Mutation Analysis in Cultured Cells of Transgenic Rodents.

To comply with guiding principles for the ethical use of animals for experimental research, the field of mutation research has witnessed a shift of interest from large-scale in vivo animal experiments to small-sized in vitro studies. Mutation assays in cultured cells of transgenic rodents constitute, in many ways, viable alternatives to in vivo mutagenicity experiments in the corresponding animals. A variety of transgenic rodent cell culture models and mutation detection systems have been developed for mutagenicity testing of carcinogens. Of these, transgenic Big Blue® (Stratagene Corp., La Jolla, CA, USA, acquired by Agilent Technologies Inc., Santa Clara, CA, USA, BioReliance/Sigma-Aldrich Corp., Darmstadt, Germany) mouse embryonic fibroblasts and the λ Select cII Mutation Detection System have been used by many research groups to investigate the mutagenic effects of a wide range of chemical and/or physical carcinogens. Here, we review techniques and principles involved in preparation and culturing of Big Blue® mouse embryonic fibroblasts, treatment in vitro with chemical/physical agent(s) of interest, determination of the cII mutant frequency by the λ Select cII assay and establishment of the mutation spectrum by DNA sequencing. We describe various approaches for data analysis and interpretation of the results. Furthermore, we highlight representative studies in which the Big Blue® mouse cell culture model and the λ Select cII assay have been used for mutagenicity testing of diverse carcinogens. We delineate the advantages of this approach and discuss its limitations, while underscoring auxiliary methods, where applicable.

Expression of epigenetic modifiers is not significantly altered by exposure to secondhand smoke.

OBJECTIVES: Secondhand smoke (SHS) is a major risk factor for lung cancer in nonsmokers. DNA damage-derived mutagenicity is a well-established mechanism of SHS-carcinogenicity; however very little is known about the impact of SHS exposure on the epigenome. MATERIALS AND METHODS: We have investigated whether exposure to SHS can modulate the expression of key epigenetic regulators responsible for the establishment and/or maintenance of DNA methylation and histone modification patterns in vivo. We have sub-chronically exposed mice to a mutagenic but non-tumorigenic dose of SHS, and subsequently determined the expression levels of major epigenetic modifiers in the lungs of SHS-exposed mice, immediately after termination of exposure and following 7-month recovery in clean air. RESULTS AND CONCLUSION: Quantification of the expression of genes encoding DNA methyltransferases (Dnmt1, Dnmt3a, Dnmt3b and Dnmt3l), methyl binding domain proteins (Mecp2, Mbd2 and Mbd3) and histone deacetylases (Hdac1 and Hdac2) by quantitative reverse-transcription polymerase chain reaction analysis showed modest but not statistically significant differences in the relative transcription of these key epigenetic regulators between SHS-exposed mice and age-matched controls. The non-significant changes in the expression of main epigenetic modifiers in SHS-exposed mice imply that SHS may predominantly induce genotoxic effects, particularly at non-tumorigenic doses, whereas epigenetic effects may only be secondary and manifest en route to tumor formation.

Exposure of mice to secondhand smoke elicits both transient and long-lasting transcriptional changes in cancer-related functional networks.

Secondhand smoke (SHS) has long been linked to lung cancer and other diseases in nonsmokers. Yet, the underlying mechanisms of SHS carcinogenicity in nonsmokers remain to be elucidated. We investigated the immediate and long-lasting effects of SHS exposure on gene expression in mice in vivo. We exposed mice whole body to SHS for 5 h/day, 5 days/week for 4 months in exposure chambers of a microprocessor-controlled smoking machine. Subsequently, we performed microarray gene expression profiling, genome-wide, to construct the pulmonary transcriptome of SHS-exposed mice, immediately after discontinuation of exposure (T0) and following 1-month (T1) and 7-month (T2) recoveries in clean air. Sub-chronic exposure of mice to SHS elicited a robust transcriptomic response, including both reversible and irreversible changes in gene expression. There were 674 differentially expressed transcripts immediately after treatment (T0), of which the majority were involved in xenobiotic metabolism, signaling, and innate immune response. Reduced, yet, substantial numbers of differentially expressed transcripts were detectable in mice after cessation of SHS-exposure (254 transcripts at T1 and 30 transcripts at T2). Top biofunctional networks disrupted in SHS-exposed mice, even after termination of exposure, were implicated in cancer, respiratory disease, and inflammatory disease. Our data show that exposure of mice to SHS induces both transient and long-lasting changes in gene expression, which impact cancer-related functional networks. The pattern of transcriptional changes in SHS-exposed mice may provide clues on the underlying mechanisms of lung tumorigenesis in nonsmokers. Our findings underscore the importance of eliminating SHS from environments where nonsmokers are unavoidably exposed to this carcinogen.

