The dietary antioxidant quercetin reduces hallmarks of bleomycin-induced lung fibrogenesis in mice.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, lethal disease of which the etiology is still not fully understood. Current treatment comprises two FDA-approved drugs that can slow down yet not stop or reverse the disease. As IPF pathology is associated with an altered redox balance, adding a redox modulating component to current therapy might exert beneficial effects. Quercetin is a dietary antioxidant with strong redox modulating capacities that is suggested to exert part of its antioxidative effects via activation of the redox-sensitive transcription factor Nrf2 that regulates endogenous antioxidant levels. Therefore, the aim of the present study was to investigate if the dietary antioxidant quercetin can exert anti-fibrotic effects in a mouse model of bleomycin-induced pulmonary fibrogenesis through Nrf2-dependent restoration of redox imbalance. METHODS: Homozygous Nrf2 deficient mice and their wildtype littermates were fed a control diet without or with 800 mg quercetin per kg diet from 7 days prior to a single 1 µg/2 µl per g BW bleomycin challenge until they were sacrificed 14 days afterwards. Lung tissue and plasma were collected to determine markers of fibrosis (expression of extracellular matrix genes and histopathology), inflammation (pulmonary gene expression and plasma levels of tumor necrosis factor-α (TNFα) and keratinocyte chemoattrachtant (KC)), and redox balance (pulmonary gene expression of antioxidants and malondialdehyde-dG (MDA)- DNA adducts). RESULTS: Mice fed the enriched diet for 7 days prior to the bleomycin challenge had significantly enhanced plasma and pulmonary quercetin levels (11.08 ± 0.73 µM versus 7.05 ± 0.2 µM) combined with increased expression of Nrf2 and Nrf2-responsive genes compared to mice fed the control diet in lung tissue. Upon bleomycin treatment, quercetin-fed mice displayed reduced expression of collagen (COL1A2) and fibronectin (FN1) and a tendency of reduced inflammatory lesions (2.8 ± 0.7 versus 1.9 ± 0.8). These beneficial effects were accompanied by reduced pulmonary gene expression of TNFα and KC, but not their plasma levels, and enhanced Nrf2-induced pulmonary antioxidant defences. In Nrf2 deficient mice, no effect of the dietary antioxidant on either histology or inflammatory lesions was observed. CONCLUSION: Quercetin exerts anti-fibrogenic and anti-inflammatory effects on bleomycin-induced pulmonary damage in mice possibly through modulation of the redox balance by inducing Nrf2. However, quercetin could not rescue the bleomycin-induced pulmonary damage indicating that quercetin alone cannot ameliorate the progression of IPF.

Limited additive value of the Ki-67 proliferative index on patient survival in World Health Organization-classified pulmonary carcinoids.

AIMS: Currently pulmonary carcinoids are separated into typical and atypical based on mitotic count and presence of necrosis, according to the World Health Organization. At variance with gastroenteropancreatic neuroendocrine tumours, which are graded based on mitotic count and Ki-67 proliferative index, the use of Ki-67 for grading pulmonary carcinoids is still under debate. METHODS AND RESULTS: In this study we evaluated the prognostic impact of Ki-67 assessment in a multicentre cohort of 201 carcinoids [147 typical carcinoids (TCs) and 54 atypical carcinoids (ACs)] using manual analysis (2000 cells counted) and digital image analysis (in-house Leica Qwin program; ≥4500 cells counted). The Ki-67 proliferative index was correlated with overall survival by means of univariate analysis and in comparison to clinical data by means of multivariable analysis. The Ki-67 index was significantly higher in ACs than in TCs for both counting methods (P ≤ 2.7e-5 ). In addition, using cut-offs of 2.5% and 4% (manual counting) or 1% and 5% (digital analysis), the highest differences in overall survival were observed (P ≤ 0.0067). Nevertheless, histopathological classification into TCs and ACs showed an equally strong association with disease outcome, although Ki-67 had some additive value within TCs. Ki-67 index was not an independent predictor of survival in multivariable analysis. CONCLUSIONS: Our study demonstrates that, although Ki-67 is a strong prognostic factor for pulmonary carcinoids, its usefulness in addition to histopathology in prediction of prognosis is limited. None the less, it may have additional value, especially in cases that are difficult to classify, in combination with histopathology and other molecular markers.

