HPV16 Tumor Associated Macrophages Suppress Antitumor T Cell Responses

Purpose: High-risk human papillomavirus (HPV) is the main etiologic factor for cervical cancer. The severity of HPV-associated cervical lesions has been correlated to the number of infiltrating macrophages. The objective of this work is to characterize the role of tumor-associated macrophages (TAM) on the immune cellular response against the tumor. Experimental Design: We used the HPV16 E6- and E7-expressing TC-1 mouse tumor model to study the effect of TAM on T-cell function in vitro, and depleted TAM, using clodronate-containing liposomes, to characterize its role in vivo. Results: TAM, characterized by the positive expression of CD45, F4/80, and CD11b, formed the major population of infiltrating tumor cells. TAM displayed high basal Arginase I activity, producing interleukin-10 (IL-10); they were resistant to iNOSll activity induction, therefore reversion to M1 phenotype, when stimulated in vitro with lipopolysaccharide/IFN gamma, indicating an M2 phentoype. In cultures of isolated TAM, TAM induced regulatory phenotype, characterized by IL-10 and Foxp3 expression, and inhibited proliferation of CD8 lymphocytes. In vivo, depletion of TAM inhibited tumor growth and stimulated the infiltration of tumors by HPV16 E7(49-57)-specific CD8 lymphocytes, whereas depletion of Gr1(+) tumor-associated cells had no effect. Conclusions: M2-like macrophages infiltrate HPV16-associated tumors causing suppression of antitumor T-cell response, thus facilitating tumor growth. Depletion or phenotype alteration of this population should be considered in immunotherapy strategies.

[(13)C(2)]- Acetaldehyde Promotes Unequivocal Formation of 1,N(2)-Propano-2 `-deoxyguanosine in Human Cells

Acetaldehyde is an environmentally widespread genotoxic aldehyde present in tobacco smoke, vehicle exhaust and several food products. Endogenously, acetaldehyde is produced by the metabolic oxidation of ethanol by hepatic NAD-dependent alcohol dehydrogenase and during threonine catabolism. The formation of DNA adducts has been regarded as a critical factor in the mechanisms of acetaldehyde mutagenicity and carcinogenesis. Acetaldehyde reacts with 2`-deoxyguanosine in DNA to form primarily N(2)-ethylidene-2`-deoxyguanosine. The subsequent reaction of N(2)-ethylidenedGuo with another molecule of acetaldehyde gives rise to 1,N(2)-propano-2`-deoxyguanosine (1,N(2)-propanodGuo), an adduct also found as a product of the crotonaldehyde reaction with dGuo. However, adducts resulting from the reaction of more than one molecule of acetaldehyde in vivo are still controversial. In this study, the unequivocal formation of 1,N(2)-propanodGuo by acetaldehyde was assessed in human cells via treatment with [(13)C(2)]-acetaldehyde. Detection of labeled 1,N(2)-propanodGuo was performed by HPLC/MS/MS. Upon acetaldehyde exposure (703 mu M), increased levels of both 1,N(2)-etheno-2`-deoxyguanosine (1,N(2)-epsilon dGuo), which is produced from alpha,beta-unsaturated aldehydes formed during the lipid peroxidation process, and 1,N(2)-propanodGuo were observed. The unequivocal formation of 1,N(2)-propanodGuo in cells exposed to this aldehyde can be used to elucidate the mechanisms associated with acetaldehyde exposure and cancer risk.

