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Induction of apoptosis in experimental human B cell lymphomas by conditional TRAIL-expressing T cells.

Ucur E, Mattern J, Wenger T, Okouoyo S, Schroth A, Debatin KM, Herr I - Br. J. Cancer (2003)

Bottom Line: Jurkat-TRAIL cells had an additive effect with cytotoxic drugs in vitro, since cell death was enhanced.To elucidate the antitumoral activity of these Jurkat-TRAIL cells in vivo, we injected them intratumorally in xenografts of human Burkitt lymphomas.Switching on expression of TRAIL by adding tetracycline to the drinking water of the mice strongly reduced tumour growth by apoptosis in a caspase-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Clinical Cooperation Unit, Molecular Oncology/Pediatrics, German Cancer Research Center, Heidelberg, Germany.

ABSTRACT
In the present study, we demonstrate the utility of a non-tumour-forming T-cell line for the inducible gene transfer of tumour necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL), which has been shown to selectively induce apoptosis in malignant but not in normal cells. To generate T cells inducible for TRAIL expression, we stably transfected Jurkat cells with TRAIL in the context of the Tet-On system. The switched on cells strongly expressed TRAIL mRNA, whose protein product was expressed on the cell surface. Paracrine induction of apoptosis in human target tumour cells was solely found for membrane-bound TRAIL. The Jurkat-TRAIL cells itself survived due to clonal selection of TRAIL-resistant cells. Jurkat-TRAIL cells had an additive effect with cytotoxic drugs in vitro, since cell death was enhanced. To elucidate the antitumoral activity of these Jurkat-TRAIL cells in vivo, we injected them intratumorally in xenografts of human Burkitt lymphomas. Switching on expression of TRAIL by adding tetracycline to the drinking water of the mice strongly reduced tumour growth by apoptosis in a caspase-dependent manner. Thus, non-tumour-forming T-cell lines offer a novel method for gene transfer and inducible expression of TRAIL in tumour therapy.

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Tet induces TRAIL overexpression in Jurkat-TR (J-TR) cells. (A) Five selected single clones of J-TR or one clone of Jurkat-CO cells (J-CO) were cultured for 48 h in the presence (+, switched on) or absence (−, switched off) of tet (2 μg ml−1). Proteins were extracted and TRAIL expression was assayed by Western blot analysis using a mouse mAb, which specifically detects the 32 kDa TRAIL protein. Expression of the 43 kDa α-ACTIN protein was examined as a control for equal conditions. (B) Jurkat-TRAIL (J-TR) clone 5 and Jurkat-CO (J-CO) cells were incubated in the presence (+) or absence (−) of increasing doses of tet (0, 0.1, 0.5, 1, 1.5, 2 μg ml−1). After 48 h, total RNA was harvested and RT–PCR was performed using TRAIL-specific primers. Assaying the RNA expression of GAPDH confirmed equal conditions. (C) Jurkat-TRAIL (J-TR) or Jurkat-CO (J-CO) cells were cultured in the presence (open histograms with dotted lines) or absence (filled histograms with solid line) of tet (2 μg ml−1) and analysed by flow cytometry. The number indicates the percentage of TRAIL-positive cells. (D) Jurkat-TRAIL (J-TR) clone 5 and Jurkat-CO cells were switched on (2 μg ml−1 tet) or left switched off. After 48 h, cytospins were prepared and the cell surface expression of TRAIL was analysed by immunofluorescence staining. The representative results shown are from one of three different experiments with similar outcomes.
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fig1: Tet induces TRAIL overexpression in Jurkat-TR (J-TR) cells. (A) Five selected single clones of J-TR or one clone of Jurkat-CO cells (J-CO) were cultured for 48 h in the presence (+, switched on) or absence (−, switched off) of tet (2 μg ml−1). Proteins were extracted and TRAIL expression was assayed by Western blot analysis using a mouse mAb, which specifically detects the 32 kDa TRAIL protein. Expression of the 43 kDa α-ACTIN protein was examined as a control for equal conditions. (B) Jurkat-TRAIL (J-TR) clone 5 and Jurkat-CO (J-CO) cells were incubated in the presence (+) or absence (−) of increasing doses of tet (0, 0.1, 0.5, 1, 1.5, 2 μg ml−1). After 48 h, total RNA was harvested and RT–PCR was performed using TRAIL-specific primers. Assaying the RNA expression of GAPDH confirmed equal conditions. (C) Jurkat-TRAIL (J-TR) or Jurkat-CO (J-CO) cells were cultured in the presence (open histograms with dotted lines) or absence (filled histograms with solid line) of tet (2 μg ml−1) and analysed by flow cytometry. The number indicates the percentage of TRAIL-positive cells. (D) Jurkat-TRAIL (J-TR) clone 5 and Jurkat-CO cells were switched on (2 μg ml−1 tet) or left switched off. After 48 h, cytospins were prepared and the cell surface expression of TRAIL was analysed by immunofluorescence staining. The representative results shown are from one of three different experiments with similar outcomes.

Mentions: Surviving single clones were assayed for inducible protein expression of TRAIL. Five clones out of 112 Jurkat-TRAIL cells in the switched on status showed strong upregulation of TRAIL, in contrast to Jurkat-CO cells in which no induction was observed (Figure 1AFigure 1


Induction of apoptosis in experimental human B cell lymphomas by conditional TRAIL-expressing T cells.

