In mammals, TUBBY-like proteins play an important role in maintenance and function of neuronal cells during postdifferentiation and development. We have identified a TUBBY-like protein gene family with 11 members in Arabidopsis, named AtTLP1-11. Although seven of the AtTLP genes are located on chromosome I, no local tandem repeats or gene clusters are identified. Except for AtTLP4, reverse transcription-PCR analysis indicates that all these genes are expressed in various organs in 6-week-old Arabidopsis. AtTLP1, 2, 3, 6, 7, 9, 10, and 11 are expressed ubiquitously in all the organs tested, but the expression of AtTLP5 and 8 shows dramatic organ specificity. These 11 family members share 30% to 80% amino acid similarities across their conserved C-terminal tubby domains. Unlike the highly diverse N-terminal region of animal TUBBY-like proteins, all AtTLP members except AtTLP8 contain a conserved F-box domain (51–57 residues). The interaction between AtTLP9 and ASK1 (Arabidopsis Skp1-like 1) is confirmed via yeast (Saccharomyces cerevisiae) two-hybrid assays. Abscisic acid (ABA)-insensitive phenotypes are observed for two independent AtTLP9 mutant lines, whereas transgenic plants overexpressing AtTLP9 are hypersensitive to ABA. These results suggest that AtTLP9 may participate in the ABA signaling pathway.

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Cancer clinics currently use high-dose stereotactic body radiation therapy as a curative treatment for several kinds of cancers. However, the contribution of vascular endothelial cells to tumor response to radiation remains controversial. Using dual-recombinase technology, we generated primary sarcomas in mice with targeted genetic mutations specifically in tumor cells or endothelial cells. We selectively mutated the proapoptotic gene Bax or the DNA damage–response gene Atm to genetically manipulate the radiosensitivity of endothelial cells in primary soft tissue sarcomas. Bax deletion from endothelial cells did not affect radiation-induced cell death in tumor endothelial cells or sarcoma response to radiation therapy. Although Atm deletion increased endothelial cell death after radiation therapy, deletion of Atm from endothelial cells failed to enhance sarcoma eradication. In contrast, deletion of Atm from tumor cells increased sarcoma eradication by radiation therapy. These results demonstrate that tumor cells, rather than endothelial cells, are critical targets that regulate sarcoma eradication by radiation therapy. Treatment with BEZ235, a small-molecule protein kinase inhibitor, radiosensitized primary sarcomas more than hearts. These results suggest that inhibiting ATM kinase during radiation therapy is a viable strategy for radiosensitization of some tumors.

Genotoxic cancer therapies, such as chemoradiation, cause hematologic toxicity primarily by activating the tumor suppressor p53. While inhibiting p53-mediated cell death during cancer therapy ameliorates hematologic toxicity, whether it also impacts carcinogenesis remains unclear. Here we utilize a mouse model of inducible p53 short hairpin RNA (shRNA) to show that temporarily blocking p53 during total-body irradiation (TBI) not only ameliorates acute toxicity, but also improves long-term survival by preventing lymphoma development. Using KrasLA1 mice, we show that TBI promotes the expansion of a rare population of thymocytes that express oncogenic KrasG12D. However, blocking p53 during TBI significantly suppresses the expansion of KrasG12D-expressing thymocytes. Mechanistically, bone marrow transplant experiments demonstrate that TBI activates p53 to decrease the ability of bone marrow cells to suppress lymphoma development through a non-cell-autonomous mechanism. Together, our results demonstrate that the p53 response to acute DNA damage promotes the development of radiation-induced lymphoma.

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SUMMARYThe site-specific recombinases Cre and Flp can mutate genes in a spatially and temporally restricted manner in mice. Conditional recombination of the tumor suppressor gene p53 using the Cre-loxP system has led to the development of multiple genetically engineered mouse models of human cancer. However, the use of Cre recombinase to initiate tumors in mouse models limits the utilization of Cre to genetically modify other genes in tumor stromal cells in these models. To overcome this limitation, we inserted FRT (flippase recognition target) sites flanking exons 2–6 of the endogenous p53 gene in mice to generate a p53FRT allele that can be deleted by Flp recombinase. We show that FlpO-mediated deletion of p53 in mouse embryonic fibroblasts impairs the p53-dependent response to genotoxic stress in vitro. In addition, using FSF-KrasG12D/+; p53FRT/FRT mice, we demonstrate that an adenovirus expressing FlpO recombinase can initiate primary lung cancers and sarcomas in mice. p53FRT mice will enable dual recombinase technology to study cancer biology because Cre is available to modify genes specifically in stromal cells to investigate their role in tumor development, progression and response to therapy.

In the process of transferring the miR-182-flox and LSL-miR-182 mice to the Jackson Laboratory, the authors realized that the description of how the LSL-miR-182 mice were generated in the manuscript contained an error. Although the miR-182-flox mice were generated by crossing the mice to a flpO deleter strain to delete the Neo cassette, as was stated in the Methods section, the R26-LSL-miR-182 mice were not crossed to a deleter strain. Instead, the R26-LSL-miR-182 mice that were utilized in this work retained the Neo cassette. Two corrected sentences for the Methods section are below.Chimeric males were mated to WT C57BL/6 females to generate heterozygotes. These mice were subsequently crossed to a flpO deleter strain to excise the frt-Neo-frt cassette to generate miR-182-flox mice.The R26-LSL-miR-182 mice that were utilized in this work retained the Neo cassette.The authors regret the error.

Purpose To develop a mouse model of cardiac injury following partial-heart irradiation (PHI) and test whether DE-microCT and 4D-microCT can be used to assess cardiac injury after PHI to complement myocardial perfusion imaging using microSPECT. Methods and Materials To study cardiac injury from tangent field irradiation in mice, we used a small-field biological irradiator to deliver a single dose of 12 Gy x-rays to approximately one-third of the left ventricle (LV) of Tie2Cre; p53FL/+ and Tie2Cre; p53FL/− mice, where one or both alleles of p53 are deleted in endothelial cells. Four and 8 weeks after irradiation, mice were injected with gold and iodinated nanoparticle-based contrast agents, and imaged with DE-microCT and 4D-microCT to evaluate myocardial vascular permeability and cardiac function, respectively. Additionally, the same mice were imaged with microSPECT to assess myocardial perfusion. Results After PHI with tangent fields, DE-microCT scans showed a time-dependent increase in accumulation of gold nanoparticles (AuNp) in the myocardium of Tie2Cre; p53FL/− mice. In Tie2Cre; p53FL/− mice, extravasation of AuNp was observed within the irradiated LV, whereas in the myocardium of Tie2Cre; p53FL/+ mice, AuNp were restricted to blood vessels. In addition, data from DE-microCT and microSPECT showed a linear correlation (R2=0.97) between the fraction of the LV that accumulated AuNp and the fraction of LV with a perfusion defect. Furthermore, 4D-microCT scans demonstrated that PHI caused Tie2Cre; p53FL/− mice to develop a markedly decreased ejection fraction, and higher end-diastolic and end-systolic volumes, which were associated with compensatory cardiac hypertrophy of the heart that was not irradiated. Conclusions Our results show that DE-microCT and 4D-microCT with nanoparticle-based contrast agents are novel imaging approaches complementary to microSPECT to non-invasively assess the change in myocardial vascular permeability and cardiac function of mice that develop myocardial injury after PHI.