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Nuclear localization of mouse Ku70 in interphase cells and focus formation of mouse Ku70 at DNA damage sites immediately after irradiation.

Koike M, Yutoku Y, Koike A - J. Vet. Med. Sci. (2015)

Bottom Line: Furthermore, our findings indicate that EYFP-mouse Ku70 accumulates with its heterodimeric partner Ku80 immediately at laser-microirradiated DSB sites.We also confirmed that the structure of Ku70 nuclear localization signal (NLS) is highly conserved among various rodent species, such as the mouse, rat, degu and ground squirrel, supporting the idea that NLS is important for the regulation of rodent Ku70 function.Collectively, these results suggest that the mechanisms of regulating the localization and accumulation of Ku70 at DSBs might be well conserved between the mouse and human species.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan.

ABSTRACT
To elucidate the mechanisms of DNA repair pathway is critical for developing next-generation radiotherapies and chemotherapeutic drugs for cancer. Ionizing radiation and many chemotherapeutic drugs kill tumor cells mainly by inducing DNA double-strand breaks (DSBs). The classical nonhomologous DNA-end joining (NHEJ) (C-NHEJ) pathway repairs a predominant fraction of DSBs in mammalian cells. The C-NHEJ pathway appears to start with the binding of Ku (heterodimer of Ku70 and Ku80) to DNA break ends. Therefore, recruitment of Ku to DSB sites might play a critical role in regulating NHEJ activity. Indeed, human Ku70 and Ku80 localize in the nuclei and accumulate at microirradiated DSB sites. However, the localization and regulation mechanisms of Ku70 and Ku80 homologues in animal models, such as mice and other species, have not been elucidated in detail, particularly in cells immediately after microirradiation. Here, we show that EYFP-tagged mouse Ku70 localizes in the interphase nuclei of mouse fibroblasts and epithelial cells. Furthermore, our findings indicate that EYFP-mouse Ku70 accumulates with its heterodimeric partner Ku80 immediately at laser-microirradiated DSB sites. We also confirmed that the structure of Ku70 nuclear localization signal (NLS) is highly conserved among various rodent species, such as the mouse, rat, degu and ground squirrel, supporting the idea that NLS is important for the regulation of rodent Ku70 function. Collectively, these results suggest that the mechanisms of regulating the localization and accumulation of Ku70 at DSBs might be well conserved between the mouse and human species.

No MeSH data available.


Related in: MedlinePlus

EYFP-mouse Ku70 accumulated rapidly at DSBs induced by laser microirradiation inmouse lung epithelial cell lines. (A) Expression of Ku70 and Ku80 in total celllysates from NIH3T3, Ku70 +/– MLE and Ku70 –/– MLE cells. Total cell lysates from thetwo lung epithelial cell lines and the NIH3T3 cell line were analyzed by Westernblotting using the anti-Ku70, anti-Ku80 or anti-β-actin antibody. (B) Imaging ofliving EYFP-mouse Ku70- or EYFP-transfected mouse lung epithelial cells before andafter microirradiation. Arrowheads indicate the microirradiated sites. (C) NLS ofmouse Ku70 (amino acids 537–554). (D) Alignment of the primary sequence among humanand rodent homologous Ku70 proteins. The basic (red) or nonbasic residues (black) areindicated in different colors for comparison. The GeneBank accession number for eachsequence is indicated.
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fig_004: EYFP-mouse Ku70 accumulated rapidly at DSBs induced by laser microirradiation inmouse lung epithelial cell lines. (A) Expression of Ku70 and Ku80 in total celllysates from NIH3T3, Ku70 +/– MLE and Ku70 –/– MLE cells. Total cell lysates from thetwo lung epithelial cell lines and the NIH3T3 cell line were analyzed by Westernblotting using the anti-Ku70, anti-Ku80 or anti-β-actin antibody. (B) Imaging ofliving EYFP-mouse Ku70- or EYFP-transfected mouse lung epithelial cells before andafter microirradiation. Arrowheads indicate the microirradiated sites. (C) NLS ofmouse Ku70 (amino acids 537–554). (D) Alignment of the primary sequence among humanand rodent homologous Ku70 proteins. The basic (red) or nonbasic residues (black) areindicated in different colors for comparison. The GeneBank accession number for eachsequence is indicated.

Mentions: Nuclear localization and accumulation of EYFP-mouse Ku70 immediately at DSBs inmouse epithelial cells with or without endogenous Ku70 expression: We analyzedwhether EYFP-mouse Ku70 localizes in the nuclei during the interphase and accumulatesrapidly at DSBs induced by laser microirradiation in mouse epithelial cells. First, byWestern blot analysis, we investigated the expression of Ku70 and Ku80 in the mousefibroblast NIH3T3 cells and the 2 mouse epithelial cell lines (Ku70+/− MLE and Ku70−/− MLE)derived from Ku70-knockout mice. As shown in Fig.4AFig. 4.


