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Dynamic changes in subcellular localization of cattle XLF during cell cycle, and focus formation of cattle XLF at DNA damage sites immediately after irradiation.

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

Bottom Line: Moreover, nuclear localization and accumulation of cattle XLF at DSB sites are dependent on 12 amino acids (288-299) of the C-terminal region of XLF (XLF CTR).Furthermore, basic amino acids on the XLF CTR are highly conserved among domestic animals including cattle, goat and horses, suggesting that the CTR is essential for the function of XLF in domestic animals.These findings might be useful to develop the molecular-targeting therapeutic drug taking XLF as a target molecule for human and domestic animals.

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
Clinically, many chemotherapeutics and ionizing radiation (IR) have been applied for the treatment of various types of human and animal malignancies. These treatments kill tumor cells by causing DNA double-strand breaks (DSBs). Core factors of classical nonhomologous DNA-end joining (C-NHEJ) play a vital role in DSB repair. Thus, it is indispensable to clarify the mechanisms of C-NHEJ in order to develop next-generation chemotherapeutics for cancer. The XRCC4-like factor (XLF; also called Cernunnos or NHEJ1) is the lastly identified core NHEJ factor. The localization of core NHEJ factors might play a critical role in regulating NHEJ activity. The localization and function of XLF have not been elucidated in animal species other than mice and humans. Domestic cattle (Bos taurus) are the most common and vital domestic animals in many countries. Here, we show that the localization of cattle XLF changes dynamically during the cell cycle. Furthermore, EYFP-cattle XLF accumulates quickly at microirradiated sites and colocalizes with the DSB marker γH2AX. Moreover, nuclear localization and accumulation of cattle XLF at DSB sites are dependent on 12 amino acids (288-299) of the C-terminal region of XLF (XLF CTR). Furthermore, basic amino acids on the XLF CTR are highly conserved among domestic animals including cattle, goat and horses, suggesting that the CTR is essential for the function of XLF in domestic animals. These findings might be useful to develop the molecular-targeting therapeutic drug taking XLF as a target molecule for human and domestic animals.

No MeSH data available.


Related in: MedlinePlus

The C-terminal region (CTR) is vital for the nuclear localization and recruitment ofcattle XLF to DSBs in vivo. (A) Extracts from cattle (MDBK) cellstransiently expressing the indicated cattle XLF deletions were prepared and subjectedto Western blotting using the anti-GFP or anti-β-actin antibody. (B, C) Identificationof essential domain of cattle XLF for nuclear localization and for accumulation atDSBs. EYFP-cattle XLF mutants were expressed in cattle (MDBK) cells. The localizationand accumulation of the chimeric proteins at laser-induced DSBs were investigated vialive cell imaging. The results are summarized on the right: Cellular localization (N,nucleus; C, cytoplasm) and formation of focus (+, accumulated at microirradiatedsites; -, not accumulated at microirradiated sites). Arrowheads indicate themicroirradiated sites (C). (D) Immunostaining of microirradiated EYFP-cattle XLF(162–287)-transfected cells with anti-γH2AX antibody. The cells were fixed and stainedwith the anti-γH2AX antibody at 5 min postirradiation. Left panel, EYFP-cattle XLF(162-287); center panel, γH2AX image; right panel, merged image. (E) Identity betweenthe cattle XLF and human XLF at the amino acid level and the CTR of cattle XLF (aminoacids 288–299). (F) The alignment of the primary sequence among homologous XLFproteins. For comparison, the basic (red) or non-basic residues (black) are shown indifferent colors. The GeneBank accession number for each sequence is mentioned. *, Thesequence of CTR of chicken XLF is from Reference [1].
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fig_004: The C-terminal region (CTR) is vital for the nuclear localization and recruitment ofcattle XLF to DSBs in vivo. (A) Extracts from cattle (MDBK) cellstransiently expressing the indicated cattle XLF deletions were prepared and subjectedto Western blotting using the anti-GFP or anti-β-actin antibody. (B, C) Identificationof essential domain of cattle XLF for nuclear localization and for accumulation atDSBs. EYFP-cattle XLF mutants were expressed in cattle (MDBK) cells. The localizationand accumulation of the chimeric proteins at laser-induced DSBs were investigated vialive cell imaging. The results are summarized on the right: Cellular localization (N,nucleus; C, cytoplasm) and formation of focus (+, accumulated at microirradiatedsites; -, not accumulated at microirradiated sites). Arrowheads indicate themicroirradiated sites (C). (D) Immunostaining of microirradiated EYFP-cattle XLF(162–287)-transfected cells with anti-γH2AX antibody. The cells were fixed and stainedwith the anti-γH2AX antibody at 5 min postirradiation. Left panel, EYFP-cattle XLF(162-287); center panel, γH2AX image; right panel, merged image. (E) Identity betweenthe cattle XLF and human XLF at the amino acid level and the CTR of cattle XLF (aminoacids 288–299). (F) The alignment of the primary sequence among homologous XLFproteins. For comparison, the basic (red) or non-basic residues (black) are shown indifferent colors. The GeneBank accession number for each sequence is mentioned. *, Thesequence of CTR of chicken XLF is from Reference [1].

Mentions: The C-terminal region (CTR) of cattle XLF is essential for the nuclear localizationand recruitment of XLF to DSBs in cattle cells: To determine the region essentialfor nuclear localization of cattle XLF, we investigated the localization of cattle XLF andits mutant. Firstly, the pEYFP-cattle XLF and its mutants were transfected into MDBK cells.As shown in Fig. 4AFig. 4.