Nuclear and Mitochondrial DNA Methylation Patterns Induced by Valproic Acid in Human Hepatocytes.

Valproic acid (VPA) is one of the most widely prescribed antiepileptic drugs in the world. Despite its pharmacological importance, it may cause liver toxicity and steatosis through mitochondrial dysfunction. The aim of this study is to further investigate VPA-induced mechanisms of steatosis by analyzing changes in patterns of methylation in nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Therefore, primary human hepatocytes (PHHs) were exposed to an incubation concentration of VPA that was shown to cause steatosis without inducing overt cytotoxicity. VPA was administered daily for 5 days, and this was followed by a 3 day washout (WO). Methylated DNA regions (DMRs) were identified by using the methylated DNA immunoprecipitation-sequencing (MeDIP-seq) method. The nDNA DMRs after VPA treatment could indeed be classified into oxidative stress- and steatosis-related pathways. In particular, networks of the steatosis-related gene EP300 provided novel insight into the mechanisms of toxicity induced by VPA treatment. Furthermore, we suggest that VPA induces a crosstalk between nDNA hypermethylation and mtDNA hypomethylation that plays a role in oxidative stress and steatosis development. Although most VPA-induced methylation patterns appeared reversible upon terminating VPA treatment, 31 nDNA DMRs (including 5 zinc finger protein genes) remained persistent after the WO period. Overall, we have shown that MeDIP-seq analysis is highly informative in disclosing novel mechanisms of VPA-induced toxicity in PHHs. Our results thus provide a prototype for the novel generation of interesting methylation biomarkers for repeated dose liver toxicity in vitro.

High-throughput data integration of RNA-miRNA-circRNA reveals novel insights into mechanisms of benzo[a]pyrene-induced carcinogenicity.

The chain of events leading from a toxic compound exposure to carcinogenicity is still barely understood. With the emergence of high-throughput sequencing, it is now possible to discover many different biological components simultaneously. Using two different RNA libraries, we sequenced the complete transcriptome of human HepG2 liver cells exposed to benzo[a]pyrene, a potent human carcinogen, across six time points. Data were integrated in order to reveal novel complex chemical-gene interactions. Notably, we hypothesized that the inhibition of MGMT, a DNA damage response enzyme, by the over-expressed miR-181a-1_3p induced by BaP, may lead to liver cancer over time.

Dynamic Interplay between the Transcriptome and Methylome in Response to Oxidative and Alkylating Stress.

In recent years, it has been shown that free radicals not only react directly with DNA but also regulate epigenetic processes such as DNA methylation, which may be relevant within the context of, for example, tumorigenesis. However, how these free radicals impact the epigenome remains unclear. We therefore investigated whether methyl and hydroxyl radicals, formed by tert-butyl hydroperoxide (TBH), change temporal DNA methylation patterns and how this interferes with genome-wide gene expression. At three time points, TBH-induced radicals in HepG2 cells were identified by electron spin resonance spectroscopy. Total 5-methylcytosine (5mC) levels were determined by liquid chromatography and tandem mass spectrometry and genome-wide changes in 5mC and gene expression by microarrays. Induced methylome changes rather represent an adaptive response to the oxidative stress-related reactions observed in the transcriptome. More specifically, we found that methyl radicals did not induce DNA methylation directly. An initial oxidative and alkylating stress-related response of the transcriptome during the early phase of TBH treatment was followed by an epigenetic response associated with cell survival signaling. Also, we identified genes of which the expression seems directly regulated by DNA methylation. This work suggests an important role of the methylome in counter-regulating primary oxidative and alkylating stress responses in the transcriptome to restore normal cell function. Altogether, the methylome may play an important role in counter-regulating primary oxidative and alkylating stress responses in the transcriptome presumably to restore normal cell function.

Interindividual variation in gene expression responses and metabolite formation in acetaminophen-exposed primary human hepatocytes.

