[Show abstract][Hide abstract] ABSTRACT: PTEN inactivation is the most frequent genetic aberration in endometrial cancer. One of the phosphatase-independent roles of PTEN is associated with homologous recombination (HR) in nucleus. Poly (ADP-ribose) polymerase (PARP) plays key roles in the repair of DNA single-strand breaks, and a PARP inhibitor induces synthetic lethality in cancer cells with HR deficiency. We examined the anti-tumor activity of olaparib, a PARP inhibitor, and its correlation between the sensitivity and status of PTEN in endometrial cancer cell lines.
The response to olaparib was evaluated using a clonogenic assay with SF50 values (concentration to inhibit cell survival to 50%) in 16 endometrial cancer cell lines. The effects of PTEN on the sensitivity to olaparib and ionizing radiation (IR) exposure were compared between parental HEC-6 (PTEN-null) and HEC-6 PTEN + (stably expressing wild-type PTEN) cells by clonogenic assay, foci formation of RAD51 and gammaH2AX, and induction of cleaved PARP. The effects of siRNA to PTEN were analyzed in cells with wild-type PTEN.
The SF50 values were 100 nM or less in four (25%: sensitive) cell lines; whereas, SF50 values were 1,000 nM or more in four (25%: resistant) cell lines. PTEN mutations were not associated with sensitivity to olaparib (Mutant [n = 12]: 746 +/- 838 nM; Wild-type [n = 4]: 215 +/- 85 nM, p = 0.26 by Student's t test). RAD51 expression was observed broadly and was not associated with PTEN status in the 16 cell lines. The number of colonies in the clonogenic assay, the foci formation of RAD51 and gammaH2AX, and the induction of apoptosis were not affected by PTEN introduction in the HEC-6 PTEN + cells. The expression level of nuclear PTEN was not elevated within 24 h following IR in the HEC-6-PTEN + cells. In addition, knocking down PTEN by siRNA did not alter the sensitivity to olaparib in 2 cell lines with wild-type PTEN.
Our results suggest that olaparib, a PARP inhibitor, is effective on certain endometrial cancer cell lines. Inactivation of PTEN might not affect the DNA repair function. Predictive biomarkers are warranted to utilize olaparib in endometrial cancer.
BMC Cancer 03/2014; 14(1):179. DOI:10.1186/1471-2407-14-179 · 3.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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. This article is protected by copyright. All rights reserved.
Cancer Science 01/2014; 105(4). DOI:10.1111/cas.12366 · 3.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The meiosis-specific synaptonemal complex protein SYCP3 has been reported to be aberrantly expressed in tumours. However, in contrast to its well-defined function in meiosis, its possible role in mitotic cells is entirely unknown. Here, we show that SYCP3 is expressed in a range of primary tumours and that it impairs chromosomal integrity in mitotic cells. Expression of SYCP3 inhibits the homologous recombination (HR) pathway mediated by RAD51, inducing hypersensitivity to DNA-damaging agents such as a poly(ADP-ribose) polymerase (PARP) inhibitor and chromosomal instability. SYCP3 forms a complex with BRCA2 and inhibits its role in HR. These findings highlight a new mechanism for chromosomal instability in cancer and extend the range of PARP-inhibitor sensitive tumours to those expressing SYCP3.
[Show abstract][Hide abstract] ABSTRACT: The human Ena/Vasp-like (EVL) protein is considered to be a bifunctional protein, involved in both actin remodeling and homologous recombination. In the present study, we found that human EVL forms heat-stable multimers of circular single-stranded DNA (ssDNA) molecules in the presence of a type I topoisomerase in vitro. An electron microscopic analysis revealed that the heat-stable ssDNA multimers formed by EVL and topoisomerase were ssDNA catemers. The ssDNA catenation did not occur when either EVL or topoisomerase was omitted from the reaction mixture. A deletion analysis revealed that the ssDNA catenation completely depended on the annealing activity of EVL. Human EVL was captured from a human cell extract by TOPO IIIα-conjugated beads, and the interaction between EVL and TOPO IIIα was confirmed by a surface plasmon resonance analysis. Purified TOPO IIIα catalyzed the ssDNA catenation with EVL as efficiently as the Escherichia coli topoisomerase I. Since the ssDNA cutting and rejoining reactions, which are the sub-steps of ssDNA catenation, may be an essential process in homologous recombination, EVL and TOPO IIIα may function in the processing of DNA intermediates formed during homologous recombination.
