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1.
EBioMedicine ; 30: 167-183, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29628341

RESUMO

BACKGROUND: Human cytomegalovirus (HCMV) establishes a persistent life-long infection and increasing evidence indicates HCMV infection can modulate signaling pathways associated with oncogenesis. Breast milk is an important route of HCMV transmission in humans and we hypothesized that mammary epithelial cells could be one of the main cellular targets of HCMV infection. METHODS: The infectivity of primary human mammary epithelial cells (HMECs) was assessed following infection with the HCMV-DB strain, a clinical isolate with a marked macrophage-tropism. The impact of HCMV-DB infection on expression of p53 and retinoblastoma proteins, telomerase activity and oncogenic pathways (c-Myc, Akt, Ras, STAT3) was studied. Finally the transformation of HCMV-DB infected HMECs was evaluated using soft agar assay. CTH cells (CMV Transformed HMECs) were detected in prolonged cultures of infected HMECs. Tumor formation was observed in NOD/SCID Gamma (NSG) mice injected with CTH cells. Detection of long non coding RNA4.9 (lncRNA4.9) gene was assessed in CTH cells, tumors isolated from xenografted NSG mice and biopsies of patients with breast cancer using qualitative and quantitative PCR. RESULTS: We found that HCMV, especially a clinical strain named HCMV-DB, infects HMECs in vitro. The clinical strain HCMV-DB replicates productively in HMECs as evidenced by detection of early and late viral transcripts and proteins. Following infection of HMECs with HCMV-DB, we observed the inactivation of retinoblastoma and p53 proteins, the activation of telomerase activity, the activation of the proto-oncogenes c-Myc and Ras, the activation of Akt and STAT3, and the upregulation of cyclin D1 and Ki67 antigen. Colony formation was observed in soft agar seeded with HCMV-DB-infected HMECs. Prolonged culture of infected HMECs resulted in the development of clusters of spheroid cells that we called CTH cells (CMV Transformed HMECs). CTH cells when injected in NOD/SCID Gamma (NSG) mice resulted in the development of tumors. We detected in CTH cells the presence of a HCMV signature corresponding to a sequence of the long noncoding RNA4.9 (lncRNA4.9) gene. We also found the presence of the HCMV lncRNA4.9 sequence in tumors isolated from xenografted NSG mice injected with CTH cells and in biopsies of patients with breast cancer using qualitative and quantitative PCR. CONCLUSIONS: Our data indicate that key molecular pathways involved in oncogenesis are activated in HCMV-DB-infected HMECs that ultimately results in the transformation of HMECs in vitro with the appearance of CMV-transformed HMECs (CTH cells) in culture. CTH cells display a HCMV signature corresponding to a lncRNA4.9 genomic sequence and give rise to fast growing triple-negative tumors in NSG mice. A similar lncRNA4.9 genomic sequence was detected in tumor biopsies of patients with breast cancer.


Assuntos
Mama/patologia , Carcinogênese/patologia , Citomegalovirus/metabolismo , Células Epiteliais/patologia , Células Epiteliais/virologia , Animais , Carcinogênese/genética , Agregação Celular , Proliferação de Células , Transformação Celular Neoplásica/patologia , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Ciclina D1/genética , Ciclina D1/metabolismo , Citomegalovirus/genética , Infecções por Citomegalovirus/patologia , Infecções por Citomegalovirus/virologia , Células Epiteliais/metabolismo , Feminino , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação , Filogenia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Esferoides Celulares/patologia , Telomerase/genética , Telomerase/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
2.
Epigenomics ; 3(4): 487-502, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22126207

RESUMO

After entry into the target cell and reverse transcription, HIV-1 genes are integrated into the host genome. It is now well established that the viral promoter activity is directly governed by its chromatin environment. Nuc-1, a nucleosome located immediately downstream of the HIV-1 transcriptional initiation site directly impedes long-terminal repeat (LTR) activity. Epigenetic modifications and disruption of Nuc-1 are a prerequisite to the activation of LTR-driven transcription and viral expression. The compaction of chromatin and its permissiveness for transcription are directly dependent on the post-translational modifications of histones such as acetylation, methylation, phosphorylation and ubiquitination. Understanding the molecular mechanisms underlying HIV-1 transcriptional silencing and activation is thus a major challenge in the fight against AIDS and will certainly lead to new therapeutic tools.


