Tp53 is the most commonly mutated tumour-suppressor gene in human cancers. In addition to the loss of tumour-suppression function, some missense mutants gain novel oncogenic activities. To elucidate the nature of the gain of function, we introduced the most common p53 cancer mutations (R248W and R273H) independently into the humanized p53 knock-in (HUPKI) allele in mice. Tumour-suppressor functions of p53 are abolished in p53-mutant mice. Several lines of evidence further indicate gain-of-function of p53 mutants in promoting tumorigenesis. p53(R248W) mice rapidly succumb to certain types of cancers not commonly observed in p53(-/-) mice. Interchromosomal translocations, a type of genetic instability rarely observed in p53(-/-) cells, are readily detectable in p53-mutant pre-tumor thymocytes. Although normal in p53(-/-) mouse cells, the G(2)-M checkpoint is impaired in p53-mutant cells after DNA damage. These acquired oncogenic properties of mutant p53 could be explained by the findings that these p53 mutants interact with the nuclease Mre11 and suppress the binding of the Mre11-Rad50-NBS1 (MRN) complex to DNA double-stranded breaks (DSBs), leading to impaired Ataxia-telangiectasia mutated (ATM) activation. Therefore, p53 gain-of-function mutants promote tumorigenesis by a novel mechanism involving active disruption of critical DNA damage-response pathways.

Although mouse models have been valuable for studying human disease, the cellular and physiological differences between mouse and human have made it increasingly important to develop more relevant human disease models for mechanistic studies and drug discovery. Human embryonic stem cells (hESCs), which can undergo unlimited self-renewal and retain the potential to differentiate into all cell types, present a possible solution. To improve the efficiency of genetic manipulation of hESCs, we have developed bacterial artificial chromosome (BAC) based approach that enables high efficiency homologous recombination. By sequentially disrupting both alleles of ATM or p53 with BAC targeting vectors, we have established ATM(-/-) and p53(-/-) hESCs as models for two major human genetic instability syndromes and used the generated cells to reveal the importance of p53 in maintaining genome stability of hESCs. Our findings suggest that it will be feasible to develop genetically modified hESCs as relevant human disease models.

The critical tumor suppressor p53 is mutated in over half of all human cancers. The majority of p53 cancer mutations are missense mutations, which can be classified into contact mutations that directly disrupt the DNA-binding motif of p53 but have modest impact on p53 conformation and structural mutations that greatly disrupt p53 conformation. Many p53 cancer mutants, including the hotspot mutations (R175H, R248W and R273H), not only lose p53-dependent tumor suppressor activities, but also acquire new oncogenic activities to promote cancer. Therefore, it is critical to elucidate the gain of oncogenic function of p53 cancer mutants. Employing humanized p53 mutant knock-in mouse models, we have identified a gain of oncogenic function shared by the most common p53 contact mutants (R273H and R248W) and structural mutant (R175H). This gain of function inactivates Mre11/ATM-dependent DNA damage responses, leading to chromosomal translocation and defective G2/M checkpoint. Considering the critical roles of ATM in maintaining genetic responses and therapeutic responses to many cancer treatments, the identification of this common gain of function of p53 cancer mutants will have important implication on the drug resistance of a significant portion of human cancers that express both the contact and structural p53 cancer mutants.

Proper cellular responses to DNA double-strand breaks (DSBs) are critical for maintaining genetic stability and for tumor suppression (38). A gene consistently mutated in genetic instability syndrome ataxia-telangiectasia (A-T), called ATM (for ataxia-telangiectasia mutated), encodes a large protein kinase that is responsible for activating cellular responses to DNA damage (44). In this context, ATM is required for homologous recombination and all three cell cycle checkpoints after DNA DSB damage. In addition, ATM deficiency leads to hypersensitivity to ␥-irradiation and multisystemic defects, including growth retardation, abolished germ cell development, immunodeficiency, and greatly increased cancer risk (43). As a protein kinase, ATM functions by phosphorylating and activating a number of DNA repair and checkpoint proteins, including p53, NBS1, H2AX, 53BP1, Brca1, Smc1, and Chk2 (43).Immediately after DNA DSB damage, histone H2AX is phosphorylated at the C-terminal Ser residues (Ser136 and Ser139) (40). This phosphorylation, called ␥-H2AX, can be detected within minutes after the introduction of DSBs and is involved in the recruitment of other known components of DNA repair, including Brca1, NBS1/Mre11/Rad50, and 53BP1, to the sites of the DNA damage (11,41,48). ␥-H2AX is mainly mediated by ATM after DNA DSB damage, suggesting that H2AX is a downstream mediator of ATM function (6,20). H2AX is also required for the recruitment of 53BP1 to sites of DNA DSB damage, and 53BP1 appears to be an upstream activator of ATM (11,39,48). Therefore, it remains unclear whether H2AX plays any role in activating ATM after DNA DSB damage. NBS1, the gene product mutated in Nijmegen breakage syndrome (NBS) patients, is the p95 component of the Mre11 complex that forms foci at the sites of DNA DSBs (8,36,47). The importance of NBS1 in DNA repair is indicated by the multisystemic defects observed in NBS patients, including growth retardation, immunodeficiency, and increased lymphoid malignancies (46). In addition, NBS cells are hypersensitive to ionizing radiation and defective in intra-S and G 2 /M checkpoints. Targeted disruption of the N terminus of NBS1 in mice recapitulates most of the systemic and cellular defects observed in NBS patients (25,50). The functional link between ATM and NBS1 is suggested by the large panel of defects shared by NBS1 and ATM mutations and is further demonstrated by the findings that ATM can phosphorylate and activate NBS1 at a consensus site, Ser343 (21,30,51,55). Therefore, NBS1 appears to function as a downstream mediator of ATM function. Recent studies show that mutation of the Mre11 complex or NBS1 leads to impaired ATM activation in human cell lines, indicating a role for NBS1 in activating ATM after DNA DSB damage (9,23,39,45). Since the disruption of this NBS1 function could account for the A-T-related defects observed in NBS patients, the contribution of the impaired adapter function of NBS1 in mediating ATM activities to the A-T-related defects in NBS1 mutant mice and human patients rema...

The complexity and dynamic nature of the Internet (and the emerging Computational Grid) demand that middleware and applications adapt to the changes in configuration and availability of resources. However, to the best of our knowledge there are no simulation tools which support systematic exploration of dynamic Grid software (or Grid resource) behavior.We describe our vision and initial efforts to build tools to meet these needs. Our MicroGrid simulation tools enable Globus applications to be run in arbitrary virtual grid resource environments, enabling broad experimentation. We describe the design of these tools, and their validation on microbenchmarks, the NAS parallel benchmarks, and an entire Grid application. These validation experiments show that the MicroGrid can match actual experiments within a few percent (2% to 4%).