scholarly journals Deoxyribonucleoside triphosphate imbalance. 5-Fluorodeoxyuridine-induced DNA double strand breaks in mouse FM3A cells and the mechanism of cell death.

1987 ◽  
Vol 262 (17) ◽  
pp. 8235-8241 ◽  
Author(s):  
A Yoshioka ◽  
S Tanaka ◽  
O Hiraoka ◽  
Y Koyama ◽  
Y Hirota ◽  
...  
Keyword(s):  
Cell Death ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Deoxyribonucleoside Triphosphate ◽  
Fm3a Cells

A Role for DNA Double Strand Breaks in the Sensitivity of AML Cells to Clofarabine: Enhanced In Vitro Sensitivity in Primary AML Cells with FLT3 Mutations.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2582-2582
Author(s):  
Monica Pallis ◽  
Martin Grundy ◽  
Claire Seedhouse ◽  
Heather Pimblett ◽  
Nigel Russell
Keyword(s):  
Cell Death ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Mitochondrial Membrane ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Cytochrome C Release ◽  
Strand Breaks ◽  
Flt3 Mutations ◽  
Kg1 Cells

Abstract Clofarabine is a purine nucleoside analogue which has been incorporated into several therapeutic trial protocols for the treatment of leukaemias including acute myeloid leukaemia (AML). The aims of the study are to ascertain mechanisms of clofarabine action in AML using cell lines and presentation samples from patients. We measured apoptosis by TdT assay, cytochrome C release and flow cytometric assays of the mitochondrial membrane potential probe DiOC6. To study the effects of clofarabine on DNA synthesis and DNA double strand breaks, we used bromodeoxyuridine (BrdU) and H2AX assays respectively. Equitoxic doses were established that caused approximately 20% cell death in the AML cell lines HL-60 (0.3 μM), KG1 (1 μM) and MV4.11 (1 μM) after 6 hours of continuous exposure. At these doses, clofarabine induced apoptotic DNA nicks, as measured by the TdT assay, and Cytochrome C release in all three cell lines. However, clofarabine-induced mitochondrial hyperpolarisation and depolarisation were found to be cell-type specific, occurring in HL-60 but not MV4.11 or KG1 cells, i.e. mitochondrial membrane depolarisation is not an essential part of the mechanism of clofarabine-induced apoptosis. Following clofarabine treatment, DNA synthesis was reduced by 91% within 1 hour in KG1 cells and by 80% in HL-60, but by only 9% in MV4.11. Out of the three cell lines, MV4.11 cells, which have a FLT3 internal tandem duplication (ITD), were the only ones to completely repair DNA double strand breaks following clofarabine removal. In 12 samples from patients with AML, the proportion of cells which incorporated BrdU in a 45 minute assay - a measure of the rate of cell cycling - differed considerably between samples, from 0.8% to 23%, median 13%. Cell death induced by clofarabine was correlated with the cycling rate of untreated cells (P=0.016). All 12 samples showed inhibition of DNA synthesis within 60 minutes of clofarabine treatment (range 20%–92% inhibition, median 54%). Mitochondrial membrane hyperpolarisation and depolarisation were not observed in patient cells. However, DNA double strand breaks were induced by clofarabine in patient cells. Paradoxically, i.e. in contrast to the results seen with the MV4.11 cell line, toxicity was greatest in samples with a FLT3 mutation (P=0.007). In conclusion, the FLT3 mutant MV4.11 cell line effectively repairs clofarabine-induced double strand breaks. Cell death in patient cells cultured with clofarabine is correlated with the presence of a FLT3 mutation. As we have previously established that AML samples with FLT3 mutations have upregulated DNA repair activity, this paradox might be explained by cycles of attempted DNA repair frustrated by renewed clofarabine incoporation into DNA, thus increasing the toxicity of the drug.

scholarly journals FBH1 co-operates with MUS81 in inducing DNA double-strand breaks and cell death following replication stress

2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Kasper Fugger ◽  
Wai Kit Chu ◽  
Peter Haahr ◽  
Arne Nedergaard Kousholt ◽  
Halfdan Beck ◽  
...  
Keyword(s):  
Cell Death ◽  
Replication Stress ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

PARP Inhibitor Selectively Induces Cell Death in E2A-Hlf Positive Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2183-2183
Author(s):  
Masatoshi Takagi ◽  
Jiuhua Piao ◽  
Takahiro Kamiya ◽  
Mitsuko Masutani ◽  
Shuki Mizutani
Keyword(s):  
Cell Death ◽  
Biological Effects ◽  
Parp Inhibitor ◽  
Double Strand Breaks ◽  
Parp Inhibition ◽  
Breast Cancers ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Daudi Cell ◽  
Increased Sensitivity