Assuntos
Metilação de DNA/fisiologia , Epigênese Genética/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Infecções por HIV/metabolismo , HIV-1/fisiologia , Histonas/metabolismo , Nucleossomos/metabolismo , Transcrição Gênica/fisiologia , Azepinas/farmacologia , Epigênese Genética/efeitos dos fármacos , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Histona Acetiltransferases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Humanos , Ésteres de Forbol/farmacologia , Piperazinas/farmacologia , Quinazolinas/farmacologia , Integração Viral/fisiologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
3.
Apoptosis ; 15(12): 1453-60, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20640890

RESUMO

One of the hallmarks of Human Immunodeficiency Virus-1 (HIV-1) infection is progressive depletion of the infected and bystander CD4+ T-cells by apoptosis. Different mitochondrial proteins have been implicated in this apoptotic process; however, the role of different subunits of mitochondrial oxidative phosphorylation (OXPHOS) complexes in apoptosis is not clearly understood. Some of the OXPHOS complex subunits seem to perform other functions in addition to their primary role in energy generating process. GRIM-19 (gene associated with retinoid-interferon-induced-mortality-19), a subunit of mitochondrial complex-I was previously implicated in Interferon-ß and retionoic acid induced apoptosis in many tumor cells. In this study we report, using differential gene expression analysis, that GRIM-19 is up-regulated in HIV-1 infected apoptotic T-cells. A temporal up regulation of this subunit was observed in different HIV-1 infected T-cell lines and human PBMC and the extent of increase correlated to increasing apoptosis and virus production. Moreover, silencing GRIM-19 in HIV-1 infected cells reduced apoptosis, indicating its involvement in HIV-1 induced T-cell death.


Assuntos
Proteínas Reguladoras de Apoptose , Apoptose , Complexo I de Transporte de Elétrons/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Infecções por HIV/enzimologia , Infecções por HIV/imunologia , Mitocôndrias/enzimologia , Mitocôndrias/virologia , NADH NADPH Oxirredutases , Linfócitos T/enzimologia , Linfócitos T/virologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Clonagem Molecular , Complexo I de Transporte de Elétrons/genética , Regulação Enzimológica da Expressão Gênica/imunologia , Inativação Gênica , Infecções por HIV/fisiopatologia , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/fisiologia , Humanos , Mitocôndrias/genética , Mitocôndrias/imunologia , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/imunologia , NADH NADPH Oxirredutases/metabolismo , Fosforilação Oxidativa , Estaurosporina/farmacologia , Linfócitos T/citologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima
4.
Apoptosis ; 15(1): 28-40, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19771519

RESUMO

Human Immunodeficiency Virus-1 (HIV-1) infection leads to CD4+ T cell depletion primarily by apoptosis employing both intrinsic and extrinsic pathways. Although extensive literature exists about the role of mitochondrial proteins in HIV induced T cell apoptosis, there is little understanding about the role of different components of mitochondrial oxidative phosphorylation (OXPHOS) system in apoptosis. The OXPHOS system comprises of five enzyme complexes (Complex I, II, III, IV, V), subunits of which have been implicated in various functions in addition to their primary role in energy generating process. Here using differential gene expression analysis, we report that Cytochrome Oxidase-II (COX-II), a subunit of Complex-IV is induced in HIV infected apoptotic T-cells. We also observe a temporal up regulation of this subunit across different T-cell lines and in human PBMCs. Further analysis indicates increase in expression of majority of Complex-IV subunits with concomitant increase in Complex-IV activity in HIV infected T cells. Silencing of COX-II expression leads to reduced apoptosis in infected T-cells, indicating its importance in apoptosis. Furthermore, our results also show that the activities of enzyme complexes I, II and III are decreased while those of Complex IV and V are increased at the time of acute infection and apoptosis. This differential regulation in activities of OXPHOS system complexes indicate a complex modulation of host cell energy generating system during HIV infection that ultimately leads to T cell apoptosis.


Assuntos
Apoptose , Complexo IV da Cadeia de Transporte de Elétrons/genética , Infecções por HIV/enzimologia , Infecções por HIV/imunologia , Mitocôndrias/metabolismo , Linfócitos T/citologia , Regulação para Cima , Linhagem Celular , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Regulação Enzimológica da Expressão Gênica , Infecções por HIV/fisiopatologia , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/fisiologia , Humanos , Mitocôndrias/enzimologia , Fosforilação Oxidativa , Linfócitos T/enzimologia , Linfócitos T/imunologia , Linfócitos T/virologia
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