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1.
Mol Oncol ; 8(8): 1747-59, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25113059

ABSTRACT

REV3, the catalytic subunit of translesion polymerase zeta (polζ), is commonly associated with DNA damage bypass and repair. Despite sharing accessory subunits with replicative polymerase δ, very little is known about the role of polζ in DNA replication. We previously demonstrated that inhibition of REV3 expression induces persistent DNA damage and growth arrest in cancer cells. To reveal determinants of this sensitivity and obtain insights into the cellular function of REV3, we performed whole human genome RNAi library screens aimed at identification of synthetic lethal interactions with REV3 in A549 lung cancer cells. The top confirmed hit was RRM1, the large subunit of ribonucleotide reductase (RNR), a critical enzyme of de novo nucleotide synthesis. Treatment with the RNR-inhibitor hydroxyurea (HU) synergistically increased the fraction of REV3-deficient cells containing single stranded DNA (ssDNA) as indicated by an increase in replication protein A (RPA). However, this increase was not accompanied by accumulation of the DNA damage marker γH2AX suggesting a role of REV3 in counteracting HU-induced replication stress (RS). Consistent with a role of REV3 in DNA replication, increased RPA staining was confined to HU-treated S-phase cells. Additionally, we found genes related to RS to be significantly enriched among the top hits of the synthetic sickness/lethality (SSL) screen further corroborating the importance of REV3 for DNA replication under conditions of RS.


Subject(s)
DNA Replication/physiology , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/metabolism , Tumor Suppressor Proteins/metabolism , Cell Line, Tumor , DNA Replication/genetics , DNA-Binding Proteins/genetics , DNA-Directed DNA Polymerase/genetics , Flow Cytometry , Humans , RNA Interference/physiology , Ribonucleoside Diphosphate Reductase , Tumor Suppressor Proteins/genetics
2.
Neoplasia ; 13(10): 961-70, 2011 Oct.
Article in English | MEDLINE | ID: mdl-22028621

ABSTRACT

REV3 is the catalytic subunit of DNA translesion synthesis polymerase ζ. Inhibition of REV3 expression increases the sensitivity of human cells to a variety of DNA-damaging agents and reduces the formation of resistant cells. Surprisingly, we found that short hairpin RNA-mediated depletion of REV3 per se suppresses colony formation of lung (A549, Calu-3), breast (MCF-7, MDA-MB-231), mesothelioma (IL45 and ZL55), and colon (HCT116 +/-p53) tumor cell lines, whereas control cell lines (AD293, LP9-hTERT) and the normal mesothelial primary culture (SDM104) are less affected. Inhibition of REV3 expression in cancer cells leads to an accumulation of persistent DNA damage as indicated by an increase in phospho-ATM, 53BP1, and phospho-H2AX foci formation, subsequently leading to the activation of the ATM-dependent DNA damage response cascade. REV3 depletion in p53-proficient cancer cell lines results in a G(1) arrest and induction of senescence as indicated by the accumulation of p21 and an increase in senescence-associated ß-galactosidase activity. In contrast, inhibition of REV3 expression in p53-deficient cells results in growth inhibition and a G(2)/M arrest. A small fraction of the p53-deficient cancer cells can overcome the G(2)/M arrest, which results in mitotic slippage and aneuploidy. Our findings reveal that REV3 depletion per se suppresses growth of cancer cell lines from different origin, whereas control cell lines and a mesothelial primary culture were less affected. Thus, our findings indicate that depletion of REV3 not only can amend cisplatin-based cancer therapy but also can be applied for susceptible cancers as a potential monotherapy.


Subject(s)
Cell Proliferation , DNA Damage , DNA-Binding Proteins/genetics , DNA-Directed DNA Polymerase/genetics , RNA Interference , Ataxia Telangiectasia Mutated Proteins , Blotting, Western , Cell Cycle Checkpoints , Cell Cycle Proteins/metabolism , Cell Division , Cell Line, Tumor , Cellular Senescence , DNA Breaks, Double-Stranded , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/metabolism , G2 Phase , Genomic Instability , HCT116 Cells , HEK293 Cells , Histones/metabolism , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Microscopy, Fluorescence , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/metabolism , Tumor Suppressor p53-Binding Protein 1
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