CD73 participates in cellular multiresistance program and protects against TRAIL-induced apoptosis

Abstract

The molecular mechanisms underlying the multiresistant phenotype of leukemic and other cancer cells are incompletely understood. We used expression arrays to reveal differences in the gene expression profiles of an apoptosis-resistant T cell leukemia clone (A4) and normally apoptosis-sensitive parental Jurkat cells. CD73 (ecto-5'-nucleotidase) was the most up-regulated gene in the resistant A4 cell clone. A4 cells displayed CD73 surface expression and significant ecto-5'-nucleotidase activity. The role of CD73 was confirmed by transfection of wild-type CD73 into native Jurkat cells, which led to specific resistance against TRAIL-induced apoptosis, but not other types of apoptosis. The protective role of CD73 was further confirmed by small interfering RNA-mediated down-regulation of CD73, restoring TRAIL sensitivity. CD73-mediated resistance was independent of enzymatic activity of CD73, but was reliant on the anchoring of the protein to the membrane via GPI. We suggest that the inhibition of TRAIL signaling works through interaction of CD73 with death receptor 5, as CD73 and death receptor 5 could be coimmunoprecipitated and were shown to be colocalized in the plasma membrane by confocal microscopy. We propose that CD73 is a component of multiresistance machinery, the transcription of which is activated under selective pressure of the immune system.

FIGURE 1

A, Immunochemical screening of antiapoptotic proteins. Lysates of parental Jurkat and A4 cells were subjected to Western blotting with Abs against HSP-90, ERK (p44/42), Bcl-2, Bcl-x_L, and BID. Reactions were developed with HRP conjugates of corresponding secondary Abs and visualized by chemiluminescence. HSC70 and Lamin B were used as loading controls. Densitometry ratios are shown below the individual graphs. The densitometric value obtained with Jurkat cells equals 1. B, Analysis of CD73 expression by flow cytometry. The elevated CD73 expression in A4 cells was confirmed by FACS analysis. Resting Jurkat, A4 and different CD73 transfectant cells were labeled with anti-CD73 Ab 4G4, triple washed and visualized with secondary Abs conjugated to Alexa 647. The cells were washed once with PBS and data from 10⁵ cells were collected on a FACScan (BD Biosciences) flow cytometer and analyzed with FCSExpress3 software. The following mean fluorescence intensity values were obtained from the FACS analyses: Jurkat wild-type labeled with nonspecific Ab, 2209; Jurkat wt, 2239; A4, 3672; β.92.20, 12294; B-NT5.1, 13345; B-TM15.1, 18289. C, Confirmation of CD73 expression on A4 cells by Western blotting. Lysates of 10⁵ resting Jurkat and A4 cells equivalent to those shown in A were subjected to Western blotting with Abs against CD73. Lysates of 2 × 10⁴ Jurkat cells stably transfected with CD73 (B-NT5.1 and B-TM15.1) were used as positive controls.

FIGURE 2

A, Jurkat cells transfected with GPI-anchored CD73 are resistant to TRAIL-induced apoptosis. Jurkat, A4, B-NT5.1 (stable transfectants with GPI-anchored human CD73), and NC-1 (stable transfectants with GPI-anchored murine CD73) cells were treated with rFasL for 4 h, 50 ng/ml TRAIL for 4 h, or 10 μM etoposide for 20 h to induce apoptosis. Cells were then stained with allophycocyanin-annexin V and Sytox Green and evaluated for apoptosis as described in Materials and Methods. Bars (mean values + SD values from five repeats, 10⁴ cells counted) labeled with one asterisk (*) represent statistically significant difference (p < 0.01) from the untreated control; bars labeled with two asterisks (**) represent statistically significant difference (p < 0.01) from untransfected Jurkat cells having the same treatment. B, The GPI anchor, but not the enzyme activity of ecto-5′-nucleotidase is required for resistance against TRAIL-induced apoptosis. Jurkat (±pretreatment with 2.5 U/ml PI-PLC), B-NT5.1 cells (±pretreatment with 1 mM AMPCP or with 2.5 U/ml PI-PLC), and B-TM15.1 cells were exposed to 50 ng/ml TRAIL for 4 h to induce apoptosis. Cells were then stained with allophycocyanin-annexin V and Sytox Green and evaluated for apoptosis as described in Materials and Methods (bars represent mean values + SD values from five repeats, 10⁴ cells counted; *, p < 0.01 between values marked with ¤ and ø).