Nuclear localization of mouse Ku70 in interphase cells and focus formation of mouse Ku70 at DNA damage sites immediately after irradiation.

Koike M, Yutoku Y, Koike A - J. Vet. Med. Sci. (2015)

EYFP-mouse Ku70 accumulated rapidly at DSBs induced by laser microirradiation inmouse lung epithelial cell lines. (A) Expression of Ku70 and Ku80 in total celllysates from NIH3T3, Ku70 +/– MLE and Ku70 –/– MLE cells. Total cell lysates from thetwo lung epithelial cell lines and the NIH3T3 cell line were analyzed by Westernblotting using the anti-Ku70, anti-Ku80 or anti-β-actin antibody. (B) Imaging ofliving EYFP-mouse Ku70- or EYFP-transfected mouse lung epithelial cells before andafter microirradiation. Arrowheads indicate the microirradiated sites. (C) NLS ofmouse Ku70 (amino acids 537–554). (D) Alignment of the primary sequence among humanand rodent homologous Ku70 proteins. The basic (red) or nonbasic residues (black) areindicated in different colors for comparison. The GeneBank accession number for eachsequence is indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591156&req=5

fig_004: EYFP-mouse Ku70 accumulated rapidly at DSBs induced by laser microirradiation inmouse lung epithelial cell lines. (A) Expression of Ku70 and Ku80 in total celllysates from NIH3T3, Ku70 +/– MLE and Ku70 –/– MLE cells. Total cell lysates from thetwo lung epithelial cell lines and the NIH3T3 cell line were analyzed by Westernblotting using the anti-Ku70, anti-Ku80 or anti-β-actin antibody. (B) Imaging ofliving EYFP-mouse Ku70- or EYFP-transfected mouse lung epithelial cells before andafter microirradiation. Arrowheads indicate the microirradiated sites. (C) NLS ofmouse Ku70 (amino acids 537–554). (D) Alignment of the primary sequence among humanand rodent homologous Ku70 proteins. The basic (red) or nonbasic residues (black) areindicated in different colors for comparison. The GeneBank accession number for eachsequence is indicated.
Mentions: Nuclear localization and accumulation of EYFP-mouse Ku70 immediately at DSBs inmouse epithelial cells with or without endogenous Ku70 expression: We analyzedwhether EYFP-mouse Ku70 localizes in the nuclei during the interphase and accumulatesrapidly at DSBs induced by laser microirradiation in mouse epithelial cells. First, byWestern blot analysis, we investigated the expression of Ku70 and Ku80 in the mousefibroblast NIH3T3 cells and the 2 mouse epithelial cell lines (Ku70+/− MLE and Ku70−/− MLE)derived from Ku70-knockout mice. As shown in Fig.4AFig. 4.

Bottom Line: Furthermore, our findings indicate that EYFP-mouse Ku70 accumulates with its heterodimeric partner Ku80 immediately at laser-microirradiated DSB sites.We also confirmed that the structure of Ku70 nuclear localization signal (NLS) is highly conserved among various rodent species, such as the mouse, rat, degu and ground squirrel, supporting the idea that NLS is important for the regulation of rodent Ku70 function.Collectively, these results suggest that the mechanisms of regulating the localization and accumulation of Ku70 at DSBs might be well conserved between the mouse and human species.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan.

ABSTRACT
To elucidate the mechanisms of DNA repair pathway is critical for developing next-generation radiotherapies and chemotherapeutic drugs for cancer. Ionizing radiation and many chemotherapeutic drugs kill tumor cells mainly by inducing DNA double-strand breaks (DSBs). The classical nonhomologous DNA-end joining (NHEJ) (C-NHEJ) pathway repairs a predominant fraction of DSBs in mammalian cells. The C-NHEJ pathway appears to start with the binding of Ku (heterodimer of Ku70 and Ku80) to DNA break ends. Therefore, recruitment of Ku to DSB sites might play a critical role in regulating NHEJ activity. Indeed, human Ku70 and Ku80 localize in the nuclei and accumulate at microirradiated DSB sites. However, the localization and regulation mechanisms of Ku70 and Ku80 homologues in animal models, such as mice and other species, have not been elucidated in detail, particularly in cells immediately after microirradiation. Here, we show that EYFP-tagged mouse Ku70 localizes in the interphase nuclei of mouse fibroblasts and epithelial cells. Furthermore, our findings indicate that EYFP-mouse Ku70 accumulates with its heterodimeric partner Ku80 immediately at laser-microirradiated DSB sites. We also confirmed that the structure of Ku70 nuclear localization signal (NLS) is highly conserved among various rodent species, such as the mouse, rat, degu and ground squirrel, supporting the idea that NLS is important for the regulation of rodent Ku70 function. Collectively, these results suggest that the mechanisms of regulating the localization and accumulation of Ku70 at DSBs might be well conserved between the mouse and human species.

No MeSH data available.


Related in: MedlinePlus