Identification of protein-coding and intronic noncoding RNAs down-regulated in clear cell renal carcinoma

The clear cell subtype of renal cell carcinoma (RCC) is the most lethal and prevalent cancer of the urinary system. To investigate the molecular changes associated with malignant transformation in clear cell RCC, the gene expression profiles of matched samples of tumor and adjacent non-neoplastic tissue were obtained from six patients. A custom-built cDNA microarray platform was used, comprising 2292 probes that map to exons of genes and 822 probes for noncoding RNAs mapping to intronic regions. Intronic transcription was detected in all normal and neoplastic renal tissues. A subset of 55 transcripts was significantly down-regulated in clear cell RCC relative to the matched nontumor tissue as determined by a combination of two statistical tests and leave-one-out patient cross-validation. Among the down-regulated transcripts, 49 mapped to untranslated or coding exons and 6 were intronic relative to known exons of protein-coding genes. Lower levels of expression of SIN3B, TRIP3, SYNJ2BP and NDE1 (P<0.02), and of intronic transcripts derived from SND1 and ACTN4 loci (P<0.05), were confirmed in clear cell RCC by Real-time RT-PCR. A subset of 25 transcripts was deregulated in additional six nonclear cell RCC samples, pointing to common transcriptional alterations in RCC irrespective of the histological subtype or differentiation state of the tumor. Our results indicate a novel set of tumor suppressor gene candidates, including noncoding intronic RNAs, which may play a significant role in malignant transformations of normal renal cells. (C) 2008 Wiley-Liss, Inc.

Base Excision Repair Activities Differ in Human Lung Cancer Cells and Corresponding Normal Controls

Oxidative damage to DNA is thought to play a role in carcinogenesis by causing Mutations, and indeed accumulation of oxidized DNA bases has been observed in samples obtained from tumors but not from surrounding tissue within the same patient. Base excision repair (BER) is the main pathway for the repair of oxidized modifications both in nuclear and mitochondrial, DNA. In order to ascertain whether diminished BER capacity might account for increased levels of oxidative DNA damage in cancer cells, the activities of BER enzymes in three different lung cancer cell lines and their non-cancerous counterparts were measured using oligonucleotide substrates with single DNA lesions to assess specific BER enzymes. The activities of four BER enzymes, OGG1, NTH1, UDG and APE1, were compared in mitochondrial and nuclear extracts. For each specific lesion, the repair activities were similar among the three cell lines used. However, the specific activities and cancer versus control comparison differed significantly between the nuclear and mitochondrial compartments. OGG1 activity, as measured by 8-oxodA incision, was upregulated in cancer cell mitochondria but down-regulated in the nucleus when compared to control cells. Similarly, NTH1 activity was also up-regulated in mitochondrial extracts from cancer cells but did not change significantly in the nucleus. Together, these results support the idea that alterations in BER capacity are associated with carcinogenesis.

The isatin-Schiff base copper(II) complex Cu(isaepy)(2) acts as delocalized lipophilic cation, yields widespread mitochondrial oxidative damage and induces AMP-activated protein kinase-dependent apoptosis

We previously demonstrated that Bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl) pyridine-N, N`] copper(II) [Cu(isaepy)(2)] was an efficient inducer of the apoptotic mitochondrial pathway. Here, we deeply dissect the mechanisms underlying the ability of Cu(isaepy)(2) to cause mitochondriotoxicity. In particular, we demonstrate that Cu(isaepy)(2) increases NADH-dependent oxygen consumption of isolated mitochondria and that this phenomenon is associated with oxy-radical production and insensitive to adenosine diphosphate. These data indicate that Cu(isaepy)(2) behaves as an uncoupler and this property is also confirmed in cell systems. Particularly, SH-SY5Y cells show: (i) an early loss of mitochondrial transmembrane potential; (ii) a decrease in the expression levels of respiratory complex components and (iii) a significant adenosine triphosphate (ATP) decrement. The causative energetic impairment mediated by Cu(isaepy)(2) in apoptosis is confirmed by experiments carried out with rho(0) cells, or by glucose supplementation, where cell death is significantly inhibited. Moreover, gastric and cervix carcinoma AGS and HeLa cells, which rely most of their ATP production on oxidative phosphorylation, show a marked sensitivity toward Cu(isaepy)(2). Adenosine monophosphate-activated protein kinase (AMPK), which is activated by events increasing the adenosine monophosphate: ATP ratio, is deeply involved in the apoptotic process because the overexpression of its dominant/negative form completely abolishes cell death. Upon glucose supplementation, AMPK is not activated, confirming its role as fuel-sensing enzyme that positively responds to Cu(isaepy)(2)-mediated energetic impairment by committing cells to apoptosis. Overall, data obtained indicate that Cu(isaepy)(2) behaves as delocalized lipophilic cation and induces mitochondrial-sited reactive oxygen species production. This event results in mitochondrial dysfunction and ATP decrease, which in turn triggers AMPK-dependent apoptosis.

