The human DEK oncogene regulates DNA damage response signaling and repair

The human DEK gene is frequently overexpressed and sometimes amplified in human cancer. Consistent with oncogenic functions, Dek knockout mice are partially resistant to chemically induced papilloma formation. Additionally, DEK knockdown in vitro sensitizes cancer cells to DNA damaging agents and induces cell death via p53-dependent and -independent mechanisms. Here we report that DEK is important for DNA double-strand break repair. DEK depletion in human cancer cell lines and xenografts was sufficient to induce a DNA damage response as assessed by detection of γH2AX and FANCD2. Phosphorylation of H2AX was accompanied by contrasting activation and suppression, respectively, of the ATM and DNA-PK pathways. Similar DNA damage responses were observed in primary Dek knockout mouse embryonic fibroblasts (MEFs), along with increased levels of DNA damage and exaggerated induction of senescence in response to genotoxic stress. Importantly, Dek knockout MEFs exhibited distinct defects in non-homologous end joining (NHEJ) when compared to their wild-type counterparts. Taken together, the data demonstrate new molecular links between DEK and DNA damage response signaling pathways, and suggest that DEK contributes to DNA repair

Ligand-Independent Regulation of Transforming Growth Factor β1 Expression and Cell Cycle Progression by the Aryl Hydrocarbon Receptor▿ †

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxic effects of its xenobiotic ligands and acts as an environmental checkpoint during the cell cycle. We expressed stably integrated, Tet-Off-regulated AHR variants in fibroblasts from AHR-null mice to further investigate the AHR role in cell cycle regulation. Ahr+/+ fibroblasts proliferated significantly faster than Ahr−/− fibroblasts did, and exposure to a prototypical AHR ligand or deletion of the ligand-binding domain did not change their proliferation rates, indicating that the AHR function in cell cycle was ligand independent. Growth-promoting genes, such as cyclin and cyclin-dependent kinase genes, were significantly down-regulated in Ahr−/− cells, whereas growth-arresting genes, such as the transforming growth factor β1 (TGF-β1) gene, extracellular matrix (ECM)-related genes, and cyclin-dependent kinase inhibitor genes, were up-regulated. Ahr−/− fibroblasts secreted significantly more TGF-β1 into the culture medium than Ahr+/+ fibroblasts did, and Ahr−/− showed increased levels of activated Smad4 and TGF-β1 mRNA. Inhibition of TGF-β1 signaling by overexpression of Smad7 reversed the proliferative and gene expression phenotype of Ahr−/− fibroblasts. Changes in TGF-β1 mRNA accumulation were due to stabilization resulting from decreased activity of TTP, the tristetraprolin RNA-binding protein responsible for mRNA destabilization through AU-rich motifs. These results show that the Ah receptor possesses interconnected intrinsic cellular functions, such as ECM formation, cell cycle control, and TGF-β1 regulation, that are independent of activation by either exogenous or endogenous ligands and that may play a crucial role during tumorigenesis

Absence of nucleolar disruption after impairment of 40S ribosome biogenesis reveals an rpL11-translation-dependent mechanism of p53 induction

Impaired ribosome biogenesis is attributed to nucleolar disruption and diffusion of a subset of 60S ribosomal proteins, particularly ribosomal protein (rp)L11, into the nucleoplasm, where they inhibit MDM2, leading to p53 induction and cell-cycle arrest. Previously, we demonstrated that deletion of the 40S rpS6 gene in mouse liver prevents hepatocytes from re-entering the cell cycle after partial hepatectomy. Here, we show that this response leads to an increase in p53, which is recapitulated in culture by rpS6-siRNA treatment and rescued by the simultaneous depletion of p53. However, disruption of biogenesis of 40S ribosomes had no effect on nucleolar integrity, although p53 induction was mediated by rpL11, leading to the finding that the cell selectively upregulates the translation of mRNAs with a polypyrimidine tract at their 5'-transcriptional start site (5'-TOP mRNAs), including that encoding rpL11, on impairment of 40S ribosome biogenesis. Increased 5'-TOP mRNA translation takes place despite continued 60S ribosome biogenesis and a decrease in global translation. Thus, in proliferative human disorders involving hypomorphic mutations in 40S ribosomal proteins, specific targeting of rpL11 upregulation would spare other stress pathways that mediate the potential benefits of p53 induction

