Noriko Hosoya

A Robust Algorithm for Copy Number Detection Using High-Density Oligonucleotide Single Nucleotide Polymorphism Genotyping Arrays

We have developed a robust algorithm for copy number analysis of the human genome using high-density oligonucleotide microarrays containing 116,204 single-nucleotide polymorphisms. The advantages of this algorithm include the improvement of signal-to-noise (S/N) ratios and the use of an optimized reference. The raw S/N ratios were improved by accounting for the length and GC content of the PCR products using quadratic regressions. The use of constitutional DNA, when available, gives the lowest SD values (0.16 +/- 0.03) and also enables allele-based copy number detection in cancer genomes, which can unmask otherwise concealed allelic imbalances. In the absence of constitutional DNA, optimized selection of multiple normal references with the highest S/N ratios, in combination with the data regressions, dramatically improves SD values from 0.67 +/- 0.12 to 0.18 +/- 0.03. These improvements allow for highly reliable comparison of data across different experimental conditions, detection of allele-based copy number changes, and more accurate estimations of the range and magnitude of copy number aberrations. This algorithm has been implemented in a software package called Copy Number Analyzer for Affymetrix GeneChip Mapping 100K arrays (CNAG). Overall, these enhancements make CNAG a useful tool for high-resolution detection of copy number alterations which can help in the understanding of the pathogenesis of cancers and other diseases as well as in exploring the complexities of the human genome.

Mouse Jagged1 physically interacts with Notch2 and other Notch receptors. Assessment by quantitative methods

The Delta/Serrate/LAG-2 (DSL) domain containing proteins are considered to be ligands for Notch receptors. However, the physical interaction between DSL proteins and Notch receptors is poorly understood. In this study, we cloned a cDNA for mouse Jagged1 (mJagged1). To identify the receptor interacting with mJagged1 and to gain insight into its binding characteristics, we established two experimental systems using fusion proteins comprising various extracellular parts of mJagged1, a “cell” binding assay and a “solid-phase” binding assay. mJagged1 physically bound to mouse Notch2 (mNotch2) on the cell surface and to a purified extracellular portion of mNotch2, respectively, in a Ca2+-dependent manner. Scatchard analysis of mJagged1 binding to BaF3 cells and to the soluble Notch2 protein demonstrated dissociation constants of 0.4 and 0.7 nm, respectively, and that the number of mJagged1-binding sites on BaF3 is 5,548 per cell. Furthermore, deletion mutant analyses showed that the DSL domain of mJagged1 is a minimal binding unit and is indispensable for binding to mNotch2. The epidermal growth factor-like repeats of mJagged1 modulate the affinity of the interaction, with the first and second repeats playing a major role. Finally, solid-phase binding assay showed that Jagged1 binds to Notch1 and Notch3 in addition to Notch2, suggesting that mJagged1 is a ligand for multiple Notch receptors.

Binding of Delta1, Jagged1, and Jagged2 to Notch2 Rapidly Induces Cleavage, Nuclear Translocation, and Hyperphosphorylation of Notch2

ABSTRACT Delta1, Jagged1, and Jagged2, commonly designated Delta/Serrate/LAG-2 (DSL) proteins, are known to be ligands for Notch1. However, it has been less understood whether they are ligands for Notch receptors other than Notch1. Meanwhile, ligand-induced cleavage and nuclear translocation of the Notch protein are considered to be fundamental for Notch signaling, yet direct observation of the behavior of the Notch molecule after ligand binding, including cleavage and nuclear translocation, has been lacking. In this report, we investigated these issues for Notch2. All of the three DSL proteins bound to endogenous Notch2 on the surface of BaF3 cells, although characteristics of Jagged2 for binding to Notch2 apparently differed from that of Delta1 and Jagged1. After binding, the three DSL proteins induced cleavage of the membrane-spanning subunit of Notch2 (Notch2TM), which occurred within 15 min. In a simultaneous time course, the cleaved fragment of Notch2TMwas translocated into the nucleus. Interestingly, the cleaved Notch2 fragment was hyperphosphorylated also in a time-dependent manner. Finally, binding of DSL proteins to Notch2 also activated the transcription of reporter genes driven by the RBP-Jκ-responsive promoter. Together, these data indicate that all of these DSL proteins function as ligands for Notch2. Moreover, the findings of rapid cleavage, nuclear translocation, and phosphorylation of Notch2 after ligand binding facilitate the understanding of the Notch signaling.

Targeting DNA damage response in cancer therapy.

