QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online March 12, 2004
doi: 10.1242/10.1242/jcs.00994

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, Y. Articles by Ma, D. Search for Related Content PubMed PubMed Citation Articles by Wang, Y. Articles by Ma, D.

An alternative form of paraptosis-like cell death, triggered by TAJ/TROY and enhanced by PDCD5 overexpression

¹ Laboratory of Medical Immunology, School of Basic Medical Science, Peking University, Xueyuan Road 38, Beijing 100083, China
² Center for Human Disease Genomics, Health Science Center, Peking University, Beijing 100083, China

View larger version (88K): [in a new window]   Fig. 1. Overexpression of TAJ/TROY induces non-apoptotic cell death. (A) Colony formation after TAJ/TROY transfection. HEK293 and HeLa cells were transfected with an empty vector or expression vectors encoding Bax or TAJ/TROY. Overexpression of TAJ/TROY significantly decreased clonogenic survival of the cells. The figure shows a representative experiment with the means and standard deviations from triplicate plates. (B) Electron micrographs of TAJ/TROY-induced cell death. HEK293, HeLa and 293T cells were transfected with a vector control or plasmids encoding Bax or TAJ/TROY along with a GFP-encoding vector. Whereas Bax-transfected 293T cells (b) displayed classic characteristics of apoptosis, including chromatin condensation and apoptosis body, TAJ/TROY-transfected HEK293 (e), HeLa (g) and 293T (c) cells showed extensive cytoplasmic vacuolization and swelling of mitochondria or ER, along with absence of nuclear fragmentation, membrane blebbing or apoptotic-body formation. The following micrographs of empty-vector-transfected HEK293 (d), HeLa (f) and 293T (a) cells provided normal cell controls. Higher-magnification images of the vacuolization suggested that some vacuoles might be formed from the remnants of the mitochondrial (h) or ER (i). The transfection efficiency of the indicated plasmids ranged from 60% to 70% as determined by the expression of co-transfected GFP. Scale bars, 2 µM (a-g), 0.2 µM (h,i).

View larger version (29K): [in a new window]   Fig. 2. Cell death induced by TAJ/TROY is accompanied by PS exposure in the membrane. 40 hours after transfection, cells were analysed by flow cytometry with FITC-labeled annexin V and propidium iodide. (bottom left) Viable cells (PI-, annexin-V/FITC-). (bottom right) Early apoptotic or paraptotic cells (PI-, annexin-V/FITC+). (top right) Non-viable, late apoptotic/necrotic cells (PI+, annexin-V/FITC+). (top left) Necrotic cells. Numbers in the quadrants indicate the proportions of cells in the corresponding areas. Data from one experiment are presented. Experiments were performed in triplicate, with similar results.

View larger version (41K): [in a new window]   Fig. 3. TAJ-induced non-apoptotic cell death is independent of caspase activation. (A) DEVDase activity assay. 293T cells were transfected with the empty vector or indicated constructs and, 40 hours after transfection, experiments were performed as described in Materials and Methods. All data are presented as mean±standard deviation (s.d.). (B) Cellular proteins were extracted from 293T cells that were transfected with mock, Bax or TAJ/TROY plasmid. Protein levels were detected by western blots with various antibodies as described in Materials and Methods.

View larger version (110K): [in a new window]   Fig. 4. Mitochondrial changes in TAJ/TROY-transfected cells. (A) Transfected cells were subjected to mitochondrial transmembrane potential assessment 30 hours after transfection using a potentiometric dye, rhodamine 123. Loss of m was visualized as a reduction in the fluorescence signal in the FL1 channel. The proportions of cells with reduced rhodamine 123 staining are shown. Data are presented from one experiment. Experiments were performed in triplicate with similar results. (B) Ultrastructural mitochondrial characteristics of TAJ/TROY-induced cell death. HEK293, HeLa and 293T cells were transfected with a vector control or plasmids encoding Bax or TAJ/TROY along with a GFP-encoding vector. Extensive mitochondria swelling was seen in TAJ/TROY-transfected HEK293 (b,c), HeLa (e) and 293T (g-i) cells. The following micrographs of empty-vector-transfected HEK293 (a), HeLa (d) and 293T (f) cells provide normal cell controls. Scale bars, 1 µM (a,b,d,e), 0.5 µM (c,f-h), 0.2 µM (i).

View larger version (34K): [in a new window]   Fig. 5. Overexpression of PDCD5 enhances TAJ-induced cell death. 293T cells were transfected with vector control and plasmids encoding TAJ/TROY, PDCD5 or both TAJ/TROY and PDCD5. Following transfection (36 hours), cells were processed for flow cytometry analysis (A) and DEVDase activity assay (B).

View larger version (33K): [in a new window]   Fig. 6. Upregulation of PDCD5 protein in TAJ/TROY-transfected cells. (A) 293T cells were transfected with empty plasmid vector or indicated plasmids. 30 hours after transfection, endogenous PDCD5 levels were measured with a FITC-labeled anti-PDCD5 monoclonal antibody by flow cytometry. Median values of fluorescence intensity represent the expression of cellular PDCD5. 293T cells transfected with PDCD5 expression plasmid were used as a positive control. Significant upregulation of endogenous PDCD5 was detected in TAJ/TROY-overexpressing cells. (B) Schematic overlap of fluorescence histograms. It helped to observe the increase of median values of fluorescence intensity in TAJ/TROY-transfected cells.