Host cell reactivation of cisplatin-treated adenovirus is reduced in nucleotide excision repair deficient mammalian cells and several human tumour cells

Author(s):  
J Bulmer ◽  
K Davis ◽  
A Rainbow
Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4475-4475
Author(s):  
Joerg Baesecke ◽  
Kai Thoms ◽  
Tina Roedling ◽  
Birgit Aut ◽  
Volker Viereck ◽  
...  

Abstract Therapy related acute myelogenous leukemia (t-AML) is frequently observed among patients who undergo a high dose chemotherapy and its incidence varies between 5 to 15% in non-myeloablative and myeloablative treatment protocols. Deficiencies in individual DNA-repair systems seem to be involved in t-AML leukemogenesis since indirect genetic markers of impaired DNA repair, e.g. microsatellite instability (mismatch repair) and polymorphisms of XRCC genes (base/nucleotide excision repair), are frequent in these patients. To directly determine the activity of different DNA repair systems we developed a modified host cell reactivation (HCR) assay which is applicable for the analysis of primary hematopoietic cells of clinical samples. This assay determines the ability of transfected host cells to repair damaged plasmid DNA as reflected in the recovery of luciferase activity. Depending on the type of DNA damage introduced to the plasmid prior to transfection, different DNA repair systems can be analysed, e.g. nucleotide excision repair (NER) or double strand break repair. We performed the HCR assay using 75 to 250 ng luciferase reporter gene plasmid (pCMV-Luc). Lymphocytes and cord blood CD34+-progenitor cells from healthy donors were collected according to the convention of Helsinki. 200,000 to 2,000,000 cells were cryopreserved, thawed and transfected using DEAE-dextran at a concentration of 0,1 mg/ml in a transfection volume of 250 μl. We obtained luciferase activities of 350-fold above background in CD34+ progenitor cells (1000-fold in lymphocytes) rendering these cells applicable for DNA repair analysis. In addition, we evaluated the normalized NER capacity (1000 J/m2 UVC irradiated plasmid vs. unirradiated control) of lymphocytes and two AML cell lineages, Kasumi-1 and HL60. Kasumi-1 and HL60 cells exhibited a significantly reduced NER capacity compared to lymphocytes (6.15% +/− 1.57% and 6.5% +/− 1.59% vs. 12.3% +/− 3.2%). Clinical AML samples are currently been investigated. Our modified HCR can be used for functional DNA repair analysis in fresh and cryopreserved patient samples of pre- and post-leukemic conditions as well as in leukemic blasts to elucidate the role of defective DNA repair during t-AML leukemogenesis. Furthermore, the modified HCR may also be used to determine the individual susceptibility for therapy related myeloid leukemia prior to chemotherapy.


2008 ◽  
Vol 80 (12) ◽  
pp. 2735-2750
Author(s):  
Ju-Pi Li ◽  
Chun-Yu Wang ◽  
Yen-An Tang ◽  
Yun-Wei Lin ◽  
Jia-Ling Yang

Arsenic and lead can induce genetic injuries and epigenetic signaling pathways in cultured mammalian cells. To test whether signaling pathways affect the extent of genetic injuries, we explored the impacts of extracellular signal-regulated kinase 1 and 2 (ERK) on nucleotide excision repair (NER), cytotoxicity, and genotoxicity following sodium arsenite [As(III)] and lead acetate [Pb(II)]. Sustained ERK activation was observed in human cells exposed to As(III) and Pb(II). As(III) inhibited the cellular NER synthesis capability; conversely, Pb(II) stimulated it. ERK activation contributed to the As(III)-induced NER inhibition and micronucleus formation. In contrast, this signal was required for inducing cellular NER activity and preventing mutagenesis following Pb(II). ERK activation by Pb(II) was dependent on protein kinase C (PKCα) that also exhibited anti-mutagenicity. Enforced expression of ERK signaling markedly elevated the cellular NER activity, which was suppressed by As(III). Nonetheless, ERK activation could counteract the cytotoxicity caused by these two metals. Together, the results indicate that pro-survival ERK signaling exhibits dual and opposing impacts on NER process following As(III) and Pb(II) exposures. The findings also suggest that ERK is an important epigenetic signaling in the determination of metal genotoxicity.


1992 ◽  
pp. 235-238
Author(s):  
G. Weeda ◽  
C. Troelstra ◽  
W. Vermeulen ◽  
D. Bootsma ◽  
J.H.J. Hoeijmakers ◽  
...  

2001 ◽  
Vol 22 (4) ◽  
pp. 661-664 ◽  
Author(s):  
Z. Matijasevic ◽  
M.L. Precopio ◽  
J.E. Snyder ◽  
D.B. Ludlum

The most versatile strategy for repair of damage to DNA, and the main process for repair of uv-induced damage, is nucleotide excision repair. In mammalian cells, the complete mechanism involves more than 20 polypeptides, and defects in many of these are associated with various forms of inherited disorders in humans. The syndrome xeroderma pigmentosum (XP) is associated with mutagen hypersensitivity and increased cancer frequency, and studies of the nucleotide excision repair defect in this disease have been particularly informative. Many of the XP proteins are now being characterized. XPA binds to DNA, with a preference for damaged base pairs. XPC activity is part of a protein complex with single-stranded DNA binding activity. The XPG protein is a nuclease.


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