RESUMEN
Cervical carcinomas result from cellular transformation by the human papillomavirus (HPV) E6 and E7 oncogenes which are constitutively expressed in cancer cells. The E6 oncogene degrades p53 thereby modulating a large set of p53 target genes as shown previously in the cervical carcinoma cell line HeLa. Here we show that the TAp63ß isoform of the p63 transcription factor is also a target of E6. The p63 gene plays an essential role in skin homeostasis and is expressed as at least six isoforms. One of these isoforms, ΔNp63α, has been found overexpressed in squamous cell carcinomas and is shown here to be constitutively expressed in Caski cells associated with HPV16. We therefore explored the role of p63 in these cells by performing microarray analyses after repression of endogenous E6/E7 expression. Upon repression of the oncogenes, a large set of p53 target genes was found activated together with many p63 target genes related to cell adhesion. However, through siRNA silencing and ectopic expression of various p63 isoforms we demonstrated that TAp63ß is involved in activation of this cell adhesion pathway instead of the constitutively expressed ΔNp63α and ß. Furthermore, we showed in cotransfection experiments, combined with E6AP siRNA silencing, that E6 induces an accelerated degradation of TAp63ß although not through the E6AP ubiquitin ligase used for degradation of p53. Repression of E6 transcription also induces stabilization of endogenous TAp63ß in cervical carcinoma cells that lead to an increased concentration of focal adhesions at the cell surface. Consequently, TAp63ß is the only p63 isoform suppressed by E6 in cervical carcinoma as demonstrated previously for p53. Down-modulation of focal adhesions through disruption of TAp63ß therefore appears as a novel E6-dependent pathway in transformation. These findings identify a major physiological role for TAp63ß in anchorage independent growth that might represent a new critical pathway in human carcinogenesis.
Asunto(s)
Transformación Celular Viral , Adhesiones Focales/metabolismo , Papillomavirus Humano 16/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Proteolisis , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/virología , Adhesión Celular , Adhesiones Focales/genética , Adhesiones Focales/virología , Células HeLa , Papillomavirus Humano 16/genética , Humanos , Proteínas Oncogénicas Virales/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
BACKGROUND: French Guiana (FG) is an ultra-peripheral European region in the Amazon, and the COVID-19 epidemic has had very different kinetics from both its giant neighbors, Brazil or mainland France. METHODS: This study summarized the epidemics of COVID-19 in FG. RESULTS: The tropical climate, multiethnicity, and remoteness of the population forced healthcare providers to accordingly adapt the management of the epidemic. Incidence and mortality have been lower than that in Europe and Latin America due to a combination of prevalence of the youth in the population and highly developed healthcare system. CONCLUSIONS: Currently, vaccine hesitancy hinders the rapid expansion of vaccine coverage.
Asunto(s)
COVID-19 , Epidemias , Adolescente , Brasil , COVID-19/epidemiología , Europa (Continente) , Guyana Francesa/epidemiología , HumanosRESUMEN
A strong epithelial specific enhancer drives transcription of the human papillomavirus type 18 (HPV18) oncogenes. Its activity depends on the formation of a higher-order nucleoprotein complex (enhanceosome) involving the sequence-specific JunB/Fra2 transcription factor and the HMG-I(Y) architectural protein. Here we show that proteins from HeLa cell nuclear extract cover almost all of the HPV18 enhancer sequences and that it contains seven binding sites for the purified HMG-I(Y) protein, providing evidence for a tight nucleoprotein structure. Binding of HMG-I(Y) and the AP1 heterodimer from HeLa nuclear extract to overlapping sites of the core enhanceosome is cooperative. The integrity of this specific HMG-I(Y) binding site is as essential as the AP1 binding site for the enhancer function, indicating the fundamental role played by this architectural protein. We demonstrate that the CBP/p300 coactivator is recruited by the HPV18 enhanceosome and that it is limiting for transcriptional activation, since it is sequestered by the adenovirus E1A protein and by the JunB/Fra2 positive factor in excess. We show the involvement of JunB and p300 in vivo in the HPV18 transcription by chromatin immunoprecipitation of HPV18 sequences in HeLa cells.