Expression of human papillomavirus type 16 E7 oncoprotein alters keratinocytes expression profile in response to tumor necrosis factor-alpha

Acute expression of E7 oncogene from human papillomavirus (HPV) 16 or HPV18 is sufficient to overcome tumor necrosis factor (TNF)-alpha cytostatic effect on primary human keratinocytes. In the present study, we investigated the molecular basis of E7-induced TNF resistance through a comparative analysis of the effect of this cytokine on the proliferation and global gene expression of normal and E7-expressing keratinocytes. Using E7 functional mutants, we show that E7-induced TNF resistance correlates with its ability to mediate pRb degradation and cell transformation. On the other hand, this effect does not depend on E7 sequences required to override DNA damage-induced cell cycle arrest or extend keratinocyte life span. Furthermore, we identified a group of 66 genes whose expression pattern differs between normal and E7-expressing cells upon cytokine treatment. These genes are mainly involved in cell cycle regulation suggesting that their altered expression may contribute to sustained cell proliferation even in the presence of a cytostatic stimulus. Differential expression of TCN1 (transcobalamin I), IFI44 (Interferon-induced protein 44), HMGB2 (high-mobility group box 2) and FUS [Fusion (involved in t(12; 16) in malignant liposarcoma)] among other genes were further confirmed by western-blot and/or real-time polymerase chain reaction. Moreover, FUS upregulation was detected in HPV-positive cervical high-grade squamous intraepithelial lesions when compared with normal cervical tissue. Further evaluation of the role of such genes in TNF resistance and HPVassociated disease development is warranted.

Quantitative Analysis of Diepoxybutane Damage within the Chicken β-globin Domain

In industrial polymer and synthetic rubber production facilities, workers are exposed to 1,3-butadiene. This compound is converted in vivo to 1,2,3,4-diepoxybutane (DEB) and has been linked to increased incidences of cancer in these individuals. Carcinogenesis has been attributed to formation of DEB induced DNA interstrand cross-links. Previous studies have demonstrated that DEB cross-links deoxyguanosine residues within 5'-GNC sequences in synthetic DNA, in restriction fragments, and in defined sequence nucleosomes. The current study utilized the polymerase chain reaction (PCR) to examine DEB damage frequencies within nuclear genes, found within "open" regions of chromatin, as compared to regions of unexpressed sequence that reside in tightly packed, "closed" chromatin, to more closely model DEB reactivity in vivo. These initial studies have been performed in chicken liver homogenates. Preliminarily, we have found a dose-dependent DEB lesion-forming response within "open" chromatin. DEB appears to have little-to-no effect upon regions of "closed" chromatin.

Mapping of Diepoxybutane Damage in the Cytochrome b Domain of Mitochondrial DNA and the β-globin Domain of Nuclear Chicken DNA Using Quantitative PCR

Diepoxybutane (DEB), a known industrial carcinogen, reacts with DNA primarily at the N7 position of deoxyguanosine residues and creates interstrand cross-links at the sequence 5'-GNC. Since N7-N7 cross-links cause DNA to fragment upon heating, quantative polymerase chain reaction (QPCR) is being used in this experiment to measure the amount of DEB damage (lesion frequency) with three different targets-mitochondrial (unpackaged), open chromatin region, and closed chromatin region. Initial measurements of DEB damage within these three targets were not consistent because the template DNA was not the limiting reagent in the PCR. Follow-up PCR trials using a limiting amount of DNA are still in progress although initial experimentation looks promising. Sequencing of these three targets to confirm the primer targets has only been successfully performed for the closed chromatin target and does not match the sequence from NIH used to design that primer pair. Further sequencing trials need to be conducted on all three targets to assure that a mitochondrial, open chromatin, and closed chromatin region are actually being amplified in this experimental series.

