Histone Deacetylase Inhibitors Selectively Target Homology Dependent DNA Repair Defective Cells and Elevate Non-Homologous Endjoining Activity

Background: We have previously used the ATAD5-luciferase high-throughput screening assay to identify genotoxic compounds with potential chemotherapeutic capabilities. The successful identification of known genotoxic agents, including the histone deacetylase inhibitor (HDACi) trichostatin A (TSA), confirmed the specificity of the screen since TSA has been widely studied for its ability to cause apoptosis in cancer cells. Because many cancers have acquired mutations in DNA damage checkpoints or repair pathways, we hypothesized that these cancers may be susceptible to treatments that target compensatory pathways. Here, we used a panel of isogenic chicken DT40 B lymphocyte mutant and human cell lines to investigate the ability of TSA to define selective pathways that promote HDACi toxicity. Results: HDACi induced a DNA damage response and reduced viability in all repair deficient DT40 mutants although ATM-nulls were least affected. The most dramatic sensitivity was observed in mutants lacking the homology dependent repair (HDR) factor BLM or the non-homologous end-joining (NHEJ) and HDR factors, KU/RAD54, suggesting an involvement of either HDR or NHEJ in HDACi-induced cell death. To extend these findings, we measured the frequencies of HDR and NHEJ after HDACi treatment and monitored viability in human cell lines comparably deficient in HDR or NHEJ. Although no difference in HDR frequency was observed between HDACi treated and untreated cells, HDR-defective human cell lines were clearly more sensitive than wild type. Unexpectedly, cells treated with HDACis showed a significantly elevated NHEJ frequency. Conclusions: HDACi targeting drugs induced significant increases in NHEJ activity in human cell lines but did not alter HDR frequency. Moreover, HDR is required for cellular resistance to HDACi therapy; therefore, NHEJ does not appear to be a critical axis for HDACi resistance. Rather, HDACi compounds induced DNA damage, most likely double strand breaks (DSBs), and HDR proficiency is correlated with cell survivalclose4

RAD18 ト ポリ ADP リボース ポリメラーゼ ワ ドクリツ ニ ヒソウドウ マッタン ケツゴウ ノ ニホンサ セツダン エ ノ アクセス オ ヨクセイシテ ソウドウ クミカエ オ ソクシンスル

京都大学0048新制・課程博士博士(医学)甲第12959号医博第3119号新制||医||951(附属図書館)UT51-2007-H232京都大学大学院医学研究科分子医学系専攻(主査)教授 藤堂 剛, 教授 松本 智裕, 教授 小松 賢志, 教授 松田 文彦学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDA

Alpha‐globin gene triplication and its effect in beta‐thalassemia carrier, sickle cell trait, and healthy individual

Abstract The genotype and phenotype correlation between coinheritance of heterozygous beta‐thalassemia with the alpha‐globin triplication is unclear. In this study we have investigated and reviewed alpha triplication frequency in beta‐thalassemia carriers, sickle cell trait, and healthy individuals and its effect on hematological and phenotypical changes. In this study, 4005 beta‐thalassemia carriers, 455 sickle cell trait, and 2000 healthy individuals were included. Molecular characterization of beta and alpha‐thalassemia was performed. The frequencies of alpha‐globin triplication in beta‐thalassemia carriers, sickle cell trait, and healthy individuals were 67 (1.67%), 4 (0.88%), and 18 (0.9%), respectively. In total, the frequency of alpha‐triplications is approximately 89 (1.39%) in Khuzestan province, South of Iran population. We have compared the average hematological parameters of beta‐thalassemia carriers, sickle cell trait, and healthy individuals with and without alpha gene triplication. This mutation did not show any significant effect on the change of blood indices, neither in healthy individuals nor in sickle cell trait and beta‐thalassemia carriers. Therefore, there is no need to take more notice of anti 3.7 mutation in beta‐thalassemia carriers is opposed with some studies reported that the presence of excess alpha‐globin genes in beta‐thalassemia carriers can lead to the phenotype of beta‐thalassemia intermedia. Therefore, not every individual with triplicated alpha globin coinherited with beta‐thalassemia trait will have a significantly lower Hb than normal, and it is highly likely that none of them will need transfusion

Identification of Three Novel Mutations in the FANCA, FANCC, and ITGA2B Genes by Whole Exome Sequencing.