Dynamic changes in subcellular localization of cattle XLF during cell cycle, and focus formation of cattle XLF at DNA damage sites immediately after irradiation.

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

The C-terminal region (CTR) is vital for the nuclear localization and recruitment ofcattle XLF to DSBs in vivo. (A) Extracts from cattle (MDBK) cellstransiently expressing the indicated cattle XLF deletions were prepared and subjectedto Western blotting using the anti-GFP or anti-β-actin antibody. (B, C) Identificationof essential domain of cattle XLF for nuclear localization and for accumulation atDSBs. EYFP-cattle XLF mutants were expressed in cattle (MDBK) cells. The localizationand accumulation of the chimeric proteins at laser-induced DSBs were investigated vialive cell imaging. The results are summarized on the right: Cellular localization (N,nucleus; C, cytoplasm) and formation of focus (+, accumulated at microirradiatedsites; -, not accumulated at microirradiated sites). Arrowheads indicate themicroirradiated sites (C). (D) Immunostaining of microirradiated EYFP-cattle XLF(162–287)-transfected cells with anti-γH2AX antibody. The cells were fixed and stainedwith the anti-γH2AX antibody at 5 min postirradiation. Left panel, EYFP-cattle XLF(162-287); center panel, γH2AX image; right panel, merged image. (E) Identity betweenthe cattle XLF and human XLF at the amino acid level and the CTR of cattle XLF (aminoacids 288–299). (F) The alignment of the primary sequence among homologous XLFproteins. For comparison, the basic (red) or non-basic residues (black) are shown indifferent colors. The GeneBank accession number for each sequence is mentioned. *, Thesequence of CTR of chicken XLF is from Reference [1].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591152&req=5

fig_004: The C-terminal region (CTR) is vital for the nuclear localization and recruitment ofcattle XLF to DSBs in vivo. (A) Extracts from cattle (MDBK) cellstransiently expressing the indicated cattle XLF deletions were prepared and subjectedto Western blotting using the anti-GFP or anti-β-actin antibody. (B, C) Identificationof essential domain of cattle XLF for nuclear localization and for accumulation atDSBs. EYFP-cattle XLF mutants were expressed in cattle (MDBK) cells. The localizationand accumulation of the chimeric proteins at laser-induced DSBs were investigated vialive cell imaging. The results are summarized on the right: Cellular localization (N,nucleus; C, cytoplasm) and formation of focus (+, accumulated at microirradiatedsites; -, not accumulated at microirradiated sites). Arrowheads indicate themicroirradiated sites (C). (D) Immunostaining of microirradiated EYFP-cattle XLF(162–287)-transfected cells with anti-γH2AX antibody. The cells were fixed and stainedwith the anti-γH2AX antibody at 5 min postirradiation. Left panel, EYFP-cattle XLF(162-287); center panel, γH2AX image; right panel, merged image. (E) Identity betweenthe cattle XLF and human XLF at the amino acid level and the CTR of cattle XLF (aminoacids 288–299). (F) The alignment of the primary sequence among homologous XLFproteins. For comparison, the basic (red) or non-basic residues (black) are shown indifferent colors. The GeneBank accession number for each sequence is mentioned. *, Thesequence of CTR of chicken XLF is from Reference [1].
Mentions: The C-terminal region (CTR) of cattle XLF is essential for the nuclear localizationand recruitment of XLF to DSBs in cattle cells: To determine the region essentialfor nuclear localization of cattle XLF, we investigated the localization of cattle XLF andits mutant. Firstly, the pEYFP-cattle XLF and its mutants were transfected into MDBK cells.As shown in Fig. 4AFig. 4.

Bottom Line: Moreover, nuclear localization and accumulation of cattle XLF at DSB sites are dependent on 12 amino acids (288-299) of the C-terminal region of XLF (XLF CTR).Furthermore, basic amino acids on the XLF CTR are highly conserved among domestic animals including cattle, goat and horses, suggesting that the CTR is essential for the function of XLF in domestic animals.These findings might be useful to develop the molecular-targeting therapeutic drug taking XLF as a target molecule for human and domestic animals.

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
Clinically, many chemotherapeutics and ionizing radiation (IR) have been applied for the treatment of various types of human and animal malignancies. These treatments kill tumor cells by causing DNA double-strand breaks (DSBs). Core factors of classical nonhomologous DNA-end joining (C-NHEJ) play a vital role in DSB repair. Thus, it is indispensable to clarify the mechanisms of C-NHEJ in order to develop next-generation chemotherapeutics for cancer. The XRCC4-like factor (XLF; also called Cernunnos or NHEJ1) is the lastly identified core NHEJ factor. The localization of core NHEJ factors might play a critical role in regulating NHEJ activity. The localization and function of XLF have not been elucidated in animal species other than mice and humans. Domestic cattle (Bos taurus) are the most common and vital domestic animals in many countries. Here, we show that the localization of cattle XLF changes dynamically during the cell cycle. Furthermore, EYFP-cattle XLF accumulates quickly at microirradiated sites and colocalizes with the DSB marker γH2AX. Moreover, nuclear localization and accumulation of cattle XLF at DSB sites are dependent on 12 amino acids (288-299) of the C-terminal region of XLF (XLF CTR). Furthermore, basic amino acids on the XLF CTR are highly conserved among domestic animals including cattle, goat and horses, suggesting that the CTR is essential for the function of XLF in domestic animals. These findings might be useful to develop the molecular-targeting therapeutic drug taking XLF as a target molecule for human and domestic animals.

No MeSH data available.


Related in: MedlinePlus