The Establishment and Characterization of Cell Lines Stably Expressing Human Ku80 Tagged with Enhanced Green Fluorescent Protein

Koike, Manabu; Koike, Aki

doi:10.1269/jrr.45.119

Cited by 18 publications

(34 citation statements)

References 42 publications

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“…X-irradiation: Cells were exposed to X-rays at room temperature, as described previously [7]. X-rays were generated at 200 kVp/20 mA and filtered through 0.5-mm Cu and Al filters, as described previously [7].…”

Section: Cell Lines and Culturesmentioning

confidence: 99%

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The Defect of Ku70 Affects Sensitivity to X-Ray and Radiation-Induced Caspase-Dependent Apoptosis in Lung Cells

Koike¹,

Yutoku

Koike³

2013

The Journal of Veterinary Medical Science

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ABSTRACT. The DNA repair protein Ku70 is a key player in chemoresistance to anticancer agents (e.g., etoposide) or radioresistance. The responses of different organs to radiation vary widely and likely depend on the cell population in the organs. Previously, we established and characterized Ku70-deficient murine lung epithelial (Ku70 −/− MLE) cells and found that these cells are more sensitive than Ku70 +/− MLE cells (control cells) to X-irradiation, as determined by clonogenic survival assay; however, the mechanism underlying this sensitivity remains unclear. In this study, we examined the mechanism by which X-irradiation triggers the death of Ku70 −/− MLE cells. Our results showed that Ku70 −/− MLE cells were more sensitive to radiation-induced apoptosis than control cells, although X-irradiation activated caspase-3 and caspase-7, and cleaved PARP in both cell lines. We also examined the expression level of phosphorylated H2AX (γH2AX), which is a marker of DSB, and observed the phosphorylation of H2AX and the elimination of γH2AX in both cell lines after X-irradiation. The elimination in Ku70 −/− MLE cells was slower than that in control cells, suggesting that DSB repair activity in the Ku70 −/− MLE cells is lower than that in control cells. These findings suggest that Ku70 might play a key role in the inhibition of apoptosis through the DSB repair pathway in lung epithelial cells. Our findings also suggest that these cell lines might be useful for the study of Ku70 functions and the Ku70-dependent DSB repair pathway in lung epithelial cells. KEY WORDS: anticancer treatment, epithelial cell, γH2AX, Ku70, Ku80.

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Section: Cell Lines and Culturesmentioning

confidence: 99%

“…X-rays were generated at 200 kVp/20 mA and filtered through 0.5-mm Cu and Al filters, as described previously [7].…”

Section: Cell Lines and Culturesmentioning

confidence: 99%

The Defect of Ku70 Affects Sensitivity to X-Ray and Radiation-Induced Caspase-Dependent Apoptosis in Lung Cells

Koike¹,

Yutoku

Koike³

2013

The Journal of Veterinary Medical Science

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“…Three lung epithelial cell lines were established from the lungs of 7-day-old Ku70-/-or Ku70+/-mice by standard procedures and SV40 immortalization. Briefly, the SV40 T antigen expression vector (pLNCLT) was transfected using Effectene (Qiagen Inc., Chatsworth, CA, U.S.A.), and stable cell lines were established using G418 (0.3 mg/mL), as described previously [16]. A cell line of a human tumor, HeLa, was cultured as described in previous studies [10,12].…”

Section: Mice Cell Lines Cultures and Transfectionmentioning

confidence: 99%

“…X-irradiation: Cells were exposed to X-rays at a dose rate of 0.86-0.91 Gy/min at room temperature [16]. X-rays were generated at 200 kVp/20 mA and filtered through 0.5-mm Cu and Al filters, as described previously [16].…”

Section: Mice Cell Lines Cultures and Transfectionmentioning

confidence: 99%

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Establishment of Ku70-Deficient Lung Epithelial Cell Lines and Their Hypersensitivity to Low-Dose X-Irradiation

Koike¹,

Yutoku

Koike

2011

The Journal of Veterinary Medical Science

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ABSTRACT. In clinical situations, cellular resistance to chemotherapy and radiotherapy is a significant component of tumor treatment failure. The DNA repair protein Ku70 is a key contributor to chemoresistance to anticancer agents, e.g., etoposide and bleomycin, or radioresistance. Ku70 plays a key role as a sensor of DNA double-strand breaks (DSBs) induced following exposure to ionizing radiation as well as treatment with some chemotherapeutic drugs. The responses of different organs to radiation vary widely and likely depend on the cell population in the organs. However, it is not clear whether Ku70 plays a role in the low-dose radioresistance of lung epithelial cells. In this study, we established Ku70-deficient epithelial cell lines from murine lungs lacking Ku70. Ku70-/-lung epithelial cells exhibited reduced Ku80 expression. Moreover, Ku70-/-lung epithelial cells were more sensitive than controls (Ku70+/-lung epithelial cells) to low-dose X-irradiation (< 0.5 Gy). We also found that consistent with the Ku70 function as a sensor of DSBs, Ku70 mainly localized in the nuclei of murine lung epithelial cells. These findings clearly indicate that Ku70 plays a key role in regulation of the Ku80 expression level in and the radioresistance of lung epithelial cells. Our data also suggest that these cell lines might be useful not only for study of Ku70 functions and the DSB repair pathway, but also for study of the molecular mechanism underlying the sensitivity to chemotherapeutic drugs and radiation in lung epithelial cells.

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Cellular Dynamics of Ku: Characterization and Purification of Ku‐eGFP

et al. 2008

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Ku is a predominantly nuclear protein that functions as a DNA double-strand-break (DSB) binding protein and regulatory subunit of the DNA-dependent protein kinase (DNA-PK). DNA-PK is involved in synapsis and remodeling of broken DNA ends during nonhomologous end-joining (NHEJ) of DNA DSBs. It has also recently been demonstrated that Ku plays roles in cytoplasmic and membrane processes, namely: interaction with matrix metalloproteinase 9, acting as a co-receptor for parvoviral infection, and also interacting with cell polarity protein, Par3. We present a method for creating stable expression of Ku-eGFP in CHO cells and extend the procedure to purify Ku-eGFP for in vitro assaying. We demonstrated that Ku-eGFP localizes to the nucleus of HeLa cells upon microinjection into the cytoplasm as well as localizing to laser induced DNA damage. We also characterized the diffusional dynamics of Ku in the nucleus and in the cytoplasm using fluorescence correlation spectroscopy (FCS). The FCS data suggest that whereas the majority of Ku (70%) in the nucleus is mobile and freely diffusing, in a cellular context, there also exists a significant slow process fraction (30%). Strikingly, in the cytoplasm, this immobile/slow moving fraction is even more pronounced (45%).

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The Establishment and Characterization of Cell Lines Stably Expressing Human Ku80 Tagged with Enhanced Green Fluorescent Protein

Cited by 18 publications

References 42 publications

The Defect of Ku70 Affects Sensitivity to X-Ray and Radiation-Induced Caspase-Dependent Apoptosis in Lung Cells

The Defect of Ku70 Affects Sensitivity to X-Ray and Radiation-Induced Caspase-Dependent Apoptosis in Lung Cells

Establishment of Ku70-Deficient Lung Epithelial Cell Lines and Their Hypersensitivity to Low-Dose X-Irradiation

Cellular Dynamics of Ku: Characterization and Purification of Ku‐eGFP

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