Asunto(s)
Proteína HMGA1a/metabolismo , Proteínas Nucleares/metabolismo , Papillomaviridae/genética , Transactivadores/metabolismo , Transcripción Genética/fisiología , Proteínas E1A de Adenovirus/metabolismo , Secuencia de Bases , Núcleo Celular/química , Núcleo Celular/metabolismo , Núcleo Celular/virología , Huella de ADN , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos/fisiología , Antígeno 2 Relacionado con Fos , Células HeLa , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Nucleoproteínas/metabolismo , Unión Proteica/fisiología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Integration of the human papillomavirus (HPV) genome into the host genome is associated with the disruption of the HPV E2 gene and with amplification and rearrangement of the viral and flanking cellular sequences. Molecular characterization of the genomic structures of coamplified HPV sequences and oncogenes provides essential information concerning the mechanisms of amplification and their roles in carcinogenesis. Using fluorescent hybridization on stretched DNA molecules in two cervical cancer-derived cell lines, we have elucidated the genomic structures of amplified regions containing HPV/myc genes over several hundreds of kilobases. Direct visualization of hybridization signals on individual DNA molecules suggests that overreplication and breakage-fusion-bridge-type mechanisms are involved in the genomic instability associated with HPV cervical cancers. Further analysis from two other genital cancer-derived cell lines reveals a recurrent motif of amplification, probably generated by a common mechanism involving overreplication upon viral integration. Interestingly, different amplification patterns seem to be correlated with the disease outcome, thus providing new insights into HPV-related cancer development and tumor progression.
Asunto(s)
Genes myc/genética , Genoma Humano , Papillomaviridae/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/virología , Femenino , Amplificación de Genes , Humanos , Familia de Multigenes , Proteínas Oncogénicas Virales/genética , Integración Viral/genéticaRESUMEN
ABSTRACT Background: French Guiana (FG) is an ultra-peripheral European region in the Amazon, and the COVID-19 epidemic has had very different kinetics from both its giant neighbors, Brazil or mainland France. Methods: This study summarized the epidemics of COVID-19 in FG. Results: The tropical climate, multiethnicity, and remoteness of the population forced healthcare providers to accordingly adapt the management of the epidemic. Incidence and mortality have been lower than that in Europe and Latin America due to a combination of prevalence of the youth in the population and highly developed healthcare system. Conclusions: Currently, vaccine hesitancy hinders the rapid expansion of vaccine coverage.
RESUMEN
More than 90% of cervical carcinomas are associated with human papillomavirus (HPV) infection. The two viral oncogenes E6 and E7 play a major role in transforming the cells by disrupting p53- and pRb-dependent cell cycle checkpoints. A hallmark of HPV-associated cervical carcinoma is loss of the expression of the viral E2 protein, often by disruption of E2-encoding gene. We showed previously that reintroduction of E2 in HPV18-associated cervical carcinoma cells induces cell cycle arrest in G(1) because of the transcriptional repression of the viral oncogenes E6 and E7 and concomitant reactivation of the p53 and pRb pathways. Here we describe global gene profiling of HeLa cells expressing different HPV18 E2 mutants to study the effects of repression of the viral oncogenes. We identified 128 genes transcriptionally regulated by the viral oncogenes in cervical carcinoma. Surprisingly, E2 repressed a subset of E2F-regulated mitotic genes in an E6/E7-dependent pathway. This was corroborated by the observation that E2 delayed mitotic progression, suggesting the involvement of a mitotic pathway in HPV carcinogenesis. These mitotic genes constitute an as yet unrecognized set of genes, which were also found deregulated in other HPV-associated cervical carcinoma cell lines and therefore represent new targets for both diagnosis and therapeutic approaches in cervical cancer.
Asunto(s)
Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/virología , Adenoviridae/genética , Femenino , Fase G1/genética , Regulación Neoplásica de la Expresión Génica , Regulación Viral de la Expresión Génica , Células HeLa , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Transcripción Genética , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patologíaRESUMEN
The Human Papillomavirus (HPV) E2 protein, which inhibits the E6 and E7 viral oncogenes, is believed to have anti-oncogenic properties. Here, we challenge this view and show that HPV-18 E2 over-activates the Spindle Assembly Checkpoint (SAC) and induces DNA breaks in mitosis followed by aneuploidy. This phenotype is associated with interaction of E2 with the Mitotic Checkpoint Complex (MCC) proteins Cdc20, MAD2 and BUBR1. While BUBR1 silencing rescues the mitotic phenotype induced by E2, p53 silencing or presence of E6/E7 (inactivating p53 and increasing BUBR1 levels respectively) both amplify it. This work pinpoints E2 as a key protein in the initiation of HPV-induced cervical cancer and identifies the SAC as a target for oncogenic pathogens. Moreover, our results suggest a role of p53 in regulating the mitotic process itself and highlight SAC over-activation in a p53-negative context as a highly pathogenic event.
