA library-derived expressed sequence tags (ESTs) present in the dbEST database maintained by the National Center for Biotechnology Information. Total RNA from two cell lines, prostate line 8.4 and melanoma line UACC903, was used to make radiolabeled probe for filter hybridizations. The absolute intensity of each individual cDNA spot was determined by phosphorimager scanning and evaluated by a bioinformatics package developed specifically for analysis of cDNA microarray experimentation. Results indicated 89% of the genes showed intensity levels above background in prostate cells compared with only 28% in melanoma cells. Replicate probe preparations yielded results with correlation values ranging from r = 0.90 to 0.93 and coefficient of variation ranging from 16 to 28%. Findings indicate that among others, the keratin 5 and vimentin genes were differentially expressed between these two divergent cell lines. Follow-up northern blot analysis verified these two expression changes, thereby demonstrating the reliability of this system. We report the development of a cDNA microarray system that is sensitive and reliable, demonstrates a low degree of variability, and is capable of determining verifiable gene expression differences between two distinct human cell lines. This system will prove useful for differential gene expression analysis in prostate-derived cells and tissue.


Assuntos
DNA Complementar/genética , DNA Complementar/isolamento & purificação , Perfilação da Expressão Gênica , Próstata , Neoplasias da Próstata/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos/métodos
9.
Nat Genet ; 21(1 Suppl): 38-41, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9915499

RESUMO

Gene expression microarrays hold great promise for studies of human disease states. There are significant technical issues specific to utilizing clinical tissue samples which have yet to be rigorously addressed and completely overcome. Precise, quantitative measurement of gene expression profiles from specific cell populations is at hand, offering the scientific community the first comprehensive view of the in vivo molecular anatomy of normal cells and their diseased counterparts. Here, we propose a model for integrating-in three dimensions-expression data obtained using the microarray.


Assuntos
Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Bases de Dados Factuais , Genoma Humano , Humanos , Masculino , Próstata/anatomia & histologia , Próstata/química , Próstata/patologia , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Neoplásico/análise , RNA Neoplásico/genética , Manejo de Espécimes
10.
Neoplasia ; 1(2): 101-6, 1999 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10933042

RESUMO

As the process of tumor progression proceeds from the normal cellular state to a preneoplastic condition and finally to the fully invasive form, the molecular characteristics of the cell change as well. These characteristics can be considered a molecular fingerprint of the cell at each stage of progression and, analogous to fingerprinting a criminal, can be used as markers of the progression process. Based on this premise, the Cancer Genome Anatomy Project was initiated with the broad goal of determining the comprehensive molecular characterization of normal, premalignant, and malignant tumor cells, thus making a reality the identification of all major cellular mechanisms leading to tumor initiation and progression ([Strausberg, R.L., Dahl, C.A., and Klausner, R.D. (1997). "New opportunities for uncovering the molecular basis of cancer." Nat. Genet., 16: 415-516.], www.ncbi.nlm.nih.gov/ncicgap/). The expectation of determining the genetic fingerprints of cancer progression will allow for 1) correlation of disease progression with therapeutic outcome; 2) improved evaluation of disease treatment; 3) stimulation of novel approaches to prevention, detection, and therapy; and 4) enhanced diagnostic tools for clinical applications. Whereas acquiring the comprehensive molecular analysis of cancer progression may take years, results from initial, short-term goals are currently being realized and are proving very fruitful.


Assuntos
Etiquetas de Sequências Expressas , Neoplasias/genética , Neoplasias/patologia , Bases de Dados Factuais , Progressão da Doença , Humanos , Internet , National Institutes of Health (U.S.) , Neoplasias/diagnóstico , Estados Unidos
11.
Curr Opin Mol Ther ; 1(6): 712-9, 1999 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19629868

RESUMO

The Human Genome Project will be completed in the near future, providing new opportunities for researchers to better understand human biology. In order to maximize the value of the genetic data, high-throughput molecular analyses will become an essential experimental methodology, allowing global views of gene expression to be produced and examined. As an example, this approach will permit investigators studying cancer to comprehensively examine the genes and gene products whose alterations underlie tumor development and progression. This information will be essential in determining the fundamental causes of human neoplasms as well as having immediate practical value in the development of clinically useful diagnostic markers and therapeutic targets.


