Replicative cell division is an energetically demanding process that can be executed only if cells have sufficient metabolic resources to support a doubling of cell mass. Here we show that proliferating mammalian cells have a cell-cycle checkpoint that responds to glucose availability. The glucose-dependent checkpoint occurs at the G(1)/S boundary and is regulated by AMP-activated protein kinase (AMPK). This cell-cycle arrest occurs despite continued amino acid availability and active mTOR. AMPK activation induces phosphorylation of p53 on serine 15, and this phosphorylation is required to initiate AMPK-dependent cell-cycle arrest. AMPK-induced p53 activation promotes cellular survival in response to glucose deprivation, and cells that have undergone a p53-dependent metabolic arrest can rapidly reenter the cell cycle upon glucose restoration. However, persistent activation of AMPK leads to accelerated p53-dependent cellular senescence. Thus, AMPK is a cell-intrinsic regulator of the cell cycle that coordinates cellular proliferation with carbon source availability.

Induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells with defined factors, hold great promise for regenerative medicine as the renewable source of autologous cells. Whereas it has been generally assumed that these autologous cells should be immune-tolerated by the recipient from whom the iPSCs are derived, their immunogenicity has not been vigorously examined. We show here that, whereas embryonic stem cells (ESCs) derived from inbred C57BL/6 (B6) mice can efficiently form teratomas in B6 mice without any evident immune rejection, the allogeneic ESCs from 129/SvJ mice fail to form teratomas in B6 mice due to rapid rejection by recipients. B6 mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs by either retroviral approach (ViPSCs) or a novel episomal approach (EiPSCs) that causes no genomic integration. In contrast to B6 ESCs, teratomas formed by B6 ViPSCs were mostly immune-rejected by B6 recipients. In addition, the majority of teratomas formed by B6 EiPSCs were immunogenic in B6 mice with T cell infiltration, and apparent tissue damage and regression were observed in a small fraction of teratomas. Global gene expression analysis of teratomas formed by B6 ESCs and EiPSCs revealed a number of genes frequently overexpressed in teratomas derived from EiPSCs, and several such gene products were shown to contribute directly to the immunogenicity of the B6 EiPSC-derived cells in B6 mice. These findings indicate that, in contrast to derivatives of ESCs, abnormal gene expression in some cells differentiated from iPSCs can induce T-cell-dependent immune response in syngeneic recipients. Therefore, the immunogenicity of therapeutically valuable cells derived from patient-specific iPSCs should be evaluated before any clinic application of these autologous cells into the patients.

The tumour suppressor p53 becomes activated in response to upstream stress signals, such as DNA damage, and causes cell-cycle arrest or apoptosis. Here we report a novel role for p53 in the differentiation of mouse embryonic stem cells (ESCs). p53 binds to the promoter of Nanog, a gene required for ESC self-renewal, and suppresses Nanog expression after DNA damage. The rapid down-regulation of Nanog mRNA during ESC differentiation correlates with the induction of p53 transcriptional activity and Ser 315 phosphorylation. The importance of Ser 315 phosphorylation was revealed by the finding that induction of p53 activity is impaired in p53(S315A) knock-in ESCs during differentiation, leading to inefficient suppression of Nanog expression. The decreased inhibition of Nanog expression in p53(S315A) ESCs during differentiation is due to an impaired recruitment of the co-repressor mSin3a to the Nanog promoter. These findings indicate an alternative mechanism for p53 to maintain genetic stability in ESCs, by inducing the differentiation of ESCs into other cell types that undergo efficient p53-dependent cell-cycle arrest and apoptosis.

ATM, the gene mutated in the inherited human disease ataxia-telangiectasia, is a member of a family of kinases involved in DNA metabolism and cell-cycle checkpoint control. To help clarify the physiological roles of the ATM protein, we disrupted the ATM gene in mice through homologous recombination. Initial evaluation of the ATM knockout animals indicates that inactivation of the mouse ATM gene recreates much of the phenotype of ataxia-telangiectasia. The homozygous mutant (ATM -/-) mice are viable, growth-retarded, and infertile. The infertility of ATM -/-mice results from meiotic failure. Meiosis is arrested at the zygotene/pachytene stage of prophase I as a result of abnormal chromosomal synapsis and subsequent chromosome fragmentation. Immune defects also are evident in A TM-/-mice, including reduced numbers of B220+CD43 -pre-B cells, thymocytes, and peripheral T cells, as well as functional impairment of T-cell-dependent immune responses. The cerebella of ATM -~-mice appear normal by histologic examination at 3 to 4 months and the mice have no gross behavioral abnormalities. The majority of mutant mice rapidly develop thymic lymphomas and die before 4 months of age. These findings indicate that the ATM gene product plays an essential role in a diverse group of cellular processes, including meiosis, the normal growth of somatic tissues, immune development, and tumor suppression.

