GAAP-1: a transcriptional activator of p53 and IRF-1 possesses pro-apoptotic activity - PubMed (original) (raw)

GAAP-1: a transcriptional activator of p53 and IRF-1 possesses pro-apoptotic activity

Christophe Lallemand et al. EMBO Rep. 2002 Feb.

Abstract

The mechanisms that regulate the transcription of the tumour suppressor genes p53 and IRF-1 are poorly understood. We have characterized a 68-kDa transcription factor, GAAP-1 (gatekeeper of apoptosis activating proteins), encoded by an alternative splice product of the PRDII-BF1 gene, that recognizes a novel regulatory element within the p53 and IRF-1 promoters. Transfection of U937 cells with GAAP-1 activates p53 and IRF-1 expression and leads to apoptosis, whereas over-expression of GAAP-1 in K562 cells that lack p53 and IRF-1 induces cell differentiation. Alterations in the 6p24 locus containing the GAAP-1 gene are frequent in acute myelogenous leukemia (AML), and AML-derived cell lines display reduced GAAP-1 mRNA levels. Together, these results suggest that GAAP-1 acts as a gatekeeper at a critical point in the tumour suppressor gene pathway.

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Figures

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Fig. 1. GAAP-1 is a differential splice product of PRDII-BF1 precursor mRNA. (A) Schematic representation of the exon composition of PRDII-BF1 and GAAP-1 transcripts. These data are inferred from the work of Xu et al. (1998) and Fan and Maniatis (1990). The nucleotide positions are as in Fan and Maniatis (1990). (B) Partial amino acid sequence of wild-type GAAP-1. The NLS is underlined. (C) Sub-cellular localization of EGFP/GAAP-1 variants. HuH7 cells were transiently transfected with the expression vector indicated. After 24 h, the nuclei were stained with Hoechst 33342 and examined by confocal microscopy.

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Fig. 2. GAAP-1 possesses a double binding specificity involving the same zinc-finger structure. (A) NF-κB and IPCS-p53 oligonucleotides. (B) EMSA using the IPCS-p53 probe and in vitro translated GAAP-1 (lane 1), GAAP-1 mtZ1 (lane 3), GAAP-1mt Z2 (lane 5) or reticulocyte lysate with the empty expression vector (lane 7). Even lanes: 50-fold molar excess of IPCS-p53 oligonucleotide was added to the reaction of the previous lane. The arrow indicates the specific complex. (C) The same as in (B), except that the IPCS-p53 probe was replaced by the NF-κB probe. Even lanes: 50-fold molar excess of NF-κB oligonucleotide. (D) Amino acid sequence of the two zinc-finger domains. The residues that are substituted in GAAP-1 mtZ1 or in GAAP-1 mtZ2 are underlined.

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Fig. 3. Transcriptional properties of GAAP-1. (A) Relative activity of the wild-type and mutated p53 and IRF-1 promoters, IPCS-SV40 chimeric promoter and the HIV-LTR in a co-transfection assay. U937 cells were transiently co-transfected with 3 µg of the reporter plasmids and 7 µg of the expression vectors indicated. The pcDNA3.1 vector was used as a control. The pRL-SV40 plasmid was used as an internal control for the normalization of transfection efficiency. The results shown in this figure represent the ratio between the luciferase activity obtained with each promoter construct in the presence of the co-transfected GAAP-1 or PRDII-BF1 plasmids and the activity obtained in their absence. The ratio between the luciferase activity obtained with the wild-type promoter constructs and the activity obtained with the mutant constructs in the absence of any co-transfected expression vector was ∼2- to 3-fold, reflecting the effect of endogenous GAAP-1 on the basal level of expression of the p53 and IRF-1 promoters. The transfected cells were harvested 8 h after transfection and assayed for luciferase activity. (B) U937 and K562 cells were stably transfected with GAAP-1 or EGFP expression vectors and selected using G418 in semi-solid methyl-cellulose medium. The number of clones was determined at 21 days. (C) Western blotting of 20 µg of cellular extracts from U937 cells or MCF7 Tet-Off cells stably transfected with either an EGFP or EGFP/GAAP-1 expression vector as indicated. In MCF7 Tet-Off cells, EGFP/GAAP-1 expression was induced by the removal of doxycycline from the culture medium. The blot was probed with anti-p53, anti-IRF-1 or anti-GFP antibodies.

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Fig. 4. Apoptotic properties of GAAP-1. (A) Spontaneous apoptosis of U937 cells stably transfected with EGFP or EGFP/GAAP-1 expression vectors was assayed by annexin V-PE/7-AAD double labelling. (B) U937 cells stably transfected with either the EGFP or EGFP/GAAP-1 fusion protein expression vectors were treated, or not, with 1 µM 5FU. (C) MCF7 Tet-Off cells stably transfected with the inducible EGFP/GAAP-1 fusion protein expression vector were treated with 100 µM 5FU, and the fusion protein was induced by the removal of doxycycline from the medium. Apoptosis was analysed by the TUNEL assay 72 h after treatment.

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Fig. 5. Effect of GAAP-1 on the differentiation of K562 cells. (A) Parental K562 cells or clones stably transfected with either the EGFP or EGFP/GAAP-1 expression vectors were left untreated or treated for 4 days with Ara-C (1 µM) or hemin (5 µM) and then assayed for hemoglobin content. (B) Total RNA (1 µg) from the AML-derived cell lines KG1, TF1, UT7 and U937, the CML-derived cell line K562 and PBMC from a healthy donor were analysed by RT–PCR. Reverse transcription was carried out using an anti-sense primer localized in exon VI, and amplification was carried out using a pair of primers localized in exon III and exon V. The amplification of GAAP-1 mRNA produces a fragment of ∼300 bp, whereas mRNA from PRDII-BF1 is not amplified under these conditions due to its size of >6 kb. The identity of the 300-bp fragment was confirmed by direct sequencing.

References

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