Mirjam M. Schaap

Mice Expressing a Mammary Gland–Specific R270H Mutation in the p53 Tumor Suppressor Gene Mimic Human Breast Cancer Development

The tumor suppressor gene p53 has an apparent role in breast tumor development in humans, as approximately 30% of sporadic tumors acquire p53 mutations and Li-Fraumeni syndrome patients carrying germ line p53 mutations frequently develop breast tumors at early age. In the present study, conditional expression of a targeted mutation is used to analyze the role of the human R273H tumor-associated hotspot mutation in p53 in mammary gland tumorigenesis. Heterozygous p53(R270H/+)WAPCre mice (with mammary gland-specific expression of the p53.R270H mutation, equivalent to human R273H, at physiologic levels) develop mammary tumors at high frequency, indicating that the R270H mutation predisposes for mammary gland tumor development and acts in a dominant-negative manner in early stages of tumorigenesis. Spontaneous tumor development in these mice is further accelerated by 7,12-dimethylbenz(a)anthracene (DMBA) treatment at young age. The majority of spontaneous and DMBA-induced carcinomas and sarcomas from p53(R270H/+)WAPCre mice is estrogen receptor alpha positive, and expression profiles of genes also implicated in human breast cancer appear similarly altered. As such, p53(R270H/+)WAPCre mice provide a well-suited model system to study the role of p53 in breast tumorigenesis and the responsiveness of mammary gland tumors to chemotherapeutics.

Dominant-Negative but not Gain-of-Function Effects of a p53.R270H Mutation in Mouse Epithelium Tissue after DNA Damage

p53 alterations in human tumors often involve missense mutations that may confer dominant-negative or gain-of-function properties. Dominant-negative effects result in inactivation of wild-type p53 protein in heterozygous mutant cells and as such in a p53 null phenotype. Gain-of-function effects can directly promote tumor development or metastasis through antiapoptotic mechanisms or transcriptional activation of (onco)genes. Here, we show, using conditional mouse technology, that epithelium-specific heterozygous expression of mutant p53 (i.e., the p53.R270H mutation that is equivalent to the human hotspot R273H) results in an increased incidence of spontaneous and UVB-induced skin tumors. Expression of p53.R270H exerted dominant-negative effects on latency, multiplicity, and progression status of UVB-induced but not spontaneous tumors. Surprisingly, gain-of-function properties of p53.R270H were not detected in skin epithelium. Apparently, dominant-negative and gain-of-function effects of mutant p53 are highly tissue specific and become most manifest upon stabilization of p53 after DNA damage.

Finding transcriptomics biomarkers for in vivo identification of (non-)genotoxic carcinogens using wild-type and Xpa/p53 mutant mouse models

The carcinogenic potential of chemicals and pharmaceuticals is traditionally tested in the chronic, 2 year rodent bioassay. This assay is not only time consuming, expensive and often with a limited sensitivity and specificity but it also causes major distress to the experimental animals. A major improvement in carcinogenicity testing, especially regarding reduction and refinement of animal experimentation, could be the application of toxicogenomics. The ultimate aim of this study is to demonstrate a proof-of-principle for transcriptomics biomarkers in various tissues for identification of (subclasses of) carcinogenic compounds after short-term in vivo exposure studies. Both wild-type and DNA repair-deficient Xpa(-/-)/p53(+/-) (Xpa/p53) mice were exposed up to 14 days to compounds of three distinct classes: genotoxic carcinogens (GTXC), non-genotoxic carcinogens (NGTXC) and non-carcinogens. Subsequently, extensive transcriptomics analyses were performed on several tissues, and transcriptomics data were screened for potential biomarkers using advanced statistical learning techniques. For all tissues analyzed, we identified multigene gene-expression signatures that are, with a high confidence, predictive for GTXC and NGTXC exposures in both mouse genotypes. Xpa/p53 mice did not perform better in the short-term bioassay. We were able to achieve a proof-of-principle for the identification and use of transcriptomics biomarkers for GTXC or NGTXC. This supports the view that toxicogenomics with short-term in vivo exposure provides a viable tool for classifying (geno)toxic compounds.

