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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

Expression of EYFP-mouse Ku70 in mouse cells. (A) Schematics of EYFP-mouse Ku70chimeric protein (EYFP-mouse Ku70) and control protein (EYFP). (B) Extracts from mouse(NIH3T3) cells transiently expressing the EYFP-mouse Ku70 or EYFP prepared andsubjected to Western blotting using the anti-Ku70, anti-GFP or anti-β-actinantibody.
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fig_001: Expression of EYFP-mouse Ku70 in mouse cells. (A) Schematics of EYFP-mouse Ku70chimeric protein (EYFP-mouse Ku70) and control protein (EYFP). (B) Extracts from mouse(NIH3T3) cells transiently expressing the EYFP-mouse Ku70 or EYFP prepared andsubjected to Western blotting using the anti-Ku70, anti-GFP or anti-β-actinantibody.

Mentions: Expression and nuclear localization of EYFP-mouse Ku70 in living mousefibroblasts: We examined the expression and subcellular localization ofEYFP-mouse Ku70 in mouse NIH3T3 fibroblasts. First, we generated cells transientlyexpressing EYFP-mouse Ku70 in NIH3T3 cells. The expression vector pEYFP-C1 containingfull-length mouse Ku70 or pEYFP-C1 alone was transfected into NIH3T3 cells (Fig. 1AFig. 1.


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)

Expression of EYFP-mouse Ku70 in mouse cells. (A) Schematics of EYFP-mouse Ku70chimeric protein (EYFP-mouse Ku70) and control protein (EYFP). (B) Extracts from mouse(NIH3T3) cells transiently expressing the EYFP-mouse Ku70 or EYFP prepared andsubjected to Western blotting using the anti-Ku70, anti-GFP or anti-β-actinantibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig_001: Expression of EYFP-mouse Ku70 in mouse cells. (A) Schematics of EYFP-mouse Ku70chimeric protein (EYFP-mouse Ku70) and control protein (EYFP). (B) Extracts from mouse(NIH3T3) cells transiently expressing the EYFP-mouse Ku70 or EYFP prepared andsubjected to Western blotting using the anti-Ku70, anti-GFP or anti-β-actinantibody.
Mentions: Expression and nuclear localization of EYFP-mouse Ku70 in living mousefibroblasts: We examined the expression and subcellular localization ofEYFP-mouse Ku70 in mouse NIH3T3 fibroblasts. First, we generated cells transientlyexpressing EYFP-mouse Ku70 in NIH3T3 cells. The expression vector pEYFP-C1 containingfull-length mouse Ku70 or pEYFP-C1 alone was transfected into NIH3T3 cells (Fig. 1AFig. 1.

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