Eli S. Williams

NBS1 knockdown by small interfering RNA increases ionizing radiation mutagenesis and telomere association in human cells

Hypomorphic mutations which lead to decreased function of the NBS1 gene are responsible for Nijmegen breakage syndrome, a rare autosomal recessive hereditary disorder that imparts an increased predisposition to development of malignancy. The NBS1 protein is a component of the MRE11/RAD50/NBS1 complex that plays a critical role in cellular responses to DNA damage and the maintenance of chromosomal integrity. Using small interfering RNA transfection, we have knocked down NBS1 protein levels and analyzed relevant phenotypes in two closely related human lymphoblastoid cell lines with different p53 status, namely wild-type TK6 and mutated WTK1. Both TK6 and WTK1 cells showed an increased level of ionizing radiation-induced mutation at the TK and HPRT loci, impaired phosphorylation of H2AX (gamma-H2AX), and impaired activation of the cell cycle checkpoint regulating kinase, Chk2. In TK6 cells, ionizing radiation-induced accumulation of p53/p21 and apoptosis were reduced. There was a differential response to ionizing radiation-induced cell killing between TK6 and WTK1 cells after NBS1 knockdown; TK6 cells were more resistant to killing, whereas WTK1 cells were more sensitive. NBS1 deficiency also resulted in a significant increase in telomere association that was independent of radiation exposure and p53 status. Our results provide the first experimental evidence that NBS1 deficiency in human cells leads to hypermutability and telomere associations, phenotypes that may contribute to the cancer predisposition seen among patients with this disease.

DNA double-strand breaks are not sufficient to initiate recruitment of TRF2.

Kelvin Y K Chan1, Vera S F Chan2, Yongxiong Chen2, Shea-Ping Yip3, Chen-Lung S Lin2& Ui-Soon Khoo1 1Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Special Administrative Region, China. 2Division of Surgery, Oncology, Reproduction Biology and Anaesthetics, Faculty of Medicine, Imperial College London, Hammersmith Hospital, London, UK. 3Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong, Special Administrative Region, China. e-mail: steve.lin@imeprial.ac.uk or uskhoo@pathology.hku.hk

Translation Initiation Factor eIF4E Is a Target for Tumor Cell Radiosensitization

A core component in the cellular response to radiation occurs at the level of translational control of gene expression. Because a critical element in translation control is the availability of the initiation factor eIF4E, which selectively enhances the cap-dependent translation of mRNAs, we investigated a regulatory role for eIF4E in cellular radiosensitivity. eIF4E silencing enhanced the radiosensitivity of tumor cell lines but not normal cells. Similarly, pharmacologic inhibition of eIF4E with ribavirin also enhanced tumor cell radiosensitivity. eIF4E attenuation did not affect cell-cycle phase distribution or radiation-induced apoptosis, but it delayed the dispersion of radiation-induced γH2AX foci and increased the frequency of radiation-induced mitotic catastrophe. Radiation did not affect 4E-BP1 phosphorylation or cap-complex formation but it increased eIF4E binding to more than 1,000 unique transcripts including many implicated in DNA replication, recombination, and repair. Taken together, our findings suggest that eIF4E represents a logical therapeutic target to increase tumor cell radiosensitivity.

Suppression of DNA-PK by RNAi has Different Quantitative Effects on Telomere Dysfunction and Mutagenesis in Human Lymphoblasts Treated with γ Rays or HZE Particles