Prevalência da expressão imunoistoquímica da proteína p21 em adenocarcinoma do esôfago

INTRODUÇÃO: No mundo ocidental, a prevalência de adenocarcinoma da junção esofagogástrica vem crescendo nas últimas décadas. Atualmente, é aceito que o adenocarcinoma do esôfago se desenvolve de uma lesão pré-maligna: esôfago de Barrett. Este carcinoma é de difícil diagnóstico nos seus estágios iniciais, o que resulta em uma mortalidade significativa. O estudo da biologia molecular tem demonstrado que grande parte dos tumores malignos tem origem na interação entre o componente hereditário e influências externas, que em indivíduos predispostos podem ocasionar alterações genéticas que influenciem o controle da diferenciação e crescimento celular. O p21 (WAF1/CIP1) tem um papel fundamental na regulação do ciclo celular, e sua expressão imunoistoquímica tem sido estudada em diversos tumores, mostrando influência no prognóstico de várias neoplasias. OBJETIVO: Verificar a prevalência da expressão da proteína p21 em pacientes com adenocarcinoma de esôfago diagnosticados nos últimos cinco anos no Grupo de Cirurgia de Esôfago e Estômago do Hospital de Clínicas de Porto Alegre (GCEE/HCPA). METODOLOGIA: A população em estudo foi constituída de 42 pacientes com adenocarcinoma de esôfago diagnosticados no GCEE/HCPA entre janeiro de 1998 e dezembro de 2002. A expressão da proteína p21 foi realizada por meio de imunoistoquímica, com anticorpo primário, p21, clone SX118, código M7202 da DAKO, e avaliada de acordo com o Sistema de Escore de Imunorreatividade (Immunoreactive scoring system – IRS). RESULTADOS: Foram estudados 42 pacientes. 83,3% eram do sexo masculino, com idade superior a 40 anos. Destes, 56,2% foram submetidos a procedimentos cirúrgicos com intenção curativa: Gastrectomia total e Esofagogastrectomia transiatal. Os demais foram submetidos à cirurgia paliativa ou não sofreram tratamento cirúrgico. Apenas cinco pacientes receberam tratamento adjuvante com quimioterapia e radioterapia, isoladas ou combinadas. Quanto ao estadiamento, 78,6% dos pacientes apresentavam doença avançada, estádios III e IV. Apenas 9 apresentaram positividade para o p21, quando considerado o Sistema de Escore de Imunorreatividade (em que p21+ é ³ 3). CONCLUSÃO: A proteína p21 esteve expressa em 9 dos 42 pacientes (21,4%) com adenocarcinoma de esôfago diagnosticados nos últimos cinco anos no Grupo de Cirurgia de Esôfago e Estômago do Hospital de Clínicas de Porto Alegre. Nessa casuística, o acúmulo de p21 não se mostrou essencial no processo de carcinogênese do adenocarcinoma esofágico.

Modelo experimental de lesões intra-epiteliais e de adenocarcinoma ductal pancreático induzidos por 7,12-dimetilbenzantraceno em camundongos