Acetaminophen (APAP) is a readily available over-the-counter drug and is one of the most commonly used analgesics/antipyretics worldwide. Large interindividual variation in susceptibility toward APAP-induced liver failure has been reported. However, the exact underlying factors causing this variability in susceptibility are still largely unknown. The aim of this study was to better understand this variability in response to APAP by evaluating interindividual differences in gene expression changes and APAP metabolite formation in primary human hepatocytes (PHH) from several donors (n = 5) exposed in vitro to a non-toxic to toxic APAP dose range. To evaluate interindividual variation, gene expression data/levels of metabolites were plotted against APAP dose/donor. The correlation in APAP dose response between donors was calculated by comparing data points from one donor to the data points of all other donors using a Pearson-based correlation analysis. From that, a correlation score/donor for each gene/metabolite was defined, representing the similarity of the omics response to APAP in PHH of a particular donor to all other donors. The top 1 % highest variable genes were selected for further evaluation using gene set overrepresentation analysis. The biological processes in which the genes with high interindividual variation in expression were involved include liver regeneration, inflammatory responses, mitochondrial stress responses, hepatocarcinogenesis, cell cycle, and drug efficacy. Additionally, the interindividual variation in the expression of these genes could be associated with the variability in expression levels of hydroxyl/methoxy-APAP and C8H13O5N-APAP-glucuronide. The before-mentioned metabolites or their derivatives have also been reported in blood of humans exposed to therapeutic APAP doses. Possibly these findings can contribute to elucidating the causative factors of interindividual susceptibility toward APAP.

Oxidative Stress Mechanisms Do Not Discriminate between Genotoxic and Nongenotoxic Liver Carcinogens.

It is widely accepted that in chemical carcinogenesis different modes-of-action exist, e.g., genotoxic (GTX) versus nongenotoxic (NGTX) carcinogenesis. In this context, it has been suggested that oxidative stress response pathways are typical for NGTX carcinogenesis. To evaluate this, we examined oxidative stress-related changes in gene expression, cell cycle distribution, and (oxidative) DNA damage in human hepatoma cells (HepG2) exposed to GTX-, NGTX-, and noncarcinogens, at multiple time points (4-8-24-48-72 h). Two GTX (azathriopine (AZA) and furan) and two NGTX (tetradecanoyl-phorbol-acetate, (TPA) and tetrachloroethylene (TCE)) carcinogens as well as two noncarcinogens (diazinon (DZN, d-mannitol (Dman)) were selected, while per class one compound was deemed to induce oxidative stress and the other not. Oxidative stressors AZA, TPA, and DZN induced a 10-fold higher number of gene expression changes over time compared to those of furan, TCE, or Dman treatment. Genes commonly expressed among AZA, TPA, and DZN were specifically involved in oxidative stress, DNA damage, and immune responses. However, differences in gene expression between GTX and NGTX carcinogens did not correlate to oxidative stress or DNA damage but could instead be assigned to compound-specific characteristics. This conclusion was underlined by results from functional readouts on ROS formation and (oxidative) DNA damage. Therefore, oxidative stress may represent the underlying cause for increased risk of liver toxicity and even carcinogenesis; however, it does not discriminate between GTX and NGTX carcinogens.

Epigenetic mechanisms underlying arsenic-associated lung carcinogenesis.

Arsenic is an established human carcinogen, but the mechanisms through which it contributes to for instance lung cancer development are still unclear. As arsenic is methylated during its metabolism, it may interfere with the DNA methylation process, and is therefore considered to be an epigenetic carcinogen. In the present study, we hypothesize that arsenic is able to induce DNA methylation changes, which lead to changes in specific gene expression, in pathways associated with lung cancer promotion and progression. A549 human adenocarcinoma lung cells were exposed to a low (0.08 µM), intermediate (0.4 µM) and high (2 µM) concentration of sodium arsenite for 1, 2 and 8 weeks. DNA was isolated for whole-genome DNA methylation analyses using NimbleGen 2.1 M deluxe promoter arrays. In addition, RNA was isolated for whole-genome transcriptomic analysis using Affymetrix microarrays. Arsenic modulated DNA methylation and expression levels of hundreds of genes in a dose-dependent and time-dependent manner. By combining whole-genome DNA methylation and gene expression data with possibly involved transcription factors, a large molecular interaction network was created based on transcription factor-target gene pairs, consisting of 216 genes. A tumor protein p53 (TP53) subnetwork was identified, showing the interactions of TP53 with other genes affected by arsenic. Furthermore, multiple other new genes were discovered showing altered DNA methylation and gene expression. In particular, arsenic modulated genes which function as transcription factor, thereby affecting target genes which are known to play a role in lung cancer promotion and progression.

