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. 2013 Oct 17;4(10):e863.
doi: 10.1038/cddis.2013.389.

Constitutive localization of DR4 in lipid rafts is mandatory for TRAIL-induced apoptosis in B-cell hematologic malignancies

M Marconi  1 , B Ascione, L Ciarlo, R Vona, T Garofalo, M Sorice, A M Gianni, S L Locatelli, C Carlo-Stella, W Malorni, P Matarrese
Affiliations

Constitutive localization of DR4 in lipid rafts is mandatory for TRAIL-induced apoptosis in B-cell hematologic malignancies

M Marconi et al. Cell Death Dis. .

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) acts as an apoptosis inducer for cancer cells sparing non-tumor cell targets. However, several phase I/II clinical trials have shown limited benefits of this molecule. In the present work, we investigated whether cell susceptibility to TRAIL ligation could be due to the presence of TRAIL death receptors (DRs) 4 and 5 in membrane microdomains called lipid rafts. We performed a series of analyses, either by biochemical methods or fluorescence resonance energy transfer (FRET) technique, on normal cells (i.e. lymphocytes, fibroblasts, endothelial cells), on a panel of human cancer B-cell lines as well as on CD19(+) lymphocytes from patients with B-chronic lymphocytic leukemia, treated with different TRAIL ligands, that is, recombinant soluble TRAIL, specific agonistic antibodies to DR4 and DR5, or CD34(+) TRAIL-armed cells. Irrespective to the expression levels of DRs, a molecular interaction between ganglioside GM3, abundant in lymphoid cells, and DR4 was detected. This association was negligible in all non-transformed cells and was strictly related to TRAIL susceptibility of cancer cells. Interestingly, lipid raft disruptor methyl-beta-cyclodextrin abrogated this susceptibility, whereas the chemotherapic drug perifosine, which induced the recruitment of TRAIL into lipid microdomains, improved TRAIL-induced apoptosis. Accordingly, in ex vivo samples from patients with B-chronic lymphocytic leukemia, the constitutive embedding of DR4 in lipid microdomains was associated per se with cell death susceptibility, whereas its exclusion was associated with TRAIL resistance. These results provide a key mechanism for TRAIL sensitivity in B-cell malignances: the association, within lipid microdomains, of DR4 but not DR5, with a specific ganglioside, that is the monosialoganglioside GM3. On these bases we suggest that lipid microdomains could exert a catalytic role for DR4-mediated cell death and that an ex vivo quantitative FRET analysis could be predictive of cancer cell sensitivity to TRAIL.