Ucur E, Mattern J, Wenger T, Okouoyo S, Schroth A, Debatin KM, Herr I - Br. J. Cancer (2003)

Tet induces TRAIL overexpression in Jurkat-TR (J-TR) cells. (A) Five selected single clones of J-TR or one clone of Jurkat-CO cells (J-CO) were cultured for 48 h in the presence (+, switched on) or absence (−, switched off) of tet (2 μg ml−1). Proteins were extracted and TRAIL expression was assayed by Western blot analysis using a mouse mAb, which specifically detects the 32 kDa TRAIL protein. Expression of the 43 kDa α-ACTIN protein was examined as a control for equal conditions. (B) Jurkat-TRAIL (J-TR) clone 5 and Jurkat-CO (J-CO) cells were incubated in the presence (+) or absence (−) of increasing doses of tet (0, 0.1, 0.5, 1, 1.5, 2 μg ml−1). After 48 h, total RNA was harvested and RT–PCR was performed using TRAIL-specific primers. Assaying the RNA expression of GAPDH confirmed equal conditions. (C) Jurkat-TRAIL (J-TR) or Jurkat-CO (J-CO) cells were cultured in the presence (open histograms with dotted lines) or absence (filled histograms with solid line) of tet (2 μg ml−1) and analysed by flow cytometry. The number indicates the percentage of TRAIL-positive cells. (D) Jurkat-TRAIL (J-TR) clone 5 and Jurkat-CO cells were switched on (2 μg ml−1 tet) or left switched off. After 48 h, cytospins were prepared and the cell surface expression of TRAIL was analysed by immunofluorescence staining. The representative results shown are from one of three different experiments with similar outcomes.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2376862&req=5

fig1: Tet induces TRAIL overexpression in Jurkat-TR (J-TR) cells. (A) Five selected single clones of J-TR or one clone of Jurkat-CO cells (J-CO) were cultured for 48 h in the presence (+, switched on) or absence (−, switched off) of tet (2 μg ml−1). Proteins were extracted and TRAIL expression was assayed by Western blot analysis using a mouse mAb, which specifically detects the 32 kDa TRAIL protein. Expression of the 43 kDa α-ACTIN protein was examined as a control for equal conditions. (B) Jurkat-TRAIL (J-TR) clone 5 and Jurkat-CO (J-CO) cells were incubated in the presence (+) or absence (−) of increasing doses of tet (0, 0.1, 0.5, 1, 1.5, 2 μg ml−1). After 48 h, total RNA was harvested and RT–PCR was performed using TRAIL-specific primers. Assaying the RNA expression of GAPDH confirmed equal conditions. (C) Jurkat-TRAIL (J-TR) or Jurkat-CO (J-CO) cells were cultured in the presence (open histograms with dotted lines) or absence (filled histograms with solid line) of tet (2 μg ml−1) and analysed by flow cytometry. The number indicates the percentage of TRAIL-positive cells. (D) Jurkat-TRAIL (J-TR) clone 5 and Jurkat-CO cells were switched on (2 μg ml−1 tet) or left switched off. After 48 h, cytospins were prepared and the cell surface expression of TRAIL was analysed by immunofluorescence staining. The representative results shown are from one of three different experiments with similar outcomes.
Mentions: Surviving single clones were assayed for inducible protein expression of TRAIL. Five clones out of 112 Jurkat-TRAIL cells in the switched on status showed strong upregulation of TRAIL, in contrast to Jurkat-CO cells in which no induction was observed (Figure 1AFigure 1

Bottom Line: Jurkat-TRAIL cells had an additive effect with cytotoxic drugs in vitro, since cell death was enhanced.To elucidate the antitumoral activity of these Jurkat-TRAIL cells in vivo, we injected them intratumorally in xenografts of human Burkitt lymphomas.Switching on expression of TRAIL by adding tetracycline to the drinking water of the mice strongly reduced tumour growth by apoptosis in a caspase-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Clinical Cooperation Unit, Molecular Oncology/Pediatrics, German Cancer Research Center, Heidelberg, Germany.

ABSTRACT
In the present study, we demonstrate the utility of a non-tumour-forming T-cell line for the inducible gene transfer of tumour necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL), which has been shown to selectively induce apoptosis in malignant but not in normal cells. To generate T cells inducible for TRAIL expression, we stably transfected Jurkat cells with TRAIL in the context of the Tet-On system. The switched on cells strongly expressed TRAIL mRNA, whose protein product was expressed on the cell surface. Paracrine induction of apoptosis in human target tumour cells was solely found for membrane-bound TRAIL. The Jurkat-TRAIL cells itself survived due to clonal selection of TRAIL-resistant cells. Jurkat-TRAIL cells had an additive effect with cytotoxic drugs in vitro, since cell death was enhanced. To elucidate the antitumoral activity of these Jurkat-TRAIL cells in vivo, we injected them intratumorally in xenografts of human Burkitt lymphomas. Switching on expression of TRAIL by adding tetracycline to the drinking water of the mice strongly reduced tumour growth by apoptosis in a caspase-dependent manner. Thus, non-tumour-forming T-cell lines offer a novel method for gene transfer and inducible expression of TRAIL in tumour therapy.

Show MeSH
Related in: MedlinePlus