Tumor suppression by p53 in the absence of Atm.

Oncogenes can induce p53 through a signaling pathway involving p19/Arf. It was recently proposed that oncogenes can also induce DNA damage, and this can induce p53 through the Atm DNA damage pathway. To assess the relative roles of Atm, Arf, and p53 in the suppression of Ras-driven tumors, we examined susceptibility to skin carcinogenesis in 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate (TPA)-treated Atm- and p53-deficient mice and compared these results to previous studies on Arf-deficient mice. Mice with epidermal-specific deletion of p53 showed increased papilloma number and progression to malignant invasive carcinomas compared with wild-type littermates. In contrast, Atm-deficient mice showed no increase in papilloma number, growth, or malignant progression. gamma-H2AX and p53 levels were increased in both Atm(+/+) and Atm(-/-) papillomas, whereas Arf(-/-) papillomas showed much lower p53 expression. Thus, although there is evidence of DNA damage, signaling through Arf seems to regulate p53 in these Ras-driven tumors. In spontaneous and radiation-induced lymphoma models, tumor latency was accelerated in Atm(-/-)p53(-/-) compound mutant mice compared with the single mutant Atm(-/-) or p53(-/-) mice, indicating cooperation between loss of Atm and loss of p53. Although p53-mediated apoptosis was impaired in irradiated Atm(-/-) lymphocytes, p53 loss was still selected for during lymphomagenesis in Atm(-/-) mice. In conclusion, in these models of oncogene- or DNA damage-induced tumors, p53 retains tumor suppressor activity in the absence of Atm

p19( Arf) Suppresses Growth, Progression, and Metastasis of Hras-Driven Carcinomas through p53-Dependent and -Independent Pathways

Ectopic expression of oncogenes such as Ras induces expression of p19(Arf), which, in turn, activates p53 and growth arrest. Here, we used a multistage model of squamous cell carcinoma development to investigate the functional interactions between Ras, p19(Arf), and p53 during tumor progression in the mouse. Skin tumors were induced in wild-type, p19(Arf)-deficient, and p53-deficient mice using the DMBA/TPA two-step protocol. Activating mutations in Hras were detected in all papillomas and carcinomas examined, regardless of genotype. Relative to wild-type mice, the growth rate of papillomas was greater in p19(Arf)-deficient mice, and reduced in p53-deficient mice. Malignant conversion of papillomas to squamous cell carcinomas, as well as metastasis to lymph nodes and lungs, was markedly accelerated in both p19 (Arf)- and p53-deficient mice. Thus, p19(Arf) inhibits the growth rate of tumors in a p53-independent manner. Through its regulation of p53, p19(Arf) also suppresses malignant conversion and metastasis. p53 expression was upregulated in papillomas from wild-type but not p19( Arf)-null mice, and p53 mutations were more frequently seen in wild-type than in p19( Arf)-null carcinomas. This indicates that selection for p53 mutations is a direct result of signaling from the initiating oncogenic lesion, Hras, acting through p19(Arf)

Plasma Proteome Profiles Associated with Inflammation, Angiogenesis, and Cancer

Tumor development is accompanied by a complex host systemic response, which includes inflammatory and angiogenic reactions. Both tumor-derived and systemic response proteins are detected in plasma from cancer patients. However, given their non-specific nature, systemic response proteins can confound the detection or diagnosis of neoplasia. Here, we have applied an in-depth quantitative proteomic approach to analyze plasma protein changes in mouse models of subacute irritant-driven inflammation, autoreactive inflammation, and matrix associated angiogenesis and compared results to previously described findings from mouse models of polyoma middle T-driven breast cancer and Pdx1-Cre KrasG12D Ink4a/Arf lox/lox -induced pancreatic cancer. Among the confounding models, approximately 1/3 of all quantified plasma proteins exhibited a significant change in abundance compared to control mice. Of the proteins that changed in abundance, the majority were unique to each model. Altered proteins included those involved in acute phase response, inflammation, extracellular matrix remodeling, angiogenesis, and TGFβ signaling. Comparison of changes in plasma proteins between the confounder models and the two cancer models revealed proteins that were restricted to the cancer-bearing mice, reflecting the known biology of these tumors. This approach provides a basis for distinguishing between protein changes in plasma that are cancer-related and those that are part of a non-specific host response

Loss of Maternal CTCF Is Associated with Peri-Implantation Lethality of Ctcf Null Embryos