Epigenetic targeting of the Nanog pathway and signaling networks during chemical carcinogenesis.

Chemical carcinogenesis has long been synonymous with genotoxicity, which entails DNA damage, genetic mutations and chromosomal abnormalities. The present study investigates a paradigm-shifting model in which epigenetic changes are key contributors to chemical carcinogenesis. Using genome-wide microarray-based analysis followed by conventional validation assays, we have progressively chronicled changes in the epigenetic landscape, as reflected in the patterns of DNA methylation, in the target organ of tumorigenesis in mice treated in vivo with a prototype chemical carcinogen (benzo[a]pyrene). Here, we demonstrate characteristic CpG island gain/loss of methylation and demethylation of repetitive DNA elements in carcinogen-treated mice, dependent on tumor progression. Alterations of the DNA methylome are accompanied by silencing of major DNA methyltransferases. Members of the Nanog pathway that establishes and maintains pluripotency in embryonic stem cells and possibly triggers uncontrolled proliferation of neoplastic cells are preferential targets of aberrant DNA methylation and concomitant gene dysregulation during chemical carcinogenesis. Several components of the MEK/ERK, JAK/STAT3, PI3K/AKT, WNT/ß- catenin and Shh signaling cascades, which are known to modulate Nanog expression, also show concurrent changes in the patterns of DNA methylation and gene expression. Our data support an epigenetic model of chemical carcinogenesis and suggest that surveillance of the epigenetic landscape, particularly at the loci and in the pathways identified in this study, may have utility for early detection and monitoring of the progression of malignancy.

Mammalian cells acquire epigenetic hallmarks of human cancer during immortalization.

Progression to malignancy requires that cells overcome senescence and switch to an immortal phenotype. Thus, exploring the genetic and epigenetic changes that occur during senescence/immortalization may help elucidate crucial events that lead to cell transformation. In the present study, we have globally profiled DNA methylation in relation to gene expression in primary, senescent and immortalized mouse embryonic fibroblasts. Using a high-resolution genome-wide mapping technique, followed by extensive locus-specific validation assays, we have identified 24 CpG islands that display significantly higher levels of CpG methylation in immortalized cell lines as compared to primary murine fibroblasts. Several of these hypermethylated CpG islands are associated with genes involved in the MEK-ERK pathway, one of the most frequently disrupted pathways in cancer. Approximately half of the hypermethylated targets are developmental regulators, and bind to the repressive Polycomb group (PcG) proteins, often in the context of bivalent chromatin in mouse embryonic stem cells. Because PcG-associated aberrant DNA methylation is a hallmark of several human malignancies, our methylation data suggest that epigenetic reprogramming of pluripotency genes may initiate cell immortalization. Consistent with methylome alterations, global gene expression analysis reveals that the vast majority of genes dysregulated during cell immortalization belongs to gene families that converge into the MEK-ERK pathway. Additionally, several dysregulated members of the MAP kinase network show concomitant hypermethylation of CpG islands. Unlocking alternative epigenetic routes for cell immortalization will be paramount for understanding crucial events leading to cell transformation. Unlike genetic alterations, epigenetic changes are reversible events, and as such, can be amenable to pharmacological interventions, which makes them appealing targets for cancer therapy when genetic approaches prove inadequate.

Whole DNA methylome profiling in mice exposed to secondhand smoke.

Aberration of DNA methylation is a prime epigenetic mechanism of carcinogenesis. Aberrant DNA methylation occurs frequently in lung cancer, with exposure to secondhand smoke (SHS) being an established risk factor. The causal role of SHS in the genesis of lung cancer, however, remains elusive. To investigate whether SHS can cause aberrant DNA methylation in vivo, we have constructed the whole DNA methylome in mice exposed to SHS for a duration of 4 mo, both after the termination of exposure and at ensuing intervals post-exposure (up to 10 mo). Our genome-wide and gene-specific profiling of DNA methylation in the lung of SHS-exposed mice revealed that all groups of SHS-exposed mice and controls share a similar pattern of DNA methylation. Furthermore, the methylation status of major repetitive DNA elements, including long-interspersed nuclear elements (LINE L1), intracisternal A particle long-terminal repeat retrotransposons (IAP-LTR), and short-interspersed nuclear elements (SINE B1), in the lung of all groups of SHS-exposed mice and controls remains comparable. The absence of locus-specific gain of DNA methylation and global loss of DNA methylation in the lung of SHS-exposed mice within a timeframe that precedes neoplastic-lesion formation underscore the challenges of lung cancer biomarker development. Identifying the initiating events that cause aberrant DNA methylation in lung carcinogenesis may help improve future strategies for prevention, early detection and treatment of this highly lethal disease.

A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens.

Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents.