Rad54 and Mus81 cooperation promotes DNA damage repair and restrains chromosome missegregation.

Rad54 and Mus81 mammalian proteins physically interact and are important for the homologous recombination DNA repair pathway; however, their functional interactions in vivo are poorly defined. Here, we show that combinatorial loss of Rad54 and Mus81 results in hypersensitivity to DNA-damaging agents, defects on both the homologous recombination and non-homologous DNA end joining repair pathways and reduced fertility. We also observed that while Mus81 deficiency diminished the cleavage of common fragile sites, very strikingly, Rad54 loss impaired this cleavage to even a greater extent. The inefficient repair of DNA double-strand breaks (DSBs) in Rad54(-/-)Mus81(-/-) cells was accompanied by elevated levels of chromosome missegregation and cell death. Perhaps as a consequence, tumor incidence in Rad54(-/-)Mus81(-/-) mice remained comparable to that in Mus81(-/-) mice. Our study highlights the importance of the cooperation between Rad54 and Mus81 for mediating DNA DSB repair and restraining chromosome missegregation.

Cooperation of Blm and Mus81 in development, fertility, genomic integrity and cancer suppression.

BLM is a DNA helicase important for the restart of stalled replication forks and for homologous recombination (HR) repair. Mutations of BLM lead to Bloom Syndrome, a rare autosomal recessive disorder characterized by elevated levels of sister chromatid exchanges (SCEs), dwarfism, immunodeficiency, infertility and increased cancer predisposition. BLM physically interacts with MUS81, an endonuclease involved in the restart of stalled replication forks and HR repair. Herein we report that loss of Mus81 in Blm hypomorph mutant mice leads to infertility, and growth and developmental defects that are not observed in single mutants. Double mutant cells and mice were hypersensitive to Mitomycin C and γ-irradiation (IR) compared with controls and their repair of DNA double-strand breaks (DSBs) mediated by HR pathway was significantly defective, whereas their non-homologous-end-joining repair was elevated compared with controls. We also demonstrate the importance of the loss of the nuclease activity of Mus81 in the defects observed in Mus81(-/-) and double mutant cells. Exacerbated IR-induced chromosomal aberration was observed in double mutant mice and despite their reduced SCE levels, these mutants showed increased tumorigenesis risks. Our data highlight the importance of Mus81 and Blm in DNA DSB repair pathways, fertility, development and cancer.

The E3 ligase PIRH2 polyubiquitylates CHK2 and regulates its turnover.

The serine threonine kinase checkpoint kinase 2 (CHK2) is a DNA damage checkpoint protein important for the ATM-p53 signaling pathway. In addition to its phosphorylation, CHK2 is also ubiquitylated, and both post-translational modifications are important for its function. However, although the mechanisms that regulate CHK2 phosphorylation are well established, those that control its ubiquitylation are not fully understood. In this study, we demonstrate that the ubiquitin E3 ligase PIRH2 (p53-induced protein with a RING (Really Interesting New Gene)-H2 domain) interacts with CHK2 and mediates its polyubiquitylation and proteasomal degradation. We show that the deubiquitylating enzyme USP28 forms a complex with PIRH2 and CHK2 and antagonizes PIRH2-mediated polyubiquitylation and proteasomal degradation of CHK2. We also provide evidence that CHK2 ubiquitylation by PIRH2 is dependent on its phosphorylation status. Cells deficient in Pirh2 displayed accumulation of Chk2 and enhanced hyperactivation of G1/S and G2/M cell-cycle checkpoints. This hyperactivation was, however, no longer observed in Pirh2-/-Chk2-/- cells, providing evidence for the importance of Chk2 regulation by Pirh2. These findings indicate that PIRH2 has central roles in the ubiquitylation of Chk2 and its turnover and in the regulation of its function.

Essential role for Bclaf1 in lung development and immune system function.

Bcl-2 associated factor 1 (Bclaf1) is a nuclear protein that was originally identified in a screen of proteins that interact with the adenoviral bcl-2 homolog E1B19K. Overexpression of Bclaf1 was shown to result in apoptosis and transcriptional repression that was reversible in the presence of Bcl-2 or Bcl-x(L). Furthermore, antiapoptotic members, but not proapoptotic members of the Bcl-2 protein family, were shown to interact with Bclaf1 and prevent its localization to the nucleus. Bclaf1 has also recently been identified as a binding partner for Emerin, a nuclear membrane protein that is mutated in X-linked recessive Emery-Dreifuss muscular dystrophy. To ascertain the in vivo function of Bclaf1, we have generated mice that carry a targeted mutation of the bclaf1 locus. In this study, we show that Bclaf1 is required for proper spatial and temporal organization of smooth muscle lineage during the saccular stage of lung development. We also show that Bclaf1 is dispensable for thymocyte development but is essential for peripheral T-cell homeostasis. Despite its postulated role as a proapoptotic protein, Bclaf1-deficient cells did not show any defect in cell death linked to development or after exposure to various apoptotic stimuli. Our findings show a critical role for Bclaf1 in developmental processes independent of apoptosis.

