| Budget Amount *help |
¥11,000,000 (Direct Cost: ¥11,000,000)
Fiscal Year 2004: ¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 2003: ¥6,700,000 (Direct Cost: ¥6,700,000)
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| Research Abstract |
Gap junctions are considered to play an important role in moderating cell death including apoptosis.
However, the basic phenomena underlying when and where the alterations of gap junctions occur during apoptosis have not been well documented. In this study, we examined the fate of Cx43 during apoptosis in order to answer the following questions : (1)Does Cx43 localization change during apoptosis? (2)If so, when? (3)Does alteration of Cx43 depend on caspase activation? To answer these questions, we analyzed the spatiotemporal changes of connexin(Cx) during UV light-induced apoptosis using Cx43-enhanced green fluorescent protein(EGFP)-expressing HeLa cells, and compared them with those of mitochondrial membrane potential(MMP) using tetramethylrhodamine ethyl ester(TMRE) and nuclear morphological observation using Hoechst 33342. At 2 hr post-UV-irradiation, a third of the cells became TMRE-negative, i.e., they showed the loss of MMP, but with slight nuclear fragmentation, and high percentages of linear Cx43-EGFP plaques were found among both TMRE-positive and TMRE-negative cells. At 4 hr post-UV-irradiation, the percentage of these linear plaques was decreased, and both punctate and diffuse localization of Cx43-EGFP were noted in the cytoplasm of TMRE-negative cells without nuclear fragmentation. At 8 hr post-irradiation, punctate cytoplasmic localization of Cx43-EGFP was noted in TMRE-negative cells with nuclear fragmentation.
Treatment with the caspase inhibitor Z-VAD-FMK blocked nuclear fragmentation and partially preserved both gap junctional plaques and MMP. These results indicate that, during apoptosis, Cx mobilization into the cytoplasm occurs after MMP depolarization but before nuclear fragmentation and that this alteration partly depends on caspase.
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