Assuntos
Perfilação da Expressão Gênica/métodos , Neoplasias/genética , Neoplasias/patologia , Biologia Computacional/métodos , Eletroforese em Gel Bidimensional , Histocitoquímica , Humanos , Informática Médica/métodos , Informática Médica/tendências , Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Proteômica/métodos
12.
Cancer Res ; 58(23): 5326-8, 1998 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-9850058

RESUMO

Here, we developed an improved method for constructing microdissected cDNA libraries, based on strand-switching properties of reverse transcriptase, followed by PCR amplification with primers to mediate unidirectional insert cloning. Using RNA from microdissected ovarian carcinoma cells, we constructed a cDNA library consisting of 1.3 x 10(6) unidirectional recombinants with an average insert size of 500 bp. Single-pass sequencing of 100 clones with the T7 primer revealed 89 inserts derived from known genes, anonymous expressed sequence tags (ESTs), or novel sequences. Among these clones were known genes and ESTs previously found in cDNA libraries from bulk ovarian tissue RNA, sequences seen for the first time in an ovarian-derived library, and novel sequences not previously seen in any cDNA library. These results demonstrate a methodology for constructing quality cDNA libraries that are cloned in a unidirectional fashion, are complex and diverse, and reflect the tissue of origin.


Assuntos
Cistadenocarcinoma Papilar/genética , DNA de Neoplasias/genética , Neoplasias Ovarianas/genética , Actinas/genética , Idoso , Clonagem Molecular , DNA de Neoplasias/análise , Feminino , Humanos , RNA Neoplásico/análise , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade
13.
Semin Radiat Oncol ; 8(3): 217-23, 1998 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9634498

RESUMO

Advances in biotechnology and bioinformatics are offering promise for new breakthroughs in gene discovery and elucidation of gene function. At present, many candidate genes related to cancer pathogenesis have been identified in several types of human cancer, yet frequently their function remains elusive. This is particularly true as it relates to the progression of human cancer. This landscape could change dramatically, however, as technological innovations and improvements continue to revolutionize these fields. High-throughput molecular approaches are emerging, which may become accurate, automated, and cost-effective. For example, DNA arrays on microchips are under development with numerous applications, including the ability to screen genes rapidly for mutations and to study patterns of gene expression on a large scale. Automated systems for microdissection and sequencing are also in their implementation stages. Commensurate with their integration and evolution, these information and technological tools have the potential to offer a more comprehensive understanding of multiple genetic and cellular alterations occurring during cancer initiation, development, and progression. Ultimately, this fundamental knowledge can provide strategies for intervention, prevention, and early diagnosis. This is a US government work. There are no restrictions on its use.


Assuntos
Dissecação , Microquímica/instrumentação , Microcirurgia , Metástase Neoplásica/genética , Neoplasias/genética , Sequência de Bases , Biotecnologia , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Aplicações da Informática Médica , Biologia Molecular , Mutação/genética , Neoplasias/diagnóstico , Neoplasias/prevenção & controle , Neoplasias/terapia , Oncogenes/genética
14.
Genomics ; 42(2): 295-301, 1997 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-9192850

RESUMO

We used a combination of sequence analysis and exon trapping in an effort to determine the complete transcript map for a cosmid (6E5) derived from 12q13.3, a region of DNA sequence amplification in human cancers. This cosmid, previously known to contain three genes (CDK4, SAS, and OS9), was sequenced, and that information was used for computer-assisted analysis. In addition, 6E5 was subjected to both internal and 3'-terminal exon-trapping protocols, and the results of these studies were used to guide cDNA cloning experiments. These studies demonstrate that this cosmid is derived from a remarkably gene-dense region and add two new transcripts (KIAA0167 and 6E5.2) to the list of sequences that are expressed in tumors bearing amplification of this region.


Assuntos
Cromossomos Humanos Par 12/genética , Amplificação de Genes , Neoplasias/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Cosmídeos , DNA Complementar/genética , Bases de Dados Factuais , Éxons , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos
15.
Cancer Res ; 56(23): 5380-3, 1996 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-8968089

RESUMO

We report the construction of a plasmid-based cDNA library made from microdissected cells derived from prostatic intraepithelial neoplasia. Total RNA was extracted and converted to blunt-ended, double-stranded cDNA by oligo(dT)-mediated reverse transcription followed by linker addition. A linker-specific primer with UDG-compatible ends was used to amplify the cDNA and the resulting PCR product was subcloned. A total of 154 clones were sequenced and results indicated that 81.5% of the clones derived from either known genes, anonymous expressed sequence tags, or novel transcripts with very little redundancy of screened clones. These results demonstrate the feasibility of constructing complex representative cDNA libraries from specific microdissected cell populations that represent microscopic precursor stages of cancer progression. This method should facilitate identification of transcripts specifically expressed in cells of a distinct histological origin and tumorigenic stage.


