JS-K, a GST-Activated Nitric Oxide Generator, Induces DNA-Double Strand Breaks and Inhibits Growth and Survival of Multiple Myeloma Cells In Vitro and In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3453-3453
Author(s):  
Tanyel Kiziltepe ◽  
Teru Hideshima ◽  
Noopur Raje ◽  
Kenji Ishitsuka ◽  
Enrique M. Ocio ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Multiple Myeloma ◽  
Double Strand Breaks ◽  
Cell Surface Expression ◽  
Dna Double Strand Breaks ◽  
Normal Peripheral Blood ◽  
Strand Breaks ◽  
Induced Apoptosis

Abstract JS-K (O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate) is a diazeniumdiolate class of prodrug which is designed to release nitric oxide (NO·) on reaction with glutathione S-transferases (GST). GST has been shown to be overexpressed in a broad spectrum of tumor cells. Therefore, JS-K can possibly turn GST overexpression to the tumor’s disadvantage by generating high intracellular concentrations of cytotoxic NO·. Multiple myeloma (MM) is currently an incurable hematological malignancy where new treatment options are urgently needed. In this study we investigated the cytotoxicity of JS-K in MM in vitro and in vivo. JS-K showed significant cytotoxicity in both conventional therapy-sensitive and -resistant MM cell lines, as well as patient MM cells (IC50: 0.3–2.5 mM). Importantly, no significant cytotoxic effects of JS-K at these doses were observed in normal peripheral blood mononuclear cells. JS-K treatment induced apoptosis in MM cells which was associated with PARP, caspase 8, and caspase 9 cleavage; increased cell surface expression of Fas/CD95; Mcl-1 cleavage; Bcl-2 phosphorylation; as well as mitochondrial cyt c, AIF, and EndoG release. Moreover, JS-K could overcome the survival and growth advantages conferred by exogenous IL-6 and IGF-1, or by adherence of MM cells to bone marrow stromal cells. Flow cytometry experiments revealed significant NO· generation in JS-K-treated MM cells. Since NO· is known to cause DNA double strand breaks (DSB), we hypothesized that JS-K induces DSB in MM cells and confirmed DSB formation by neutral comet assay. We further showed that JS-K also activated DNA damage response pathways as evidenced by H2AX, Chk2 and p53 phosphorylation. In addition, JNK was also activated by JS-K treatment in MM cells, and inhibition of JNK significantly decreased JS-K-induced cytotoxicity, suggesting that JS-K induced apoptosis is mediated via JNK signaling. Finally, JS-K was also significantly effective in inhibiting tumor growth and prolonging median survival (p < 0.01) in a human plasmacytoma xenograft mouse model. Analysis of tumors harvested from treated animals showed that JS-K induced apoptosis and decreased angiogenesis in vivo. Taken together, these data provide the preclinical rationale for the clinical evaluation of JS-K to improve patient outcome in MM.

scholarly journals JS-K, a GST-activated nitric oxide generator, induces DNA double-strand breaks, activates DNA damage response pathways, and induces apoptosis in vitro and in vivo in human multiple myeloma cells

Blood ◽  
2007 ◽  
Vol 110 (2) ◽  
pp. 709-718 ◽  
Author(s):  
Tanyel Kiziltepe ◽  
Teru Hideshima ◽  
Kenji Ishitsuka ◽  
Enrique M. Ocio ◽  
Noopur Raje ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Multiple Myeloma ◽  
Dna Damage ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Endonuclease G ◽  
Human Multiple Myeloma

Abstract Here we investigated the cytotoxicity of JS-K, a prodrug designed to release nitric oxide (NO•) following reaction with glutathione S-transferases, in multiple myeloma (MM). JS-K showed significant cytotoxicity in both conventional therapy-sensitive and -resistant MM cell lines, as well as patient-derived MM cells. JS-K induced apoptosis in MM cells, which was associated with PARP, caspase-8, and caspase-9 cleavage; increased Fas/CD95 expression; Mcl-1 cleavage; and Bcl-2 phosphorylation, as well as cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (EndoG) release. Moreover, JS-K overcame the survival advantages conferred by interleukin-6 (IL-6) and insulin-like growth factor 1 (IGF-1), or by adherence of MM cells to bone marrow stromal cells. Mechanistic studies revealed that JS-K–induced cytotoxicity was mediated via NO• in MM cells. Furthermore, JS-K induced DNA double-strand breaks (DSBs) and activated DNA damage responses, as evidenced by neutral comet assay, as well as H2AX, Chk2 and p53 phosphorylation. JS-K also activated c-Jun NH2-terminal kinase (JNK) in MM cells; conversely, inhibition of JNK markedly decreased JS-K–induced cytotoxicity. Importantly, bortezomib significantly enhanced JS-K–induced cytotoxicity. Finally, JS-K is well tolerated, inhibits tumor growth, and prolongs survival in a human MM xenograft mouse model. Taken together, these data provide the preclinical rationale for the clinical evaluation of JS-K to improve patient outcome in MM.