Caspase-12 compensates for lack of caspase-2 and caspase-3 in female germ cells.

Previously, we analyzed mice lacking either caspase-2 or caspase-3 and documented a role for caspase-2 in developmental and chemotherapy-induced apoptosis of oocytes. Those data also revealed dispensability of caspase-3, although we found this caspase critical for ovarian granulosa cell death. Because of the mutual interdependence of germ cells and granulosa cells, herein we generated caspase-2 and -3 double-mutant (DKO) mice to evaluate how these two caspases functionally relate to each other in orchestrating oocyte apoptosis. No difference was observed in the rate of spontaneous oocyte apoptosis between DKO and wildtype (WT) females. In contrast, the oocytes from DKO females were more susceptible to apoptosis induced by DNA damaging agents, compared with oocytes from WT females. This increased sensitivity to death of DKO oocytes appears to be a specific response to DNA damage, and it was associated with a compensatory upregulation of caspase-12. Interestingly, DKO oocytes were more resistant to apoptosis induced by methotrexate (MTX) than WT oocytes. These results revealed that in female germ cells, insults that directly interfere with their metabolic status (e.g. MTX) require caspase-2 and caspase-3 as obligatory executioners of the ensuing cell death cascade. However, when DNA damage is involved, and in the absence of caspase-2 and -3, caspase-12 becomes upregulated and mediates apoptosis in oocytes.

CD95 apoptosis resistance in certain cells can be overcome by noncanonical activation of caspase-8.

CD95 apoptosis resistance of tumor cells is often acquired through mutations in the death domain (DD) of one of the CD95 alleles. Furthermore, Type I cancer cells are resistant to induction of apoptosis by soluble CD95 ligand (CD95L), which does not induce efficient formation of the death-inducing signaling complex (DISC). Here, we report that tumor cells expressing a CD95 allele that lacks a functional DD, splenocytes from heterozygous lpr(cg) mice, which express one mutated CD95 allele, and Type I tumor cells stimulated with soluble CD95L can all die through CD95 when protein synthesis or nuclear factor kappa B is inhibited. This noncanonical form of CD95-mediated apoptosis is dependent on the enzymatic activity of procaspase-8 but does not involve fully processed active caspase-8 subunits. Our data suggest that it is possible to overcome the CD95 apoptosis resistance of many tumor cells that do not efficiently form a DISC through noncanonical activation of the caspase-8 proenzyme.

Animal models of tumor-suppressor genes.

The development of cancer requires multiple genetic alterations perturbing distinct cellular pathways. In human cancers, these alterations often arise owing to mutations in tumor-suppressor genes whose normal function is to either inhibit the proliferation, apoptosis, or differentiation of cells, or maintain their genomic integrity. Mouse models for tumor suppressors frequently provide definitive evidence for the antitumorigenic functions of these genes. In addition, animal models permit the identification of previously unsuspected roles of these genes in development and differentiation. The availability of null and tissue-specific mouse mutants for tumor-suppressor genes has greatly facilitated our understanding of the mechanisms leading to cancer. In this review, we describe mouse models for tumor-suppressor genes.

Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death.

Programmed cell death is a fundamental requirement for embryogenesis, organ metamorphosis and tissue homeostasis. In mammals, release of mitochondrial cytochrome c leads to the cytosolic assembly of the apoptosome-a caspase activation complex involving Apaf1 and caspase-9 that induces hallmarks of apoptosis. There are, however, mitochondrially regulated cell death pathways that are independent of Apaf1/caspase-9. We have previously cloned a molecule associated with programmed cell death called apoptosis-inducing factor (AIF). Like cytochrome c, AIF is localized to mitochondria and released in response to death stimuli. Here we show that genetic inactivation of AIF renders embryonic stem cells resistant to cell death after serum deprivation. Moreover, AIF is essential for programmed cell death during cavitation of embryoid bodies-the very first wave of cell death indispensable for mouse morphogenesis. AIF-dependent cell death displays structural features of apoptosis, and can be genetically uncoupled from Apaf1 and caspase-9 expression. Our data provide genetic evidence for a caspase-independent pathway of programmed cell death that controls early morphogenesis.

BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore.

Many apoptotic signaling pathways are directed to mitochondria, where they initiate the release of apoptogenic proteins and open the proposed mitochondrial permeability transition (PT) pore that ultimately results in the activation of the caspase proteases responsible for cell disassembly. BNIP3 (formerly NIP3) is a member of the Bcl-2 family that is expressed in mitochondria and induces apoptosis without a functional BH3 domain. We report that endogenous BNIP3 is loosely associated with mitochondrial membrane in normal tissue but fully integrates into the mitochondrial outer membrane with the N terminus in the cytoplasm and the C terminus in the membrane during induction of cell death. Surprisingly, BNIP3-mediated cell death is independent of Apaf-1, caspase activation, cytochrome c release, and nuclear translocation of apoptosis-inducing factor. However, cells transfected with BNIP3 exhibit early plasma membrane permeability, mitochondrial damage, extensive cytoplasmic vacuolation, and mitochondrial autophagy, yielding a morphotype that is typical of necrosis. These changes were accompanied by rapid and profound mitochondrial dysfunction characterized by opening of the mitochondrial PT pore, proton electrochemical gradient (Deltapsim) suppression, and increased reactive oxygen species production. The PT pore inhibitors cyclosporin A and bongkrekic acid blocked mitochondrial dysregulation and cell death. We propose that BNIP3 is a gene that mediates a necrosis-like cell death through PT pore opening and mitochondrial dysfunction.

Executionary pathway for apoptosis: lessons from mutant mice.

Apoptosis or programmed cell death (PCD) is an evolutionarily conserved cellular process that is essential for normal development and homeostasis of multicellular organisms. Defects in the apoptosis signaling result in many diseases including autoimmune diseases and cancer. The apoptosis signaling pathway was first described genetically in the nematode Caenorhabditis elegans which serves as a framework for the more complex apoptotic pathways that exist in mammals. In this review, we will discuss the apoptotic pathways that are emerging in mammals as elucidated by studies of gene-targeted mutant mice.

Brca1 required for T cell lineage development but not TCR loci rearrangement.

Brca1 (breast cancerl, early onset) deficiency results in early embryonic lethality. As Brca1 is highly expressed in the T cell lineage, a T cell-specific disruption of Brca1 was generated to assess the role of Brca1 in relation to T lymphocyte development. We found that thymocyte development in Brca1-/- mice was impaired not as a result of V(D)J T cell receptor (TCR) recombination but because thymocytes had increased expression of tumor protein p53. Chromosomal damage accumulation and abnormal cell death were observed in mutant cells. We found that cell death inhibitor Bcl-2 overexpression, or p53-/- backgrounds, completely restored survival and development of Brca1-/- thymocytes; peripheral T cell numbers were not totally restored in Brcal-/- p53-/- mice; and that a mutant background for p21 (cyclin-dependent kinase inhibitor 1A) did not restore Brca1-/- thymocyte development, but partially restored peripheral T cell development. Thus, the outcome of Brca1 deficiency was dependent on cellular context, with the major defects being increased apoptosis in thymocytes, and defective proliferation in peripheral T cells.

In vivo evidence that caspase-3 is required for Fas-mediated apoptosis of hepatocytes.

Caspase-3 is essential for Fas-mediated apoptosis in vitro. We investigated the role of caspase-3 in Fas-mediated cell death in vivo by injecting caspase-3-deficient mice with agonistic anti-Fas Ab. Wild-type controls died rapidly of fulminant hepatitis, whereas the survival of caspase-3-/- mice was increased due to a delay in hepatocyte cell death. Bcl-2 expression in the liver was dramatically decreased in wild-type mice following anti-Fas injection, but was unchanged in caspase-3-/- mice. Hepatocytes from anti-Fas-injected wild-type, but not caspase-3-/-, mice released cytochrome c into the cytoplasm. Western blotting confirmed the lack of caspase-3-mediated cleavage of Bcl-2. Presumably the presence of intact Bcl-2 in caspase-3-/- hepatocytes prevents the release of cytochrome c from the mitochondria, a required step for the mitochondrial death pathway. We also show by Western blot that Bcl-xL, caspase-9, caspase-8, and Bid are processed by caspase-3 in injected wild-type mice but that this processing does not occur in caspase-3-/- mice. This study thus provides novel in vivo evidence that caspase-3, conventionally known for its downstream effector function in apoptosis, also modifies Bcl-2 and other upstream proteins involved in the regulation of Fas-mediated apoptosis.