Asunto(s)
Aneuploidia , Papillomavirus Humano 18/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Proteínas Oncogénicas Virales/metabolismo , Infecciones por Papillomavirus/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Huso Acromático/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Regulación de la Expresión Génica , Células HeLa , Interacciones Huésped-Patógeno , Papillomavirus Humano 18/genética , Humanos , Queratinocitos/metabolismo , Queratinocitos/virología , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Proteínas Oncogénicas Virales/genética , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/virología , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Transducción de Señal , Huso Acromático/genética , Huso Acromático/virología , Factores de Tiempo , Transfección , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/virologíaRESUMEN
The E2 protein is expressed in the early stage of human papillomavirus (HPV) infection that is associated with cervical lesions. This protein plays important roles in regulation of viral replication and transcription. To characterize the role of E2 protein in modulation of cellular gene expression in HPV infected cells, genome-wide expression profiling of human primary keratinocytes (HPK) harboring HPV16 E2 and HPV18 E2 was investigated using microarray. The Principle Components Analysis (PCA) revealed that the expression data of HPV16 E2 and HPV18 E2-transduced HPKs were rather closely clustered. The Venn diagram of modulated genes showed an overlap of 10 common genes in HPV16 E2 expressing HPK and HPV18 E2 expressing HPK. These genes were expressed with significant difference by comparison with control cells. In addition, the distinct sets of modulated genes were detected 14 and 34 genes in HPV16 E2 and HPV18 E2 expressing HPKs, respectively.
RESUMEN
In the early stages of human papillomavirus (HPV) infection, the viral proteins elicit specific immune responses that can participate to regression of ano-genital lesions. HPV E6 protein for instance can reduce type I interferon (IFN) including IFN-κ that is involved in immune evasion and HPV persistence. To evaluate the role of E2 protein in innate immunity in HPV16-associated cervical lesions, genome-wide expression profiling of human primary keratinocytes (HPK) transduced by HPV16 E2 was investigated using microarrays and innate immunity associated genes were specifically analyzed. The analyses showed that the expression of 779 genes was modulated by HPV16E2 and 92 of them were genes associated with innate immunity. Notably IFN-κ and STING were suppressed in HPK expressing the E2 proteins of HPV16 or HPV18 and the trans-activation amino-terminal domain of E2 was involved in the suppressive effect. The relationship between STING, IFN-κ and interferon stimulated genes (ISGs) in HPK was confirmed by gene silencing and real time PCR. The expression of STING and IFN-κ were further determined in clinical specimens by real time PCR. STING and IFN-κ were down-modulated in HPV positive low grade squamous intraepithelial lesions compared with HPV negative controls. This study demonstrates that E2 proteins of high risk HPV reduce STING and IFN-κ transcription and its downstream target genes that might be an immune evasion mechanism involved in HPV persistence and cervical cancer development.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Interferón Tipo I/genética , Queratinocitos/metabolismo , Proteínas de la Membrana/genética , Proteínas Oncogénicas Virales/metabolismo , Transcripción Genética , Adenoviridae/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/química , Regulación hacia Abajo/efectos de los fármacos , Femenino , Redes Reguladoras de Genes , Genoma Humano/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Interferón Tipo I/metabolismo , Queratinocitos/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Proteínas Oncogénicas Virales/química , Poli I-C/farmacología , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Recombinación Genética , Transcripción Genética/efectos de los fármacos , Transducción GenéticaRESUMEN
Palmoplantar keratodermas (PPKs) are a group of disorders that are diagnostically and therapeutically problematic in dermatogenetics. Punctate PPKs are characterized by circumscribed hyperkeratotic lesions on the palms and soles with considerable heterogeneity. In 18 families with autosomal dominant punctate PPK, we report heterozygous loss-of-function mutations in AAGAB, encoding α- and γ-adaptin-binding protein p34, located at a previously linked locus at 15q22. α- and γ-adaptin-binding protein p34, a cytosolic protein with a Rab-like GTPase domain, was shown to bind both clathrin adaptor protein complexes, indicating a role in membrane trafficking. Ultrastructurally, lesional epidermis showed abnormalities in intracellular vesicle biology. Immunohistochemistry showed hyperproliferation within the punctate lesions. Knockdown of AAGAB in keratinocytes led to increased cell division, which was linked to greatly elevated epidermal growth factor receptor (EGFR) protein expression and tyrosine phosphorylation. We hypothesize that p34 deficiency may impair endocytic recycling of growth factor receptors such as EGFR, leading to increased signaling and cellular proliferation.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular , Proteínas Portadoras/genética , Haploinsuficiencia , Poroqueratosis/genética , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Mapeo Cromosómico , Citosol/ultraestructura , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Queratinocitos/metabolismo , Queratinocitos/patología , Linaje , Poroqueratosis/metabolismo , Unión Proteica , Proteínas/genética , Proteínas/metabolismoRESUMEN
The papillomavirus (PV) E2 proteins have been shown to exert many functions in the viral cycle including pivotal roles in transcriptional regulation and in viral DNA replication. Besides these historical roles, which rely on their aptitude to bind to specific DNA sequences, E2 has also been shown to modulate the host cells through direct protein interactions mainly through its amino terminal transactivation domain. We will describe here some of these new functions of E2 and their potential implication in the HPV-induced carcinogenesis. More particularly we will focus on E2-mediated modulation of the host cell cycle and consequences to cell transformation. In all, the HPV E2 proteins exhibit complex functions independent of transcription that can modulate the host cells in concert with the viral vegetative cycle and which could be involved in early carcinogenesis.
