The Cancer/Testes (CT) Antigen HORMAD1 is germ cell-restricted and plays developmental roles in generation and processing of meiotic DNA Double Strand Breaks (DSB). Many tumors aberrantly overexpress ...HORMAD1 yet the potential impact of this CT antigen on cancer biology is unclear. We tested a potential role of HORMAD1 in genome maintenance in lung adenocarcinoma cells. We show that HORMAD1 re-distributes to nuclear foci and co-localizes with the DSB marker γH2AX in response to ionizing radiation (IR) and chemotherapeutic agents. The HORMA domain and C-term disordered oligomerization motif are necessary for localization of HORMAD1 to IR-induced foci (IRIF). HORMAD1-depleted cells are sensitive to IR and camptothecin. In reporter assays, Homologous Recombination (HR)-mediated repair of targeted ISce1-induced DSBs is attenuated in HORMAD1-depleted cells. In Non-Homologous End Joining (NHEJ) reporter assays, HORMAD1-depletion does not affect repair of ISce1-induced DSB. Early DSB signaling events (including ATM phosphorylation and formation of γH2AX, 53BP1 and NBS1 foci) are intact in HORMAD1-depleted cells. However, generation of RPA-ssDNA foci and redistribution of RAD51 to DSB are compromised in HORMAD1-depleted cells, suggesting that HORMAD1 promotes DSB resection. HORMAD1-mediated HR is a neomorphic activity that is independent of its meiotic partners (including HORMAD2 and CCDC36. Bioinformatic analysis of TCGA data show that similar to known HR pathway genes HORMAD1 is overexpressed in lung adenocarcinomas. Overexpression of HR genes is associated with specific mutational profiles (including copy number variation). Taken together, we identify HORMAD1-dependent DSB repair as a new mechanism of radioresistance and a probable determinant of mutability in lung adenocarcinoma.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Trans-lesion synthesis (TLS) is an important DNA-damage tolerance mechanism that permits ongoing DNA synthesis in cells harbouring damaged genomes. The E3 ubiquitin ligase RAD18 activates TLS by ...promoting recruitment of Y-family DNA polymerases to sites of DNA-damage-induced replication fork stalling. Here we identify the cancer/testes antigen melanoma antigen-A4 (MAGE-A4) as a tumour cell-specific RAD18-binding partner and an activator of TLS. MAGE-A4 depletion from MAGE-A4-expressing cancer cells destabilizes RAD18. Conversely, ectopic expression of MAGE-A4 (in cell lines lacking endogenous MAGE-A4) promotes RAD18 stability. DNA-damage-induced mono-ubiquitination of the RAD18 substrate PCNA is attenuated by MAGE-A4 silencing. MAGE-A4-depleted cells fail to resume DNA synthesis normally following ultraviolet irradiation and accumulate γH2AX, thereby recapitulating major hallmarks of TLS deficiency. Taken together, these results demonstrate a mechanism by which reprogramming of ubiquitin signalling in cancer cells can influence DNA damage tolerance and probably contribute to an altered genomic landscape.
Accurate DNA replication is crucial for cell survival and the maintenance of genome stability. Cells have developed mechanisms to cope with the frequent genotoxic injuries that arise from both ...endogenous and environmental sources. Lesions encountered during DNA replication are often tolerated by post-replication repair mechanisms that prevent replication fork collapse and avert the formation of DNA double strand breaks. There are two predominant post-replication repair pathways, trans-lesion synthesis (TLS) and template switching (TS). TLS is a DNA damage-tolerant and low-fidelity mode of DNA synthesis that utilizes specialized 'Y-family' DNA polymerases to replicate damaged templates. TS, however, is an error-free 'DNA damage avoidance' mode of DNA synthesis that uses a newly synthesized sister chromatid as a template in lieu of the damaged parent strand. Both TLS and TS pathways are tightly controlled signaling cascades that integrate DNA synthesis with the overall DNA damage response and are thus crucial for genome stability. This review will cover the current knowledge of the primary mediators of post-replication repair and how they are regulated in the cell.
Formaldehyde induces micronuclei (MN) in mouse erythropoietic cells and suppresses the expansion of human erythroid progenitor cells (EPC). (BM: bone marrow, PBMC: peripheral blood mononuclear cells, ...GF: growth factor.)