Background Various blood diseases are caused by mutations in the FANCA, FANCC, and ITGA2B genes. Exome sequencing is a suitable method for identifying single-gene disease and genetic heterogeneity complaints. Methods Among families who were referred to Narges Genetic and PND Laboratory in 2015-2017, five families with a history of blood diseases were analyzed using the whole exome sequencing (WES) method. Results We detected two novel mutations (c.190-2A>G and c.2840C>G) in the FANCA gene, c. 1429dupA mutation in the FANCC gene, and c.1392A>G mutation in the ITGA2B gene. The prediction of variant pathogenicity has been done using bioinformatics tools such as Mutation taster PhD-SNP and polyphen2 and were confirmed by Sanger sequencing. Conclusions WES could be as a precise tool for identifying the pathologic variants in affected patient and heterozygous carriers among families. This highly successful technique will remain at the forefront of platelet and blood genomic research

An essential role for Cdk1 in S phase control is revealed via chemical genetics in vertebrate cells.

In vertebrates Cdk1 is required to initiate mitosis; however, any functionality of this kinase during S phase remains unclear. To investigate this, we generated chicken DT40 mutants, in which an analog-sensitive mutant cdk1 as replaces the endogenous Cdk1, allowing us to specifically inactivate Cdk1 using bulky ATP analogs. In cells that also lack Cdk2, we find that Cdk1 activity is essential for DNA replication initiation and centrosome duplication. The presence of a single Cdk2 allele renders S phase progression independent of Cdk1, which suggests a complete overlap of these kinases in S phase control. Moreover, we find that Cdk1 inhibition did not induce re-licensing of replication origins in G2 phase. Conversely, inhibition during mitosis of Cdk1 causes rapid activation of endoreplication, depending on proteolysis of the licensing inhibitor Geminin. This study demonstrates essential functions of Cdk1 in the control of S phase, and exemplifies a chemical genetics approach to target cyclin-dependent kinases in vertebrate cells

Culturing in serum-free culture medium on collagen type-I-coated plate increases expression of CD133 and retains original phenotype of HT-29 cancer stem cell

Background: A sub-population of tumor cells termed cancer stem cells (CSCs) has an important role in tumor initiation, progression, and recurrence. Selecting a suitable procedure for isolation and enrichment of CSCs is the biggest challenge in the study of CSCs. In the present study, the role of the combination of stem cell culture medium and collagen type-I was evaluated for successful isolation and enrichment of HT-29 CSCs. Materials and Methods: HT-29 cells were cultured in serum-containing medium (parental culture medium: Medium + 10% fetal bovine serum) and serum-free medium (stem cell culture medium); both on collagen-coated plates. Spheres forming ability and CD133 expression, as a potential marker of colorectal CSCs, were evaluated in two culture mediums. Results: The results show spheroids usually give rise completely within 15 days in the stem cell culture medium on the collagen-coated plate. CD133 expression in spheroid cells (84%) is extensively higher than in parental cells (25%). Moreover, relative to parental cells, spheroid cells were more radioresistance. Conclusion: Finding of this study suggested that CSCs derived from colon cancer cell line (HT-29) can be propagated and form colonospheres in serum-free culture medium on collagen type-I. According to maintenance of their original phenotype in these conditions, it seems serum-free culture medium on collagen type-I is a suitable way to drug screening of HT-29 CSCs

Parp-1 protects homologous recombination from interference by ku and ligase iv in vertebrate cells

Parp-1 and Parp-2 are activated by DNA breaks and have been implicated in the repair of DNA single-strand breaks (SSB). Their involvement in double-strand break (DSB) repair mediated by homologous recombination (HR) or nonhomologous end joining (NHEJ) remains unclear. We addressed this question using chicken DT40 cells, which have the advantage of carrying only a PARP-1 gene but not a PARP-2 gene. We found that PARP-1(-/-) DT40 mutants show reduced levels of HR and are sensitive to various DSB-inducing genotoxic agents. Surprisingly, this phenotype was strictly dependent on the presence of Ku, a DSB-binding factor that mediates NHEJ. PARP-1/KU70 double mutants were proficient in the execution of HR and displayed elevated resistance to DSB-inducing drugs. Moreover, we found deletion of Ligase IV, another NHEJ gene, suppressed the camptothecin of PARP-1(-/-) cells. Our results suggest a new critical function for Parp in minimizing the suppressive effects of Ku and the NHEJ pathway on HR