Cancer chemotherapy and radiotherapy are designed to kill cancer cells mostly by inducing DNA damage. DNA damage is normally recognized and repaired by the intrinsic DNA damage response machinery. If the damaged lesions are successfully repaired, the cells will survive. In order to specifically and effectively kill cancer cells by therapies that induce DNA damage, it is important to take advantage of specific abnormalities in the DNA damage response machinery that are present in cancer cells but not in normal cells. Such properties of cancer cells can provide biomarkers or targets for sensitization. For example, defects or upregulation of the specific pathways that recognize or repair specific types of DNA damage can serve as biomarkers of favorable or poor response to therapies that induce such types of DNA damage. Inhibition of a DNA damage response pathway may enhance the therapeutic effects in combination with the DNA‐damaging agents. Moreover, it may also be useful as a monotherapy when it achieves synthetic lethality, in which inhibition of a complementary DNA damage response pathway selectively kills cancer cells that have a defect in a particular DNA repair pathway. The most striking application of this strategy is the treatment of cancers deficient in homologous recombination by poly(ADP‐ribose) polymerase inhibitors. In this review, we describe the impact of targeting the cancer‐specific aberrations in the DNA damage response by explaining how these treatment strategies are currently being evaluated in preclinical or clinical trials.

Long‐term ultra‐low‐dose acyclovir against varicella‐zoster virus reactivation after allogeneic hematopoietic stem cell transplantation

To evaluate the efficacy of long‐term prophylaxis with ultra‐low‐dose acyclovir against varicella‐zoster virus (VZV) reactivation, we analyzed the records of 242 Japanese adult patients who underwent allogeneic hematopoietic stem cell transplantation for the first time from 1995 to 2006 at our hospital. We started long‐term oral acyclovir at 200 mg/day in July 2001. Acyclovir was continued until the end of immunosuppressive therapy and at least 1 year after transplantation. Sixty‐six patients developed VZV reactivation at a median of 248 days after HSCT, with a cumulative incidence of 34.7%. Only one breakthrough reactivation occurred during long‐term acyclovir, which responded well to therapeutic dose of valacyclovir. The use of long‐term acyclovir was the only independent determinant that significantly decreased the overall incidence of VZV reactivation (20% vs. 50%, P < 0.0001). With this prophylaxis, visceral dissemination and serious complications other than post‐herpetic neuralgia was completely eliminated, and thereby need for hospitalization was significantly reduced (21% vs. 71%, P = 0.0034). Fifteen of the 57 patients who discontinued acyclovir developed VZV reactivation, with a cumulative incidence of 32.1%. VZV reactivation following discontinuation tended to occur in patients who were receiving immunosuppressive therapy at the cessation of acyclovir. These findings suggested that long‐term prophylaxis of ultra‐low‐dose acyclovir resulted in a successful prevention of severe VZV‐related symptoms and death, with a significantly decreased overall incidence of VZV reactivation. Prolongation of prophylactic acyclovir on profound immunosuppression might be important for thorough suppression of VZV reactivation. Am. J. Hematol., 2008.

Genomewide screening of DNA copy number changes in chronic myelogenous leukemia with the use of high-resolution array-based comparative genomic hybridization

Chronic myelogenous leukemia (CML) evolves from an indolent chronic phase (CP) characterized by the Philadelphia chromosome. Without effective therapy, it progresses to an accelerated phase (AP) and eventually to a fatal blast crisis (BC). To identify the genes involved in stage progression in CML, we performed a genomewide screening of DNA copy number changes in a total of 55 CML patients in different stages with the use of the high‐resolution array‐based comparative genomic hybridization (array CGH) technique. We constructed Human 1M arrays that contained 3,151 bacterial artificial chromosome (BAC) DNAs, allowing for an average resolution of 1.0 Mb across the entire genome. In addition to common chromosomal abnormalities, array CGH analysis unveiled a number of novel copy number changes. These alterations included losses in 2q26.2–q37.3, 5q23.1–q23.3, 5q31.2–q32, 7p21.3–p11.2, 7q31.1–q31.33, 8pter‐p12(p11.2), 9p, and 22q13.1–q13.31 and gains in 3q26.2–q29, 6p22.3, 7p15.2–p14.3, 8p12, 8p21.3, 8p23.2, 8q24.13–q24.21, 9q, 19p13.2–p12, and 22q13.1–q13.32 and occurred at a higher frequency in AP and BC. Minimal copy number changes affecting even a single BAC locus were also identified. Our data suggests that at least a proportion of CML patients carry still‐unknown cryptic genomic alterations that could affect a gene or genes of importance in the disease progression of CML. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045‐2257/suppmat.