INTRODUÇÃO: O adenocarcinoma de pâncreas apresenta um mau prognóstico. A utilização de modelos experimentais é necessária para a compreensão do comportamento biológico tumoral, principalmente das lesões precoces (neoplasias intra-epiteliais pancreáticas - NIPan) e para o desenvolvimento de opções terapêuticas. OBJETIVO: Avaliar a carcinogênese pancreática induzida por 7,12-dimetilbenzantraceno (DMBA), em camundongos, aplicando a classificação das neoplasias intra-epiteliais pancreáticas. MÉTODOS: 90 camundongos machos, mus musculus, da cepa CF1, foram submetidos à laparotomia mediana e 1 mg de DMBA foi implantado na porção cefálica do pâncreas. Os animais foram divididos em dois grupos, com eutanásia em 30 e 60 dias. Em seguida, o pâncreas foi retirado, fixado em formalina e foram confeccionadas lâminas coradas com hematoxilina eosina. Os cortes histológicos foram avaliados por dois patologistas de acordo com os seguintes critérios: pâncreas normal, hiperplasia reacional, NIPan 1A, NIPan 1B, NIPan 2, NIPan 3 e carcinoma. As alterações inflamatórias também foram analisadas. RESULTADOS: A avaliação patológica evidenciou, no grupo de 30 dias: 4 (16,7%) animais com hiperplasia reativa, 16 (66,6%) com NIPan e 4 (16,7%) com adenocarcinoma. No grupo de 60 dias: 10 (27,1%) animais com hiperplasia reativa, 13 (35,1%) com NIPan e 14 (37,8%) com adenocarcinoma. A diferença entre os grupos apresentou significância estatistística (P < 0,05 – teste exato de Fisher). A prevalência de alterações inflamatórias em 30 dias foi: pancreatite aguda (n=11), pancreatite crônica (n=5) e inflamação dependente da bolsa (n=8). No grupo de 60 dias 11 espécimes apresentavam pancreatite aguda e 26 pancreatite crônica. CONCLUSÕES: O modelo experimental com DMBA em camundongos, induz neoplasia intra-epitelial pancreática e adenocarcinoma ductal histologicamente semelhantes ao carcinoma pancreático em humanos. Este modelo pode ser utilizado na investigação da carcinogênese com enfoque na progressão molecular das lesões precursoras até o adenocarcinoma.

Chromosomal aberrations induced by 5-azacytidine combined with VP-16 (etoposide) in CHO-K1 and XRS-5 cell lines

A cytogenetic study was carried out with 5-azacytidine (5-azaC) and etoposide (VP-16) in CHO-K1 and XRS-5 (mutant cells deficient for double-strand break rejoining) cell lines to verify the interaction effects of the drugs in terms of induction of chromosomal aberrations. 5-azaC is incorporated into DNA causing DNA hypomethylation, and VP-16 (inhibitor of topoisomerase 11 enzyme) is a potent clastogenic agent. Cells in exponential growth were treated with 5-azaC for I h, following incubation for 7 h, and posttreatment with VP16 for the last 3 h. In K1 cells, the combined treatments induced a significant reduction in the aberrations induced in the X and A (autosome) chromosomes, which are the main target for 5-azaC. However, in XRS-5 cells, the drug combination caused a significant increase in the aberrations induced in those chromosomes, but with a concomitant reduction in the randomly induced-aberrations. In addition, each cell line presented characteristic cell cycle kinetics; while the combined treatment induced an S-arrest in K1 cells, alterations in cell cycle progression were not found for XRS-5, although each drug alone caused a G2-arrest. The different cell responses presented by the cell lines may be explained on the basis of the evidence that alterations in chromatin structure caused by 5-aza-C probably occur to a different extent in K1 and XRS-5 cells, since the mutant cells present a typical hyper-condensed chromosome structure (especially the X- and A chromosomes), but, alternatively, 5-aza-C could induce reactivation of DNA repair genes in XRS-5 cells. Teratogenesis Carcinog. Mutagen. Suppl. 1:171-186, 2003. (C) 2003 Wiley-Liss, Inc.