Chromosome stability in tonsillar squamous cell carcinoma is associated with HPV16 integration and indicates a favorable prognosis.

Tonsillar squamous cell carcinoma (TSCC) is frequently associated with human papillomavirus (HPV) and chromosome instability. Data from cellular model systems are, however, controversial concerning a relation between HPV and chromosome instability development. Here we studied this association in 77 primary TSCC with known clinical outcome and cell cycle protein expression profiles. Thirty-two tumors (42%) showed HPV16-integration. All 77 cases were analyzed by fluorescence in situ hybridization using chromosome 1- and 7-specific centromere DNA probes to detect chromosome instability, indicated by the presence of chromosome imbalances and/or polyploidization for these chromosomes. In addition, eight HPV-positive dysplasias, seven of which were adjacent to a carcinoma, were analyzed. Disomy for chromosome 1 and 7 was present in 29 out of 77 TSCC (38%), of which 19 were HPV16-positive (p = 0.002). Aneusomy was observed in the remaining 48 TSCC, of which 13 were HPV-positive. Aneusomies correlated significantly with tobacco- and alcohol consumption (p = 0.001 and p = 0.016, respectively) and a higher T-stage (p = 0.018). Both HPV-positivity and chromosome disomy were significantly associated with a favorable disease-free survival (p = 0.001 and p = 0.025, respectively). Particularly in the HPV16-positive group chromosome instability is a very strong indicator for an unfavorable prognosis (p = 0.032). In the dysplasias an identical HPV and chromosome copy number status was identified as in the adjacent tumors. We conclude that HPV-positive TSCC and their precursor lesions are more often genetically stable than HPV-negative lesions and that these tumors are associated with a favorable prognosis. Chromosome instability is an indicator for unfavorable prognosis, particularly in the HPV-positive patient group.

Deletions of 11q22.3-q25 are associated with atypical lung carcinoids and poor clinical outcome.

Carcinoids are slow-growing neuroendocrine tumors that, in the lung, can be subclassified as typical (TC) or atypical (AC). To identify genetic alterations that improve the prediction of prognosis, we investigated 34 carcinoid tumors of the lung (18 TCs, 15 ACs, and 1 unclassified) by using array comparative genomic hybridization (array CGH) on 3700 genomic bacterial artificial chromosome arrays (resolution ≤1 Mb). When comparing ACs with TCs, the data revealed: i) a significant difference in the average number of chromosome arms altered (9.6 versus 4.2, respectively; P = 0.036), with one subgroup of five ACs having more than 15 chromosome arms altered; ii) chromosomal changes in 30% of ACs or more with additions at 9q (≥1 Mb) and losses at 1p, 2q, 10q, and 11q; and iii) 11q deletions in 8 of 15 ACs versus 1 of 18 TCs (P = 0.004), which was confirmed via fluorescence in situ hybridization. The four critical regions of interest in 45% ACs or more comprised 11q14.1, 11q22.1-q22.3, 11q22.3-q23.2, and 11q24.2-q25, all telomeric of MEN1 at 11q13. Results were correlated with patient clinical data and long-term follow-up. Thus, there is a strong association of 11q22.3-q25 loss with poorer prognosis, alone or in combination with absence of 9q34.11 alterations (P = 0.0022 and P = 0.00026, respectively).

'Omics analysis of low dose acetaminophen intake demonstrates novel response pathways in humans.

