DNA double-strand breaks (DSBs) are extremely dangerous lesions with severe consequences for cell survival and the maintenance of genomic stability. In higher eukaryotic cells, DSBs in chromatin promptly initiate the phosphorylation of the histone H2A variant, H2AX, at Serine 139 to generate γ-H2AX. This phosphorylation event requires the activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs, ATM, and ATR, and serves as a landing pad for the accumulation and retention of the central components of the signaling cascade initiated by DNA damage. Regions in chromatin with γ-H2AX are conveniently detected by immunofluorescence microscopy and serve as beacons of DSBs. This has allowed the development of an assay that has proved particularly useful in the molecular analysis of the processing of DSBs. Here, we first review the role of γ-H2AX in DNA damage response in the context of chromatin and discuss subsequently the use of this modification as a surrogate marker for mechanistic studies of DSB induction and processing. We conclude with a critical analysis of the strengths and weaknesses of the approach and present some interesting applications of the resulting methodology.

Gene-modified autologous hematopoietic stem cells (HSC) can provide ample clinical benefits to subjects suffering from X-linked chronic granulomatous disease (X-CGD), a rare inherited immunodeficiency characterized by recurrent, often life-threatening bacterial and fungal infections. Here we report on the molecular and cellular events observed in two young adults with X-CGD treated by gene therapy in 2004. After the initial resolution of bacterial and fungal infections, both subjects showed silencing of transgene expression due to methylation of the viral promoter, and myelodysplasia with monosomy 7 as a result of insertional activation of ecotropic viral integration site 1 (EVI1). One subject died from overwhelming sepsis 27 months after gene therapy, whereas a second subject underwent an allogeneic HSC transplantation. Our data show that forced overexpression of EVI1 in human cells disrupts normal centrosome duplication, linking EVI1 activation to the development of genomic instability, monosomy 7 and clonal progression toward myelodysplasia. 1 Institute for Biomedical Research, Georg-Speyer-Haus, Frankfurt, Germany. 2 Department of Hematology/Oncology, University Medical School, Frankfurt, Germany. 3 Institute of Human Genetics, University Hospital Heidelberg, Heidelberg, Germany. 4 Molecular Epidemiology Group, German Cancer Research Center, Heidelberg, Germany. 5 University Women's Clinic, Division Molecular Biology of Breast Cancer, Heidelberg, Germany. 6 Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany. 7 Clinical Cooperation Unit Molecular Hematology/Oncology, German Cancer Research Center and Department of Internal Medicine V, University of Heidelberg, Heidelberg, Germany. 7 Pediatric Hematology, Oncology and Hemostaseology, University Medical School, Frankfurt, Germany. 8 Department of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany. 9 Department of Hematology, Oncology and Transfusion Medicine, Charité, Campus Benjamin Franklin, Berlin, Germany. 10 Institute of Pathology, Heinrich-Heine University, Düsseldorf, Germany. 11 EUFETS AG, Idar-Oberstein, Germany. 12 Centre for Immunodeficiency, UCL Institute of Child Health, and Great Ormond Street Hospital for Children NHS Trust London, UK. 13 Division of Immunology/Hematology, University Children's Hospital Zurich, Zurich, Switzerland. 15 These authors contributed equally to this work. a r t i c l e sThe subject received daily granulocyte colonystimulating factor (G-CSF) support (5 µg per kg body weight per day) from months 18 to 20 and months 24 to 26, as well as multiple red blood and platelet transfusions. Following a dental abscess and a febrile episode requiring antibiotic and antimycotic treatment, subject 1 was noted to have extensive splenomegaly and underwent splenectomy at month 25 to avoid spontaneous rupture. Histopathological examination of the spleen revealed extramedullary hematopoiesis and siderosis in the red pulp, without signs of dys...

The multispanning membrane protein Ste6, a member of the ABC-transporter family, is transported to the yeast vacuole for degradation. To identify functions involved in the intracellular trafficking of polytopic membrane proteins, we looked for functions that block Ste6 transport to the vacuole upon overproduction. In our screen, we identified several known vacuolar protein sorting (VPS) genes (SNF7/VPS32, VPS4, and VPS35) and a previously uncharacterized open reading frame, which we named MOS10 (more of Ste6). Sequence analysis showed that Mos10 is a member of a small family of coiled-coil-forming proteins, which includes Snf7 and Vps20. Deletion mutants of all three genes stabilize Ste6 and show a "class E vps phenotype." Maturation of the vacuolar hydrolase carboxypeptidase Y was affected in the mutants and the endocytic tracer FM4-64 and Ste6 accumulated in a dot or ring-like structure next to the vacuole. Differential centrifugation experiments demonstrated that about half of the hydrophilic proteins Mos10 and Vps20 was membrane associated. The intracellular distribution was further analyzed for Mos10. On sucrose gradients, membrane-associated Mos10 cofractionated with the endosomal t-SNARE Pep12, pointing to an endosomal localization of Mos10. The growth phenotypes of the mutants suggest that the "Snf7-family" members are involved in a cargo-specific event.

The RUNX1/ETO (RE) fusion protein, which originates from the t(8;21) chromosomal rearrangement, is one of the most frequent translocation products found in de novo acute myeloid leukemia (AML). In RE leukemias, activated forms of the c-KIT tyrosine kinase receptor are frequently found, thereby suggesting oncogenic cooperativity between these oncoproteins in the development and maintenance of t(8;21) malignancies. In this report, we show that activated c-KIT cooperates with a C-terminal truncated variant of RE, REtr, to expand human CD34+ hematopoietic progenitors ex vivo. CD34+ cells expressing both oncogenes resemble the AML-M2 myeloblastic cell phenotype, in contrast to REtr-expressing cells which largely undergo granulocytic differentiation. Oncogenic c-KIT amplifies REtr-depended clonogenic growth and protects cells from exhaustion. Activated c-KIT reverts REtr-induced DNA damage and apoptosis. In the presence of activated c-KIT, REtr-downregulated DNA-repair genes are re-expressed leading to an enhancement of DNA-repair efficiency via homologous recombination. Together, our results provide new mechanistic insight into REtr and c-KIT oncogenic cooperativity and suggest that augmented DNA repair accounts for the increased chemoresistance observed in t(8;21)-positive AML patients with activated c-KIT mutations. This cell-protective mechanism might represent a new therapeutic target, as REtr cells with activated c-KIT are highly sensitive to pharmacological inhibitors of DNA repair.

We looked for membrane-associated Dubs (deubiquitinating enzymes) among the 16 yeast members of the ubiquitinspeci¢c processing protease (Ubp) family to identify potential regulators of ubiquitin-dependent processes at membranes. For each of the Ubps examined, a certain fraction was found to be membrane associated. This fraction was only small for most Ubps but quite substantial for some Ubps. For Ubp4/Doa4 almost 40% of the protein was found in the membrane fraction suggesting that this protein performs a major function at membranes, probably at endosomes. Among the proteins tested, only one protein (Ubp16) was exclusively membrane associated. By cell fractionation and immuno£uorescence experiments, we could show that Ubp16 is localized to mitochondria. Ubp16 contains an N-terminal hydrophobic domain that is similar to N-terminal sequences of other yeast outer mitochondrial membrane proteins. The presence of this putative signal sequence and the result of protease protection experiments suggest that Ubp16 is an integral membrane protein of the outer mitochondrial membrane with an N in^Cout orientation. Phenotypic characterization of the v vubp16 mutant and overexpression studies further suggest that Ubp16 is probably not important for the general functioning of mitochondria, but that it rather performs a more specialized function at mitochondria. ß