Nucleic Acids Research 11/2010; 38(21):7579-86. DOI:10.1093/nar/gkq630 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
International Journal of Cancer 06/2009; 124(12):2997-3001. DOI:10.1002/ijc.24268 · 5.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The efficacy of cancer chemotherapy and radiotherapy relies on generation of DNA damage. Since intrinsic DNA repair pathways
enable cancer cells to survive by repairing these damaged lesions, inactivation of DNA repair coupled with chemotherapy and
radiotherapy has a potential to enhance the effect of these therapies. Small molecule compounds that inhibit specific DNA
repair proteins have been developed, and early clinical trials are ongoing. While DNA repair inhibitors have been tried mostly
as a part of combination therapies with cytotoxic agents, recent reports highlighted a new concept in cancer therapy where
DNA repair inhibitors could be used as single agents to selectively kill cancer cells. This concept is based on the findings
that cancer cells are frequently defective in particular DNA repair pathway(s) and the presumption that inhibition of the
compensatory repair pathway(s) in such cells might be useful to kill them. For example, poly(ADP-ribose) polymerase (PARP)
plays a critical role in DNA base-excision repair, and inactivation of this protein increases the number of single-strand
breaks, leading to double-strand breaks that require to be repaired by homologous recombination (HR) mediated by BRCA1 and
BRCA2. Recently, BRCA1- or BRCA2-defective tumour cells were shown to be sensitive to PARP inhibitors alone. This treatment
may be tumour-specific because only the BRCA1−/− or BRCA2−/− tumours in the BRCA1+/− or BRCA2+/− patients are completely defective
in HR repair. The following short review aims at summarizing the basic mechanisms of DNA repair and the therapeutic options
using DNA repair inhibitors in cancer therapy.
memo - Magazine of European Medical Oncology 01/2009; 2(1):9-14. DOI:10.1007/s12254-008-0081-7
[Show abstract][Hide abstract] ABSTRACT: Neurological complications during the treatment of hematological malignancies have a wide range of causes. Treatment-related leukoencephalopathy has been recognized as a major complication of combined chemotherapy and radiotherapy for central nervous system (CNS) lymphoma, and can complicate the diagnosis of CNS infection. Herein, we present a patient with diffuse large B-cell lymphoma who developed herpes simplex encephalitis (HSE) and subsequent cytomegalovirus encephalitis after chemoradiotherapy for CNS relapse. Although cerebrospinal fluid examination (CSF) showed no significant pleocytosis, brain magnetic resonance imaging and polymerase chain reaction analysis of the CSF were useful in the diagnosis. With a review of the literature on the association between HSE and radiotherapy for CNS malignancies, our case suggests that an awareness of viral encephalitis is important in the differential diagnosis of acute neurologic disturbance during chemoradiotherapy for CNS lymphoma.
International Journal of Hematology 07/2008; 87(5):538-41. DOI:10.1007/s12185-008-0083-6 · 1.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
American Journal of Hematology 06/2008; 83(6):472-6. DOI:10.1002/ajh.21152 · 3.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
International Journal of Hematology 05/2008; 87(3):310-8. DOI:10.1007/s12185-008-0051-1 · 1.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although Aspergillus galactomannan (GM) antigen detection is widely applied in the diagnosis of invasive aspergillosis (IA), false-positive reactions with fungus-derived antibiotics, other fungal genera or the passage of dietary GM through injured mucosa are a matter of concern. The aim of this study was to investigate the cumulative incidence and risk factors for false-positive GM antigenaemia.
The records of 157 adult allogeneic haematopoietic stem cell transplantation (HSCT) recipients were retrospectively analysed. Episodes of positive GM antigenaemia, defined as two consecutive GM results with an optical density index above 0.6, were classified into true, false and inconclusive GM antigenaemia by reviewing the clinical course.
Twenty-five patients developed proven or probable IA with a 1 year cumulative incidence of 12.9%, whereas 50 experienced positive GM antigenaemia with an incidence of 32.2%. Among the total 58 positive episodes of the 50 patients, 29 were considered false-positive. The positive predictive value (PPV) was lower during the first 100 days than beyond 100 days after HSCT (37.5% versus 58.8%). Gastrointestinal chronic graft-versus-host disease (GVHD) was identified as the only independent significant factor for the increased incidence of false-positive GM antigenaemia (PPV 0% versus 66.7%, P = 0.02).
GM antigen results must be considered cautiously in conjunction with other diagnostic procedures including computed tomography scans, especially during the first 100 days after HSCT and in patients with gastrointestinal chronic GVHD.