Acetaminophen is the primary cause of acute liver toxicity in Europe/USA, which led the FDA to reconsider recommendations concerning safe acetaminophen dosage/use. Unfortunately, the current tests for liver toxicity are no ideal predictive markers for liver injury, i.e. they only measure acetaminophen exposure after profound liver toxicity has already occurred. Furthermore, these tests do not provide mechanistic information. Here, 'omics techniques (global analysis of metabolomic/gene-expression responses) may provide additional insight. To better understand acetaminophen-induced responses at low doses, we evaluated the effects of (sub-)therapeutic acetaminophen doses on metabolite formation and global gene-expression changes (including, for the first time, full-genome human miRNA expression changes) in blood/urine samples from healthy human volunteers. Many known and several new acetaminophen-metabolites were detected, in particular in relation to hepatotoxicity-linked, oxidative metabolism of acetaminophen. Transcriptomic changes indicated immune-modulating effects (2g dose) and oxidative stress responses (4g dose). For the first time, effects of acetaminophen on full-genome human miRNA expression have been considered and confirmed the findings on mRNA level. 'Omics techniques outperformed clinical chemistry tests and revealed novel response pathways to acetaminophen in humans. Although no definitive conclusion about potential immunotoxic effects of acetaminophen can be drawn from this study, there are clear indications that the immune system is triggered even after intake of low doses of acetaminophen. Also, oxidative stress-related gene responses, similar to those seen after high dose acetaminophen exposure, suggest the occurrence of possible pre-toxic effects of therapeutic acetaminophen doses. Possibly, these effects are related to dose-dependent increases in levels of hepatotoxicity-related metabolites.

Performance of in vitro γH2AX assay in HepG2 cells to predict in vivo genotoxicity.

The γH2AX assay has recently been suggested as a new in vitro assay for detecting genotoxic (GTX) properties of chemicals. This assay is based on the phosphorylation of H2AX histone in response to DNA damage [i.e. induction of double-strand breaks (DSBs)]. Quantification of γH2AX foci using flow cytometry can rapidly detect DNA damage induced by chemicals that cause DNA DSBs. Up to now, only few compounds have been tested with this assay. The main goal of this study was to compare the performance of this automated γH2AX assay with that of standard in vitro genotoxicity assays in predicting in vivo genotoxicity. HepG2 cells were exposed to 64 selected compounds with known GTX properties and subsequently analysed for induction of γH2AX foci. The results of this assay were compared with public data from standard in vitro genotoxicity tests. Accuracy, sensitivity and specificity in predicting in vivo genotoxicity, using the γH2AX assay alone or in combinations with conventional assays, were calculated. Both the γH2AX assay and the bacterial mutagenicity test (Ames) were highly specific for in vivo GTX, whereas chromosomal aberration/micronucleus test (CA/MN) resulted in highest sensitivity. The currently widely used in vitro genotoxicity test battery-Ames test, mouse lymphoma assay (MLA) and CA/MN test-resulted in low accuracy (55-65%) to predict in vivo genotoxicity. Interestingly, the inclusion of γH2AX assay in the standard battery, instead of MLA assay, resulted in higher accuracy (62-70%) compared with other combinations. Advantage of the γH2AX assay in HepG2 cells is its high sensitivity to detect DNA-reactive GTX compounds, although the reduced sensitivity for compounds that require metabolic activation needs to be improved. In conclusion, the automated γH2AX assay can be a useful, fast and cost-effective human cell-based tool for early screening of compounds for in vivo genotoxicity.

Decreased nucleotide excision repair in steatotic livers associates with myeloperoxidase-immunoreactivity.

Chronic inflammation is characterized by the influx of neutrophils and is associated with an increased production of reactive oxygen species that can damage DNA. Oxidative DNA damage is generally thought to be involved in the increased risk of cancer in inflamed tissues. We previously demonstrated that activated neutrophil mediated oxidative stress results in a reduction in nucleotide excision repair (NER) capacity, which could further enhance mutagenesis. Inflammation and oxidative stress are critical factors in the progression of nonalcoholic fatty liver disease that is linked with enhanced liver cancer risk. In this report, we therefore evaluated the role of neutrophils and the associated oxidative stress in damage recognition and DNA repair in steatotic livers of 35 severely obese subjects with either nonalcoholic steatohepatitis (NASH) (n=17) or steatosis alone (n=18). The neutrophilic influx in liver was assessed by myeloperoxidase (MPO) staining and the amount of oxidative DNA damage by measuring M(1)dG adducts. No differences in M(1)dG adduct levels were observed between patients with or without NASH and also not between individuals with high or low MPO immunoreactivity. However, we found that high expression of MPO in the liver, irrespective of disease status, reduced the damage recognition capacity as determined by staining for histone 2AX phosphorylation (γH2AX). This reduction in γH2AX formation in individuals with high MPO immunoreactivity was paralleled by a significant decrease in NER capacity as assessed by a functional repair assay, and was not related to cell proliferation. Thus, the observed reduction in NER capacity upon hepatic inflammation is associated with and may be a consequence of reduced damage recognition. These findings suggest a novel mechanism of liver cancer development in patients with nonalcoholic fatty liver disease.