scholarly journals A Role for DEAD Box 1 at DNA Double-Strand Breaks

2008 ◽  
Vol 28 (20) ◽  
pp. 6413-6425 ◽  
Author(s):  
Lei Li ◽  
Elizabeth A. Monckton ◽  
Roseline Godbout
Keyword(s):  
Ionizing Radiation ◽  
Neuroblastoma Cell ◽  
Rnase H ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Rna Helicases ◽  
Strand Breaks ◽  
Dead Box

ABSTRACT DEAD box proteins are a family of putative RNA helicases associated with all aspects of cellular metabolism involving the modification of RNA secondary structure. DDX1 is a member of the DEAD box protein family that is overexpressed in a subset of retinoblastoma and neuroblastoma cell lines and tumors. DDX1 is found primarily in the nucleus, where it forms two to four large aggregates called DDX1 bodies. Here, we report a rapid redistribution of DDX1 in cells exposed to ionizing radiation, resulting in the formation of numerous foci that colocalize with γ-H2AX and phosphorylated ATM foci at sites of DNA double-strand breaks (DSBs). The formation of DDX1 ionizing-radiation-induced foci (IRIF) is dependent on ATM, which was shown to phosphorylate DDX1 both in vitro and in vivo. The treatment of cells with RNase H prevented the formation of DDX1 IRIF, suggesting that DDX1 is recruited to sites of DNA damage containing RNA-DNA structures. We have shown that DDX1 has RNase activity toward single-stranded RNA, as well as ADP-dependent RNA-DNA- and RNA-RNA-unwinding activities. We propose that DDX1 plays an RNA clearance role at DSB sites, thereby facilitating the template-guided repair of transcriptionally active regions of the genome.

scholarly journals The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions

2018 ◽  
Vol 12 (11) ◽  
pp. e0006875 ◽  
Author(s):  
Danielle Gomes Passos Silva ◽  
Selma da Silva Santos ◽  
Sheila C. Nardelli ◽  
Isabela Cecília Mendes ◽  
Anna Cláudia Guimarães Freire ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

scholarly journals Induction and repair of DNA double-strand breaks in hippocampal neurons of miсe of different age after exposure to 60Со γ-rays in vivo and in vitro

2018 ◽  
Vol 177 ◽  
pp. 06001
Author(s):  
R.A. Kozhina ◽  
V.N. Chausov ◽  
E.A. Kuzmina ◽  
A.V. Boreyko
Keyword(s):  
Central Nervous System ◽  
Dna Damage ◽  
Nervous System ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Age Related ◽  
Γ Rays

One of the central problems of modern radiobiology is the study of DNA damage induction and repair mechanisms in central nervous system cells, in particular, in hippocampal cells. The study of the regularities of molecular damage formation and repair in the hippocampus cells is of special interest, because these cells, unlike most cells of the central nervous system (CNS), keep proliferative activity, i.e. ability to neurogenesis. Age-related changes in hippocampus play an important role, which could lead to radiosensitivity changes in neurons to the ionizing radiation exposure. Regularities in DNA double-strand breaks (DSB) induction and repair in different aged mice hippocampal cells in vivo and in vitro under the action of γ-rays 60Со were studied with DNA comet-assay. The obtained dose dependences of DNA DSB induction are linear both in vivo and in vitro. It is established that in young animals' cells, the degree of DNA damage is higher than in older animals. It is shown that repair kinetics is basically different for exposure in vivo and in vitro.

scholarly journals A novel IKK- and proteasome-independent mechanism of RelA activation triggers senescence associated secretome via transcriptional repression of NFKBIA

2019 ◽  
Author(s):  
Marina Kolesnichenko ◽  
Nadine Mikuda ◽  
Uta E. Höpken ◽  
Maja Milanovic ◽  
A. Bugra Tufan ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Physiological Mechanism ◽  
Double Strand Breaks ◽  
Second Phase ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Iκb Kinase ◽  
Apoptosis And Inflammation

