Localization, trafficking, and temperature-dependence of the Aequorea green fluorescent protein in cultured vertebrate cells.

Ogawa, Hidesato; Inouye, Satoshi; Tsuji, Frederick I.; Yasuda, Kazuaki; Umesono, Kazuhiko

doi:10.1073/pnas.92.25.11899

Cited by 227 publications

(161 citation statements)

References 29 publications

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“…After removal of¯oating cells,¯at cells remaining on the plate did not express NPM-MLF1 proteins. Aequorea green¯uorescent protein (GFP) (Ogawa et al, 1995) was used as a transfection marker to comparatively analyse the kinetics of apoptosis induced by ectopic overexpression of NPM-MLF1, cMyc or E2F-1 (Evan et al, 1992;Askew et al, 1991;Wu and Levine, 1994;Qin et al, 1994;Shan and Lee, 1994) followed by serum starvation. Figure 2b shows that NPM-MLF1 transfectants died gradually during 72 h after serum starvation, while most of the c-Myc and E2F-1 transfectants rapidly lost their viability within 24 h. These observations indicate that ectopic overexpression of NPM-MLF1 in mouse ®broblasts induced apoptotic cell death upon serum deprivation with slower kinetics than did other well-known apoptotic inducers such as c-Myc or E2F-1.…”

Section: Resultsmentioning

confidence: 99%

Apoptosis induced by the myelodysplastic syndrome-associated NPM-MLF1 chimeric protein

1999

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The NPM-MLF1 chimeric protein is produced by the t(3;5)(q25.1;q34) chromosomal translocation, which is associated with myelodysplastic syndrome (MDS) prior to progression into acute myeloid leukemia (AML). Here we report that K562 human leukemia cells ectopically expressing NPM-MLF1, but not those with wild-type MLF1, were gradually eliminated from the culture by undergoing apoptosis. NIH3T3 mouse ®broblasts engineered to overexpress NPM-MLF1 grew normally but serum deprivation triggered apoptotic cell death with slower kinetics than did other well-known apoptotic inducers such as c-Myc or E2F-1. Quantitative analysis of apoptotic induction con®rmed that, neither NPM nor MLF1, but the NPM-MLF1 fusion protein was able to induce apoptosis. Analyses using a variety of deletion mutants of NPM-MLF1 revealed that induction of apoptosis required the N-terminal domain of MLF1 and the NPM domain containing nuclear localization signal and that removal of the NPM dimerization domain markedly impaired the ability to induce apoptosis. Co-expression of Bcl-2 rescued NIH3T3 ®broblasts from NPM-MLF1-mediated cell death without a ecting the expression level or the subcellular localization of NPM-MLF1 and enabled cells to progress into S phase in low serum. These ®ndings provide an NPM-MLF1-mediated novel mechanism of apoptotic induction and imply that NPM-MLF1 in collaboration with anti-apoptotic oncoproteins may play an important role in multi-step progression from MDS to AML.

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Section: Resultsmentioning

confidence: 99%

Apoptosis induced by the myelodysplastic syndrome-associated NPM-MLF1 chimeric protein

1999

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“…Cells in six-well plates were transfected with 0.2 mg expression vector for EGFP 39 together with 0.8 mg pSUPER vector for RNAi against FLIP or with pCMV-Tag2B (Stratagene) encoding FLIP-S (0.025 mg) and FLIP-L (0.025 mg) using Lipofectamine Plus (Invitrogen), in accordance with the manufacturer's instructions. The total amount of DNA for each transfection was adjusted to 1.0 mg per sample by adding empty vector.…”

Section: Analysis Of Caspase-3 Activation In Various Transfectantsmentioning

confidence: 99%

Sensitization of osteosarcoma cells to death receptor-mediated apoptosis by HDAC inhibitors through downregulation of cellular FLIP

2004

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Fas-mediated apoptosis plays an important role in elimination of tumor cells in vivo, but some tumor-derived cells are resistant to this mechanism. Here, we show that treatment with the histone deacetylase (HDAC) inhibitor FR901228 renders Fas-resistant osteosarcoma cell lines sensitive to Fas-mediated apoptosis by downregulating expression of cellular FLIP (cellular FLICE-inhibitory protein), an inhibitor of Fas-mediated activation of caspase-8. Moreover, sensitization to Fas-mediated apoptosis was also induced in Fas-resistant osteosarcoma cells by suppressing FLIP expression using FLIP-specific RNA interference. HDAC inhibitors including FR901228 were shown to induce downregulation of cellular FLIP through inhibiting generation of FLIP mRNA, rather than stimulating degradation at either protein or mRNA level, and the inhibition was independent of de novo protein synthesis. These results clearly indicate that some tumor cells exhibit a phenotype resistant to death receptor-mediated apoptosis by expressing cellular FLIP, and that HDAC inhibitors sensitize such resistant tumor cells by directly downregulating cellular FLIP mRNA.

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“…GFP-fusion DED and CARDs, including Casp8NC-DED (1-202 aa), Casp2CARD (1-152 aa of human caspase-2), Casp9CARD (1-172 aa of human caspase-9), Apaf-1CARD (1-97 aa of human Apaf-1 (Zou et al 1997)) and Apaf-1530 (1-530 aa of human Apaf-1) were prepared by PCR. The PCR products were digested by appropriate restriction enzymes and ligated to an expression vector for GFP (green fluorescent protein)-fusion proteins, pCMX-SAH/Y145F (Ogawa et al 1995). The proper construction of all of the plasmids was confirmed by DNA sequencing.…”

Section: Plasmid Constructionsmentioning

confidence: 99%

Requirement of cooperative functions of two repeated death effector domains in caspase‐8 and in MC159 for induction and inhibition of apoptosis, respectively

Tsukumo

Yonehara

1999

Genes to Cells

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Localization, trafficking, and temperature-dependence of the Aequorea green fluorescent protein in cultured vertebrate cells.

Abstract: The localization, trafficking, and fluorescence ofAequorea green fluorescent protein (GFP)

Cited by 227 publications

References 29 publications

Apoptosis induced by the myelodysplastic syndrome-associated NPM-MLF1 chimeric protein

Apoptosis induced by the myelodysplastic syndrome-associated NPM-MLF1 chimeric protein

Sensitization of osteosarcoma cells to death receptor-mediated apoptosis by HDAC inhibitors through downregulation of cellular FLIP

Requirement of cooperative functions of two repeated death effector domains in caspase‐8 and in MC159 for induction and inhibition of apoptosis, respectively

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