HPV infection, a described cause for human cancer: HPV detection and molecular mechanisms of HPV carcinogenesis (Review).

[en]Human papillomavirus (HPV) belongs to the Papillomaviridae virus family and it is one of the most common sexual transmission infections. HPV genome is composed of eight genes, including two early genes and six late genes. Among these late genes, E6 and E7 code for proteins that trigger cell-cycle re-entry in infected cells, which can lead to cervical cancer development. The IARC (International Agency for Research Cancer) proposed a guideline based on Hill’s criteria to determine whether the relation between HPV infection and cervical cancer is causal or not. Epidemiological studies have demonstrated that HPV infection is a necessary but non-sufficient cause for cervical cancer. Furthermore, HPV infection is considered the first necessary cause described of a human cancer, being HPV16 and 18 carcinogenic to humans and the most studied types. Cervical cancer is the second leading cause of cancer death among women worldwide. Different screening programs are carried out with the aim of preventing cervical cancer; such as cytologies and HPV tests. There are two main methods which are equally usable to detect HPV: the real-time PCR assays and the array assays. Regarding the molecular mechanisms of HPV mediated malignancies, E2, E6 and E7 proteins of HPV16 lead to immune response evasion, inducing IL-10 and TGF-β1 gene expression. Besides, E6 and E7 proteins allow cell-cycle reentry, phosphorylating RB and ubiquitinating p53 respectively. HPV genome integration in host genome leads to the alteration of host and viral genes expression, including oncogenes and tumor suppressor genes. However, the differences of E6 and E7 oncoproteins in different HPV types is poorly known due to the fact that almost the most studied HPV type has been HPV16.

Avaliação do efeito do inibidor de histona deacetilase Panobinostat (LBH589) no metabolismo energético de células derivadas de câncer de pulmão

Os mecanismos de ação citotóxica do inibidor de histona deacetilase LBH589 foram investigados em associação com o quimioterápico cisplatina (CDDP) em duas linhagens derivadas de câncer de pulmão de não pequenas células (CPNPC). Os resultados foram analisados em relação ao tipo de morte celular associada às alterações em enzimas relacionadas ao metabolismo energético e a via glicolítica. Para realização do trabalho, foram utilizadas as linhagens tumorais A549 (selvagem para o gene de p53) e Calu-1 (nulo para o gene de p53) tratadas com LBH589 em combinação ou não com CDDP. Foram realizadas curvas de tempo e dose-resposta com as drogas isoladamente pelo ensaio de viabilidade celular (MTT) nas duas linhagens para a escolha das melhores condições para o nosso estudo. As condições dos tratamentos isolados com redução da viabilida celular menores que o IC50 de cada fármaco foram selecionados para realização dos tratamentos combinados. As avaliações de apoptose foram realizadas por citometria de fluxo pelo ensaio de Anexina V/PI, e com a marcação de proteínas por Western Blotting. As proteínas relacionadas a via glicolítica foram avaliadas por Western Blotting e a expressão de RNAm por qPCR. Os resultados demonstraram que o LBH589 combinado a CDDP foi capaz de induzir apoptose em 70% das células (Calu-1) e 54,9% (A549) no tempo de 24 horas, e 90% (calu-1) e 62,1% (A549) em 48 horas, independendo, portanto, do status da p53. Os níveis de expressão de enzimas relacionadas com o metabolismos energético também sofreram alterações nos tratamentos estudados. O LBH589 induziu aumento de cerca de 4x dos níveis de RNAm de HK isoformas I e II em ambas as linhagens. Houve também um aumento na expressão proteica das isoformas de HK I e II. Outras enzimas relacionadas a via glicolítica como PFKP, PKM2 e LDHA foram analisadas e apresentaram redução da expressão proteica, principalmente na presença do LBH589. A combinação da CDDP com LBH589 parece ser promissora para o tratamento de CPNPC induzindo apoptose através de alterações no metabolismo energético tumoral.

