scholarly journals Application of Host Cell Reactivation in Evaluating the Effects of Anticancer Drugs and Environmental Toxicants on Cellular DNA Repair Activity in Head and Neck Cancer

10.5772/24472 ◽  
2011 ◽  
Author(s):  
Yi-Shan Tsai ◽  
Jau-Ling Huang ◽  
Chang-Shen Li
Keyword(s):  
Dna Repair ◽  
Head And Neck Cancer ◽  
Head And Neck ◽  
Host Cell ◽  
Anticancer Drugs ◽  
Environmental Toxicants ◽  
Cell Reactivation ◽  
Repair Activity ◽  
Cellular Dna ◽  
Host Cell Reactivation

scholarly journals Exploring new potential role of DDB2 by host cell reactivation assay in human tumorigenic cells

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Elisabetta Bassi ◽  
Paola Perucca ◽  
Isabella Guardamagna ◽  
Ennio Prosperi ◽  
Lucia A. Stivala ◽  
...  
Keyword(s):  
Dna Repair ◽  
Host Cell ◽  
Human Cells ◽  
Mutant Form ◽  
Cell Reactivation ◽  
Cellular Environment ◽  
Dna Plasmid ◽  
Host Cell Reactivation ◽  
Damaged Dna

Abstract Background The Host Cell Reactivation assay (HCR) allows studying the DNA repair capability in different types of human cells. This assay was carried out to assess the ability in removing UV-lesions from DNA, thus verifying NER efficiency. Previously we have shown that DDB2, a protein involved in the Global Genome Repair, interacts directly with PCNA and, in human cells, the loss of this interaction affects DNA repair machinery. In addition, a mutant form unable to interact with PCNA (DDB2PCNA-), has shown a reduced ability to interact with a UV-damaged DNA plasmid in vitro. Methods In this work, we have investigated whether DDB2 protein may influence the repair of a UV-damaged DNA plasmid into the cellular environment by applying the HCR method. To this end, human kidney 293 stable clones, expressing DDB2Wt or DDB2PCNA-, were co-transfected with pmRFP-N2 and UV-irradiated pEGFP-reported plasmids. Moreover, the co-localization between DDB2 proteins and different NER factors recruited at DNA damaged sites was analysed by immunofluorescence and confocal microscopy. Results The results have shown that DDB2Wt recognize and repair the UV-induced lesions in plasmidic DNA transfected in the cells, whereas a delay in these processes were observed in the presence of DDB2PCNA-, as also confirmed by the different extent of co-localization of DDB2Wt and some NER proteins (such as XPG), vs the DDB2 mutant form. Conclusion The HCR confirms itself as a very helpful approach to assess in the cellular context the effect of expressing mutant vs Wt NER proteins on the DNA damage response. Loss of interaction of DDB2 and PCNA affects negatively DNA repair efficiency.

scholarly journals Defining the function of xeroderma pigmentosum group F protein in psoralen interstrand cross-link-mediated DNA repair and mutagenesis

2004 ◽  
Vol 379 (1) ◽  
pp. 71-78 ◽  
Author(s):  
Zhiwen CHEN ◽  
Xiaoxin Susan XU ◽  
Jason HARRISON ◽  
Gan WANG
Keyword(s):  
Dna Repair ◽  
Host Cell ◽  
Xeroderma Pigmentosum ◽  
Normal Fibroblast ◽  
Fibroblast Cells ◽  
Cross Link ◽  
Cell Reactivation ◽  
Xeroderma Pigmentosum Group F ◽  
Host Cell Reactivation

Many commonly used drugs, such as psoralen and cisplatin, can generate a very unique type of DNA damage, namely ICL (interstrand cross-link). An ICL can severely block DNA replication and transcription and cause programmed cell death. The molecular mechanism of repairing the ICL damage has not been well established. We have studied the role of XPF (xeroderma pigmentosum group F) protein in psoralen-induced ICL-mediated DNA repair and mutagenesis. The results obtained from our mutagenesis studies revealed a very similar mutation frequency in both human normal fibroblast cells and XPF cells. The mutation spectra generated in both cells, however, were very different: most of the mutations generated in the normal fibroblast cells were T167→A transversions, whereas most of the mutations generated in the XPF cells were T167→G transversions. When a wild-type XPF gene cDNA was stably transfected into the XPF cells, the T167→A mutations were increased and the T167→G mutations were decreased. We also determined the DNA repair capability of the XPF cells using both the host-cell reactivation and the in vitro DNA repair assays. The results obtained from the host-cell reactivation experiments revealed an effective reactivation of a luciferase reporter gene from the psoralen-damaged plasmid in the XPF cells. The results obtained from the in vitro DNA repair experiments demonstrated that the XPF nuclear extract is normal in introducing dual incisions during the nucleotide excision repair process. These results suggest that the XPF protein has important roles in the psoralen ICL-mediated DNA repair and mutagenesis.

A Host Cell Reactivation Assay for DNA Repair System Analysis in Primary Hematopoietic Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4475-4475
Author(s):  
Joerg Baesecke ◽  
Kai Thoms ◽  
Tina Roedling ◽  
Birgit Aut ◽  
Volker Viereck ◽  
...  
Keyword(s):  
Dna Repair ◽  
Host Cell ◽  
Progenitor Cells ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Hematopoietic Cells ◽  
Cell Reactivation ◽  
Nucleotide Excision ◽  
Repair Systems ◽  
Host Cell Reactivation

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.

A modified host-cell reactivation assay to quantify DNA repair capacity in cryopreserved peripheral lymphocytes

DNA Repair ◽  
2011 ◽  
Vol 10 (6) ◽  
pp. 603-610 ◽  
Author(s):  
Pedro Mendez ◽  
Miquel Taron ◽  
Teresa Moran ◽  
Marco A. Fernandez ◽  
Gerard Requena ◽  
...  
Keyword(s):  
Dna Repair ◽  
Host Cell ◽  
Repair Capacity ◽  
Cell Reactivation ◽  
Dna Repair Capacity ◽  
Peripheral Lymphocytes ◽  
Host Cell Reactivation

DNA repair in cells sensitive and resistant to cis-diamminedichloroplatinum(II): host-cell reactivation of damaged plasmid DNA

Biochemistry ◽  
1989 ◽  
Vol 28 (7) ◽  
pp. 3120-3124 ◽  
Author(s):  
Nader Sheibani ◽  
Margaretha M. Jennerwein ◽  
Alan Eastman
Keyword(s):  
Dna Repair ◽  
Host Cell ◽  
Plasmid Dna ◽  
Cell Reactivation ◽  
Host Cell Reactivation ◽  
In Cells
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