Pathogenesis of aryl hydrocarbon receptor-mediated development of lymphoma is associated with increased cyclooxygenase-2 expression

Abstract

Epidemiological studies indicate that exposure to environmental pollutants such as pesticides and dioxins leads to the pathogenesis of lymphoma and leukemia. Here, we show that activation of the aryl hydrocarbon receptor (AhR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in loss of the programmed cell death (apoptosis) response in three different lymphoma cell lines, which plays a key role in the development of cancer, especially lymphoma and leukemia. The AhR-mediated inhibition of apoptosis in vitro was associated with a clear increase of cyclooxygenase-2 (COX-2) and deregulation of genes of the B-cell lymphoma-2 (Bcl-2) family involved in apoptosis including Bcl-xl and Mcl-1 in several lymphoma cell lines. Treatment with the COX-2 inhibitor NS-398 and the AhR antagonist 3'-methoxy-4'-nitroflavone abolished the TCDD-induced resistance of apoptosis in vitro. Furthermore, using micropositron emission tomography imaging, in vivo findings demonstrated that exposure to TCDD promotes the development of lymphoma in superficial lymph nodes of C57BL/10J mice, which was associated with a marked increase of COX-2 expression in the affected lymph nodes. The results indicate that AhR activation and COX-2 overexpression likely represent a mechanism of resistance to apoptosis in lymphoma cell lines that might be relevant for the development of lymphoma in vivo.

Figure 1

Effect of TCDD on UV-induced apoptosis in lymphoma cell lines. U937, Namalwa, and DOHH-2 cells were treated with 10 nmol/L TCDD for 48 hours. Then, apoptosis was induced by UV light, and the number of apoptotic cells was determined after 4 hours. Data are shown as percent inhibition versus control. In parallel experiments, cells were co-cultured with the COX-2 inhibitor NS-398 or the AhR antagonist 3′-methoxy-4′-nitroflavone. Data are means ± SD of results from three independent experiments performed. *Significantly different from control P ≤ 0.01, and **significantly different from cells treated with TCDD alone P ≤ 0.01.

Figure 2

Effect of AhR, C/EBPβ, and COX-2 gene silencing on TCDD-mediated apoptotic resistance in U937 cells. Cells were pretreated with TCDD or dimethyl sulfoxide (control) before apoptosis was induced by UV light. To block the TCDD- and AhR-mediated effect on apoptosis U937 cells were transfected with siRNA specific for AhR, C/EBPβ, or COX-2 for 24 hours before cells were treated with TCDD for 48 hours. Then, apoptosis was induced by UV light, and the number of apoptotic cells was determined after 4 hours. Values are averages of duplicates from two different experiments. *Significantly different from control P ≤ 0.01.

Figure 3

Treatment of the U937 lymphoma cell line with TCDD results in increased levels of Bcl-xl and Mcl-1 mRNA levels. Cells were treated with 10 nmol/L TCDD for 48 hours, and mRNA expression of Bax, Bcl-2, Bcl-w, Bcl-xl, and Mcl-1 was analyzed by real-time PCR. These data are shown as fold induction relative to control cells. Data are means ± SD of results from three independent experiments performed in duplicates. *Significantly different from control P ≤ 0.01.

Figure 4

Induction of C/EBPβ and COX-2 by TCDD is AhR-dependent. To block the TCDD-induced expression of COX-2, C/EBPβ, and Bcl-xl, U937 cells were transfected with siRNA specific for AhR, C/EBPβ, or COX-2 for 24 hours before cells were treated with TCDD for 48 hours. Data are means ± SD of results from three independent experiments. *Significantly increased compared with control P ≤ 0.01, and **significantly decreased compared with control P ≤ 0.01.

Figure 5

Time-dependent increased level of COX-2 protein in U937 cells treated with TCDD. Whole cell lysates from the U937 lymphoma cell line were prepared after treatment with 10 nmol/L TCDD for 12, 24, 48, and 72 hours and analyzed by immunoblotting using a polyclonal human COX-2-specific antibody. A representative result of three independent experiments is shown.

Figure 6

Development of lymphomas in axillary and inguinal lymph nodes of TCDD-treated C57BL/10J mice is associated with overexpression of COX-2. A and B: MicroPET imaging of control (A) and TCDD-treated (B) C57BL/10J mice using [¹⁸F]fluorodeoxyglucose (FDG). Mice received an initial dose of 20 μg of TCDD/kg b.wt. and a biweekly maintenance dose as described in Materials and Methods. Control mice received the corresponding amount of corn oil. MicroPET imaging was taken 140 days after initial treatment. C: Increased expression level of COX-2, C/EBPβ, and Bcl-xl mRNA in lymphoma tissue of TCDD-treated C57BL/10J mice. RNA was isolated from fresh lymph nodes and spleen 140 days after initial treatment and analyzed by real-time PCR. The induced mRNA expression is given relative to the values of control animals. Data are means ± SD of results from three animals of each group. *Significantly different from control P ≤ 0.05.

Figure 7

H&E staining and expression of the B-cell marker Pax5 in lymph node biopsies. Biopsies from lymph nodes of C57BL/10J mice were analyzed using H&E staining and by immunohistochemistry. A: H&E-stained lymph node from control mouse. B: H&E-stained lymph node from TCDD-treated mouse. C: Pax5 expression in lymph node from control mouse. D: Pax5 expression in lymph node from TCDD-treated mice. Inset depicts strong positive staining for Pax5. A representative sample is shown. Scale bar = 300 μm. Original magnifications: ×25 (A–D); ×500 (D, inset).