Concentration-dependent gene expression responses to flusilazole in embryonic stem cell differentiation cultures.

The murine embryonic stem cell test (EST) is designed to evaluate developmental toxicity based on compound-induced inhibition of embryonic stem cell (ESC) differentiation into cardiomyocytes. The addition of transcriptomic evaluation within the EST may result in enhanced predictability and improved characterization of the applicability domain, therefore improving usage of the EST for regulatory testing strategies. Transcriptomic analyses assessing factors critical for risk assessment (i.e. dose) are needed to determine the value of transcriptomic evaluation in the EST. Here, using the developmentally toxic compound, flusilazole, we investigated the effect of compound concentration on gene expression regulation and toxicity prediction in ESC differentiation cultures. Cultures were exposed for 24 h to multiple concentrations of flusilazole (0.54-54 µM) and RNA was isolated. In addition, we sampled control cultures 0, 24, and 48 h to evaluate the transcriptomic status of the cultures across differentiation. Transcriptomic profiling identified a higher sensitivity of development-related processes as compared to cell division-related processes in flusilazole-exposed differentiation cultures. Furthermore, the sterol synthesis-related mode of action of flusilazole toxicity was detected. Principal component analysis using gene sets related to normal ESC differentiation was used to describe the dynamics of ESC differentiation, defined as the 'differentiation track'. The concentration-dependent effects on development were reflected in the significance of deviation of flusilazole-exposed cultures from this transcriptomic-based differentiation track. Thus, the detection of developmental toxicity in EST using transcriptomics was shown to be compound concentration-dependent. This study provides further insight into the possible application of transcriptomics in the EST as an improved alternative model system for developmental toxicity testing.

Dystrophin in the Neonatal and Adult Rat Intestine.

BACKGROUND: Gastrointestinal (GI) complaints are frequently noted in aging dystrophinopathy patients, yet their underlying molecular mechanisms are largely unknown. As dystrophin protein isoform 71 (Dp71) is particularly implicated in the development of smooth muscle cells, we evaluated its distribution in the neonatal and adult rat intestine in this study. METHODS: Dp71 expression levels were assessed in the proximal (duodenum, jejunum and ileum) and distal (caecum, colon and rectum) intestine by Western blotting and qPCR. In addition, the cellular distribution of total Dp was evaluated in the duodenum and colon by immunohistochemical colocalization studies with alpha-smooth muscle actin (aSMA), Hu RNA binding proteins C and D (HuC/HuD) for neurons and vimentin (VIM) for interstitial cells. RESULTS: In neonatal and adult rats, the distal intestine expressed 2.5 times more Dp71 protein than the proximal part (p < 0.01). This regional difference was not observed in Dp71 mRNA. During both stages, Dp-immunoreactivity was predominant in the muscularis propria, where it co-localized with aSMA and HuC/HuD. CONCLUSIONS: In neonatal and adult rats, Dp71 was expressed highest in the distal intestine. Together with the observation that Dp may be expressed by myenteric neurons, this warrants a paradigm shift in the treatment of GI comorbidities.

Oxidative stress in pancreatic alpha and beta cells as a selection criterion for biocompatible biomaterials.

