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Functional differences between wild-type and mutant-type BRCA1-associated protein 1 tumor suppressor against malignant mesothelioma cells.

Hakiri S, Osada H, Ishiguro F, Murakami H, Murakami-Tonami Y, Yokoi K, Sekido Y - Cancer Sci. (2015)

Bottom Line: Transduction of the WT BAP1 vector into MM cells with homozygous deletion at the BAP1 3' side resulted in both inhibition of cell proliferation and anchorage-independent cell growth, whereas BAP1 mutants of a missense or C-terminal truncated form showed impaired growth inhibitory effects.Furthermore, using the MM cells with BAP1 deletion, we found that WT BAP1, and even a missense mutant, conferred a higher survival rate after IR compared to the control vector.Our results suggested that, whereas WT BAP1 suppresses MM cell proliferation and restores cell survival after IR damage, some mutant BAP1 may also moderately retain these functions.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Oncology, Aichi Cancer Center Research Institute, Nagoya, Japan.

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Related in: MedlinePlus

Irradiation (IR) induces phosphorylation of BRCA1-associated protein 1 (BAP1). (a) Western blot analysis with anti-pS592-BAP1 antibody revealed that endogenous WT BAP1 was phosphorylated after IR at 7.5 Gy. (b) BAP1 phosphorylation levels increased in proportion to IR doses. *Non-specific bands. (c) Immunofluorescence analysis showed dominant nuclear localization of phosphorylated-BAP1 protein at 1 h post-IR (7.5 Gy). (d) Western blot analysis of nuclear/cytoplasmic fractionation tested the localization of exogenously transduced BAP1 and its phosphorylated form in malignant mesothelioma cells with BAP1 deletion. HD, homozygous deletion.
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fig04: Irradiation (IR) induces phosphorylation of BRCA1-associated protein 1 (BAP1). (a) Western blot analysis with anti-pS592-BAP1 antibody revealed that endogenous WT BAP1 was phosphorylated after IR at 7.5 Gy. (b) BAP1 phosphorylation levels increased in proportion to IR doses. *Non-specific bands. (c) Immunofluorescence analysis showed dominant nuclear localization of phosphorylated-BAP1 protein at 1 h post-IR (7.5 Gy). (d) Western blot analysis of nuclear/cytoplasmic fractionation tested the localization of exogenously transduced BAP1 and its phosphorylated form in malignant mesothelioma cells with BAP1 deletion. HD, homozygous deletion.

Mentions: As one of the major cellular functions of BAP1 is thought to be DNA repair, we induced DNA damage in MM cells with X-ray irradiation to reveal how BAP1 is involved in DNA repair in MM cells. First, when MeT-5A cells, which harbor WT BAP1, were irradiated, we observed the strongest BAP1 phosphorylation level 1 h after IR, which then gradually decreased (Fig.4a). The phosphorylation level of WT BAP1 was also confirmed to be increased in proportion to IR doses (Fig.4b). In addition, with immunofluorescence analysis, the strong phosphorylation of the exogenously transduced WT BAP1 into the MM cell with BAP1 deletion was also observed in the nucleus after IR exposure, in the same manner as with endogenous BAP1 (Fig.4c). Using the MM cell with BAP1 deletion to confirm these results, we then carried out cellular fractionation and found that phospho-mutant-type BAP1 proteins tended to localize in the nucleus after IR like WT BAP1, whereas total mutant-type BAP1 proteins dominantly localized in the cytoplasm (Fig.4d). These results suggested that several types of mutant BAP1 proteins as well as the WT form might be recruited to IR-induced DSB sites, although the precise mechanism is yet to be determined.


Functional differences between wild-type and mutant-type BRCA1-associated protein 1 tumor suppressor against malignant mesothelioma cells.

Hakiri S, Osada H, Ishiguro F, Murakami H, Murakami-Tonami Y, Yokoi K, Sekido Y - Cancer Sci. (2015)

Irradiation (IR) induces phosphorylation of BRCA1-associated protein 1 (BAP1). (a) Western blot analysis with anti-pS592-BAP1 antibody revealed that endogenous WT BAP1 was phosphorylated after IR at 7.5 Gy. (b) BAP1 phosphorylation levels increased in proportion to IR doses. *Non-specific bands. (c) Immunofluorescence analysis showed dominant nuclear localization of phosphorylated-BAP1 protein at 1 h post-IR (7.5 Gy). (d) Western blot analysis of nuclear/cytoplasmic fractionation tested the localization of exogenously transduced BAP1 and its phosphorylated form in malignant mesothelioma cells with BAP1 deletion. HD, homozygous deletion.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556387&req=5

fig04: Irradiation (IR) induces phosphorylation of BRCA1-associated protein 1 (BAP1). (a) Western blot analysis with anti-pS592-BAP1 antibody revealed that endogenous WT BAP1 was phosphorylated after IR at 7.5 Gy. (b) BAP1 phosphorylation levels increased in proportion to IR doses. *Non-specific bands. (c) Immunofluorescence analysis showed dominant nuclear localization of phosphorylated-BAP1 protein at 1 h post-IR (7.5 Gy). (d) Western blot analysis of nuclear/cytoplasmic fractionation tested the localization of exogenously transduced BAP1 and its phosphorylated form in malignant mesothelioma cells with BAP1 deletion. HD, homozygous deletion.
Mentions: As one of the major cellular functions of BAP1 is thought to be DNA repair, we induced DNA damage in MM cells with X-ray irradiation to reveal how BAP1 is involved in DNA repair in MM cells. First, when MeT-5A cells, which harbor WT BAP1, were irradiated, we observed the strongest BAP1 phosphorylation level 1 h after IR, which then gradually decreased (Fig.4a). The phosphorylation level of WT BAP1 was also confirmed to be increased in proportion to IR doses (Fig.4b). In addition, with immunofluorescence analysis, the strong phosphorylation of the exogenously transduced WT BAP1 into the MM cell with BAP1 deletion was also observed in the nucleus after IR exposure, in the same manner as with endogenous BAP1 (Fig.4c). Using the MM cell with BAP1 deletion to confirm these results, we then carried out cellular fractionation and found that phospho-mutant-type BAP1 proteins tended to localize in the nucleus after IR like WT BAP1, whereas total mutant-type BAP1 proteins dominantly localized in the cytoplasm (Fig.4d). These results suggested that several types of mutant BAP1 proteins as well as the WT form might be recruited to IR-induced DSB sites, although the precise mechanism is yet to be determined.

Bottom Line: Transduction of the WT BAP1 vector into MM cells with homozygous deletion at the BAP1 3' side resulted in both inhibition of cell proliferation and anchorage-independent cell growth, whereas BAP1 mutants of a missense or C-terminal truncated form showed impaired growth inhibitory effects.Furthermore, using the MM cells with BAP1 deletion, we found that WT BAP1, and even a missense mutant, conferred a higher survival rate after IR compared to the control vector.Our results suggested that, whereas WT BAP1 suppresses MM cell proliferation and restores cell survival after IR damage, some mutant BAP1 may also moderately retain these functions.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Oncology, Aichi Cancer Center Research Institute, Nagoya, Japan.

Show MeSH
Related in: MedlinePlus