Yonsei Med J.  2006 Feb;47(1):55-62. 10.3349/ymj.2006.47.1.55.

The Combination of TRAIL Treatment and Cancer Cell Selective Expression of TRAIL-Death Receptor DR4 Induces Cell Death in TRAIL-Resistant Cancer Cells

• Choi E
• Kim Y
• Kim K

• KMID: 1715873
• DOI: http://doi.org/10.3349/ymj.2006.47.1.55

Abstract

The human telomerase reverse transcriptase (hTERT) promoter can be used for the tumor-specific expression of transgenes in order to induce selective cancer cell death. The hTERT core promoter is active in cancer cells but not in normal cells. To examine whether the combination of TNF-related apoptosis inducing ligand (TRAIL) treatment and cancer cell-selective expression of the TRAIL-death receptor could induce cell death in TRAIL-resistant cancer cells, we generated a death receptor-4 (DR4)-expressing adenovirus (Ad-hTERT-DR4), in which the expression of DR4 is driven by the hTERT promoter. Upon infection, DR4 expression was slightly increased in cancer cell lines, and cell death was observed in TRAIL-resistant cancer cell lines but not in normal human cells when DR4 infection was combined with TRAIL treatment. We also generated an adenovirus that expresses a secretable isoleucine zipper (ILZ)-fused, extracellular portion of TRAIL (Ad-ILZ-TRAIL). In cells infected with Ad-ILZ-TRAIL, TRAIL was expressed, secreted, oligomerized and biologically active in the induction of apoptosis in TRAIL-sensitive cancer cells. When Ad-hTERT-DR4 infected TRAIL-resistant HCE4 cells and Ad-ILZ-TRAIL infected TRAIL-resistant HCE7 cells were co-cultured, cell deaths were evident 24 h after co-culture. Taken together, our results reveal that the combination of TRAIL and cancer cell-specific expression of DR4 has the potential to overcome the resistance of cancer cells to TRAIL without inducing significant cell death in normal cells.

Keyword

Neoplasms; TNF-related apoptosis-inducing ligand; human telomerase reverse transcriptase promoter; death receptor-4; adenoviridae

MeSH Terms

Tumor Necrosis Factor-alpha/genetics/*pharmacology/secretion
Telomerase/genetics
TNF-Related Apoptosis-Inducing Ligand
Receptors, Tumor Necrosis Factor/genetics/*metabolism
Promoter Regions (Genetics)
Neoplasms/genetics/metabolism/pathology
Membrane Glycoproteins/genetics/*pharmacology/secretion
Humans
Drug Resistance, Neoplasm
DNA-Binding Proteins/genetics
Cell Line
Apoptosis Regulatory Proteins/genetics/*pharmacology/secretion
Apoptosis/*drug effects
Antineoplastic Agents/*pharmacology
Adenoviridae/genetics

Figure

• 

  Fig. 1 hTERT core promoter activity in normal cells and cancer cells. The luciferase activity assay was performed using extracts of cells transiently transfected with pGL3-basic, pGL3-control, or pGL3-hTERT vectors for 24 h. pCMV-β-galactosidase was co-transfected in order to standardize the transfection efficiency. The data are expressed as the mean ± SD for triplicate conditions, and similar results were obtained from two independent experiments.

• 

  Fig. 2 Expression-, secretion-, and apoptosis-inducing activity of adenoviral-encoded TRAIL. (A) Schematic diagrams of Ad-GFP and Ad-ILZ-TRAIL. (B) Expression and secretion of TRAIL after infection. TRAIL-resistant TE5 cells were infected with either Ad-GFP or Ad-ILZ-TRAIL (Ad-ILZ). Western blot analysis was performed using lysates and culture media 48 h after infection. The arrowhead indicates the un-processed signal peptide containing TRAIL and the arrow represents the fully processed form of TRAIL. (C) Oligomerization of secreted TRAIL. The same media used in (B) was used to confirm the oligomerization of secreted TRAIL. To maintain oligomerization of secreted TRAIL during SDS-PAGE, EGS (final 1 mM) was added into the media. (D) Apoptosis-inducing activities of expressed TRAIL. TRAIL-sensitive SW480 cells were infected with either Ad-GFP (GFP) or Ad-ILZ-TRAIL (TR). After 48 h, the cells were observed under bright field (B) or fluorescent (F) microscopy (× 400). (E) Western blot analysis was performed using the lysates obtained from the same cells as in (D). z-VAD-fmk (20 µM final) was added 1 h after infection.

• 

  Fig. 3 Cancer cell death induced by the combination of TRAIL treatment and AdhTERT-DR4 infection. (A) Schematic diagram of Ad-hTERT DR4. (B) The expression of DR4 and induction of apoptosis in cancer cell lines in the presence of TRAIL. Western blot analysis was performed using extracts of CasKi (top left) or HCE4 (top right) cells that had been infected with either Ad-GFP or Ad-hTERT DR4 for 24 h and subsequently treated with human recombinant TRAIL for 24 h. Flow cytometric analysis for sub-G1 content. CasKi or HCE4 cells were infected and treated as in (B), and subsequently harvested for flow cytometry. The sub-G1 content of 10,000 cells was examined for each sample (bottom left). This experiment was performed in triplicate. Percent of sub-G1 was plotted (bottom right). (C) Resistance of normal primary cervical keratinocytes. Normal keratinocytes were infected and treated, and cell death was quantified by flow cytometric detection as in (B). This experiment was performed in triplicate. Percent of sub-G1 was plotted.

• 

  Fig. 4 The bystander effect of adenoviral-encoded TRAIL. (A) Morphologic observation of apoptosis. TRAIL-resistant HCE4 cells were infected with either Ad-GFP (GFP) or Ad-hTERT DR4 (DR4), and TRAIL-resistant HCE7 cells were infected with either Ad-GFP or Ad-ILZ-TRAIL (TR). One day after the infection, the cells were harvested and co-cultured for 24 h. The cells were observed under bright field (B) or fluorescent (F) microscopy (× 100). (B) Biochemical detection of apoptosis. Western blot analysis was performed using the extracts obtained from the cells described in (A).

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