The clinical success rate of islet transplantation, namely independence from insulin injections, is limited by factors that lead to graft failure, including inflammation, acute ischemia, acute phase response, and insufficient vascularization. The ischemia and insufficient vascularization both lead to high levels of oxidative stress, which are further aggravated by islet encapsulation, inflammation, and undesirable cell-biomaterial interactions. To identify biomaterials that would not further increase damaging oxidative stress levels and that are also suitable for manufacturing a beta cell encapsulation device, we studied five clinically approved polymers for their effect on oxidative stress and islet (alpha and beta cell) function. We found that 300 poly(ethylene oxide terephthalate) 55/poly(butylene terephthalate) 45 (PEOT/PBT300) was more resistant to breakage and more elastic than other biomaterials, which is important for its immunoprotective function. In addition, it did not induce oxidative stress or reduce viability in the MIN6 beta cell line, and even promoted protective endogenous antioxidant expression over 7 days. Importantly, PEOT/PBT300 is one of the biomaterials we studied that did not interfere with insulin secretion in human islets.

Spectral karyotypic and comparative genomic analysis of the endocrine pancreatic tumor cell line BON-1.

BON-1 is a human serotonin-producing endocrine pancreatic tumor (EPT) cell line, which has been used for various studies of tumorigenesis and treatment. Because its genotype, phenotype and degree of differentiation may underlie events that are instrumental to the development of endocrine tumors and, moreover, may vary between labs and over time, we decided to comprehensively characterize the chromosomal constitution of BON-1 by applying conventional GTG-banding, spectral karyotyping (SKY), comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). BON-1 cells proved to be hyperdiploid containing a modal chromosome number of 57 (range 56-64). SKY identified a stemline containing 6 clonal aberrations including del(1p), t(9;12)del(9p)x2, der(10)t(5;10), der(19)t(8;19), der(14)t(9;14)t(9;10), and a sideline harboring an additional del(12q). CGH and FISH confirmed the SKY results and, in addition, highlighted the chromosomal regions involved in the rearrangements. Moreover, they identified a homozygous deletion of the key tumor suppressor genes CDKN2A and CDKN2B at 9p21.3, in accordance with absence of p16(INK4A) and p14(ARF) expression as revealed by immunocytochemistry. Apart from deregulation of the cell cycle and p53 pathway this finding indicates escape from replicative senescence (induced by mutated NRAS) and detachment-induced apoptosis as molecular mechanisms underlying the tumorigenesis of BON-1 cells. Immunostaining results for p53, MDM2 and pRb expression were consistent with previously published data using Western analysis. In conclusion, we provide here a comprehensive cytogenetic profile of BON-1. This cell line harbors both numerical and structural genomic alterations indicative for malignant EPTs.

Genetic signatures of HPV-related and unrelated oropharyngeal carcinoma and their prognostic implications.

PURPOSE: Patients with human papillomavirus (HPV)-containing oropharyngeal squamous cell carcinomas (OSCC) have a better prognosis than patients with HPV-negative OSCC. This may be attributed to different genetic pathways promoting cancer. EXPERIMENTAL DESIGN: We used comparative genomic hybridization to identify critical genetic changes in 60 selected OSCC, 28 of which were associated with HPV-16 as determined by HPV-specific PCR and fluorescence in situ hybridization analysis and positive p16(INK4A) immunostaining. The results were correlated with HPV status and clinical data from patients. RESULTS: Two thirds of OSCC harbored gain at 3q26.3-qter irrespective of HPV status. In HPV-negative tumors this alteration was associated with advanced tumor stage (P=0.013). In comparison with HPV-related OSCC, the HPV-negative tumors harbored: (a) a higher number of chromosomal alterations and amplifications (P=0.03 and 0.039, respectively); (b) significantly more losses at 3p, 5q, 9p, 15q, and 18q, and gains/amplifications at 11q13 (P=0.002, 0.03; <0.001, 0.02, 0.004, and 0.001, respectively); and (c) less often 16q losses and Xp gains (P=0.02 and 0.03). Survival analysis revealed a significantly better disease-free survival for HPV-related OSCC (P=0.02), whereas chromosome amplification was an unfavorable prognostic indicator for disease-free and overall survival (P=0.01 and 0.05, respectively). Interestingly, 16q loss, predominantly identified in HPV-related OSCC, was a strong indicator of favorable outcome (overall survival, P=0.008; disease-free survival, P=0.01) and none of these patients had a tumor recurrence. CONCLUSIONS: Genetic signatures of HPV-related and HPV-unrelated OSCC are different and most likely underlie differences in tumor development and progression. In addition, distinct chromosomal alterations have prognostic significance.