•We tested formaldehyde (FA) toxicity in liquid in vitro erythroid culture systems.•FA significantly induced micronuclei in cultured mouse polychromatic erythrocytes.•FA suppressed human erythroid progenitor cell (EPC) expansion.•FA increased proliferation in EPCs.•These data confirm that FA is toxic to hematopoietic stem and progenitor cells.
Although formaldehyde (FA) has been classified as a human leukemogen, the mechanisms of leukemogenesis remain elusive. Previously, using colony-forming assays in semi-solid media, we showed that FA exposure in vivo and in vitro was toxic to human hematopoietic stem/progenitor cells. In the present study, we have applied new liquid in vitro erythroid expansion systems to further investigate the toxic effects of FA (0–150μM) on cultured mouse and human hematopoietic stem/progenitor cells. We determined micronucleus (MN) levels in polychromatic erythrocytes (PCEs) differentiated from mouse bone marrow. We measured cell growth, cell cycle distribution, and chromosomal instability, in erythroid progenitor cells (EPCs) expanded from human peripheral blood mononuclear cells. FA significantly induced MN in mouse PCEs and suppressed human EPC expansion in a dose-dependent manner, compared with untreated controls. In the expanded human EPCs, FA slightly increased the proportion of cells in G2/M at 100μM and aneuploidy frequency in chromosomes 7 and 8 at 50μM. Our findings provide further evidence of the toxicity of FA to hematopoietic stem/progenitor cells and support the biological plausibility of FA-induced leukemogenesis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Trans-lesion synthesis (TLS) is a DNA damage-tolerant and error-prone mode of DNA replication. Recent work shows that many cancer cells coopt an aberrantly expressed germ cell protein, melanoma ...antigen-A4 (MAGE-A4), to activate TLS. MAGE-A4-induced "pathological TLS" provides a potential mechanism through which neoplastic cells can tolerate intrinsic and therapeutic genotoxicity while acquiring mutability.
Cancer cells rely on DNA damage tolerance pathways to cope with intrinsic oncogenic stresses and evade DNA-damaging environmental and therapeutic agents. However, the mechanisms by which neoplastic ...cells hijack tightly controlled DNA damage tolerance-signaling cascades to promote mutagenesis and chemoresistance are not understood. Thus, limitations in our knowledge of DNA damage tolerance and mutagenesis impede effective prevention and treatment of cancer. We have discovered two unique regulators of RAD18 and replication-associated DNA damage tolerance that are overexpressed in cancer: RNF168 (an apical mediator of double strand break signaling) and MAGEA4 (a cancer cell-specific protein with no known function). RNF168 is mutated in human RIDDLE syndrome, a disease characterized by severe immunodeficiency, developmental defects, radiosensitivity and a predisposition to cancer. We show here that RNF168 is a novel component of the RAD18 complex, facilitating its recruitment to stalled replication forks and promoting damage tolerance following replication stress. We have also identified the cancer/testis antigen (CTA) MAGEA4 as a stabilizing binding partner of RAD18 that promotes trans-lesion DNA synthesis. Thus, the findings in this thesis offer neomorphic cancer cell-specific roles for regulators of DNA damage tolerance. Identification of mechanisms of DNA damage tolerance that drive carcinogenesis and confer chemoresistance will allow for the development of more effective cancer treatment regimens. CTAs are absent from normal somatic cells but aberrantly overexpressed in many cancers. Interestingly, CTAs have been correlated with chemotherapeutic resistance and poor prognostic outcomes, though their contributions to carcinogenesis are not understood. We have found that depletion of several CTAs (MAGEA4, MAGEA10, or HORMAD1) sensitizes non-small cell lung cancer (NSCLC) cells to DNA-damaging therapies. These studies identify novel mechanisms by which NSCLC cells aberrantly overexpress germ cell proteins to alter genome maintenance, offering a cancer cell-specific mechanism by which neoplastic cells acquire chemoresistance and evade therapy. Accordingly, these CTAs are promising therapeutic targets whose inhibition should be innocuous to normal somatic cells while greatly sensitizing cancer cells to existing DNA damaging chemotherapeutic agents.
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