Gene targeting in the analysis of mammalian apoptosis and TNF receptor superfamily signaling.

Apoptosis, or programmed cell death (PCD), is the subject of much current investigative interest. Developing embryos and many adult organ systems require the tight coupling of cellular proliferation and PCD to ensure proper organogenesis and optimal tissue function. Over the past decade, our knowledge of the genetic basis underlying the execution of apoptosis in mammals has progressed enormously, thanks largely to groundbreaking studies performed in the nematode Caenorhabditis elegans. In contrast, the components of the signaling apparatus that links the various death stimuli and the receptors they stimulate to the execution mechanism remain relatively unknown. It is only in the past 4 years that studies of signal transduction via members of the tumor necrosis factor (TNF) receptor superfamily have identified a plethora of novel signaling proteins, including molecules that are directly involved in apoptosis signaling, and others that regulate the induction of cell death. This two-part review focuses on the biology of apoptosis and signaling through members of the TNF receptor superfamily as revealed by the study of gene-targeted "knockout" mice. These genetic mutant animals are invaluable tools not only for confirming or refuting a proposed function of a particular gene in an in vivo setting, but also for uncovering novel functions for a gene that were not anticipated from conventional in vitro experiments. In the field of apoptosis, as for many other areas of biomedical research, knockout mice and cell lines can be used as models for studying human disease, with the ultimate goal of developing therapeutic strategies.

Apaf-1 and caspase-9 in p53-dependent apoptosis and tumor inhibition.

The ability of p53 to promote apoptosis in response to mitogenic oncogenes appears to be critical for its tumor suppressor function. Caspase-9 and its cofactor Apaf-1 were found to be essential downstream components of p53 in Myc-induced apoptosis. Like p53 null cells, mouse embryo fibroblast cells deficient in Apaf-1 and caspase-9, and expressing c-Myc, were resistant to apoptotic stimuli that mimic conditions in developing tumors. Inactivation of Apaf-1 or caspase-9 substituted for p53 loss in promoting the oncogenic transformation of Myc-expressing cells. These results imply a role for Apaf-1 and caspase-9 in controlling tumor development.

Apaf1 is required for mitochondrial pathways of apoptosis and brain development.

Apoptosis is essential for the precise regulation of cellular homeostasis and development. The role in vivo of Apaf1, a mammalian homolog of C. elegans CED-4, was investigated in gene-targeted Apaf1-/- mice. Apaf1-deficient mice exhibited reduced apoptosis in the brain and striking craniofacial abnormalities with hyperproliferation of neuronal cells. Apaf1-deficient cells were resistant to a variety of apoptotic stimuli, and the processing of Caspases 2, 3, and 8 was impaired. However, both Apaf1-/- thymocytes and activated T lymphocytes were sensitive to Fas-induced killing, showing that Fas-mediated apoptosis in these cells is independent of Apaf1. These data indicate that Apaf1 plays a central role in the common events of mitochondria-dependent apoptosis in most death pathways and that this role is critical for normal development.

Differential requirement for caspase 9 in apoptotic pathways in vivo.

Mutation of Caspase 9 (Casp9) results in embryonic lethality and defective brain development associated with decreased apoptosis. Casp9-/- embryonic stem cells and embryonic fibroblasts are resistant to several apoptotic stimuli, including UV and gamma irradiation. Casp9-/- thymocytes are also resistant to dexamethasone- and gamma irradiation-induced apoptosis, but are surprisingly sensitive to apoptosis induced by UV irradiation or anti-CD95. Resistance to apoptosis is accompanied by retention of the mitochondrial membrane potential in mutant cells. In addition, cytochrome c is translocated to the cytosol of Casp9-/- ES cells upon UV stimulation, suggesting that Casp9 acts downstream of cytochrome c. Caspase processing is inhibited in Casp9-/- ES cells but not in thymocytes or splenocytes. Comparison of the requirement for Casp9 and Casp3 in different apoptotic settings indicates the existence of at least four different apoptotic pathways in mammalian cells.

Essential contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes.