Abstract Background: Defects in homologous recombination repair (HRR) have long been known to contribute to genomic instability leading to tumor development. Poly (ADP-ribose) polymerase (PARP) exerts various cell biological effects, such as maintenance of genomic stability, energy metabolism and cell death. PARP is indispensable in DNA repair machinery, especially in base excision repair (BER). PARP inhibition convert DNA double strand breaks from DNA single strand breaks induced by alkylating agents. These DNA double strand breaks can be repaired by HRR. Therefore, PARP inhibitor induces synthetic lethality in HRR defective cancer cells. Such lethality was successfully shown in BRCA1 or 2 mutated breast cancers. However, only a limited study has been performed other than breast cancers. Some tumors including hematological malignancies are defective in HRR function leading to a possibility to be sensitized to PARP inhibitor. Methods: Sensitivity to PARP inhibitor was screened using 28 leukemia cell lines. HRR activity was measured by DR-GFP HRR assay. Expression of proteins involves HRR was evaluated by cDNA microarray analysis and western blotting. Results: E2A-HLF positive leukemia showed susceptibility to PARP inhibitor. This experiment suggests that expression of E2A-HLF chimeric messenger RNA sensitize the leukemic cell to PARP inhibitor. To elucidate whether E2A-HLF genuinely sensitize the cell for PARP inhibition, E2A-HLF was transduced into PARP inhibitor resistant Burkitt cell line, Daudi, using retrovirus. Compared with mock infected Daudi cell, E2A-HLF expressed Daudi cell showed increased sensitivity to PARP inhibitor. This experiment suggests that E2A-HLF expressed cell is defective HRR pathway. To test this hypothesis, HRR assay using DR-GFP construct was employed. HRR between the two nonfunctional GFP genes to generate a functional GFP gene can be triggered by transient transfection of the I-SceI expression vector, which introduces a DNA double-strand break (DSB) in one of the two GFP genes. HRR proficiency can be determined by the number of cells expressing the GFP protein. DR-GFP HRR assay exhibited defect of HRR function in E2A-HLF expressed cell. Interestingly, expression of BRCA1 was decreased in E2A-HLF transfected cell, which presumably link with decreased HRR activity. Conclusions: Increased sensitivity to PARP inhibitor in E2A-HLF positive leukemia was caused by decreased HRR activity by E2A-HLF expression. PARP inhibitor will be a novel therapeutic approach for refractory leukemia, especially with E2A-HLF translocation. While PARP inhibitor monotherapy is an attractive proposition for treating such as HRR defective E2A-HLF expressed leukemia, combination of HRR inhibitor will be a universal strategy for various types of leukemia. Disclosures No relevant conflicts of interest to declare.

Delayed repletion of O6-methylguanine—DNA methyltransferase resulting in failure to protect the human glioblastoma cell line SF767 from temozolomide-induced cytotoxicity

2003 ◽  
Vol 98 (3) ◽  
pp. 591-598 ◽  
Author(s):  
Yuichi Hirose ◽  
Emiko L. Kreklau ◽  
Leonard C. Erickson ◽  
Mitchel S. Berger ◽  
Russell O. Pieper
Keyword(s):  
Cell Death ◽  
Dna Methyltransferase ◽  
Glioma Cells ◽  
Double Strand Breaks ◽  
Single Strand ◽  
Dna Double Strand Breaks ◽  
Content Type ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Fine Print

Object. Temozolomide (TMZ)-induced O6-methylguanine (MG) DNA lesions, if not removed by MG—DNA methyltransferase (MGMT), mispair with thymine, trigger rounds of futile mismatch repair (MMR), and in glioma cells lead to prolonged G2—M arrest and ultimately cell death. Depletion of MGMT by O6-benzylguanine (BG) sensitizes tumor cells to TMZ, and this combination is currently used in clinical trials. The use of the TMZ+BG combination in gliomas, however, is complicated by the prolonged TMZ-induced G2—M arrest, which may delay activation of poorly defined cell death pathways and allow for MGMT repletion and reversal of toxicity. Methods. To address these issues, the actions of TMZ were monitored in DNA MMR-proficient SF767 glioma cells depleted of MGMT by BG, and in cells in which BG was removed at various times after TMZ exposure. In MGMT-depleted cells, TMZ exposure led to DNA single-strand breaks and phosphorylation of cdc2, followed by G2—M arrest, induction of p53/p21, and DNA double-strand breaks. Although DNA single-strand breaks, phosphorylation of cdc2, and G2—M arrest could be reversed by repletion of MGMT up to 5 days after TMZ exposure, TMZ-induced cytotoxicity could only be prevented if MGMT was replenished within 24 hours of the onset of G2—M arrest, and before the creation of DNA double-strand breaks. Conclusions. These results indicate that although SF767 glioma cells undergo a prolonged G2—M arrest in response to TMZ, their ability to escape TMZ-induced cytotoxicity by MGMT repletion is limited to an approximately 24-hour period after the onset of G2—M arrest.

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