Large parts of mammalian genomes are transcriptionally inactive and enriched with various classes of interspersed and tandem repeats. Here we show that the tumor suppressor protein p53 cooperates with DNA methylation to maintain silencing of a large portion of the mouse genome. Massive transcription of major classes of short, interspersed nuclear elements (SINEs) B1 and B2, both strands of near-centromeric satellite DNAs consisting of tandem repeats, and multiple species of noncoding RNAs was observed in p53-deficient but not in p53 wild-type mouse fibroblasts treated with the DNA demethylating agent 5-aza-2'-deoxycytidine. The abundance of these transcripts exceeded the level of β-actin mRNA by more than 150-fold. Accumulation of these transcripts, which are capable of forming double-stranded RNA (dsRNA), was accompanied by a strong, endogenous, apoptosis-inducing type I IFN response. This phenomenon, which we named "TRAIN" (for "transcription of repeats activates interferon"), was observed in spontaneous tumors in two models of cancer-prone mice, presumably reflecting naturally occurring DNA hypomethylation and p53 inactivation in cancer. These observations suggest that p53 and IFN cooperate to prevent accumulation of cells with activated repeats and provide a plausible explanation for the deregulation of IFN function frequently seen in tumors. Overall, this work reveals roles for p53 and IFN that are key for genetic stability and therefore relevant to both tumorigenesis and the evolution of species.interferon alpha-beta receptor | RNA sequencing | epigenetic repression | microarray hybridization

The TOR (Target of Rapamycin) pathway accelerates cellular and organismal aging. Similar to rapamycin, p53 can inhibit the mTOR pathway in some mammalian cells. Mice lacking one copy of p53 (p53+/− mice) have an increased cancer incidence and a shorter lifespan. We hypothesize that rapamycin can delay cancer in heterozygous p53+/− mice. Here we show that rapamycin (given in a drinking water) extended the mean lifespan of p53+/− mice by 10% and when treatment started early in life (at the age less than 5 months) by 28%. In addition, rapamycin decreased the incidence of spontaneous tumors. This observation may have applications in management of Li-Fraumeni syndrome patients characterized by heterozygous mutations in the p53 gene.

It has been shown that genetic inhibition of p53 leads to enhanced proliferation of hematopoietic stem cells (HSCs). This could, in theory, contribute to the increased frequency of tumor development observed in p53-defcient mice and humans. In our previous work, we identified chemical p53 inhibitors (PFTs) that suppress the transactivation function of p53 and protect cultured cells and mice from death induced by gamma irradiation (IR). Here we found that when applied to bone marrow cells in vitro or injected into mice, PFTβ impeded IR-induced reduction of hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) population sizes. In addition, we showed that PFTβ stimulated HSC and HPC proliferation in the absence of IR in vitro and in vivo and mobilized HSCs to the peripheral blood. Importantly, however, PFTβ treatment did not affect the timing or frequency of tumor development in irradiated p53 heterozygous mice used as a model for determination of carcinogenicity. Thus, although PFTβ administration led to increased numbers of HSCs and HPCs, it was not carcinogenic in mice. These findings suggest that chemical p53 inhibitors may be clinically useful as safe and effective stimulators of hematopoiesis.

No abstract

shRNA-mediated gene-silencing technology paired with cell-based functional readouts reveals potential targets directly, providing an opportunity to identify drugs against the target without knowing the precise role of the target in the pathophysiological processes of interest. By screening a lentiviral shRNA library targeting for major components of human signaling pathways and known drug targets, we identified and validated both canonical as well as 52 novel mediators of FAS and TNF ligand-induced apoptosis. Presence of potential therapeutic targets among these mediators was confirmed by demonstration of in vivo activity of siRNAs against four identified target candidates that protected mice from acute liver failure (ALF), a life-threatening disease with known involvement of death receptor (DR)-mediated apoptosis. Network-based modeling was used to predict small-molecule inhibitors for several candidate apoptosis mediators, including somatostatin receptor 5 (SSTR5) and a regulatory subunit of PP2A phosphatase, PPP2R5A. Remarkably, pharmacological inhibition of either SSTR5 or PPP2R5A reduced apoptosis induced by either FASL or TNF in cultured cells and dramatically improved survival in several mouse models of ALF. These results demonstrate the utility of loss-of-function genetic screens and network-based drug-repositioning methods for expedited identification of targeted drug candidates and revealed pharmacological agents potentially suitable for treatment of DR-mediated pathologies.