P21 Cip1/WAF1 expression is strongly associated with HPV-positive tonsillar carcinoma and a favorable prognosis.

Human papillomavirus is involved in the carcinogenesis of tonsillar squamous cell carcinomas. Here, we investigated the expression and the prognostic value of key cell cycle proteins in the pRb and p53 pathways in both human papillomavirus type 16-positive and -negative tonsillar squamous cell carcinomas. Using immunohistochemistry, 77 tonsillar squamous cell carcinomas with known human papillomavirus type 16 status and clinical outcome were analyzed for expression of Ki67, p16(INK4A,) cyclin D1, pRb, p14(ARF), MDM2, p53, p21(Cip1/WAF1), and p27(KIP1). Results were correlated with each other and with clinical and demographic patient data. A total of 35% of tonsillar carcinomas harbored integrated human papillomavirus type 16 DNA and p16(INK4A) overexpression, both being considered essential features for human papillomavirus association. These tumors also showed the overexpression of p14(ARF) (P<0.0001) and p21(Cip1/WAF1) (P=0.001), and downregulation of pRb (P<0.0001) and cyclin D1 (P=0.027) compared with the human papillomavirus-negative cases. Univariate Cox regression analyses revealed a favorable survival rate for non-smokers (P=0.006), as well as for patients with T1-2 tumors (P<0.0001) or tumors showing low expression of cyclin D1 (P=0.028), presence of human papillomavirus and overexpression of p16(INK4A) (P=0.01), p14(ARF) (P=0.02) or p21(Cip1/WAF1) (P=0.004). In multivariate regression analyses, smoking and tumor size, as well as expression of cyclin D1 and p21(Cip1/WAF1), were found to be independent prognostic markers. We conclude that human papillomavirus positivity in tonsillar squamous cell carcinomas strongly correlates with p21(Cip1/WAF1) and p14(ARF) overexpression and downregulation of pRb and cyclin D1. In particular p21(Cip1/WAF1) overexpression is an excellent favorable prognosticator in tonsillar squamous cell carcinomas.

Molecular evidence for a clonal relationship between multiple lesions in patients with unknown primary adenocarcinoma.

Unknown primary adenocarcinoma (UPA) comprises a group of heterogeneous cancers of great clinical and biological interest. UPA presents as metastatic disease without a detectable primary site after medical workup. Here we investigated whether or not a clonal relationship exists between multiple tumors within individual UPA patients. A molecular resemblance would argue for an early clonal outgrowth of tumor cells from the primary lesion, a mutual feature observed within this group of neoplasms. In 14 patients with UPA multiple tumors, obtained at autopsy, were analyzed by molecular allelotyping and immunohistochemistry. In addition, tumors of 4 patients could be analyzed by comparative genomic hybridization (CGH). Similar genetic and phenotypic profiles were used as indicator for a clonal relationship, whereas different profiles implicate independent tumors. The molecular data indicated that the multiple lesions in the 14 UPA patients, including the primary tumors, are clonally related. In agreement with the theory of tumor progression, some metastatic lesions showed additional genetic alterations besides the characteristics that were shared with the primary tumor. Furthermore, 8 UPA patients had tumors with a high frequency of allelic loss and/or imbalance (FALI; 43-71%), while 6 patients demonstrated a lower FALI (14-29%), suggesting the occurrence of chromosomal instability in the former group. Our data provide molecular evidence for a clonal relationship between multiple metastases and the primary tumor within individual UPA patients, independent of the anatomical origin of the cancer. This finding is in agreement with the suggestion that tumor progression is rapid in UPA patients, limiting the chance of clonal divergence. The identification of 2 groups of UPAs with either a high or low FALI indicates that chromosomal instability is not the only driving force behind early tumor cell dissemination. Thus, other molecular mechanisms must underlie the common biology of these tumors.