Comparison of the metabolic activation of environmental carcinogens in mouse embryonic stem cells and mouse embryonic fibroblasts.

We compared mouse embryonic stem (ES) cells and fibroblasts (MEFs) for their ability to metabolically activate the environmental carcinogens benzo[a]pyrene (BaP), 3-nitrobenzanthrone (3-NBA) and aristolochic acid I (AAI), measuring DNA adduct formation by (32)P-postlabelling and expression of xenobiotic-metabolism genes by quantitative real-time PCR. At 2 µM, BaP induced Cyp1a1 expression in MEFs to a much greater extent than in ES cells and formed 45 times more adducts. Nqo1 mRNA expression was increased by 3-NBA in both cell types but induction was higher in MEFs, as was adduct formation. For AAI, DNA binding was over 450 times higher in MEFs than in ES cells, although Nqo1 and Cyp1a1 transcriptional levels did not explain this difference. We found higher global methylation of DNA in ES cells than in MEFs, which suggests higher chromatin density and lower accessibility of the DNA to DNA damaging agents in ES cells. However, AAI treatment did not alter DNA methylation. Thus mouse ES cells and MEFs have the metabolic competence to activate a number of environmental carcinogens, but MEFs have lower global DNA methylation and higher metabolic capacity than mouse ES cells.

Germline allele-specific expression of DAPK1 in chronic lymphocytic leukemia.

We previously reported a rare germline variant (c.1-6531) that resulted in allele-specific expression (ASE) of death-associated protein kinase 1 (DAPK1) and predisposition to chronic lymphocytic leukemia (CLL). We investigated a cohort of CLL patients lacking this mutation for the presence of ASE of DAPK1. We developed a novel strategy that combines single-nucleotide primer extension (SNuPE) with MALDI-TOF mass spectrometry, and detected germline DAPK1 ASE in 17 out of 120 (14.2%) CLL patients associated with a trend towards younger age at diagnosis. ASE was absent in 63 healthy controls. Germline cells of CLL patients with ASE showed increased levels of DNA methylation in the promoter region, however, neither genetic nor further epigenetic aberrations could be identified in the DAPK1 5' upstream regulatory region, within distinct exons or in the 3'-UTR. We identified B-lymphoid malignancy related cell line models harboring allelic imbalance and found that allele-specific methylation in DAPK1 is associated with ASE. Our data indicate that ASE at the DAPK1 gene locus is a recurrent event, mediated by epigenetic mechanisms and potentially predisposing to CLL.

Efficient introduction of specific TP53 mutations into mouse embryonic fibroblasts and embryonic stem cells.

This protocol describes a rapid, precise method for generating sets of embryonic stem (ES) cells or mouse embryonic fibroblasts (MEFs) harboring point mutations in the p53 tumor suppressor gene (officially known as Trp53). The strategy uses cells from the Trp53 (p53-null) 'platform' mouse, which allows site-specific integration of plasmid DNA into the Trp53 locus. Simple PCR protocols identify correctly targeted clones and immunoblots verify re-expression of the protein. We also present protocol modifications needed for efficient recovery of MEF clones expressing p53 constructs that retain wild-type function, including growth at low (3%) oxygen and transient downregulation of p53 regulators to forestall cell senescence of primary MEFs. A library of cell lines expressing various p53 mutants derived from the same population of primary fibroblasts or platform ES cells can be acquired and screened in less than 1 month.

Rapid derivation of genetically related mutants from embryonic cells harboring a recombinase-specific Trp53 platform.

Recombinase-mediated cassette exchange (RMCE) is a powerful method for achieving gene targeting repeatedly at a single mammalian locus. This approach could be applied to the efficient establishment of genetically related cell lines harboring different p53 mutations found in human tumors. To this end we generated a mouse strain called p53 Platform mice (PLF mice), containing PhiC31 integrase-specific attP sequences at the Trp53 locus. The attP sites flank a PGK-neo cassette that has replaced exons 2 to 9 of the endogenous murine p53 gene, generating a null allele. Electroporation of a fluorescence indicator plasmid into embryonic stem (ES) cell lines from PLF mice demonstrated that PhiC31 integrase-mediated cassette exchange (IMCE) can be achieved at > 60% efficiency without selecting against random insertion. To produce somatic cell lines with endogenously controlled expression of mutant p53, we performed IMCE in PLF murine embryonic fibroblasts (MEFs) with plasmid constructs containing human p53 gene sequences carrying specific tumor-associated missense mutations (A138V; G245S). The MEF cell lines produce the expected mutated mRNA transcripts and express p53 protein that is phosphorylated at serine 15 following DNA damage. Within a few weeks one can thus acquire a family of p53 mutant cell lines from the same population of primary cells, but each harboring a different mutation.

Differential enhancement of a cutaneous HPV promoter by DeltaNP63alpha, Jun and mutant p53.

The mechanism through which cutaneous papillomaviruses induce lesions is largely unknown. Ectopic expression of the DeltaNp63alpha isoform highly increased the viral promoter activity. The co-expression of c-Jun mediated and increased the DeltaNp63alpha activity by binding to the AP-1 site in an enhancer region of the HPV 20 URR. This strong activation by DeltaNp63alpha is diminished in the presence of wtp53 and abolished by the simultaneous expression of "hot-spot" mutant p53 R248W. We demonstrate that c-Jun is responsible for the viral promoter activation through its direct interaction with both DeltaNp63alpha and wtp53. The downregulation by p53 mutant R248W is accompanied by reduced protein levels of DeltaNp63alpha and phosphorylated c-Jun. The data presented in this study provide insight into a possible mechanism through which these cellular proteins may modulate a cutaneous papillomavirus genome to induce viral replication, latent infection or malignant transformation.