Prostaglandin D(2), its metabolite 15-d-PGJ(2), and peroxisome proliferator activated receptor-gamma agonists induce apoptosis in transformed, but not normal, human T lineage cells

Abstract

Prostaglandin D(2) (PGD(2)) is abundantly produced by mast cells, platelets, and alveolar macrophages and has been proposed as a key immunoregulatory lipid mediator. 15-Deoxy-Delta(12,14)-PGJ(2) (15-d-PGJ(2)), a key PGD(2) metabolite, is under intense study as a potential anti-inflammatory mediator. Little is known about PGD(2) or the role of 15-d-PGJ(2), if any, in regulating the activities of human T lineage cells. In this report we demonstrate that both PGD(2) and 15-d-PGJ(2) have potent antiproliferative effects, and in fact kill human T lymphocyte lines derived from malignant cells by an apoptotic mechanism. Interestingly, normal human T cells were not similarly affected. Although the T lymphocyte lines express mRNA for the PGD(2) receptor (DP-R), a potent DP receptor agonist, BW245C, did not inhibit the proliferation or viability of the cells, suggesting an alternative mechanism of action. PGD(2) and 15-d-PGJ(2) can bind to the peroxisome proliferator activated receptor-gamma (PPAR-gamma) which is implicated in lipid metabolism and apoptosis. Exposure to synthetic PPAR-gamma ligands (e.g. ciglitazone, troglitazone) mimicked the inhibitory responses of PGD(2) and 15-d-PGJ(2), and induced apoptosis in the transformed T cells consistent with a PPAR-gamma-dependent mechanism. These observations suggest that PPAR-gamma ligands (which may include PGD2) provide strong apoptotic signals to transformed, but not normal T lymphocytes. Thus, the efficacy of utilizing PPAR-gamma and its ligands as therapeutics for human T cell cancers needs to be further evaluated.

Figure 1

PGD₂ and its metabolite 15-d-PGJ₂ inhibit the proliferation of transformed but not normal human T cells. CCRF-CEM (a), J-Jahn (b), Jurkat (c), or normal human T cells (d) were incubated for 48 hr in the presence of either PGD₂ or 15-d-PGJ₂. Non-transformed cells were also treated with anti-CD3 and anti-CD28 as described in Materials and Methods. Cells were pulsed with 1 µCi/µl of ³H-thymidine for the final 8 hr of culture. Data is graphed as percent DMSO control response. Each individual experiment was performed in triplicate, and repeated three times with similar results each time. Asterisks indicate P < 0·05 based on a two-tailed Student's t-test.

Figure 2

PGD₂ and its metabolite 15-d-PGJ₂ Inhibit the viability of transformed but not normal human T cells. CCRF-CEM (a), J-Jahn (b), Jurkat (c), or normal human T cells (d) were incubated for 48 hr in the presence of either PGD₂ or 15-d-PGJ₂. Non-transformed T cells were additionally treated with anti-CD3 and anti-CD28 as described in Materials and Methods. Cells were treated with MTT for the final 8 hr of culture. Data is graphed as percentage DMSO control response. Each individual experiment was performed in triplicate, and repeated three times with similar results each time. Asterisks indicate P < 0·05 in a two-tailed Student's t-test.

Figure 3

PGD₂ and 15-d-PGJ₂ induce apoptosis in Jurkat cells. DMSO treated (a, d), 15-d-PGJ₂ treated (b, e) at 10 µm, or PGD₂ treated (c, f) at 25 µm were stained for annexin–FITC (a–c) or TUNEL (d–e). For annexin, cells were incubated for 18 hr with the indicated concentration, then stained according to manufacturer's instructions, and analyzed on a BD FACSscan. TUNEL staining was performed according to manufacturers directions after 24-h incubation with prostaglandins. Cells were photographed under bright field.

Figure 4

Malignant human T cell lines, but not normal T cells, express mRNA for the DP-R. Total RNA was isolated from cells and reverse transcribed into cDNA. The cDNA was amplified with primers specific for β-actin (as a control) or DP-R. HMC-1 (human mast cell line) was used as a positive control for DP-R. Reverse transcriptase (−) samples were negative in all cases.

Figure 5

PGD₂ does not inhibit proliferation and viability of T cells through the DP-R. CCRF-CEM (a), J-Jahn (b), or Jurkat (c) cells were cultured for 48 hr with either PGD₂ or BW245C. For proliferation assays (a(i)–c(i)) cells were pulsed with 1 µCi/µl of ³H-thymidine for the final 8 h of incubation. For MTT (b(i)–c(i)) cells were treated with MTT for the final 8 hr of culture. Data is graphed as percentage DMSO control response. Each individual experiment was performed in triplicate, and repeated three times with similar results each time. Asterisks indicate P < 0·05 based on a two-tailed Student's t-test.

Figure 6

Malignant human T-cell lines, but not normal T cells, express PPAR-γ mRNA. Total RNA was isolated and reverse transcribed into cDNA as discussed in Materials and Methods. The cDNA was amplified with control primers (β-actin) or primers specific for PPAR-γ. L828 human lung fibroblasts were used as a positive control for PPAR-γ. Reverse transcriptase (−) samples were negative in all cases.

Figure 7

Malignant human T-cell lines express PPAR-γ protein. Malignant human T cells were spread onto slides and stained for PPAR-γ as described in Materials and Methods. Cells were then viewed and photographed under bright field. mIgG1 was used as an isotype control.

Figure 8

Stimulation of peripheral human T cells from normal donors modestly induces PPAR-γ expression. CD4 positive T lymphocytes were isolated from buffy coats of normal donors as described in Materials and Methods. Cells were stimulated with or without anti-CD3 and anti-CD28 for 24 and 48 hr. Cells were cytospun, stained for PPAR-γ expression, then photographed under bright field. Unstimulated cells did not express PPAR-γ at any time point analysed.

Figure 9

PPAR-γ agonists induce apoptosis in Jurkat cells. Jurkat cells were treated as described in Materials and Methods and stained for annexin–FITC (a) or TUNEL (b). For annexin staining, cells were incubated for 18 h with the treatments shown above, stained according to the manufacturer's instructions, and analysed by flow cytometry. For TUNEL staining, cells were treated for 24 h prior to staining, then viewed and photographed under bright field.