CTCF is a highly conserved, multifunctional zinc finger protein involved in critical aspects of gene regulation including transcription regulation, chromatin insulation, genomic imprinting, X-chromosome inactivation, and higher order chromatin organization. Such multifunctional properties of CTCF suggest an essential role in development. Indeed, a previous report on maternal depletion of CTCF suggested that CTCF is essential for pre-implantation development. To distinguish between the effects of maternal and zygotic expression of CTCF, we studied pre-implantation development in mice harboring a complete loss of function Ctcf knockout allele. Although we demonstrated that homozygous deletion of Ctcf is early embryonically lethal, in contrast to previous observations, we showed that the Ctcf nullizygous embryos developed up to the blastocyst stage (E3.5) followed by peri-implantation lethality (E4.5–E5.5). Moreover, one-cell stage Ctcf nullizygous embryos cultured ex vivo developed to the 16–32 cell stage with no obvious abnormalities. Using a single embryo assay that allowed both genotype and mRNA expression analyses of the same embryo, we demonstrated that pre-implantation development of the Ctcf nullizygous embryos was associated with the retention of the maternal wild type Ctcf mRNA. Loss of this stable maternal transcript was temporally associated with loss of CTCF protein expression, apoptosis of the developing embryo, and failure to further develop an inner cell mass and trophoectoderm ex vivo. This indicates that CTCF expression is critical to early embryogenesis and loss of its expression rapidly leads to apoptosis at a very early developmental stage. This is the first study documenting the presence of the stable maternal Ctcf transcript in the blastocyst stage embryos. Furthermore, in the presence of maternal CTCF, zygotic CTCF expression does not seem to be required for pre-implantation development

DNA-PK suppresses a p53-independent apoptotic response to DNA damage

p53 is required for DNA damage-induced apoptosis, which is central to its function as a tumour suppressor. Here, we show that the apoptotic defect of p53-deficient cells is nearly completely rescued by inactivation of any of the three subunits of the DNA repair holoenzyme DNA-dependent protein kinase (DNA-PK). Intestinal crypt cells from p53 nullizygous mice were resistant to radiation-induced apoptosis, whereas apoptosis in DNA-PKcs/p53, Ku80/p53 and Ku70/p53 double-null mice was quantitatively equivalent to that seen in wild-type mice. This p53-independent apoptotic response was specific to the loss of DNA-PK, as it was not seen in ligase IV (Lig4)/p53 or ataxia telangiectasia mutated (Atm)/p53 double-null mice. Furthermore, it was associated with an increase in phospho-checkpoint kinase 2 (CHK2), and cleaved caspases 3 and 9, the latter indicating engagement of the intrinsic apoptotic pathway. This shows that there are two separate, but equally effective, apoptotic responses to DNA damage: one is p53 dependent and the other, engaged in the absence of DNA-PK, does not require p53

Tumor Multiplicity and Proliferative Index in <i>p19^Arf /p53</i> Compound Mutant Mice

<div><p>(A) Average number of papillomas (more than 2 mm in diameter) per mouse is plotted against the number of weeks post-initiation.</p> <p>(B) Image of wild-type, <i>p19^Arf (Arf)^−/−, p53^−/−,</i> and <i>p19^Arf−/−p53^−/−</i> mice with skin tumors at time of sacrifice. Wild-type mice show large exophytic tumors, while both <i>p19^Arf</i>- and p53-deficient mice have endophytic tumors. Note larger tumors in <i>p19^Arf /p53</i> compound mutant mice relative to <i>p53</i> single mutants.</p> <p>(C) BrdU-positive cells in papillomas from wild-type<i>, p53^−/−, p19^Arf−/−,</i> and <i>p19^Arf−/−p53^−/−</i> mice at 10 wk postinitiation. (Bars represent average counts ± standard deviation from ten fields and five mice). <i>p53^−/−</i> tumors show significantly fewer BrdU-positive cells than either <i>p19^Arf−/−</i> or wild-type tumors (<i>p</i> < 0.05, Wilcoxon one-sided t-test).</p></div

LOH of Wild-Type <i>p19^Arf</i> Allele in <i>p19^Arf+/−</i> Tumors

<div><p>(A) LOH analysis by semiquantitative PCR of the wild-type <i>p19^Arf</i> allele in <i>p19^Arf+/−</i> papillomas and carcinomas. Gradient made from kidney DNA used for quantitation of wt/mu ratio (top row). wt, wild-type allele; mu, knockout allele; asterisk, loss or reduction of <i>p19^Arf</i> wild-type band.</p> <p>(B) Western blot analysis of nuclear lysates from papillomas (PA) and carcinomas (CA) from <i>p19^Arf+/+, p19^Arf+/−,</i> and <i>p19^Arf−/−</i> mice.</p></div