Dissecting modes of action of non-genotoxic carcinogens in primary mouse hepatocytes

Under REACH, the European Community Regulation on chemicals, the testing strategy for carcinogenicity is based on in vitro and in vivo genotoxicity assays. Given that non-genotoxic carcinogens are negative for genotoxicity and chronic bioassays are no longer regularly performed, this class of carcinogens will go undetected. Therefore, test systems detecting non-genotoxic carcinogens, or even better their modes of action, are required. Here, we investigated whether gene expression profiling in primary hepatocytes can be used to distinguish different modes of action of non-genotoxic carcinogens. For this, primary mouse hepatocytes were exposed to 16 non-genotoxic carcinogens with diverse modes of action. Upon profiling, pathway analysis was performed to obtain insight into the biological relevance of the observed changes in gene expression. Subsequently, both a supervised and an unsupervised comparison approach were applied to recognize the modes of action at the transcriptomic level. These analyses resulted in the detection of three of eight compound classes, that is, peroxisome proliferators, metalloids and skin tumor promotors. In conclusion, gene expression profiles in primary hepatocytes, at least in rodent hepatocytes, appear to be useful to detect some, certainly not all, modes of action of non-genotoxic carcinogens.

In vivo murine hepatic microRNA and mRNA expression signatures predicting the (non-)genotoxic carcinogenic potential of chemicals.

There is a high need to improve the assessment of, especially non-genotoxic, carcinogenic features of chemicals. We therefore explored a toxicogenomics-based approach using genome-wide microRNA and mRNA expression profiles upon short-term exposure in mice. For this, wild-type mice were exposed for seven days to three different classes of chemicals, i.e., four genotoxic carcinogens (GTXC), seven non-genotoxic carcinogens (NGTXC), and five toxic non-carcinogens. Hepatic expression patterns of mRNA and microRNA transcripts were determined after exposure and used to assess the discriminative power of the in vivo transcriptome for GTXC and NGTXC. A final classifier set, discriminative for GTXC and NGTXC, was generated from the transcriptomic data using a tiered approach. This appeared to be a valid approach, since the predictive power of the final classifier set in three different classifier algorithms was very high for the original training set of chemicals. Subsequent validation in an additional set of chemicals revealed that the predictive power for GTXC remained high, in contrast to NGTXC, which appeared to be more troublesome. Our study demonstrated that the in vivo microRNA-ome has less discriminative power to correctly identify (non-)genotoxic carcinogen classes. The results generally indicate that single mRNA transcripts do have the potential to be applied in risk assessment, but that additional (genomic) strategies are necessary to correctly predict the non-genotoxic carcinogenic potential of a chemical.

Benzo[a]pyrene-induced transcriptomic responses in primary hepatocytes and in vivo liver: Toxicokinetics is essential for in vivo–in vitro comparisons

The traditional 2-year cancer bioassay needs replacement by more cost-effective and predictive tests. The use of toxicogenomics in an in vitro system may provide a more high-throughput method to investigate early alterations induced by carcinogens. Recently, the differential gene expression response in wild-type and cancer-prone Xpa−/−p53+/− primary mouse hepatocytes after exposure to benzo[a]pyrene (B[a]P) revealed downregulation of cancer-related pathways in Xpa−/−p53+/− hepatocytes only. Here, we investigated pathway regulation upon in vivo B[a]P exposure of wild-type and Xpa−/−p53+/− mice. In vivo transcriptomics analysis revealed a limited gene expression response in mouse livers, but with a significant induction of DNA replication and apoptotic/anti-apoptotic cellular responses in Xpa−/−p53+/− livers only. In order to be able to make a meaningful in vivo–in vitro comparison we estimated internal in vivo B[a]P concentrations using DNA adduct levels and physiologically based kinetic modeling. Based on these results, the in vitro concentration that corresponded best with the internal in vivo dose was chosen. Comparison of in vivo and in vitro data demonstrated similarities in transcriptomics response: xenobiotic metabolism, lipid metabolism and oxidative stress. However, we were unable to detect cancer-related pathways in either wild-type or Xpa−/−p53+/− exposed livers, which were previously found to be induced by B[a]P in Xpa−/−p53+/− primary hepatocytes. In conclusion, we showed parallels in gene expression responses between livers and primary hepatocytes upon exposure to equivalent concentrations of B[a]P. Furthermore, we recommend considering toxicokinetics when modeling a complex in vivo endpoint with in vitro models.

