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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

Localization of EYFP-cattle XLF in living cattle cells. (A) Schematics of EYFP-cattleXLF chimeric protein and control protein (EYFP). (B) Extracts from cattle (MDBK) cellstransiently expressing the EYFP-cattle XLF or EYFP prepared and subjected to Westernblotting using the anti-XLF, anti-GFP or anti-β-actin antibody. (C) Imaging of livingEYFP-cattle XLF-transfected cells. Living MDBK cells transiently expressingEYFP-cattle XLF or EYFP were analyzed by confocal laser microscopy. EYFP images forthe same cells are shown alone (left panel) or merged (right panel) with differentialinterference contrast images (DIC) (center panel).
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fig_002: Localization of EYFP-cattle XLF in living cattle cells. (A) Schematics of EYFP-cattleXLF chimeric protein and control protein (EYFP). (B) Extracts from cattle (MDBK) cellstransiently expressing the EYFP-cattle XLF or EYFP prepared and subjected to Westernblotting using the anti-XLF, anti-GFP or anti-β-actin antibody. (C) Imaging of livingEYFP-cattle XLF-transfected cells. Living MDBK cells transiently expressingEYFP-cattle XLF or EYFP were analyzed by confocal laser microscopy. EYFP images forthe same cells are shown alone (left panel) or merged (right panel) with differentialinterference contrast images (DIC) (center panel).

Mentions: To elucidate the localization of XLF in cattle cells, we studied the distribution of XLF byconfocal laser microscopy (Fig. 1B and 1C).Indirect immunofluorescence staining using the anti-XLF antibody showed that fluorescencewas detected in the nucleoplasm of MDBK cells during the interphase. On the other hand, thefluorescence was detected throughout the cytoplasm of MDBK cells during the mitotic phase,but not in the condensed chromosomes of the mitotic cells. These observations indicate thatthe localization of cattle XLF changes dynamically during the cell cycle. To clarify thelocalization of XLF in living cattle cells during the interphase, we examined the expressionand localization of EYFP-cattle XLF in MDBK cells. We generated cells transiently expressingEYFP-cattle XLF in MDBK cells. The expression vector pEYFP-C1 containing cattle XLF(pEYFP-cattle XLF) was transfected into MDBK cells (Fig. 2AFig. 2.


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)

Localization of EYFP-cattle XLF in living cattle cells. (A) Schematics of EYFP-cattleXLF chimeric protein and control protein (EYFP). (B) Extracts from cattle (MDBK) cellstransiently expressing the EYFP-cattle XLF or EYFP prepared and subjected to Westernblotting using the anti-XLF, anti-GFP or anti-β-actin antibody. (C) Imaging of livingEYFP-cattle XLF-transfected cells. Living MDBK cells transiently expressingEYFP-cattle XLF or EYFP were analyzed by confocal laser microscopy. EYFP images forthe same cells are shown alone (left panel) or merged (right panel) with differentialinterference contrast images (DIC) (center panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig_002: Localization of EYFP-cattle XLF in living cattle cells. (A) Schematics of EYFP-cattleXLF chimeric protein and control protein (EYFP). (B) Extracts from cattle (MDBK) cellstransiently expressing the EYFP-cattle XLF or EYFP prepared and subjected to Westernblotting using the anti-XLF, anti-GFP or anti-β-actin antibody. (C) Imaging of livingEYFP-cattle XLF-transfected cells. Living MDBK cells transiently expressingEYFP-cattle XLF or EYFP were analyzed by confocal laser microscopy. EYFP images forthe same cells are shown alone (left panel) or merged (right panel) with differentialinterference contrast images (DIC) (center panel).
Mentions: To elucidate the localization of XLF in cattle cells, we studied the distribution of XLF byconfocal laser microscopy (Fig. 1B and 1C).Indirect immunofluorescence staining using the anti-XLF antibody showed that fluorescencewas detected in the nucleoplasm of MDBK cells during the interphase. On the other hand, thefluorescence was detected throughout the cytoplasm of MDBK cells during the mitotic phase,but not in the condensed chromosomes of the mitotic cells. These observations indicate thatthe localization of cattle XLF changes dynamically during the cell cycle. To clarify thelocalization of XLF in living cattle cells during the interphase, we examined the expressionand localization of EYFP-cattle XLF in MDBK cells. We generated cells transiently expressingEYFP-cattle XLF in MDBK cells. The expression vector pEYFP-C1 containing cattle XLF(pEYFP-cattle XLF) was transfected into MDBK cells (Fig. 2AFig. 2.

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