Assuntos
Adenocarcinoma/genética , Carcinoma in Situ/genética , DNA Complementar/genética , DNA de Neoplasias/genética , Biblioteca Gênica , Neoplasias da Próstata/genética , Adenocarcinoma/química , Carcinoma in Situ/patologia , Progressão da Doença , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias da Próstata/química , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Homologia de Sequência do Ácido Nucleico
16.
Mol Carcinog ; 5(3): 205-12, 1992.
Artigo em Inglês | MEDLINE | ID: mdl-1375030

RESUMO

Although it is known that cells transformed by ras and other oncogenes show reduced gap junction function, to date there has been no investigation of the quantitative relationship between intracellular levels of ras oncoprotein and loss of cell-cell communication. Using the rat liver epithelial cell line MTR6, which carries a zinc-inducible metallothionein ras T24 (MTrasT24) fusion gene, we showed a direct correlation between the accumulation of ras T24 protein and the loss of dye transfer as measured by interactive laser cytometry. After stimulation with zinc sulfate, changes in both parameters were rapid and measurable by 24 h. Similarly, there was a dose-response relationship between loss of gap junction function and increase in ras T24 protein. Northern analysis of two gap junction proteins (connexins 43 and 32) showed no differences between cells that expressed high levels of ras and control cells. These data demonstrate that the degree of loss of gap junction function is dependent on the amount of increase in ras T24 protein levels, but the mechanism by which these changes are effected remains unclear.


Assuntos
Comunicação Celular , Fígado/metabolismo , Proteínas de Membrana/análise , Proteínas Proto-Oncogênicas p21(ras)/análise , Animais , Linhagem Celular , Conexinas , Relação Dose-Resposta a Droga , Epitélio/metabolismo , Imunofluorescência , Genes , Genes ras , Junções Intercelulares , Metalotioneína/análogos & derivados , Metalotioneína/genética , RNA/análise , Ratos , Sulfatos/farmacologia , Fatores de Tempo , Zinco/farmacologia , Sulfato de Zinco
17.
Oncogene ; 6(9): 1575-82, 1991 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-1681491

RESUMO

We have been studying the effect of oncogenes on differentiation using the human ovarian teratoma-derived cell line PA-1. From this study we have characterized variants representing four stages relevant to multistage carcinogenesis, two non-tumorigenic and two tumorigenic. The two non-tumorigenic cell variants differ in that one is resistant to transformation by ras oncogenes whereas the other can be transformed to tumorigenicity. When these non-tumorigenic PA-1 variants are treated with retinoic acid (RA), a morphogen, they stop dividing, begin to express homeobox genes, and change in morphology. Transfection of an activated N-ras oncogene into ras-resistant non-tumorigenic PA-1 cells does not alter the RA responsiveness of the cells, indicating that expression of the activated oncogene is not sufficient for blocking RA-induced differentiation. Spontaneous activation of an N-ras oncogene leading to tumorigenic transformants and gene transfer-induced N-ras transformants are resistant to these effects of RA. However, another spontaneous transformant of PA-1 cells that does not contain an activated N-ras is responsive to RA. We prepared somatic cell hybrids of the RA-non-responsive, N-ras-transformed and tumorigenic PA-1 cell and the RA-responsive, ras-resistant non-tumorigenic PA-1 cell; the hybrid cell lines continue to express the oncogene but are non-tumorigenic. These non-tumorigenic hybrids are responsive to RA with regard to morphological changes, growth arrest and induction of homeobox gene expression. Tumorigenic revertants of these hybrids arise as a result of the loss of some chromosomes; these hybrid cells express the oncogene but have lost RA responsiveness. These results indicate that tumorigenic transformation in general is not sufficient to induce RA resistance, and resistance to differentiation may be oncogene-specific. In addition, the expression of an activated N-ras oncogene alone is insufficient to induce resistance to RA and ras-induced tumorigenicity is necessary. Therefore, some feature of cellular metabolism that is altered by and discordantly segregates with tumorigenic transformation controls responsiveness to RA. This controlling element is presumably a tumor suppressor.


