Summary A new non-invasive method for monitoring apoptosis has been developed using high frequency (40 MHz) ultrasound imaging. Conventional ultrasound backscatter imaging techniques were used to observe apoptosis occurring in response to anticancer agents in cells in vitro, in tissues ex vivo and in live animals. The mechanism behind this ultrasonic detection was identified experimentally to be the subcellular nuclear changes, condensation followed by fragmentation, that cells undergo during apoptosis. These changes dramatically increase the high frequency ultrasound scattering efficiency of apoptotic cells over normal cells (25-to 50-fold change in intensity). The result is that areas of tissue undergoing apoptosis become much brighter in comparison to surrounding viable tissues. The results provide a framework for the possibility of using high frequency ultrasound imaging in the future to non-invasively monitor the effects of chemotherapeutic agents and other anticancer treatments in experimental animal systems and in patients.

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J.Abraham and B.Lemmers contributed equally to this workYeast and human Eme1 protein, in complex with Mus81, constitute an endonuclease that cleaves branched DNA structures, especially those arising during stalled DNA replication. We identi®ed mouse Eme1, and show that it interacts with Mus81 to form a complex that preferentially cleaves 3¢-¯ap structures and replication forks rather than Holliday junctions in vitro. We demonstrate that Eme1 ±/± embryonic stem (ES) cells are hypersensitive to the DNA cross-linking agents mitomycin C and cisplatin, but only mildly sensitive to ionizing radiation, UV radiation and hydroxyurea treatment. Mammalian Eme1 is not required for the resolution of DNA intermediates that arise during homologous recombination processes such as gene targeting, gene conversion and sister chromatid exchange (SCE). Unlike Blm-de®cient ES cells, increased SCE was seen only following induced DNA damage in Eme1-de®cient cells. Most importantly, Eme1 de®ciency led to spontaneous genomic instability. These results reveal that mammalian Eme1 plays a key role in DNA repair and the maintenance of genome integrity.

Disruption of Brca1 results in cellular demise or tumorigenesis depending on cellular context. Inactivation of p53 contributes to Brca1-associated tumor susceptibility. However the activation of p53-dependent checkpoint/apoptotic signaling in the absence of Brca1 is poorly understood. Here, we show that Chk2 inactivation is partially equivalent to p53 inactivation, in that Chk2 deficiency facilitates the development, survival, and proliferation of Brca1-deficient T cells at the expense of genomic integrity. Brca1 deficiency was found to result in Chk2 phosphorylation and the Chk2-dependent accumulation and activation of p53. Furthermore, inactivation of Chk2 and Brca1 was cooperative in breast cancer. Our findings identify a critical role for Chk2 as a component of the DNA damage-signaling pathway activated in response to Brca1 deficiency.[Keywords: Breast cancer; T cell; genomic instability; apoptosis; cell cycle; proliferation] Supplemental material is available at http://www.genesdev.org. BRCA1 germline mutations predispose women to early onset, familial breast and ovarian cancer (Scully and Livingston 2000;Welcsh et al. 2000;Venkitaraman 2002). Despite its role in maintenance of genome integrity (Kinzler and Vogelstein 1997), transcriptional regulation (Scully et al. 1997a;Somasundaram et al. 1997), and chromatin remodeling (Yarden and Brody 1999;Bochar et al. 2000;Pao et al. 2000;Wang et al. 2000;Welcsh et al. 2000), the exact mechanism of tumor suppression by BRCA1 remains to be defined. BRCA1 associates with proteins that function in DNA replication and repair (Scully et al. 1997b;Wang et al. 2000), transcriptional activation (Chapman and Verma 1996;Monteiro et al. 1996;Scully et al. 1997a;Li et al. 2000), and the DNA damage response (Scully et al. 1997b;Zhong et al. 1999;Li et al. 2000;Wang et al. 2000;Welcsh et al. 2000). Cells with mutant Brca1 display defects in survival and proliferation (Gowen et al. 1996;Hakem et al. 1996;Ludwig et al. 1997;Shen et al. 1998), radiosensitivity (Shen et al. 1998;Welcsh et al. 2000), chromosomal abnormalities (Xu et al. 1999;Mak et al. 2000;Welcsh et al. 2000), p53 activation (Hakem et al. 1996), G2/M checkpoint loss (Larson et al. 1997;Xu et al. 1999), and impaired homologous recombination repair (Moynahan et al. 1999). Accordingly, the pleiotropic effects of BRCA1 mutation could be attributed to its involvement in DNA repair and transcriptional regulation.Brca1-targeted disruption in mice results in embryonic lethality (Gowen et al. 1996;Hakem et al. 1996;Liu et al. 1996;Ludwig et al. 1997). To circumvent this limitation, conditional targeting strategies have been employed that facilitate the study of the consequences of Brca1 disruption in vivo and ex vivo (Xu et al. 1999;Mak et al. 2000). Mice that are tBrca1 −/− ) carry a targeted null mutation of Brca1 restricted to the T-cell lineage, thus bypassing the associated lethality of Brca1 germline disruption. Brca1 disruption in the T-cell compartment results in a drastic depletion of thymocytes and peripheral T cell...

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The tumour suppressor gene product, p53, is involved in mediating cellular responses to DNA damage including growth arrest and/or apoptosis. The mechanism by which p53 protein senses the presence of damaged DNA is not understood. The possibility that p53 may be posttranslationally modi®ed by enzymes that are activated in response to DNA damage including DNA-dependent protein kinase (DNA-PK), poly(ADP-ribose) polymerase and stress activated protein kinase has received considerable attention. Recent studies have indicated that DNA-PK is not required for the transactivation or apoptosis-promoting activities of p53 protein. However, the possibility that other functions of p53 may be dependent on phosphorylation by DNA-PK has not been explored. Here we describe a series of experiments that compares the expression, function and phosphorylation status of p53 protein in normal and DNA-PK-de®cient scid cells. While several novel p53 phosphoforms are generated in response to DNA damage in normal cells, the same phosphoforms are observed in scid cells.

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