乳腺增生病p53基因第6外显子突变检测

目的 :探讨p5 3基因在乳腺癌发生早期的作用及早期诊断乳腺癌的分子病理指标。方法 :用PCR -SSCP检测36例乳腺单纯性增生、 31例不典型增生、 30例乳腺癌中p5 3基因第 6外显子突变 ,用DNA直接测序技术确定突变的碱基及其所在的密码子。结果 :乳腺单纯性增生、不典型增生、乳腺癌中p5 3基因第 6外显子的突变率分别为 0、6 5 % (2 / 31)、 13 3% (4/ 30 )。 6个点突变均为碱基替换 ,其中 4个发生于第 192密码子 (CAG→TAG) ,2个发生于第2 13密码子 (CGA→TGA) ,两者均导致多肽链合成提前终止。结论 :乳腺癌不典型增生中存在p5 3基因第 6外显子突变 ,该突变可能在乳腺不典型增生发展到乳腺癌过程中起重要作用 ,可作为早期诊断乳腺癌的辅助指标。

乳腺增生病p53基因第7外显子突变检测

目的探讨p53基固在乳腺癌发生早期的作用及早期诊断乳晾瘟的分子病理指标。方法用PCR—sso 法检测36例乳腺单纯性增生、31例不典型增生、14例原位癌和16例程润癌中p53基因第7外显子突变，井用叭A 直接测序技术确定突变的碱基及其所在的密码子。结果乳腺单纯性增生、不典型增生、原位癌、浸润癌中|】53基 因第7外显于的突变率分别为0％、6 5％(2／31)、21 4％(3／14)、18．8％(3／16)。8例突变中，3例发生于251密码 子，4例发生手248密码子，I例发生于236密码子。表现为碱基替换(5例)、缺失(2例)、插入(J例)共二种突变方 式。并在I例原位瘟中首次发现了251密码子的同义突变(ATC斗ATr，异亮氨酸一异亮氮酸)。结论乳腺癌不典 型增生中存在053基因第7外显子突变，该突变可能在乳腺不典型增生发展到乳腺癌的过程中起霞要作用，可作为 早期诊断乳腺癌的辅助指标。

乳腺增生病p53基因第5外显子突变及其蛋白表达

目的 :探讨 p5 3基因在乳腺癌发生早期的作用。 方法 :用免疫组化方法检测 36例乳腺单纯性增生、31例不典型增生、14例原位癌和 16例浸润癌中 p5 3蛋白的表达 ,用PCR SSCP检测了上述组织中 p5 3基因第 5外显子突变。 结果 :p5 3蛋白在单纯性增生、不典型增生、导管内癌、浸润癌中的表达率分别为 0、2 2 6 % (7/ 31)、42 8% (6 / 14)、5 0 % (8/ 16 ) ,PCR SSCP在各组中均未检测到该基因第 5外显子突变。结论 :乳腺癌发生早期阶段有 p5 3基因的参与 ,但与第 5外显子突变无明显关系

Pseudogenization of the tumor-growth promoter angiogenin in a leaf-eating monkey

Physiological functions of human genes may be studied by gene-knockout experiments in model organisms such as the mouse. This strategy relies on the existence of one-to-one gene orthology between the human and mouse. When lineage-specific gene duplication occurs and paralogous genes share a certain degree of functional redundancy, knockout mice may not provide accurate functional information on human genes. Angiogenin is a small protein that stimulates blood-vessel growth and promotes tumor development. Humans and related primates only have one angiogenin gene, while mice have three paralogous genes. This makes it difficult to generate angiogenin-knockout mice and even more difficult to interpret the genotype-phenotype relation from such animals should they be generated. We here show that in the douc langur (Pygathrix nemaeus), an Asian leaf-eating colobine monkey, the single-copy angiogenin gene has a one-nucleotide deletion in the sixth codon of the mature peptide, generating a premature stop codon. This nucleotide deletion is found in five unrelated individuals sequenced, and therefore is likely to have been fixed in the species. Five colobine species that are closely related to the douc langur have intact angiogenin genes, suggesting that the pseudogenization event was recent and unique to the douc langur lineage. This natural knockout experiment suggests that primate angiogenin is dispensable even in the wild. Further physiological studies of douc largurs may offer additional information on the role of this cancer-related gene in normal physiology of primates, including humans. Our findings also provide a strong case for the importance of evolutionary analysis in biomedical studies of gene functions. (C) 2003 Elsevier Science B.V. All rights reserved.

