CD30-induced signaling is absent in Hodgkin's cells but present in anaplastic large cell lymphoma cells

Abstract

High CD30 expression in classical Hodgkin's lymphoma and anaplastic large cell lymphoma (ALCL) suggests an important pathogenic role of this cytokine receptor. To test this hypothesis, we investigated CD30 signaling in Hodgkin's and ALCL cell lines by different approaches: 1) CD30 stimulation, 2) CD30 down-regulation, and 3) a combination of both. The effects were determined at the RNA (microarray and real-time quantitative RT-PCR), protein (electrophoretic mobility shift analysis, immunoblot, and flow cytometry), and cellular/functional (proliferation and apoptosis) levels. We demonstrate that Hodgkin's cells are virtually CD30 unresponsive. Neither CD30 stimulation nor CD30 silencing of Hodgkin's cells had any significant effect. In contrast, CD30 stimulation of ALCL cells activated nuclear transcription factor-kappaB (NF-kappaB), induced major transcriptional changes, and decreased proliferation. These effects could be abrogated by down-regulation of CD30. Stimulation of CD30 in ALCL cells, stably transfected with a dominant-negative NF-kappaB inhibitor, induced pronounced caspase activation and massive apoptosis. Our data indicate that 1) CD30 signaling is not effective in Hodgkin's cell lines but is effective in ALCL cell lines, 2) CD30 is probably not significantly involved in the pathogenesis of classical Hodgkin's lymphoma, and 3) CD30 stimulation triggers two competing effects in ALCL cells, namely activation of caspases and NF-kappaB-mediated survival. These data suggest that CD30-targeted therapy in ALCL should be combined with NF-kappaB inhibitors to induce effective cell killing.

Figure 1

CD30 stimulation does not induce canonical and noncanonical NF-κB activation in Hodgkin’s cells but in ALCL cells. A: Immunoblot analysis of IκBα after 30 minutes of CD30-stimulated or nonstimulated Hodgkin’s cells (L1236, L428, KM-H2, and L591) and ALCL cells (Karpas 299, JB6, SU-DHL1, and FE-PD). The decrease of IκBα expression (37 kDa) represents canonical NF-κB pathway activation. B: Immunoblot analysis of p100 and p52 in cells after 16-hour CD30 stimulation and control treatment. The appearance of p52 denotes noncanonical NF-κB pathway activation, ie, processing of p100 to p52. Unspecific signal is indicated. β-actin served as loading control. Experiments were repeated three times; representative results are shown.

Figure 2

CD30 stimulation does not regulate gene expression in Hodgkin’s cells, but it does in ALCL cells. Gene expression microarray analyses of CD30-stimulated and nonstimulated Hodgkin’s cells (L1236) in comparison with ALCL cells (Karpas 299) at 4, 16, and 72 hours. The top 250 probe sets, ranked by the differential induction score, are shown. Rows represent probe sets; columns represent samples. Relative gene expression in comparison with an untreated control (medium) of Karpas 299 is displayed. CD30 stimulation strongly induced (top panel)/repressed (bottom panel) gene expression in ALCL but not in Hodgkin’s cells.

Figure 3

CD30 stimulation does not activate the expression of selected genes in Hodgkin’s cells but induced major changes of gene expression in ALCL cells. Expression of 10 genes [ATF3, A20, cIAP2, c-FLIP, CXCR4, IEX-1, RAD1, RGS1, PTGER4, and HPRT (new CD30 targets underlined)] was analyzed by RT-RQ-PCR using 4- and 16-hour CD30-stimulated and nonstimulated Hodgkin’s cells (L1236, L428, KM-H2, and L591) and ALCL cells (Karpas 299, JB6, SU-DHL1, and FE-PD). One of two experiments is shown. Relative, HPRT-normalized gene expression, measured in triplicates, is displayed.