Abstract Zhang, Q., Williams, E. S., Askin, K. F., Peng, Y., Bedford, J. S., Liber, H. L. and Bailey, S. M. Suppression of DNA-PK by RNAi has Different Quantitative Effects on Telomere Dysfunction and Mutagenesis in Human Lymphoblasts Treated with γ Rays or HZE Particles. Radiat. Res. 164, 497–504 (2005). Basic to virtually all relevant biological effects of ionizing radiation is the underlying damage produced in DNA and the subsequent cellular processing of such damage. The damage can be qualitatively different for different kinds of radiations, and the genetics of the biological systems exposed can greatly affect damage processing and ultimate outcome—the biological effect of concern. The accurate repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity and function. Incorrect repair of such lesions results in chromosomal rearrangements and mutations that can lead to cancer and heritable defects in the progeny of irradiated parents. We have focused on the consequent phenotypic effects of faulty repair by examining connections between cellular radiosensitivity phenotypes relevant for carcinogenesis after exposure to ionizing radiation, and deficiencies in various components of the non-homologous end-joining (NHEJ) system. Here we produced deficiencies of individual components of the DNA-dependent protein kinase (DNA-PK) holoenzyme (Ku86 and the catalytic subunit, DNA-PKcs), both singly and in combination, using RNA interference (RNAi) in human lymphoblastoid cell lines. Exposure of cells exhibiting reduced protein expression to either γ rays or 1 GeV/nucleon iron particles demonstrated differential effects on telomere dysfunction and mutation frequency as well as differential effects between radiation qualities.

22q11.2 deletion syndrome presenting with early-onset Parkinson's disease.

Velocardiofacial syndrome, also known as DiGeorge syndrome, is a multisystem genetic disorder caused by microdeletion of 3.0 megabases (Mb) of DNA on the long arm of chromosome 22. Recently, 22q11.2 deletion syndrome (22q11.2DS) has been recognized as a potential risk factor for the development of Parkinson’s disease (PD). We report on a patient with 22q11.2DS who was identified when he presented with early-onset PD. A 37-year-old man presented for PD treatment. He first noted a right rest tremor at age 30. He subsequently developed a similar tremor in his left hand with noted difficulty manipulating objects and utensils. He was diagnosed with PD at age 31 and had improvement with levodopa initiation. Over time, he required increasing doses of L-dopa to manage his motor symptoms and developed motor fluctuations and dyskinesias. His medications were carbidopa/L-dopa 25/ 100 mg, 1 tablet 5 times a day; entacapone 200 mg, 1 tablet 4 times a day; and amantadine 100 mg, 1 tablet 3 times a

Chromosome orientation fluorescence in situ hybridization (CO-FISH).

Cold Spring Harb Protoc; Eli S. Williams and Susan M. Bailey Chromosome Orientation Fluorescence In Situ Hybridization (CO-FISH) Service Email Alerting click here. Receive free email alerts when new articles cite this article Categories Subject Cold Spring Harbor Protocols. Browse articles on similar topics from (341 articles) Visualization, general (463 articles) Visualization (64 articles) Probes (30 articles) Non-isotopically Labeled Probes (1009 articles) Molecular Biology, general (80 articles) In Situ Hybridization (1037 articles) Cell Biology, general

Mixed Gonadal Germ Cell Tumor Composed of a Spermatocytic Tumor-Like Component and Germinoma Arising in Gonadoblastoma in a Phenotypic Woman With a 46, XX Peripheral Karyotype: Report of the First Case

We report a unique case of gonadal mixed germ cell tumor (GCT) composed of a predominantly spermatocytic tumor (ST)-like component and a minor component of germinoma arising in gonadoblastoma in a phenotypic woman with a 46, XX peripheral karotype. The patient was a 24-year-old woman (gravida 2, para 1) found to have a 7 cm pelvic mass during routine obstetric ultrasound examination at 20 weeks gestational age. She underwent a left salpingo-gonadectomy at gestational age 23 and 2/7 weeks. She recovered well and delivered a healthy baby at full term. The resected gonadal tumor measured 7.5 cm and microscopically was composed of 3 morphologically distinct components: gonadoblastoma (1%), germinoma (1%) and a ST-like component (98%). The ST-like component was composed of 3 populations of tumor cells: small cells, intermediate and large sized cells, similar to testicular ST. Scattered binucleated and multinucleated cells were present. Immunohistochemically the ST-like component was positive for pan-GCT markers SALL4 and LIN28 but with weaker staining than the germinoma. It was negative for OCT4 and TCL1. Only rare tumor cells were positive for SOX17. In contrast, the germinoma cells were diffusely and strongly positive for SALL4, LIN28, OCT4, SOX17, and TCL1. CD117 was positive in both the germinoma and ST-like component but with fewer tumor cells positive in the latter. Flurorescence in situ hybridization study demonstrated isochromosome 12p in the germinoma component but not in the gonadoblastoma and ST-like component. This patient did not receive further chemoradiation therapy after the surgery. She has been free of disease for 10 years and 1 month since her surgery. To our knowledge, this is the first case report of a ST-like GCT in a phenotypic female.

