Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 4 de 4
Filter
Add more filters










Database
Language
Publication year range
1.
Nat Commun ; 9(1): 746, 2018 02 21.
Article in English | MEDLINE | ID: mdl-29467415

ABSTRACT

PARP1 regulates the repair of DNA single-strand breaks generated directly, or during base excision repair (BER). However, the role of PARP2 in these and other repair mechanisms is unknown. Here, we report a requirement for PARP2 in stabilising replication forks that encounter BER intermediates through Fbh1-dependent regulation of Rad51. Whereas PARP2 is dispensable for tolerance of cells to SSBs or homologous recombination dysfunction, it is redundant with PARP1 in BER. Therefore, combined disruption of PARP1 and PARP2 leads to defective BER, resulting in elevated levels of replication-associated DNA damage owing to an inability to stabilise Rad51 at damaged replication forks and prevent uncontrolled DNA resection. Together, our results demonstrate how PARP1 and PARP2 regulate two independent, but intrinsically linked aspects of DNA base damage tolerance by promoting BER directly, and by stabilising replication forks that encounter BER intermediates.


Subject(s)
DNA Helicases/metabolism , DNA Repair , DNA-Binding Proteins/metabolism , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Rad51 Recombinase/metabolism , Cell Line , DNA Damage , DNA Helicases/antagonists & inhibitors , DNA Helicases/genetics , DNA Replication , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Homologous Recombination , Humans , Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitors , Poly (ADP-Ribose) Polymerase-1/genetics , Poly(ADP-ribose) Polymerases/genetics , Protein Stability , RNA, Small Interfering/genetics , S Phase
2.
Mol Ther Nucleic Acids ; 1: e30, 2012 Jun 19.
Article in English | MEDLINE | ID: mdl-23344082

ABSTRACT

RNA-directed antisense and interference therapeutics are a promising treatment option for cancer. The demonstration of depletion of target proteins within human tumors in vivo using validated methodology will be a key to the application of this technology. Here, we present a flow cytometric-based approach to quantitatively determine protein levels in solid tumor material derived by fiber optic brushing (FOB) of non-small cell lung cancer (NSCLC) patients. Focusing upon the survivin protein, and its depletion by an antisense oligonucleotide (ASO) (LY2181308), we show that we can robustly identify a subpopulation of survivin positive tumor cells in FOB samples, and, moreover, detect survivin depletion in tumor samples from a patient treated with LY2181308. Survivin depletion appears to be a result of treatment with this ASO, because a tumor treated with conventional cytotoxic chemotherapy did not exhibit a decreased percentage of survivin positive cells. Our approach is likely to be broadly applicable to, and useful for, the quantification of protein levels in tumor samples obtained as part of clinical trials and studies, facilitating the proof-of-principle testing of novel targeted therapies.

3.
Clin Cancer Res ; 16(24): 6150-8, 2010 Dec 15.
Article in English | MEDLINE | ID: mdl-21041181

ABSTRACT

PURPOSE: Enhanced tumor cell survival through expression of inhibitors of apoptosis (IAP) is a hallmark of cancer. Survivin, an IAP absent from most normal tissues, is overexpressed in many malignancies and associated with a poorer prognosis. We report the first-in-human dose study of LY2181308, a second-generation antisense oligonucleotide (ASO) directed against survivin mRNA. PATIENTS AND METHODS: A dose-escalation study evaluating the safety, pharmacokinetics, and pharmacodynamics of LY2181308 administered intravenously for 3 hours as a loading dose on 3 consecutive days and followed by weekly maintenance doses. Patients were eligible after signing informed consent, had exhausted approved anticancer therapies and agreed to undergo pre- and posttreatment tumor biopsies to evaluate reduction of survivin protein and gene expression. RESULTS: A total of 40 patients were treated with LY2181308 at doses of 100 to 1,000 mg. Twenty-six patients were evaluated at the recommended phase 2 dose of 750 mg, at which level serial tumor sampling and [(11)C]LY2183108 PET (positron emission tomography) imaging demonstrated that ASO accumulated within tumor tissue, reduced survivin gene and protein expression by 20% and restored apoptotic signaling in tumor cells in vivo. Pharmacokinetics were consistent with preclinical modeling, exhibiting rapid tissue distribution, and terminal half-life of 31 days. CONCLUSIONS: The tumor-specific, molecularly targeted effects demonstrated by this ASO in man underpin confirmatory studies evaluating its therapeutic efficacy in cancer.


Subject(s)
Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/genetics , Neoplasms/genetics , Neoplasms/therapy , Oligonucleotides/administration & dosage , Oligonucleotides/pharmacology , Adult , Aged , Aged, 80 and over , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Dose-Response Relationship, Drug , Down-Regulation/drug effects , Evidence-Based Practice , Female , Gene Expression Regulation, Neoplastic/drug effects , Genetic Therapy/methods , Humans , Inhibitor of Apoptosis Proteins , Male , Middle Aged , Molecular Targeted Therapy , Oligonucleotides/pharmacokinetics , Oligonucleotides, Antisense/pharmacology , Pilot Projects , Survivin
4.
Mol Cell Biol ; 29(24): 6427-37, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19805513

ABSTRACT

Interstrand cross-links (ICLs) prevent DNA strand separation and, therefore, transcription and replication, making them extremely cytotoxic. The precise mechanism by which ICLs are removed from mammalian genomes largely remains elusive. Genetic evidence implicates ATR, the Fanconi anemia proteins, proteins required for homologous recombination, translesion synthesis, and at least two endonucleases, MUS81-EME1 and XPF-ERCC1. ICLs cause replication-dependent DNA double-strand breaks (DSBs), and MUS81-EME1 facilitates DSB formation. The subsequent repair of these DSBs occurs via homologous recombination after the ICL is unhooked by XPF-ERCC1. Here, we examined the effect of the loss of either nuclease on FANCD2 monoubiquitination to determine if the nucleolytic processing of ICLs is required for the activation of the Fanconi anemia pathway. FANCD2 was monoubiquitinated in Mus81(-/-), Ercc1(-/-), and XPF-deficient human, mouse, and hamster cells exposed to cross-linking agents. However, the monoubiquitinated form of FANCD2 persisted longer in XPF-ERCC1-deficient cells than in wild-type cells. Moreover, the levels of chromatin-bound FANCD2 were dramatically reduced and the number of ICL-induced FANCD2 foci significantly lower in XPF-ERCC1-deficient cells. These data demonstrate that the unhooking of an ICL by XPF-ERCC1 is necessary for the stable localization of FANCD2 to the chromatin and subsequent homologous recombination-mediated DSB repair.


Subject(s)
DNA Damage , DNA-Binding Proteins/metabolism , Endonucleases/metabolism , Fanconi Anemia Complementation Group D2 Protein/metabolism , Fanconi Anemia/metabolism , Animals , Cell Line , Chromatin/metabolism , Cricetinae , Cricetulus , DNA Breaks, Double-Stranded , DNA Repair , DNA-Binding Proteins/genetics , Endonucleases/genetics , Fanconi Anemia Complementation Group D2 Protein/genetics , Fanconi Anemia Complementation Group Proteins/genetics , Fanconi Anemia Complementation Group Proteins/metabolism , Gene Deletion , Humans , Mice , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Ubiquitin/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...