Diferentes papéis do óxido nítrico com ênfase nas neoplasias

O Óxido Nítrico (ON) é gerado por uma família de isoenzimas, através da catálise enzimática do aminoácido essencial L-arginina, que resulta na formação de L-citrulina e ON. O on está envolvido em muitos processos fisiológicos dos mamíferos, que incluem a neurotransmissão, controle da pressão sangüínea, inflamação, reações imunológicas e nos mecanismos de defesa contra microorganisnos e tumores. O descontrole na síntese de on está implicado na patogênese de doenças cardiovasculares, autoimunidade, rejeição de transplantes, doenças degenerativas, na sépsis, na genotoxicidade e no surgimento de neoplasias. O on também foi incriminado como agente de iniciação da carcinogênese, que, associado a outros fatores, poderia levar ao descontrole da citoestase e da diferenciação celular. A diversidade de efeitos do on parece estar relacionada às concentrações de on gerados, à sensibilidade individual das células e à duração do fenômeno.

BUBR1 expression in benign oral lesions and squamous cell carcinomas: Correlation with human papillomavirus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Inhibitory effect of propolis and bee venom on the mutagenicity of some direct- and indirect-acting mutagens

The antimutagenic effect of ethanolic extract of propolis (EEP) and honeybee (Apis mellifera) venom, both collected in the State of Sb Paulo, Brazil, was assessed by the Salmonella/microsome assay upon direct- and indirect-acting mutagens. EEP had inhibitory effect (in an ascending order) on the mutagenicity power of daunomycin (TA102), benzo(a)pyrene (TA100), and aflatoxin B-1(TA98) and the venom acted against the mutagenicity of 4-nitro-o-phenylenediamine (TA98) and daunomycin (TA102). (C) 1999 Wiley-Liss, Inc.

Análise comparativa da imunoexpressão das proteínas hmlh1 e hmsh2 em carcinomas epidermóides de lábio inferior e queilites actínicas com graus variados de displasia

Lip squamous cell carcinoma (SCC) may develop from a premalignant condition, actinic cheilitis (AC) in 95% of the cases. Both premalignant and neoplastic lip diseases are caused mainly by chronic exposure to the ultraviolet component of solar radiation, especially UVB. This exposure causes disruption of the cell cycle and damage to DNA repair systems, like mismatch repair, altering proteins repair as hMLH1 and hMSH2. This research aimed to investigate the immunohistochemical expression of hMLH1 and hMSH2 proteins in lower lip SCCs and ACs, providing additional information about carcinogenesis of the lower lip. The sample consisted 40 cases of ACs and 40 cases of lower lip SCCs. Histological sections of 3 μm were submitted to immunoperoxidase method, for immunohistochemical analysis of lesions were counted in 1000 cells (positive and negative), data were evaluated both in absolute numbers and percentage of immunostained cells, the latter by assigning scores. Associations of the variables and comparative analysis of biomarker expression were performed by Fisher s exact and Pearson s chi-square, "t" student, one-way ANOVA, Mann- Whitney e Kruskal-Wallis tests. The level of significance was 5%. It was found that, in lower lip SCC, the mean of the proteins was higher in female patients (hMLH1= 369,80 + 223,98; hMHS2 = 534,80 + 343,62), less than 50 years old (hMLH1 = 285,50 + 190,65; hMHS2 = 540,00 + 274,79) and classified as low-grade malignancy (hMLH1 = 264,59 + 179,21; hMHS2 = 519,32 + 302,58), in these data only to sex, for hMLH1 protein, was statistically significant (p=0.034). Comparing the different lesions, we observed that for both hMLH1 and hMSH2 protein, the average of positive epithelial cells decreased as the lesion was graded at later stages. The ACs classified without dysplasia or mild dysplasia had the highest average of immunostained cells (hMLH1 = 721.23 + 88.116; hMHS2 = 781.50 + 156.93). The ACs classified as moderate or severe dysplasia had intermediate values (hMLH1 = 532,86 + 197,72; hMHS2 = 611,14 + 172,48) and SSCs of the lower lip had the lowest averages (hMLH1 = 255,03 + 199,47; hMHS2 = 518,38 + 265,68). There was a statistically significant difference between groups (p<0.001). In conclusion, our data support the hypothesis that changes in immunoexpression of these proteins is related to the process of carcinogenesis of the lower lip