Clinical features of late cytomegalovirus infection after hematopoietic stem cell transplantation

Late cytomegalovirus (CMV) disease beyond day 100 after hematopoietic stem cell transplantation (HSCT) has become an increasing problem after the introduction of preemptive ganciclovir (GCV) administration. To clarify the risk factors and outcome for late CMV reactivation and disease, we retrospectively analyzed the records of 101 Japanese adult patients who underwent allogeneic HSCT between 1998 and 2005 at our hospital. Fifty-one developed late positive CMV antigenemia, with a cumulative incidence of 53%. Recipient CMV seropositivity, the use of alemtuzumab, chronic GVHD, and high-dose steroids were significantly associated with late positive antigenemia. Eight patients developed late CMV disease, with a cumulative incidence of 8%, including retinitis and gastrointestinal disease. None progressed to a fatal disease. The use of alemtuzumab was identified as an independent significant risk factor for late CMV disease, although it was not associated with increased non-relapse mortality. Among the 51 patients with late positive antigenemia, 28 had consistently less than three positive cells, 25 of whom showed negative conversion without antiviral agents. In conclusion, late CMV antigenemia appeared to develop frequently, especially in patients with profound immune suppression; however, a fatal outcome could be prevented by optimal preemptive therapy. Low-level antigenemia may not require antiviral treatments.

Identification of a SRC-like tyrosine kinase gene, FRK, fused with ETV6 in a patient with acute myelogenous leukemia carrying a t(6;12)(q21;p13) translocation.

The SRC family of kinases is rarely mutated in primary human tumors. We report the identification of a SRC‐like tyrosine kinase gene, FRK (Fyn‐related kinase), fused with ETV6 in a patient with acute myelogenous leukemia carrying t(6;12)(q21;p13). Both reciprocal fusion transcripts, ETV6/FRK and FRK/ETV6, were expressed. In ETV6/FRK, exon 4 of ETV6 was fused in‐frame to exon 3 of FRK, producing a chimeric protein consisting of the entire oligomerization domain of ETV6 and the kinase domain of FRK. The ETV6/FRK protein was shown to be constitutively autophosphorylated on its tyrosine residues. ETV6/FRK phosphorylated histones H2B and H4 in vitro to a greater extent than did FRK, suggesting it had elevated kinase activity. ETV6/FRK could transform both Ba/F3 cells and NIH3T3 cells, which depended on its kinase activity. Moreover, ETV6/FRK inhibited ETV6‐mediated transcriptional repression in a dominant‐negative manner. This report provides the first evidence that a SRC‐like kinase gene, FRK fused with ETV6, could directly contribute to leukemogenesis by producing an oncoprotein, ETV6/FRK, with dual functions: constitutive activation of the ETV6/FRK tyrosine kinase and dominant‐negative modulation of ETV6‐mediated transcriptional repression.

Functional evidence for Eme1 as a marker of cisplatin resistance.

The ability to predict cisplatin sensitivity in tumors has been expected to greatly improve the outcome of cancer therapy, because the drug is frequently used in a variety of tumors. Although ERCC1 and other repair proteins have been investigated as markers of cisplatin resistance, reliable markers are still needed. Here, we demonstrate that Eme1 levels can predict cisplatin sensitivity more accurately than ERCC1 or Rad51 levels in a variety of human cancer cell lines. Eme1 forms a heterodimeric protein complex with Mus81 and functions as a structure‐specific endonuclease. Haploinsufficiency of Eme1 led to hypersensitivity to cisplatin in the colon cancer cell line HCT116. On the basis of this finding, we examined the relationships between levels of proteins involved in the repair of interstrand cross‐links and cisplatin sensitivity in human tumor cell lines with a variety of origins. Although ERCC1, Rad51 and Mus81 levels correlated with sensitivity to some extent, the clearest correlation was observed with Eme1. Tumors with low Eme1 levels were more sensitive to the drug than tumors with high levels. This suggests that the measurement of Eme1 in tumors may be more informative for cisplatin‐based chemotherapy than that of the currently available markers.

Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL

The RAD51 protein is a central player in homologous recombinational repair. The RAD51B protein is one of five RAD51 paralogs that function in the homologous recombinational repair pathway in higher eukaryotes. In the present study, we found that the human EVL (Ena/Vasp-like) protein, which is suggested to be involved in actin-remodeling processes, unexpectedly binds to the RAD51 and RAD51B proteins and stimulates the RAD51-mediated homologous pairing and strand exchange. The EVL knockdown cells impaired RAD51 assembly onto damaged DNA after ionizing radiation or mitomycin C treatment. The EVL protein alone promotes single-stranded DNA annealing, and the recombination activities of the EVL protein are further enhanced by the RAD51B protein. The expression of the EVL protein is not ubiquitous, but it is significantly expressed in breast cancer-derived MCF7 cells. These results suggest that the EVL protein is a novel recombination factor that may be required for repairing specific DNA lesions, and that may cause tumor malignancy by its inappropriate expression.