[Show abstract][Hide abstract] ABSTRACT: Cyclosporine A (CsA) is the mainstay of pharmacologic prevention of acute graft-versus-host disease (GVHD). We previously reported that continuous infusion of CsA with a target blood level between 250 and 400 ng/ml significantly increased the incidence of acute GVHD compared to twice-daily infusion with a target trough level between 150 and 300 ng/ml. Thus, we raised the target level of CsA continuous infusion to 450-550 ng/ml. We treated 33 patients with the higher target level (CsA500) and compared the efficacy and toxicity with those in the 33 historical control patients (CsA300 group). Other transplantation procedures were not changed. The patients' characteristics were equivalent. The average CsA concentration was adjusted around 500 ng/ml and the actual daily dose was maintained at the initial dose (CsA 3mg/kg/day). Toxicities were equivalently observed among the two groups. The incidence of grades II-IV acute GVHD was significantly lower in the CsA500 group (27 vs. 52%, P = 0.033). The target level of CsA was identified as an independent significant risk factor for grades II-IV acute GVHD (P = 0.039), adjusted for the presence of HLA mismatch. The incidence of chronic GVHD was also decreased in the CsA500 group (47 vs. 73%, P = 0.016). We conclude that the toxicity of the continuous CsA infusion with a target level of 450-550 ng/ml is acceptable and the efficacy to prevent acute GVHD is significant. A larger comparative study is warranted to confirm these findings.
American Journal of Hematology 03/2008; 83(3):226-32. DOI:10.1002/ajh.21087 · 3.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hemolytic anemia and pure red cell aplasia (PRCA) after allogeneic hematopoietic stem cell transplantation (HSCT) have been reported to be mainly related to ABO-incompatibility between donor and recipient. Autoimmune hemolytic anemia (AIHA) without ABO-incompatibility has been also reported after allogeneic HSCT, especially with T-cell depletion. However, optimal management of AIHA or PRCA remains unclear. A 54-year-old male with myelodysplastic syndrome (MDS) underwent haploidentical human leukocyte antigen-mismatched HSCT using in vivo alemtuzumab and developed AIHA and PRCA simultaneously 15 months after transplantation, following the administration of cidofovir and probenecid for persistent cytomegalovirus (CMV) antigenemia and retinitis. AIHA was successfully treated with rituximab, and subsequently PRCA with cyclosporine without relapse of MDS or recurrence of CMV infection. The clinical course suggested that AIHA was mainly caused by humoral immune response, while PRCA was mainly caused by cell-mediated immune response in this patient, although these immune responses might be related to each other.
American Journal of Hematology 03/2008; 83(3):247-9. DOI:10.1002/ajh.21086 · 3.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The onset of invasive aspergillosis (IA) after allogeneic haematopoietic stem cell transplantation (HSCT) is bimodal. However, IA early after HSCT has become less frequent due to the shortened neutropenic period, and the clinical significance of empirical treatment for aspergillosis based on persistent febrile neutropenia (FN) became less clear. Therefore, we started a presumptive treatment strategy, in which anti-Aspergillus agents were started when patients developed positive serum test and/or infiltrates or nodules on X-ray or CT-scan associated with persistent FN, in 2002.
We retrospectively reviewed the records of 114 adult patients who underwent allogeneic HSCT between September 2002 and December 2005 in high-efficiency particulate air-filtered clean rooms. Fluconazole was given as anti-Candida prophylaxis. The primary endpoint was the development of early IA, which was defined as probable or proven IA according to the EORTC/MSG criteria that developed between the day of HSCT and 7 days after engraftment.
Among 73 patients who experienced persistent FN for 7 days or longer, anti-Aspergillus agents were empirically started in 13 patients at the discretion of attending physicians, whereas 60 patients actually followed presumptive treatment strategy. Only 4 of 60 patients received anti-Aspergillus agents. Two patients in the presumptive group developed early IA, but were successfully treated with anti-Aspergillus agents started after the diagnosis of IA.
These findings suggested the feasibility of a presumptive treatment strategy for aspergillosis in HSCT recipients. A randomized controlled trial is warranted to compare empirical and presumptive anti-Aspergillus strategy in allogeneic HSCT recipients.
[Show abstract][Hide abstract] ABSTRACT: 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.
Genes Chromosomes and Cancer 05/2006; 45(5):482-94. DOI:10.1002/gcc.20303 · 4.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Cancer Research 08/2005; 65(14):6071-9. DOI:10.1158/0008-5472.CAN-05-0465 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Genes Chromosomes and Cancer 03/2005; 42(3):269-79. DOI:10.1002/gcc.20147 · 4.04 Impact Factor