LacZ mouse embryonic fibroblasts detect both clastogens and mutagens

The clastogenic effects of MMC and BLM and the mutagenic effects of B[a]P, N-ac-AAF and ENU were studied in mouse embryonic fibroblasts derived from wild-type (WT) and Rad54/Rad54B-deficient mice. Clastogens as well as mutagens showed a statistically significant induction of mutations in the lacZ reporter gene both in a WT and Rad54/Rad54B-deficient genetic background. Rad54/Rad54B MEFs appeared equally sensitive to the clastogens compared to WT MEFs, except for MMC. The type of mutations induced by the different compounds was investigated further by hybridizing the mutant colonies with total mouse DNA. An obvious increased number of mouse DNA positive clones was observed after BLM and MMC exposure, indicating that after these treatments genome rearrangements/translocations had occurred. In this hybridization assay, Rad54/Rad54B MEFs did not show more rearrangements/translocations than WT MEFs. As expected, the mutagens used showed no increase in chromosomal rearrangements or transloctions in MEFs derived from both genotypes. These results show that WT MEFs carrying the lacZ reporter gene on a plasmid are capable to detect both clastogenic as well as mutagenic effects of compounds in vitro. Deletion of the Rad54 and Rad54B genes did not further enhance the sensitivity of MEFs towards clastogens.

A novel toxicogenomics-based approach to categorize (non-)genotoxic carcinogens

Alternative methods to detect non-genotoxic carcinogens are urgently needed, as this class of carcinogens goes undetected in the current testing strategy for carcinogenicity under REACH. A complicating factor is that non-genotoxic carcinogens act through several distinctive modes of action, which makes prediction of their carcinogenic property difficult. We have recently demonstrated that gene expression profiling in primary mouse hepatocytes is a useful approach to categorize non-genotoxic carcinogens according to their modes of action. In the current study, we improved the methods used for analysis and added mouse embryonic stem cells as a second in vitro test system, because of their features complementary to hepatocytes. Our approach involved an unsupervised analysis based on the 30 most significantly up- and down-regulated genes per chemical. Mouse embryonic stem cells and primary mouse hepatocytes were exposed to a selected set of chemicals and subsequently subjected to gene expression profiling. We focused on non-genotoxic carcinogens, but also included genotoxic carcinogens and non-carcinogens to test the robustness of this approach. Application of the optimized comparison approach resulted in improved categorization of non-genotoxic carcinogens. Mouse embryonic stem cells were a useful addition, especially for genotoxic substances, but also for detection of non-genotoxic carcinogens that went undetected by primary hepatocytes. The approach presented here is an important step forward to categorize chemicals, especially those that are carcinogenic.

Comparison of clastogen-induced gene expression profiles in wild-type and DNA repair-deficient Rad54/Rad54B cells

BackgroundPreviously we found that Rad54/Rad54B cells are more sensitive towards mitomycin C (MMC) as compared to wild-type (WT) cells. This difference in sensitivity was absent upon exposure to other clastogens like bleomycin (BLM) and γ-radiation. In order to get further insight into possible underlying mechanisms, gene expression changes in WT and Rad54/Rad54B MEFs (mouse embryonic fibroblasts) after exposure to the clastogens MMC and BLM were investigated. Exposures of these cells to mutagens (N-ac-AAF and ENU) and vehicle were taken as controls.ResultsMost exposures resulted in an induction of DNA damage signaling and apoptosis genes and a reduced expression of cell division genes in cells of both genotypes. As expected, responses to N-ac-AAF were very similar in both genotypes. ENU exposure did not lead to significant gene expression changes in cells of both genotypes, presumably due to its short half-life. Gene expression responses to clastogens, however, showed a genotype-dependent effect for BLM and MMC. MMC treated Rad54/Rad54B MEFs showed no induction of p53-signaling, DNA damage response and apoptosis as seen for all the other treatments.ConclusionThese data support our finding that different types of clastogens exist and that responses to these types depend on the DNA repair status of the cells.

DNA-repair-deficient Rad54/Rad54B mice are more sensitive to clastogens than wild-type mice

The sensitivity of DNA-repair-deficient Rad54/Rad54B mice for clastogens was studied and compared to that of wild-type mice. LacZ mutant frequencies (MF) in Rad54/Rad54B mice, after treatment with mitomycin C (MMC), bleomycin (BLM) and gamma-irradiation, were compared to those of the wild-type mice following the same treatments. While none of the clastogens showed an induction of the lacZ MF in the wild-type mice, there was a significant increase of the lacZ MF in the bone marrow of the Rad54/Rad54B mice after treatment with BLM and gamma-irradiation and in the spleen after MMC treatment. As expected, the positive control ENU showed a significant increase in the lacZ MF in all tested organs in wild-type mice. Mutant colonies were hybridized with total mouse DNA in order to discriminate between small gene mutations and large DNA rearrangements and translocations (size-change mutations). The hybridization studies showed a significant increase in mouse DNA positive clones 4 days after treatment with MMC and BLM in the bone marrow of the wild-type mice, which is indicative for chromosomal rearrangements and translocations to occur. An even more pronounced increase was seen 28 days after treatment with the same compounds in the Rad54/Rad54B mice.