RESUMEN
The WHIM syndrome, which features high susceptibility to human papillomavirus (HPV) infection, is a rare immunodeficiency associated with autosomal dominant heterozygous mutations of the CXCR4 chemokine receptor. CXCL12 and its receptors, CXCR4 and CXCR7, are linked to tumorigenesis, and we reported that abnormal expression of CXCL12 in epidermal keratinocytes correlates with HPV infection. However, the HPV-related pathologies observed in WHIM patients remain mechanistically unexplained. We show that keratinocytes immortalized by oncogenic HPV16 or HPV18 upregulate CXCL12 and its receptors in a manner dependent upon expression of the viral proteins E6 and E7. Autocrine signaling activated by CXCL12-engagement of its receptors controls motility and survival of the infected cells. Strikingly, expression of a WHIM syndrome-related gain-of-function CXCR4 mutant confers transforming capacity to HPV18-immortalized keratinocytes. These results establish a pivotal role for CXCL12 signaling in HPV-mediated transformation and provide a mechanistic basis for understanding HPV pathogenesis in WHIM syndrome.
Asunto(s)
Quimiocina CXCL12/fisiología , Papillomavirus Humano 16/fisiología , Papillomavirus Humano 18/fisiología , Queratinocitos/virología , Infecciones por Papillomavirus/virología , Receptores CXCR/biosíntesis , Animales , Movimiento Celular , Transformación Celular Viral , Quimiocina CXCL12/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Femenino , Humanos , Síndromes de Inmunodeficiencia/patología , Síndromes de Inmunodeficiencia/virología , Recién Nacido , Masculino , Ratones , Ratones Desnudos , Proteínas Oncogénicas Virales/biosíntesis , Proteínas E7 de Papillomavirus/biosíntesis , Infecciones por Papillomavirus/patología , Enfermedades de Inmunodeficiencia Primaria , Receptores CXCR/fisiología , Receptores CXCR4/biosíntesis , Proteínas Represoras/biosíntesis , Verrugas/patología , Verrugas/virologíaRESUMEN
Cervical carcinoma is associated with certain types of human papillomaviruses expressing the E6 and E7 oncogenes, which are involved in carcinogenesis through their interactions with the p53 and pRB pathways, respectively. A critical event on the path to malignant transformation is often manifested by the loss of expression of the viral E2 transcription factor due to the integration into the host genome of the viral DNA. Using microarrays, we have previously shown that reintroduction of a functional E2 in the HeLa cervical carcinoma cell line activates a cluster of p53 target genes while at the same time severely repressing a group of E2F target genes. In the present study, using new high-density microarrays containing more than 22,000 human cDNA sequences, we identified a novel p63 pathway among E2-activated genes and 38 new mitotic genes repressed by E2. We then sought to determine the pathways through which these genes were modulated and used an approach that relies on small interfering RNA to demonstrate that the p63 target genes were activated through silencing of the E6/E6AP pathway while the mitotic genes were mainly repressed through E7 silencing. Importantly, a subset of the mitotic genes was shown to be significantly induced in biopsies of stage IV cervical cancers, which points to a prominent E7 pathway in cervical carcinoma.