ABSTRACT
Telomerase negative cancer cell types use the Alternative Lengthening of Telomeres (ALT) pathway to elongate telomeres ends. Here, we show that silencing human DNA polymerase (Pol λ) in ALT cells represses ALT activity and induces telomeric stress. In addition, replication stress in the absence of Pol λ, strongly affects the survival of ALT cells. In vitro, Pol λ can promote annealing of even a single G-rich telomeric repeat to its complementary strand and use it to prime DNA synthesis. The noncoding telomeric repeat containing RNA TERRA and replication protein A negatively regulate this activity, while the Protection of Telomeres protein 1 (POT1)/TPP1 heterodimer stimulates Pol λ. Pol λ associates with telomeres and colocalizes with TPP1 in cells. In summary, our data suggest a role of Pol λ in the maintenance of telomeres by the ALT mechanism.
Subject(s)
Aminopeptidases/metabolism , DNA Polymerase beta/metabolism , G-Quadruplexes , Serine Proteases/metabolism , Telomere Homeostasis , Telomere-Binding Proteins/metabolism , Cell Line, Tumor , Humans , Multiprotein Complexes , Replication Protein A/metabolism , Shelterin Complex , Telomere/chemistry , Telomere/metabolismABSTRACT
Human DNA polymerase (pol) λ functions in base excision repair and non-homologous end joining. We have previously shown that DNA pol λ is involved in accurate bypass of the two frequent oxidative lesions, 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine during the S phase. However, nothing is known so far about the relationship of DNA pol λ with the S phase DNA damage response checkpoint. Here, we show that a knockdown of DNA pol λ, but not of its close homologue DNA pol ß, results in replication fork stress and activates the S phase checkpoint, slowing S phase progression in different human cancer cell lines. We furthermore show that DNA pol λ protects cells from oxidative DNA damage and also functions in rescuing stalled replication forks. Its absence becomes lethal for a cell when a functional checkpoint is missing, suggesting a DNA synthesis deficiency. Our results provide the first evidence, to our knowledge, that DNA pol λ is required for cell cycle progression and is functionally connected to the S phase DNA damage response machinery in cancer cells.
Subject(s)
DNA Polymerase beta/physiology , DNA Replication , S Phase Cell Cycle Checkpoints , Cell Line, Tumor , Cell Survival , Checkpoint Kinase 1 , DNA Damage , DNA Polymerase beta/antagonists & inhibitors , DNA Polymerase beta/metabolism , HeLa Cells , Humans , Hydroxyurea/pharmacology , Oxidative Stress , Protein Kinases/metabolism , RNA Interference , Recombinational DNA Repair , Stress, Physiological/geneticsABSTRACT
A hit optimization protocol applied to the first nonnucleoside inhibitor of the ATPase activity of human DEAD-box RNA helicase DDX3 led to the design and synthesis of second-generation rhodanine derivatives with better inhibitory activity toward cellular DDX3 and HIV-1 replication. Additional DDX3 inhibitors were identified among triazine compounds. Biological data were rationalized in terms of structure-activity relationships and docking simulations. Antiviral activity and cytotoxicity of selected DDX3 inhibitors are reported and discussed. A thorough analysis confirmed human DDX3 as a valid anti-HIV target. The compounds described herein represent a significant advance in the pursuit of novel drugs that target HIV-1 host cofactors.
Subject(s)
Anti-HIV Agents/chemical synthesis , DEAD-box RNA Helicases/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Anti-HIV Agents/chemistry , Anti-HIV Agents/toxicity , Cell Line, Tumor , Computer Simulation , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Drug Design , Drug Evaluation, Preclinical , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/toxicity , Gene Knockdown Techniques , HIV-1/drug effects , HIV-1/enzymology , Humans , MicroRNAs/metabolism , Rhodanine/chemical synthesis , Rhodanine/chemistry , Rhodanine/toxicity , Structure-Activity Relationship , Triazines/chemical synthesis , Triazines/chemistry , Triazines/toxicity , Virus Replication/drug effectsABSTRACT
Childhood cutaneous melanoma is a rare disease with increasing incidence. It is not clear whether it differs from adult melanoma in etiology and clinical evolution. To genetically characterize childhood melanoma, 21 pediatric patients were studied by germ-line analysis of CDKN2A, CDK4, and MC1R genes. In addition, alterations in CDKN2A, c-Kit, BRAF, and NRAS genes were evaluated at the somatic level by direct gene sequencing, fluorescence in situ hybridization analysis, and immunohistochemistry. As a control group of susceptible patients, we studied patients from 23 melanoma-prone families. At the germ-line level, CDKN2A and MC1R gene variants were detected in 2/21 and 12/21 pediatric patients and in 9/23 and 19/22 in familial patients. At the somatic level, most lesions (9/14) from pediatric patients showed CDKN2A locus homozygous deletions and a null p16 immunophenotype, whereas most lesions (5/8) from familial patients were disomic and immunoreactive. A c-Kit low-polysomy profile seems to parallel CDKN2A homozygous deletions in pediatric melanoma whereas the single activating mutation observed segregates with familial patients. Loss of KIT protein expression was frequent (7/14) in pediatric melanomas, where metastatic cases were prevalent. BRAF(V600E) mutation occurred at a similar rate (approximately 50%) in lesions from pediatric and familial patients, whereas no NRAS mutations were detected.