ABSTRACTThe IκB kinase (IKK) - NF-κB pathway is activated as part of the DNA damage response and controls both resistance to apoptosis and inflammation. How these different functions are achieved remained unknown. We demonstrate here that DNA double strand breaks elicit two subsequent phases of NF-κB activation in vivo and in vitro, which are mechanistically and functionally distinct. RNA-sequencing reveals that the first phase controls anti-apoptotic gene expression, while the second drives expression of senescence-associated secretory phenotype (SASP) genes. The first, rapidly activated phase is driven by the ATM-PARP1-TRAF6-IKK cascade, which triggers proteasomal destruction of IκBα and is terminated through IκBα (NFKBIA) re-expression. The second phase is activated days later in senescent cells but is independent of IKK and the proteasome. An altered phosphorylation status of p65, in part driven by GSK3β, results in transcriptional silencing of NFKBIA and IKK-independent, constitutive activation of NF-κB in senescence. Collectively, our study reveals a novel physiological mechanism of NF-κB activation with important implications for genotoxic cancer treatment.

DNA Double Strand Breaks Induction by Zalypsis, a Novel Marine Compound, Results in Potent Antimyeloma Activity

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 245-245
Author(s):  
Enrique M Ocio ◽  
Patricia Maiso ◽  
Xi Chen ◽  
Mercedes Garayoa ◽  
Stela Álvarez-Fernández ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Plasma Cells ◽  
Double Strand Breaks ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

Abstract Background and Aims: Although recent therapeutic advances have led to an improvement in the outcome of Multiple Myeloma (MM), it still remains an incurable disease, and therefore, new drugs with novel mechanisms of action are needed for myeloma patients. Zalypsis is a new synthetic alkaloid derived from certain marine compounds which has demonstrated significant in vitro and in vivo antitumor activity in different malignancies. It is currently under late Phase I development in solid tumours, with preliminary evidence of activity. In this study, we have analysed the preclinical activity and mechanism of action of Zalypsis in MM. Material and methods: Nine different MM cell lines and BM samples from MM patients and normal donors were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, Western-blotting and gene expression profile analysis. The in vivo activity was explored in a human subcutaneous plasmocytoma model and immunohistochemistry was performed in selected tumours. Results: Zalypsis turned out to be the most potent antimyeloma agent we have tested so far in our laboratory, with IC50s in picomolar or low nanomolar ranges depending on the cell lines studied. Interestingly, the sensitivity to Zalypsis was independent of the pattern of resistance of the cell lines to conventional antimyeloma agents such as Dexamethasone or Melphalan. It also showed remarkable ex vivo potency in freshly isolated plasma cells from six patients (including two with plasma cell leukemia) and synergized with many other antimyeloma compounds, being the combination of Zalypsis + Lenalidomide + Dexamethasone particularly attractive. Regarding toxicity, Zalypsis preserved the CD34+ hematopoietic progenitor cells from MM and normal donor BM samples. This remarkable activity prompted us to investigate the mechanism of action of the drug. Besides the induction of apoptosis and cell cycle arrest, Zalypsis provoked DNA double strand breaks, which were evidenced by an increase in phospho Histone H2AX and phospho CHK2, followed by a striking overexpression of p53 in MM cell lines bearing wild type forms of this protein. Of note, no other compound currently used in the MM clinic induced such an increase in p53 protein levels. In addition, in a subset of MM cell lines in which p53 was mutated, Zalypsis also provoked DNA double strand breaks and induced cell death, although higher concentrations were required. Changes in the gene expression profile of MM cells treated with Zalypsis were concordant with these results, since important genes involved in DNA damage response were deregulated. This include genes implicated in the ATM repair pathway, such as TLK2, ATR, CHEK2, RAD5 and BRIP1 and other mRNAs related to DNA repair, such as RAD23B, XPC, XRCC1, XRCC5 and GADD45A. These results were confirmed in vivo in a model of human subcutaneous plasmocytoma in SCID mice. Zalypsis (0.8 and 1 mg/Kg) decreased tumour growth and improved survival of mice implanted with MM1S (wild type p53) and OPM-1 (mutated p53) plasmocytomas. Immunohistochemical studies in tumours from treated animals also demonstrated DNA damage with H2AX phosphorylation and p53 overexpression. Conclusion: The potent in vitro and in vivo antimyeloma activity and the singular mechanism of action of Zalypsis uncovers the high sensitivity of tumour plasma cells to double strand breaks, and strongly supports the potential use of this compound in multiple myeloma patients.

Involvement of Ku80 in microhomology-mediated end joining for DNA double-strand breaks in vivo

DNA Repair ◽  
2007 ◽  
Vol 6 (5) ◽  
pp. 639-648 ◽  
Author(s):  
Yukitaka Katsura ◽  
Shigeru Sasaki ◽  
Masanori Sato ◽  
Kiyoshi Yamaoka ◽  
Kazumi Suzukawa ◽  
...  
Keyword(s):  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks
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