Figure 4

CD30 stimulation does not induce protein expression in Hodgkin’s cells, but it does in ALCL cells. A: Flow cytometric analysis of CD30-induced CXCR4, OX40, CD95, CD137, and PTGER4 expression (novel CD30 targets underlined) in Hodgkin’s cells (L1236 and L428) and ALCL cells (Karpas 299 and JB6). Data show the overlay of histograms, representing individual antibody-specific fluorescence of 16-hour CD30-stimulated (solid line/open area) and nonstimulated cells (dotted line/shaded area). One of five experiments is shown. B: Secretion of IL-4, IL-8, and TNF-α was determined in the supernatant of 8-, 16-, and 48-hour CD30-stimulated or nonstimulated L428 and Karpas 299 cells (measured in triplicate). SD is indicated. One of three experiments is shown. C: Immunoblot analysis of MnSOD, ATF3 using 16-hour CD30-stimulated and nonstimulated Hodgkin’s cell lines (L1236 and L428) and ALCL cell lines (Karpas 299 and JB6). β-Actin served as loading control. Experiments were repeated three times; representative results are shown.

Figure 5

CD30 stimulation of Hodgkin’s cells does not change proliferation but reduces proliferation of ALCL cells. Hodgkin’s cells (L1236 and L428) and ALCL cells (Karpas 299 and JB6) were CD30-stimulated or nonstimulated for 0, 24, 48, and 72 hours, trypan blue stained, and counted. Counts of viable and dead cells are shown. Experiments were performed at least three times in triplicates, bars indicate SD.

Figure 6

Down-regulation of CD30 does not change constitutive gene expression of Hodgkin’s cells but inhibits CD30 signaling of ALCL cells. A: CD30 down-regulation of Hodgkin’s cells (KM-H2, L1236, and L428) and ALCL cells (Karpas 299) by CD30 siRNA was verified by immunoblotting. NPM-ALK expression was analyzed in CD30 down-regulated and nonstimulated and untreated ALCL cells by immunoblotting. β-Actin served as loading control. B: Flow cytometric analysis of cells, analyzed in A. Data show the overlay of histograms, representing isotype IgG₃-control antibody-specific fluorescence (black dotted line), CD30 antibody-specific fluorescence of CD30 siRNA-transfected (red line), control siRNA-transfected (green line), and CD30 antibody-specific fluorescence of untreated cells (black line). One of two analyses is shown. C: Gene expression microarray analysis of CD30 siRNA- or control siRNA-transfected, 16-hour CD30-stimulated and nonstimulated Hodgkin’s cells (KM-H2, L1236, and L428) and ALCL cells (Karpas 299). Aliquots of cells that were analyzed in A and B were used for subsequent CD30 stimulation experiments and gene expression profiling. We performed two identical experiments and analyzed the established “CD30 signaling gene set” (Figure 2). The average of fold-change (scale bar) is displayed. Rows denote probe sets. The top part depicts the 70 most CD30-induced probe sets in Karpas 299. The bottom part represents the 35 most CD30-repressed probe sets in Karpas 299. Each column represents one sample. Treatments are indicated.

Figure 7

CD30 down-regulation does not influence proliferation of Hodgkin’s or ALCL cells. CD30 expression of Hodgkin’s cells (L428, KM-H2, and L1236) and ALCL cells (Karpas 299) was down-regulated by CD30 siRNA. Proliferation of control siRNA (black line) and CD30 siRNA-treated cells (gray line) was measured by WST-1 assay after 24, 48, and 72 hours. Experiments were performed at least three times in triplicates; bars indicate SD.

Figure 8

Simultaneous NF-κB inhibition and CD30 stimulation of ALCL cells activate caspases and lead to apoptosis. Mock-transfected ALCL cells (Karpas 299) and Karpas 299 cells, stably transfected with a dominant-negative inhibitor of NF-κB (IκBαΔN Karpas 299), were CD30-stimulated or nonstimulated for 16 hours (A and B) or 48 hours (C). A: Annexin V-FITC/PI-stained cells were analyzed by flow cytometry. B: Cytospins of 16-hour-treated cells were fixed and stained for active caspase-3 (top panel). Cleaved PARP was analyzed by immunoblotting. C: Immunoblot analysis of active caspase-8, active caspase-9, and active BID. IκBα and IκBαΔN expression was analyzed in B and C; β-actin served as loading control. Three independent experiments were performed; representative results are shown.