Association of early growth response-1 gene polymorphisms with total IgE and atopy in asthmatic children

Early growth response-1 (Egr-1) is expressed in human airways and found to modulate tumor necrosis factor, immunoglobulin E (IgE), airway responsiveness, and interleukin-13-induced inflammation in mice. We investigated the effects of Chinese-tagging singl

Elongation Factor ELL (Eleven-Nineteen Lysine-rich Leukemia) Acts as a Transcription Factor for Direct Thrombospondin-1 Regulation

The eleven-nineteen lysine-rich leukemia (ELL) gene undergoes translocation and fuses in-frame to the multiple lineage leukemia gene in a substantial proportion of patients suffering from acute forms of leukemia. Studies show that ELL indirectly modulates transcription by serving as a regulator for transcriptional elongation as well as for p53, U19/Eaf2, and steroid receptor activities. Our in vitro and in vivo data demonstrate that ELL could also serve as a transcriptional factor to directly induce transcription of the thrombospondin-1 (TSP-1) gene. Experiments using ELL deletion mutants established that full-length ELL is required for the TSP-1 up-regulation and that the trans-activation domain likely resides in the carboxyl terminus. Moreover, the DNA binding domain may localize to the first 45 amino acids of ELL. Not surprisingly, multiple lineage leukemia-ELL, which lacks these amino acids, did not induce expression from the TSP-1 promoter. In addition, the ELL core-response element appears to localize in the -1426 to -1418 region of the TSP-1 promoter. Finally, studies using zebrafish confirmed that ELL regulates TSP-1 mRNA expression in vivo, and ELL could inhibit zebrafish vasculogenesis, at least in part, through up-regulating TSP-1. Given the importance of TSP-1 as an anti-angiogenic protein, our findings may have important ramifications for better understanding cancer.

Developmental toxicity and alteration of gene expression in zebrafish embryos exposed to PFOS

Perfluorooetanesulfonate (PFOS) is a persistent organic pollutant, the potential toxicity of which is causing great concern. In the present study, we employed zebrafish embryos to investigate the developmental toxicity of this compound. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to 0.1, 0.5, 1, 3 and 5 mg/L PFOS. Hatching was delayed and hatching rates as well as larval survivorship, were significantly reduced after the embryos were exposed to 1, 3 and 5 mg/L PFOS until 132 hpf. The fry displayed gross developmental malformations, including epiboly deformities, hypopigmentation, yolk sac edema, tail and heart malformations and spinal curvature upon exposure to PFOS concentrations of I mg/L or greater. Growth (body length) was significantly reduced in the 3 and 5 mg/L PFOS-treated groups. To test whether developmental malformation was mediated via apoptosis, flow cytometry analysis of DNA content, acridine orange staining and TUNEL assay was used. These techniques indicated that more apoptotic cells were present in the PFOS-treated embryos than in the control embryos. Certain genes related to cell apoptosis, p53 and Bax, were both significantly up-regulated upon exposure to all the concentrations tested. In addition, we investigated the effects of PFOS on marker genes related to early thyroid development (hhex and pax8) and genes regulating the balance of androgens and estrogens (cyp19a and cyp19b). For thyroid development, the expression of hhex was significantly up-regulated at all concentrations tested, whereas pax8 expression was significantly up-regulated only upon exposure to lower concentrations of PFOS (0.1, 0.5, 1 mg/L). The expression of cyp19a and of cyp19b was significantly down-regulated at all exposure concentrations. The overall results indicated that zebrafish embryos constitute a reliable model for testing the developmental toxicity of PFOS, and the gene expression patterns in the embryos were able to reveal some potential mechanisms of developmental toxicity. (C) 2008 Elsevier Inc. All rights reserved.