Molecular Cytogenetics Guides Massively Parallel Sequencing of a Radiation-Induced Chromosome Translocation in Human Cells

Chromosome rearrangements are large-scale structural variants that are recognized drivers of oncogenic events in cancers of all types. Cytogenetics allows for their rapid, genome-wide detection, but does not provide gene-level resolution. Massively parallel sequencing (MPS) promises DNA sequence-level characterization of the specific breakpoints involved, but is strongly influenced by bioinformatics filters that affect detection efficiency. We sought to characterize the breakpoint junctions of chromosomal translocations and inversions in the clonal derivatives of human cells exposed to ionizing radiation. Here, we describe the first successful use of DNA paired-end analysis to locate and sequence across the breakpoint junctions of a radiation-induced reciprocal translocation. The analyses employed, with varying degrees of success, several well-known bioinformatics algorithms, a task made difficult by the involvement of repetitive DNA sequences. As for underlying mechanisms, the results of Sanger sequencing suggested that the translocation in question was likely formed via microhomology-mediated non-homologous end joining (mmNHEJ). To our knowledge, this represents the first use of MPS to characterize the breakpoint junctions of a radiation-induced chromosomal translocation in human cells. Curiously, these same approaches were unsuccessful when applied to the analysis of inversions previously identified by directional genomic hybridization (dGH). We conclude that molecular cytogenetics continues to provide critical guidance for structural variant discovery, validation and in “tuning” analysis filters to enable robust breakpoint identification at the base pair level.

Phase determination using chromosomal microarray and fluorescence in situ hybridization in a patient with early onset Parkinson disease and two deletions in PRKN

Mutations in the parkin gene (PRKN) are the most commonly identified genetic factors in early onset Parkinson disease (EOPD), with biallelic mutations, resulting in a clinical phenotype. However, normal variation is also common in PRKN, particularly in the form of copy number variation (CNV), challenging interpretation of genetic testing results. Here we report a case of a 29‐year‐old male with EOPD and two deletions in PRKN detected by chromosomal microarray (CMA).

Updates in the Pathology of Precursor Lymphoid Neoplasms in the Revised Fourth Edition of the WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues

Purpose of ReviewAcute lymphoblastic leukemias (ALL) are malignant disorders of immature B or T cells that occur characteristically in children, usually under the age of 6 (75%). Approximately 6000 new cases of ALL are diagnosed each year in the USA, 80–85% of which represent B-ALL forms. Most presentations of B-ALL are leukemic, whereas T-ALL presents with a mediastinal mass, with or without leukemic involvement. The revised fourth edition of the World Health Organization (WHO) classification (2017) has introduced some changes in both B and T-ALL. Here, we summarize the categories of lymphoblastic leukemia/lymphomas as defined by the WHO and recent developments in the understanding of this group of hematologic malignancy.Recent FindingsTwo provisional categories of B-ALL have now been identified including B-ALL, BCR-ABL1-like, and B-ALL with iAMP21. The Philadelphia chromosome-like B-ALL includes forms of the disease that shares the expression profiling of B-ALL with t(9;22) but lack such rearrangement. The second one shows amplification of part of the chromosome 21. Both entities are associated with worse prognosis. Within the T-ALL group, an early precursor T cell form has now been introduced as a provisional category. Such group demonstrates expression of stem cell and myeloid markers in conjunction with the T cell antigens.SummaryThe current review summarizes the recent updates to the WHO classification.