Transplantation of bone marrow (BM) is made possible by the differential sensitivity of its stromal and hematopoietic components to preconditioning by radiation and/or chemotherapeutic drugs. These genotoxic treatments eliminate host hematopoietic precursors by inducing p53-mediated apoptosis but keep the stromal niche sufficiently intact for the engraftment of donor hematopoietic cells. We found that p53-null mice cannot be rescued by BM transplantation (BMT) from even the lowest lethal dose of total body irradiation (TBI). We compared structural changes in BM stroma of mice differing in their p53 status to understand why donor BM failed to engraft in the irradiated p53-null mice. Irradiation did not affect the general structural integrity of BM stroma and induced massive expression of alpha-smooth muscle actin in mesenchymal cells followed by increased adiposity in p53 wild-type mice. In contrast, none of these events were found in p53-null mice, whose BM stroma underwent global structural damage following TBI. Similar differences in response to radiation were observed in in vitro-grown bone-adherent mesenchymal cells (BAMC): p53-null cells underwent mitotic catastrophe while p53 wild-type cells stayed arrested but viable. Supplementation with intact BAMC of either genotype enabled donor BM engraftment and significantly extended longevity of irradiated p53-null mice. Thus, successful preconditioning depends on the p53-mediated protection of cells critical for the functionality of BM stroma. Overall, this study reveals a dual positive role of p53 in BMT: it drives apoptotic death of hematopoietic cells and protects BM stromal cells essential for its functionality.

Technological advances in radiation therapy have greatly improved our ability to deliver a precise dose of radiation to a defined volumetric target with high accuracy. However, local recurrence of cancer, especially that in the previously irradiated area continues to be a significant cause of morbidity and mortality in patients who initially receive radiation therapy. It has been known for some time that certain agents, mostly chemotherapeutic drugs, are capable of enhancing the effects of radiation when given in conjunction with radiation therapy. Concurrent use of these agents (including cis-platinum and 5-fluorouracil, to name a few) with radiation has been shown to result in improved disease control in a wide variety of cancers. Even though these agents are highly beneficial to many patients who receive them, their use is often limited due to their inherent toxicities as well as their ability to enhance radiation induced normal tissue reactions. Previously, we have reported that quinacrine, an old antimalarial drug known to induce p53 activity and suppress NF-κB activity in cells, are capable of radiation sensitization in vitro. Since that time, we have been studying a group of compounds we named Curaxins for its radiation sensitizing qualities. Curaxins are carbazole compounds that were selected for their ability to induce p53 activity and reduce NF-κB activity in a variety of human cancer cell lines. These compounds are known to have much higher potency than quinacrine. Though they were originally developed as single-agent anticancer agents, our recent investigations have shown that many of these Curaxins are also very potent radiation sentizers in vitro and in vivo. We have recently completed a preliminary screen of our existing Curaxins for their ability to enhance cytotoxicity of ionizing radiation, utilizing B16 murine melanoma cell line as our model system. Clonogenic survival assays were peformed in presence of various doses of Curaxins ranging from 2 nM to 200 nM. Of twelve Curaxins screened for this purpose, nine were found to be very potent sensitizers at either 2 nM and/or 20 nM. We also studied the effect of two of the Curaxins for their radiosensitizing activities in vivo, using a B16 lung metastasis model in C57BL/6 mice. Combination of Curaxins to thoracic irradiation resulted in greater tumor control (indicated by reduced lung weight) and longer mean survival compred to irradiation alone or Curaxins alone. It is notable that in vitro radiosensitization by these two Curaxins occurs at concentrations not known to cause p53 induction in B16 cells, suggesting that this effect may not be entirely dependent on the p53 pathway. We are currently in the process of further characterizing the radiosensitizing qualities of the remaining seven radiosensitizing Curaxins both in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-166.