TRAIL in the mandarin fish Siniperca chuatsi: Gene and its apoptotic effect in HeLa cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is one of the TNF superfamily members, participating in many biological processes including cell proliferation and apoptotic death. In this study, a TRAIL gene was cloned from a perciform fish, the mandarin fish Siniperca chuatsi, a major cultured fish in China's aquaculture, and is named as SCTRAIL for S. chuatsi TRAIL. The full-length cDNA of SCTRAIL is 1359 bp, encoding a 283-amino-acid protein. This deduced protein contains the CYS231, a 23-mer fragment of transmembrane region, a glycosylation site and a TNF family signature, all of which are conserved among TRAIL members. SCTRAIL gene consists of six exons, with five intervening introns, spaced over approximately 9 kb of genomic sequence. Southern blotting demonstrated that the SCTRAIL gene is present as a single copy in mandarin fish genome. A 620 bp promoter region obtained by genome walking contains a number of putative transcription factor binding sites, such as Oct-1, Sp-1, NF-1, RAP-1, C/EBPaLp, NF-kappa B and AP-1. The SCTRAIL is constitutively expressed in all the analyzed tissues, as revealed by RT-PCR, which is confirmed by Western blotting analysis using polyclonal antibody against bacteria-derived recombinant SCTRAIL protein. As an apoptosis-inducing ligand, the overexpression of SCTRAIL but not the mutant SCTRAIL-C203S in HeLa cells induced changes characteristic of apoptosis, including chromatin condensation, nucleus fragmentation, DNA ladder, and increase of sub-G0/G1 cells in FACS analysis. (c) 2007 Elsevier Ltd. All rights reserved.

Molecular identification and expression analysis of tumor necrosis factor receptor-associated factor 2 in grass carp Ctenopharyngodon idella

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is a crucial component of almost the entire tumor necrosis factor receptor superfamily signaling pathway. In the present study, a TRAF2 gene has been cloned from grass carp (Ctenopharyngodon idella) by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The full-length cDNA is 3162 bp, including a 60 bp 5' untranslated region (UTR), a 1611 bp open reading frame, and a 1491 bp 3' UTR. The polyadenylation signal (AATAAA) and the mRNA instability motifs (ATTTTA, ATTTA) were followed by a poly(A) tail in the 3' UTR. No signal peptide or transmembrane region has been found in the putative amino acids of grass carp TRAF2 (gcTRAF2). Phylogenetic tree analysis clearly showed that gcTRAF2 is nearest to the TRAF2 gene of goldfish. The identity of gcTRAF2 with its homologs in other vertebrates ranges from 56% to 97%. It is characterized by one RING-type signature at the N-terminus, one zinc finger in the middle part, and one conserved TRAF domain consisting of a C-proximal (TRAF-C) subdomain and a N-proximal (TRAF-N) subdomain. The identity of TRAF-C among all TRAF2 homologs in vertebrates varies from 78% to 97%, whereas the identity of TRAF-N ranges from 56% to 100%. The recombinant gcTRAF2 has been expressed in Escherichia coli using pET-32a expression vector. The rabbit anti-gcTRAF2 polyclonal antibody was obtained. The expression of gcTRAF2 in different organs was examined by real-time quantitative polymerase chain reaction and Western blot analysis. It was widely distributed in heart, head kidney, thymus, brain, gill, liver, spleen, and trunk kidney. This is the first report of a TRAF2 homolog molecule in fish.

