Rituximab induces different but overlapping sets of genes in human B-lymphoma cell lines

Abstract

The therapeutic unconjugated anti-CD20 Mab rituximab is used for the treatment of B-non-Hodgkin's lymphomas. We have studied the direct biological effects, signalling and gene expression profiles induced by rituximab in two human B-lymphoma cell lines, DHL4 and BJAB, using microarray, quantitative PCR and gel shift analysis. Rituximab alone inhibited thymidine uptake and induced homotypic adhesion in DHL4 only, but not BJAB. Analysis of Affymetrix microchips carrying probes for about 10,000 human cDNAs, allowed us to identify 16 genes in DHL4 and 12 in BJAB induced by rituximab at 4 h. Eleven and seven of these genes were specific for DHL4 and BJAB, respectively; whereas the remaining five were up-regulated in both cell lines. Mean induction ranged from 2- to 16-fold. Real time PCR analysis allowed us to confirm up-regulation of all genes identified, except one in BJAB. Time course of induction of eight genes was studied, showing peak induction in most cases at 4 h. The up-regulation of 5/5 genes was also observed with the F(ab')(2) fragment of rituximab. Analysis of three further B-cell lymphoma lines showed that gene induction is not restricted to BJAB and DHL4. Finally, we show that rituximab alone can induce AP1 activation in both cell lines and provide evidence that the ERK1/2 pathway is involved in the rituximab-mediated up-regulation of gene expression. These data demonstrate that rituximab alone has direct signalling capacity in different B-lymphoma lines, inducing distinct but overlapping sets of genes which may play a role in the biological and/or therapeutic effect of the antibody.

Fig. 1

Rituximab inhibits thymidine uptake by DHL4 but not BJAB cells. a Exponentially growing DHL4 (solid circles) or BJAB lymphoma cells (open circles) were plated in presence or absence of increasing concentrations of rituximab and [³H]-thymidine uptake was measured at 64 h. b DHL4 cells were cultured in presence or absence of 10 μg/ml rituximab, daclizumab or campath-1H. c DHL4 cells were plated in presence or absence of 10 μg/ml rituximab and/or monoclonal antihuman IgG1 (1:500). d DHL4 cells were plated in presence or absence of 10 μg/ml rituximab, the rituximab F(ab′)₂ fragment or campath-1H antibody. [³H]-Thymidine uptake was measured at 64 h in all cases. All results shown are the mean and standard deviation of triplicate or quadruplicate wells and are representative of three independent experiments.

Fig. 2

Rituximab induces homotypic adhesion of DHL4 but not BJAB cells. Exponentially growing DHL4 or BJAB cells were plated in presence or absence of 10 μg/ml rituximab or control antibody daclizumab. Cells were examined microscopically 24 h later. a DHL4 + daclizumab, b DHL4 + rituximab, c BJAB + daclizumab, d BJAB + rituximab. The results are representative of at least five experiments.

Fig. 3

Real-time PCR analysis of genes induced by rituximab. Exponentially growing DHL4 (a) or BJAB (b) were plated in presence or absence of 10 μg/ml rituximab, and total RNA was extracted after 4 h. Fold induction of the indicated genes by rituximab over control was determined by real-time PCR using specific oligonucleotides (striped bars) and are compared with the fold inductions obtained by chip analysis (black bars). The results of PCR analysis are the mean and standard deviation of two to seven independent experiments.

Fig. 4

Both rituximab whole antibody and F(ab′)₂ fragment can up-regulate gene expression. Exponentially growing DHL4 (a) or BJAB (b) cells were plated in presence of 10 μg/ml rituximab or its F(ab′)₂ fragment. Total RNA was extracted after 4 h. Fold induction of the indicated genes by either rituximab or F(ab′)₂ fragment over control was determined by real-time PCR using specific oligonucleotides. The results shown are the mean and standard deviation obtained in two independent experiments.

Fig. 5

Time course of gene expression after exposure to rituximab. Exponentially growing DHL4 (a) or BJAB (b) cells were plated in presence or absence of 10 μg/ml rituximab antibody, and total RNA was extracted at 1, 4, 24 or 48 h. Induction of the indicated genes by rituximab relative to control antibody was determined by real-time PCR using specific oligonucleotides. The results are the mean and standard deviation of triplicate samples.

Fig. 6

Pattern of gene induction in B-lymphoma cell lines. Exponentially growing WSU-NHL (black bars), ESIII (thin-striped bars) and Raji (thick-striped bars) B-lymphoma cells were plated in presence or absence of 10 μg/ml rituximab antibody, and total RNA was extracted at 4 h. Induction of the indicated genes by rituximab relative to control antibody was determined by real-time PCR using specific oligonucleotides. The results are the mean and standard deviation of triplicate samples.

Fig. 7

Rituximab activates AP1 in both cell lines. Exponentially growing DHL4 or BJAB cells were incubated for 4 h in presence or absence of 10 μg/ml rituximab or daclizumab, or in presence of 20 ng/ml PMA. Nuclear extracts were prepared and used in electrophoretic mobility shift assays with a labelled AP1 probe. a The retarded bands obtained with unstimulated DHL4 (lane 1), or after stimulation with PMA (lane 2), rituximab (R, lane 2) or daclizumab (D, lane 4) or with unstimulated BJAB (lane 5) or BJAB stimulated with rituximab (lane 6) are shown. The results are representative of at least three independent experiments. In b, EMSA was performed using DHL4 extracts in absence (lanes 1–2) or presence of antibodies against the fos (lane 3) and jun proteins (lane 4), or in presence of excess specific (lane 5) or nonspecific unlabelled (cold) AP1 probe (lane 6). The results are representative of at least three independent experiments.

Fig. 8

Rituximab induces gene expression through the MEK1/MEK2-ERK1 pathway. Exponentially growing DHL4 cells were preincubated for 10 min in absence (black bars) or presence of 10 μM (light grey bars) or 30 μM (dark grey bars) PD98059 (MEK1/2 inhibitor) or 10 μM (thin-striped bars) or 30 μM (thick-striped bars) PD169316 (p38 inhibitor), followed by 4 h in presence or absence of 10 μg/ml rituximab. Total RNA was extracted, and expression of the indicated genes was analysed by real-time PCR. The results are the mean and standard deviation of three independent experiments.