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Ataxia telangiectasia (AT) is a rare human autosomal recessive disorder with pleiotropic phenotypes, including neuronal degeneration, immune dysfunction, premature ageing and increased cancer risk. The gene mutated in AT, ATM, encodes a putative lipid or protein kinase. Most of the human AT patient phenotypes are recapitulated in Atm-deficient mice. Cells derived from Atm-/- mice, like those from AT patients, exhibit abnormal response to ionizing radiation. One of the known responses to ionizing radiation is the activation of a nuclear tyrosine kinase encoded by the c-abl proto-oncogene. Ionizing radiation does not activate c-Abl in cells from AT patients or in thymocytes or fibroblasts from the Atm-deficient mice. Ectopic expression of a functional ATM kinase domain corrects this defect, as it phosphorylates the c-Abl tyrosine kinase in vitro at Ser 465, leading to the activation of c-Abl. A mutant c-Abl with Ser 465 changed to Ala 465 is not activated by ionizing radiation or ATM kinase in vivo. These findings identify the c-Abl tyrosine kinase as a downstream target of phosphorylation and activation by the ATM kinase in the cellular response to ionizing radiation.

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.

Dynamic changes in histone modification are critical for regulating DNA double-strand break (DSB) repair. Activation of the Tip60 acetyltransferase by DSBs requires interaction of Tip60 with histone H3 methylated on lysine 9 (H3K9me3). However, how H3K9 methylation is regulated during DSB repair is not known. Here, we demonstrate that a complex containing kap-1, HP1, and the H3K9 methyltransferase suv39h1 is rapidly loaded onto the chromatin at DSBs. Suv39h1 methylates H3K9, facilitating loading of additional kap-1/HP1/suv39h1 through binding of HP1's chromodomain to the nascent H3K9me3. This process initiates cycles of kap-1/HP1/ suv39h1 loading and H3K9 methylation that facilitate spreading of H3K9me3 and kap-1/HP1/suv39h1 complexes for tens of kilobases away from the DSB. These domains of H3K9me3 function to activate the Tip60 acetyltransferase, allowing Tip60 to acetylate both ataxia telangiectasia-mutated (ATM) kinase and histone H4. Consequently, cells lacking suv39h1 display defective activation of Tip60 and ATM, decreased DSB repair, and increased radiosensitivity. Importantly, activated ATM rapidly phosphorylates kap-1, leading to release of the repressive kap-1/HP1/suv39h1 complex from the chromatin. ATM activation therefore functions as a negative feedback loop to remove repressive suv39h1 complexes at DSBs, which may limit DSB repair. Recruitment of kap-1/HP1/suv39h1 to DSBs therefore provides a mechanism for transiently increasing the levels of H3K9me3 in open chromatin domains that lack H3K9me3 and thereby promoting efficient activation of Tip60 and ATM in these regions. Further, transient formation of repressive chromatin may be critical for stabilizing the damaged chromatin and for remodeling the chromatin to create an efficient template for the DNA repair machinery.histone methylation | homologous recombination D NA double-strand breaks (DSBs) are toxic and must be repaired to maintain genomic stability. Detection of DSBs requires recruitment of the mre11-rad50-nbs1 (MRN) complex to the DNA ends (1). MRN then recruits and activates the ataxia telangiectasia-mutated (ATM) kinase (2, 3) through a mechanism that also requires the Tip60 acetyltransferase (3). Tip60 directly acetylates and activates ATM's kinase activity (4-6) and functions, in combination with MRN, to promote ATM-dependent phosphorylation of DSB repair proteins (3), including histone H2AX. This process creates domains of phosphorylated H2AX (γH2AX) extending for hundreds of kilobases along the chromatin (7,8). Mdc1 then binds to γH2AX, providing a landing pad for other DSB repair proteins, including the RNF8/RNF168 ubiquitin ligases (1, 3, 9, 10). Tip60 also plays a critical role in regulating chromatin structure at DSBs as part of the NuA4-Tip60 complex (11). NuA4-Tip60 catalyzes histone exchange (via the p400 ATPase subunit) and acetylation of histone H4 (by Tip60) at DSBs (12-15), leading to the formation of open, flexible chromatin domains adjacent to the break (12, 13). These open chromatin structures then facilitate histone ub...