Characterization and expression analysis of TNF-related apoptosis inducing ligand (TRAIL) in grass carp Ctenopharyngodon idella

TRAIL (Apo2 ligand) described as a type II transmembrame protein belonging to the TNF superfamily can induce apoptotic cell death in a variety of cell types. In the present study, a putative cDNA sequence encoding the 299 amino acids of TRAIL (GC-TRAIL) and its genomic organization were identified in grass carp Ctenopharyngodon idella. The predicted GC-TRAIL sequence showed 44 and 41% identities to chicken and human TRAILs, respectively. In a domain search, a tumor necrosis factor homology domain (THD) was identified in the C-terminal portion of TRAILs. The GC-TRAIL gene consists of five exons, with four intervening introns, spaced over approximately 4 kb of genomic sequence. Analysis of GC-TRAlL promoter region revealed the presence of a number of putative transcription factor binding sites, such as Sp1, NF-kappaB, AP-1, GATA, NFAT, HNF, STAT, P53 and IRFI sequences which are important for the expression of other TNF family members. Phylogenetic analysis placed GC-TRAIL and the putative zebrafish (Danio rerio) TRAIL obtained from searching the zebrafish database into one separate cluster near mammalian TRAIL genes, but apart from the reported zebrafish TRAIL-like protein, indicating that the GC-TRAIL is an authentic fish TRAIL. Expression analysis revealed that GC-TRAIL is expressed in many tissues, such as in gills, liver, trunk kidney, head kidney, intestine and spleen. (c) 2005 Elsevier B.V. All rights reserved.

Molecular cloning of TRAF2 binding protein gene and its promoter region from the grass carp Ctenopharyngodon idellus

A tumor necrosis factor receptor-associated factor 2 binding protein (T2BP) gene was isolated from the grass carp (Ctenopharyngodon idellus) by utilizing suppression subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE). The grass carp T2BP (GT2BP) gene contains an open reading frame of 579 nucleotide(s) (nt), encoding 193 amino acids, with 23 nt 5'-untranslated region and a long 3'-untranslated region of 434 nt including poly (A), 1 AUUUA motif and 4 AUUUUA motifs. No signal peptide has been detected in the predicted GT2BP, but a characteristic forkhead associated domain is present. The GT2BP mRNA shares 83% identity with the zebrafish DNA sequence, and they both have no introns in the genomic DNA. The putative transcription factor binding sites of GT2BP include two C/EBP alpha binding sites, and one c-Jun binding, one AP-1 binding, and one nuclear factor kappa B (NF kappa B) binding sites. Southern blot analysis revealed that the GT2BP was a single-copy gene. Individual difference was observed in GT2BP expression in examined organs of healthy grass carp. However, the expression of GT2BP in all examined organs in a fish with the highest copepod infection level and the significantly higher expression level in spleen and liver in infected fish may indicate its up-regulation with the parasite infection. (c) 2005 Elsevier B.V. All rights reserved.

Effect of p53 on lung carcinoma cells irradiated by carbon ions or X-rays

The study is to investigate the feasibility and advantages of heavy ion beams on radiotherapy. The cellular cycle and apoptosis, cell reproductive death and p53 expression evaluated with flow cytometry, clonogenic survival assays and Western blot analysis were examined in lung carcinoma cells after exposure to 89.63 MeV/u carbon ion and 6 MV X-ray irradiations, respectively. The results showed that the number colonyforming assay of A549 was higher than that of H1299 cells in two radiation groups; A549 cellular cycle was arrested in G(2)/M in 12 It and the percentage of apoptosis ascended at each time point of carbon ion radiation with doses, the expression of p53 upregulated with doses exposed to X-ray or carbon ion. The cell number in G(2)/M of H1299 and apoptosis were increasing at all time points with doses in C-12(6+) ion irradiation group. The results suggested that the effects of carbon ions or X rays irradiation on lung carcinoma cells were different, C-12(6+) ion irradiation could have more effect on upregulating the expression of p53 than X-ray, and the upregulated expression of p53 might produce the cellular cycle G(2)/M arrested, apoptosis increasing; and p53 gene might affect the lung cancer cells radiosensitivity.