Expression of the gene in double transgenic mice during spermatogenesis

Whole mount X-gal staining of testes collected from 20-(), 30-(), and 60 day-old () bitransgenic mice showed positive reaction in primary and secondary spermatocytes and spermatids. No β-gal activity was found in adult single transgenic testes and 10-day-old testes of double mice (data not shown). Leydig cells (arrowheads) have endogenous β-gal activity. Testicular cell suspensions prepared from a mouse were subjected to May–Grünwald-Giemsa stain after X-gal staining. Positive X-gal staining (arrow) was observed in the primary spermatocytes (), primary spermatocytes in meiotic division (), spermatids (), and round spermatids ().<p><b>Copyright information:</b></p><p>Taken from "Promiscuous recombination of alleles during gametogenesis in cornea driver mice"</p><p></p><p>Molecular Vision 2008;14():562-571.</p><p>Published online 20 Mar 2008</p><p>PMCID:PMC2274927.</p><p></p

-mediated recombination of the allele in spermatogenic cells of double transgenic mouse

() Histograms of flow cytometry of testicular cell suspension obtained from a 60-day-old double transgenic mouse showed a distribution of 65%, 9%, and 17% for 1N, 2N, and 4N cells, respectively. PCR of genomic DNA from 1N and 4N cells showed excision of the allele but not from 2N cells. () Histogram showed enrichment of 2N cells by Percoll gradients as described in Methods. PCR analysis of these enriched 2N cells demonstrated excision of the allele. Primers b and c identify wild type (390 bp) and floxed allele (450 bp) whereas primers a and c identify the excised allele (500 bp). TC, testicular cells; M, DNA marker.<p><b>Copyright information:</b></p><p>Taken from "Promiscuous recombination of alleles during gametogenesis in cornea driver mice"</p><p></p><p>Molecular Vision 2008;14():562-571.</p><p>Published online 20 Mar 2008</p><p>PMCID:PMC2274927.</p><p></p

Melanocortin 1 receptor genotype: an important determinant of the damage response of melanocytes to ultraviolet radiation

The melanocortin 1 receptor gene is a main determinant of human pigmentation, and a melanoma susceptibility gene, because its variants that are strongly associated with red hair color increase melanoma risk. To test experimentally the association between melanocortin 1 receptor genotype and melanoma susceptibility, we compared the responses of primary human melanocyte cultures naturally expressing different melanocortin 1 receptor variants to α-melanocortin and ultraviolet radiation. We found that expression of 2 red hair variants abolished the response to α-melanocortin and its photoprotective effects, evidenced by lack of functional coupling of the receptor, and absence of reduction in ultraviolet radiation-induced hydrogen peroxide generation or enhancement of repair of DNA photoproducts, respectively. These variants had different heterozygous effects on receptor function. Microarray data confirmed the observed differences in responses of melanocytes with functional vs. nonfunctional receptor to α-melanocortin and ultraviolet radiation, and identified DNA repair and antioxidant genes that are modulated by α-melanocortin. Our findings highlight the molecular mechanisms by which the melanocortin 1 receptor genotype controls genomic stability of and the mutagenic effect of ultraviolet radiation on human melanocytes.—Kadekaro, A. L., Leachman, S., Kavanagh, R. J., Swope, V., Cassidy, P., Supp, D., Sartor, M., Schwemberger, S., Babcock, G., Wakamatsu, K., Ito, S., Koshoffer, A., Boissy, R. E., Manga, P., Sturm, R. A., Abdel-Malek, Z. A. Melanocortin 1 receptor genotype: an important determinant of the damage response of melanocytes to ultraviolet radiation