RESUMO
We have recently found that primary rat embryonic fibroblasts (REFs) could be immortalized by overexpression of the human mitochondrial ribosomal protein MRPS18-2 (S18-2). A derived cell line, designated 18IM, expressed the embryonic stem cell markers SSEA-1 and Sox2. Upon inoculation into severe combined immunodeficiency mice, 18IM cells differentiated to express pan-keratin. They were not tumorigenic. Here we report the gene profiling of 18IM, compared with REF cells. Pathways involved in oxidative phosphorylation, ubiquinone (Coenzyme Q 10) biosynthesis, fatty acid elongation in mitochondria, PI3K/AKT signaling, a characteristic of rapidly proliferating cells, were upregulated in 18IM. Genes involved in the transcription/translation machinery and redox reactions, like elongation factors, ATP synthases, NADH dehydrogenases, mitogen activated kinases were upregulated as well. 18IM cells produced more pyruvate, indicating enhanced ATP synthesis. The expression of Oct4, Sox2, and Nanog that can contribute to the experimental induction of pluripotency in primary fibroblasts was also elevated, in contrast to Klf4 and C-myc that were downregulated. Subsequently, three new immortalized cell lines were produced by S18-2 overexpression in order to check the representativeness of 18IM. All of them showed anchorage-independent growth pattern. Two of three clones lost vimentin and smooth muscle actin, and expressed Sox2 and Oct4. We suggest that S18-2 is involved in the developmental regulation.
Assuntos
Linhagem Celular Transformada , Células-Tronco Embrionárias/metabolismo , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Proteínas Ribossômicas/genética , Animais , Diferenciação Celular , Proliferação de Células , Células-Tronco Embrionárias/citologia , Fibroblastos/citologia , Regulação da Expressão Gênica , Humanos , Queratinas/biossíntese , Fator 4 Semelhante a Kruppel , Antígenos CD15/genética , Antígenos CD15/metabolismo , Camundongos , Camundongos SCID , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos , Ratos , Proteínas Ribossômicas/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Transplante HeterólogoRESUMO
Epstein - Barr virus (EBV) is a lymphotropic virus that infects more than 90% of the human population, and targets B cells for infection. Infection of human B cells leads to the malignant transformation and eventual immortalization. In latency III infection six EBV-encoded nuclear antigens (EBNAs) and three latent membrane proteins (LMPs) are expressed in the transformed cells that can grow as a lymphoblastoid cell lines in vitro . These proteins hijack the normal B cell growth pathways by activating the constitutive growth promotion and external survival signals. We have determined a set of the nuclear receptors that are up- (and down-) regulated in the latency III infected cells at the mRNA level. In the present paper we discussed the possible role of these receptors in B cell transformation upon EBV infection based on the literature data.
Assuntos
Linfócitos B/virologia , Transformação Celular Neoplásica/genética , Infecções por Vírus Epstein-Barr/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Infecções por Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Expressão Gênica , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/genética , Humanos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
BACKGROUND: Infection of human B cells with Epstein - Barr virus (EBV) induces metabolic activation, morphological transformation, cell proliferation and eventual immortalization. AIM: To identify the nuclear receptors, which are the cellular interaction partners of EBNAs, that will help to elucidate the mechanism of B cell transformation. METHODS: We have compared the nuclear receptor profile in the naïve and EBV-transformed B-lymphocytes, using TaqMan LDA microfluidic card technology. RESULTS: Out of 48 nuclear receptor, 17 showed differential expression at the mRNA level. The expression of 5 genes was elevated in EBV-transformed cells, whereas 12 genes were downregulated in lymphoblastoid cells (LCLs). 7 genes were studied at the protein level; 2 genes were up regulated (Nr2F2 and RARA) and 4 genes were down regulated (ERB, NUR77, PPARG, and VDR) in LCLs. CONCLUSION: The nuclear receptor profiling on EBV infected B cells showed alterations of nuclear receptors expression at both mRNA and protein levels compared with non infected peripheral blood cells. Further analysis on a possible role of each nuclear receptor in EBV induced cell transformation should be performed.
Assuntos
Linfócitos B/virologia , Transformação Celular Neoplásica/genética , Infecções por Vírus Epstein-Barr/genética , Receptores Citoplasmáticos e Nucleares/genética , Western Blotting , Transformação Celular Neoplásica/metabolismo , Infecções por Vírus Epstein-Barr/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Expressão Gênica , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Humanos , RNA Mensageiro/análise , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
New comparative genome hybridization technology on NotI-microarrays is presented (Karolinska Institute International Patent WO02/086163). The method is based on comparative genome hybridization of NotI-probes from tumor and normal genomic DNA with the principle of new DNA NotI-microarrays. Using this method 181 NotI linking loci from human chromosome 3 were analyzed in 200 malignant tumor samples from different organs: kidney, lung, breast, ovary, cervical, prostate. Most frequently (more than in 30%) aberrations--deletions, methylation,--were identified in NotI-sites located in MINT24, BHLHB2, RPL15, RARbeta1, ITGA9, RBSP3, VHL, ZIC4 genes, that suggests they probably are involved in cancer development. Methylation of these genomic loci was confirmed by methylation-specific PCR and bisulfite sequencing. The results demonstrate perspective of using this method to solve some oncogenomic problems.