Caspases are fundamental components of the mammalian apoptotic machinery, but the precise contribution of individual caspases is controversial. CPP32 (caspase 3) is a prototypical caspase that becomes activated during apoptosis. In this study, we took a comprehensive approach to examining the role of CPP32 in apoptosis using mice, embryonic stem (ES) cells, and mouse embryonic fibroblasts (MEFs) deficient for CPP32. CPP32(ex3-/-) mice have reduced viability and, consistent with an earlier report, display defective neuronal apoptosis and neurological defects. Inactivation of CPP32 dramatically reduces apoptosis in diverse settings, including activation-induced cell death (AICD) of peripheral T cells, as well as chemotherapy-induced apoptosis of oncogenically transformed CPP32(-/-) MEFs. As well, the requirement for CPP32 can be remarkably stimulus-dependent: In ES cells, CPP32 is necessary for efficient apoptosis following UV- but not gamma-irradiation. Conversely, the same stimulus can show a tissue-specific dependence on CPP32: Hence, TNFalpha treatment induces normal levels of apoptosis in CPP32 deficient thymocytes, but defective apoptosis in oncogenically transformed MEFs. Finally, in some settings, CPP32 is required for certain apoptotic events but not others: Select CPP32(ex3-/-) cell types undergoing cell death are incapable of chromatin condensation and DNA degradation, but display other hallmarks of apoptosis. Together, these results indicate that CPP32 is an essential component in apoptotic events that is remarkably system- and stimulus-dependent. Consequently, drugs that inhibit CPP32 may preferentially disrupt specific forms of cell death.

Developmental studies of Brca1 and Brca2 knock-out mice.

In humans, the inheritance of mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 increases the risk of developing breast and ovarian cancer. To study their biological function and to create animal models for these cancer susceptibility genes, several strains of mice mutated in the homologous genes Brca1 and Brca2 have been generated by gene targeting. Analyses of these "knock-out" mouse mutants have provided invaluable knowledge about the function of these genes. Brca1 and Brca2 null mutants are similar in phenotype: mutations in both genes result in embryonic lethality and the developing embryos show signs of a cellular proliferation defect associated with activation of the p53 pathway. The significance of this activation, as well as the role of these cancer susceptibility genes in DNA damage repair, is discussed.

Partial rescue of Brca1 (5-6) early embryonic lethality by p53 or p21 null mutation.

Mutations in the mouse Brca1 gene cause lethality at different embryonic stages. We have shown that Brca1 mutant embryos, in which the fifth and sixth exons of Brca1 are deleted die before E7.5 and show decreased cellular proliferation. Brca1 mutants also show decreased expression of mdm2, a gene encoding an inhibitor of p53 activity. Thus, we have proposed that the reduction in mdm2 expression in Brca1 (5-6) mutants might lead to increased p53 activity. Consistent with this finding, the expression of p21, which encodes a G1 cell cycle inhibitor and is a target for p53 transcriptional activation was dramatically increased in the Brca1 (5-6) mutants, suggesting that impaired cellular proliferation could be due to a G1 cell-cycle arrest, caused by increased p21 levels. To test this hypothesis, we generated mice double mutant for Brca1 (5-6) and p53, or Brca1 (5-6) and p21. Mutation in either p53 or p21 prolonged the survival of Brca1 (5-6) mutant embryos from E7.5 to E9.5. The development of most Brca1 (5-6): p21 double-mutant embryos was comparable to that of their wild-type littermates, although no mutant survived past E10.5. The fact that mutation of neither p53 nor p21 completely rescued Brca1 (5-6) embryos suggests that their lethality is likely due to a multi-factorial process.

Brca2 is required for embryonic cellular proliferation in the mouse.

Mutations of the tumor suppressor gene BRCA2 are associated with predisposition to breast and other cancers. Homozygous mutant mice in which exons 10 and 11 of the Brca2 gene were deleted by gene targeting (Brca2(10-11)) die before day 9.5 of embryogenesis. Mutant phenotypes range from severely developmentally retarded embryos that do not gastrulate to embryos with reduced size that make mesoderm and survive until 8.5 days of development. Although apoptosis is normal, cellular proliferation is impaired in Brca2(10-11) mutants, both in vivo and in vitro. In addition, the expression of the cyclin-dependent kinase inhibitor p21 is increased. Thus, Brca2(10-11) mutants are similar in phenotype to Brca1(5-6) mutants but less severely affected. Expression of either of these two genes was unaffected in mutant embryos of the other. This study shows that Brca2, like Brca1, is required for cellular proliferation during embryogenesis. The similarity in phenotype between Brca1 and Brca2 mutants suggests that these genes may have cooperative roles or convergent functions during embryogenesis.