FIGURE 3

The roles of CD73 in TRAIL resistance. A, siRNAs against CD73 efficiently down-regulate its endogenous expression. HEC cells were transiently transfected with three different CD73 siRNAs, scrambled synthetic siRNA, and a mixture of 3 siRNAs (1:1:1) and CD73 expression was evaluated after 44 h by Western blotting. Western blotting was also performed with Abs to HSC70 (lower lane) to demonstrate equivalent protein loading. B, siRNAs against CD73 down-regulate its expression in B-NT5.1 cells and resensitize cells to TRAIL-induced apoptosis. B-NT5.1 cells were transiently triple transfected with a mixture of three CD73 siRNAs (1:1:1) or with scrambled siRNA. After 20 h, they were incubated with 50 ng/ml TRAIL, 50 ng/ml rFasL, for 4 h or 10 μM etoposide for 16 h and then stained with Cy2 anti-CD73 and allophycocyanin-annexin V. Cells with low and high surface expression of CD73 were analyzed separately for annexin labeling as were cells transfected with scrambled siRNA. Bars indicate mean values + SD from three repeats. C, DISC analysis reveals no caspase-8 processing in B-NT5.1 cells. Cells were exposed to 200 ng/ml FLAG-tagged rTRAIL and cross-linking M2 Abs for designated time. Then cells were spinned down for 30 s at 5,000 × g + 4C and cell pellet were lysed with Pierce M-prot lysis buffer. Lysate was clarified by centrifugation 2 min at 12,000 × g + 4C and supernatant exposed to protein G Sepharose for 15 min. Sepharose slurry washed with lysing buffer and boiled with Laemmli buffer and subjected to Western blotting. D, BID is not cleaved in B-NT5.1 cells. The cell lysates for 0 and 60 min TRAIL stimulation described above were subjected to Western blot and developed with primary mAbs against epitope of BID neighboring to caspase-8 cleavage site.

FIGURE 4

Confocal microscopy reveals colocalization of TRAIL-receptor DR5 and CD73 in TRAIL-resistant transfectants. A, Jurkat, B-NT5.1, NC-1, B-TM15.1, and β92.20 cells were labeled with Cy2-conjugated anti-CD73 (green) plus Cy3B-conjugated anti-DR5 (red) for 1 h at 20°C and processed as described in Materials and Methods. In a separate experiment, B-NT5.1 was pretreated with 2.5 U/ml PI-PLC. Separate, as well as merged images are shown, with the pixel correlation shown over the merged images (mean values ± SD from 10 ratio measurements over 10 individual cells). B, B-NT5.1 and NC-1 cells were incubated with Cy2-conjugated rat anti-mouse CD73 for 3 h at 37°C, followed by 1 h with goat anti-rat Ig to allow cross-linking and capping of CD73. The cells were then washed, fixed with 3% formaldehyde, washed again and stained with Cy3B-conjugated anti-DR5 or Cy3B-conjugated isotype control Ab (data not shown) in the presence of 10% normal goat serum. Individual images as well as merged images are shown. C, Ab controls: Jurkat cells were stained with a mixture of Cy2-conjugated CD73-specific Abs (upper left, green) and Cy3B-conjugated isotype-matched mAb against nodularin (upper right, red). Phase contrast image showing cells (lower left); merged image (lower right). D, TRAIL-receptor DR5 coimmunoprecipitates with CD73. Resting B-NT5.1, β92.20, and Jurkat cells were treated with Abs against either DR5 or CD73 or species/class matching control Abs, washed, lysed with hypotonic buffer and Ab complexes extracted with protein G Sepharose. Beads were boiled with sample Laemmli buffer and the supernatant subjected to SDS-PAGE. The figure shows Western blots developed with Abs against CD73 and DR5, respectively.

FIGURE 5

Colocalization of DR5 with caveolin 1-rich regions. Viable B-NT5.1 and Jurkat cells were stained with primary Abs against DR5 (DJR2-1) and caveolin 1 (ab2910) in suspension, then washed three times with PBS and developed with corresponding (Alexa 405 goat anti-mouse IgG and Alexa 547 goat anti-rabbit IgG) secondary Abs. Stained cells were washed three times with PBS, fixed with 4% paraformaldehyde, and subjected to confocal microscopy. A, Images were taken at objective magnification ×40 with a metadetector registering emission wavelengths 411–507 nm (laser emission 405 nm) represented as the green channel, and 550–753 nm (laser emission 543 nm) for the red channel. B, B-NT5.1 cell stained as described above at objective magnification ×100. Total Z-stack projection from the optical slice of maximal diameter to the top of the cell.

FIGURE 6

Model for the role of CD73 in TRAIL resistance. A, Ectopic or acquired expression of CD73 linked via GPI to the surface of cells protects them against TRAIL-induced apoptosis by interaction with molecules such as DR5. B, Although the protection is independent of ecto-5′-nucleotidase enzymatic activity, it is dependent on the GPI-anchoring of CD73 to the cell membrane, as cleaved CD73 loses its protective property.