Assuntos
Genes ras , Neoplasias Ovarianas/genética , Teratoma/genética , Northern Blotting , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Bandeamento Cromossômico , Feminino , Expressão Gênica , Genes Homeobox , Humanos , Células Híbridas/citologia , Cariotipagem , Neoplasias Ovarianas/patologia , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Teratoma/patologia , Transfecção , Tretinoína/farmacologia
18.
Oncogene ; 6(5): 713-20, 1991 May.
Artigo em Inglês | MEDLINE | ID: mdl-1646984

RESUMO

The human teratocarcinoma cell line PA-1 was derived from culturing ascites fluid cells from a patient with an ovarian germ line tumor. We previously described a non-neoplastic variant cloned from the PA-1 human teratocarcinoma cell line, clone 6, which at passage 40 was resistant to transformation by activated ras oncogenes. However, these cells could be transformed by a plasmid containing both myc and ras. Another PA-1 cell variant, clone 1, isolated at passage 63 and used 50 passages later becomes tumorigenic in nude mice after transfection with an activated ras oncogene (Tainsky et al., Anticancer Res., 8, 899-914, 1988). We report here that the progression from ras resistance to ras susceptibility occurs in both clone 1 and clone 6 cells during 25 passages in culture. In the presence of epidermal growth factor, transforming growth factor-alpha, and basic fibroblast growth factor, the ras-transformable cells exhibit anchorage independent growth, whereas the ras-resistant cells can not be growth stimulated by these growth factors. Similarly, ornithine decarboxylase (ODC) activity was inducible in ras susceptible and ras transformed cells by these growth factors, but not in the ras resistant cells. These differences are not due to the level and activity of epidermal growth factor receptor or to the level of expression of 25 proto-oncogenes.


Assuntos
Transformação Celular Neoplásica , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/fisiologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Genes ras , Proteínas Tirosina Quinases/metabolismo , Receptores de Superfície Celular/fisiologia , Transdução de Sinais , Northern Blotting , Linhagem Celular , Células Clonais , Indução Enzimática , Receptores ErbB/efeitos dos fármacos , Humanos , Immunoblotting , Oncogenes , Ornitina Descarboxilase/biossíntese , Plasmídeos , Proteínas Proto-Oncogênicas p21(ras)/análise , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Fatores de Crescimento de Fibroblastos , Teratoma
19.
Somat Cell Mol Genet ; 16(1): 15-27, 1990 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-2408157

RESUMO

We have been using PA-1 human teratocarcinoma cells to study mechanisms by which oncogenes induce transformation. Tumorigenic PA-1 cells at passages greater than 100 (greater than P100) contain a spontaneously activated N-ras oncogene, while earlier-passage preneoplastic cells contain only the germ-line protooncogene and are nontumorigenic. One preneoplastic cell clone of PA-1 cells can be transformed by introduction of the cloned PA-1 N-ras in gene-transfer experiments, while another earlier-passage clonal cell line cannot be transformed. The goal of this investigation was to determine how human cells progress from resistance to susceptibility to ras oncogene-induced transformation. Somatic cell hybridization experiments described in this report indicate that the resistance of the low-passage cells to transformation is a dominant trait suppressing transformation. Loss of chromosomes from hybrid segregants suggested that tumor suppressors exist on chromosomes 1, 4, and 11. Extended in vitro passaging of somatic cell hybrids also resulted in the loss of chromosomes. Chromosome 1 was lost in these populations of cells, implying that reduction of this chromosome may promote proliferation and not specifically affect tumor formation.


Assuntos
Transformação Celular Neoplásica/genética , Genes ras/genética , Supressão Genética/genética , Southern Blotting , Criança , Deleção Cromossômica , Cromossomos Humanos Par 1 , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 4 , Células Clonais , Feminino , Humanos , Células Híbridas , Cariotipagem , Fenótipo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras)
20.
Mol Carcinog ; 3(5): 264-7, 1990.
Artigo em Inglês | MEDLINE | ID: mdl-2123107

RESUMO

We used a series of rat liver epithelial (RLE) cell lines that carry a zinc-regulatable metallothionein/rasT24 fusion gene (MTrasT24) to investigate the relation of ras oncogene expression to steady-state RNA levels of the jun family of genes. In these cells, steady-state RNA levels of c-jun, jun-B, and jun-D were unrelated to rasT24 RNA levels or the phenotypic changes induced by the ras oncogene. Steady-state levels of the three jun mRNAs varied among different rasT24 transformed clones, and, although some clones exhibited concomitant induction of rasT24 and jun mRNAs, other clones exhibited no such correlation. We conclude that the effects of rasT24 in transformed RLE cells do not appear to be mediated by c-jun, jun-B, or jun-D and that studies examining only a single transformed clone may give misleading results with respect to the role of various oncogenes in the transformation process.


Assuntos
Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/genética , Genes ras , Fígado/fisiologia , Fatores de Transcrição/genética , Animais , Northern Blotting , Linhagem Celular , Epitélio/fisiologia , Expressão Gênica , Engenharia Genética , Proteínas Proto-Oncogênicas c-jun , RNA Mensageiro/genética , Ratos
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