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Figures

Figure 1
Figure 1
(a) Surface expression of TRAIL receptors in transformed and non-transformed cells. Flow cytometry analysis of surface expression level of DR4 and DR5 in four Burkitt lymphoma cell lines. Histograms obtained in a representative experiment are shown. Numbers represent the median fluorescence intensity. Results obtained in a representative experiment are shown. (b) Graphs showing the surface expression level of DR4 and DR5 in four Burkitt lymphoma cell lines (left panel) and in non-transformed cells PBL, fibroblasts and HUVEC (right panel). Mean±S.D. of the median fluorescence intensity obtained in four different experiments is reported. (c) Soluble TRAIL-induced apoptosis in transformed and non-transformed cells. Biparametric flow cytometry analysis of sTRAIL-induced apoptosis in four Burkitt lymphoma cell lines after double staining with annexin V-FITC/Trypan blue. Numbers represent the percentage of dead cells either annexin V/Trypan blue double-positive or annexin V single-positive. Results obtained in a representative experiment are shown. (d) Bar graphs showing the amount of apoptosis in four Burkitt lymphoma cell lines (left panel) and in non-transformed PBL, fibroblasts and HUVEC (right panel) after sTRAIL administration. Mean±S.D. of the percentages of annexin V-positive cells obtained in four different experiments is reported. Significant differences (P<0.01) were detected between control and treated cancer cells
Figure 2
Figure 2
Constitutive localization of TRAIL receptors into lipid rafts. Left panels. Western blot analysis of sucrose gradient fractions. Fractions obtained after sucrose density gradient were analyzed by using anti-DR4 antibodies (first line) and anti-DR5 antibodies (second line). The third lane shows flotillin 2 (FLOT2) distribution, known to be enriched in fractions corresponding to lipid rafts (fractions 4–6). Right panels. IVM analysis after triple cell staining with anti-DR4/anti-GM3/Hoechst (upper panel) or with anti-DR5/anti-GM3/Hoechst. The yellow fluorescence areas indicate the co-localization. Bottom boxes. Quantitative evaluation of GM3/DR4 and GM3/DR5 association by FRET technique, as revealed by flow cytometry analysis. Numbers represent the FRET efficiency (calculated by using Rieman algorithm) and indicate that GM3/DR4 association was higher in Ramos cells, very low in Namalwa cells and negligible in PBL. GM3/DR5 association was absent in Ramos, Namalwa and PBL. Results obtained in one experiment representative of three are shown. (a) Ramos lymphoma cell line, (b) Namalwa lymphoma cell line and (c) freshly isolated human lymphocytes (PBL). Note different scales in FE bar graphs
Figure 3
Figure 3
Lipid rafts and TRAIL-induced apoptosis. Left panels. Bar graphs showing the amount of sTRAIL-induced apoptosis in cells pretreated with MBC or perifosine. Mean±S.D. of the percentages of annexin V-positive cells obtained by flow cytometry in four different experiments is reported. Right panels. IVM analysis after triple cell staining with anti-DR4/anti-GM3/Hoechst in control, MBC- or perifosine-treated cells. The yellow fluorescence areas indicate the co-localization. (a) Ramos lymphoma cell line, (b) Namalwa lymphoma cell line and (c) freshly isolated human lymphocytes. * indicates P<0.01 ° indicates P<0.05 versus sTRAIL samples. (d) Quantitative evaluation of GM3/DR4 and GM3/DR5 association by FRET technique, as revealed by flow cytometry analysis. Numbers represent the FRET efficiency (calculated by using Riemann algorithm). Note different scales
Figure 4
Figure 4
Apoptotic induction by DR4 and DR5 agonist antibodies. Bar graphs showing the amount of apoptosis induced by anti-DR4 (left panels) and anti-DR5 (right panels) agonist antibodies in control cells and in cells pretreated with MBC or perifosine before apoptotic triggering. Mean±S.D. of the percentages of annexin V-positive cells obtained by flow cytometry in four different experiments is reported. (a) Ramos lymphoma cell line; (b) Namalwa lymphoma cell line and (c) freshly isolated human lymphocytes. * indicates P<0.01 versus anti-DR4 agonist Ab samples
Figure 5
Figure 5
(a) Ex vivo analyses of lymphocytes isolated from patients with CLL. Flow cytometry analysis of surface expression level of CD19 in lymphocytes freshly isolated from a representative HD among six or from two pathological subjects among six (Pt1 and Pt2) affected by B leukemia. Numbers represent the percentage of CD19-positive cells. Results obtained in a representative experiment are shown. (b) Surface expression of TRAIL receptors. Flow cytometry analysis of surface expression level of DR4 and DR5 in lymphocytes freshly isolated from a healthy donor or from two pathological subjects affected by B leukemia. Numbers represent the median fluorescence intensity. Results obtained in a representative experiment are shown. (c) Apoptosis induction by sTRAIL, mTRAIL and agonist antibodies to DR4 and DR5. Biparametric flow cytometry analysis of apoptosis induced by sTRAIL or by mTRAIL, that is, coculturing CD34+-armed cells or CD34+-mock with target cells (1 : 1 ratio). Numbers represent the percentage of apoptotic cells either annexin V/Trypan blue double-positive or annexin V single-positive. Results obtained in a representative experiment are shown. Analyses shown in b and c were restricted to CD19-positive cells
Figure 6
Figure 6
Ex vivo analyses of lymphocytes isolated from patients with CLL. Flow cytometry analysis of lymphocytes freshly isolated from a representative HD among six or from two pathological subjects among six affected by B leukemia (Pt1 and Pt2) triggered for apoptosis. Bar graphs showing the amount of apoptosis induced by (a) sTRAIL, (b) anti-DR4 or anti-DR5 agonist antibodies or (c) coculturing CD34+-armed cells with target cells (1 : 1 ratio) in control cells and in cells pretreated with perifosine before apoptotic triggering. Note that representative Pt1 was susceptible to all the pro-apoptotic stimuli mentioned above. Mean±S.D. of the percentages of annexin V-positive cells obtained in four different experiments is reported. * indicates P<0.01; ° indicates P<0.05 versus sTRAIL sample (a), versus anti-DR4 agonist Ab sample (b) or versus CD34+-armed sample (c)
Figure 7
Figure 7
Localization of TRAIL receptors into lipid rafts. (a) Flow cytometry analysis of GM3/DR4 or GM3/DR5 association by FRET technique in lymphocytes freshly isolated from a representative HD among six or from two pathological subjects among six affected by B leukemia (Pt1, susceptible, and, Pt2, resistant). Analyses were performed in control cells and in cells treated with perifosine. Numbers indicate the percentage of FL3-positive events obtained in one experiment representative of three. (b) Bar graphs showing evaluation of FE, according to the Riemann's algorithm, of GM3/DR4 and GM3/DR5 association. Results shown represent the mean±S.D. from three independent experiments. Statistical analyses indicated that: (i) GM3/DR4 association was significant in lymphocytes isolated from Pt1 (susceptible to pro-apoptotic stimuli, see Figure 6) but not in lymphocytes from HD and Pt2 (resistant to pro-apoptotic stimuli, see Figure 6) (black columns); and that (ii) GM3/DR5 association was irrelevant either in lymphocytes from HD or from pathological subjects (gray columns). Note different scales. * indicates P<0.05 versus control samples
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