Characterization of the Resistance Mechanisms of HL60 Leukemia Cell Derivatives to Apo2L/TRAIL.

Abstract Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a recently identified member of the TNF superfamily. Recombinant Apo2L/TRAIL is a promising immunotherapeutic agent for treating malignant diseases since this molecule preferentially induces apoptosis in a variety of tumor cells with apparently little toxicity to normal cells. However, it has also been shown that some tumor cells are resistant to this molecule. We hypothesized that resistance to Apo2L/TRAIL occurs through defects in the Apo2L/TRAIL-mediated apoptotic signaling pathway. To address this hypothesis, we developed several Apo2L/TRAIL-resistant HL60 derivatives (HL60/TR) by exposure of Apo2L/TRAIL-sensitive HL60 human leukemia cells to escalating levels of Apo2L/TRAIL, followed by subcloning. Two of these resistant clones (a moderately resistant clone-R1 and a highly resistant clone-R3) were selected for further study. Molecules in the Apo2L/TRAIL-mediated apoptotic pathway of R1 and R3 cells were analyzed by Western blot analysis, flow cytometry and gene sequencing and compared to those in parental HL60 cells. In the R1 cells, the activation of caspase-8 and -10 by Apo2L/TRAIL was significantly inhibited. However, R1 cells were still sensitive to Fas agonistic monoclonal antibody treatment, indicating that the FAS-mediated apoptosis-inducing pathway was intact. In the R3 cells, caspase-8 expression was completely lost and activation of caspase-10 in response to Apo2L/TRAIL was totally inhibited; R3 cells were therefore also resistant to FAS antibody treatment. Although the total protein level of death receptors DR4 and DR5 was equal in HL60 cells and in the Apo2L/TRAIL-resistant derivatives, the cell surface levels of DR4 were significantly decreased in both R1 and R3 cells, while the surface expression level of DR5 in these two clones was comparable to that on HL60 cells. No mutation in either the DR4 or DR5 genes was found in these cells. These results suggest that defective targeting of DR4 molecules to the cell surface occurs in these Apo2L/TRAIL resistant cells. Blocking cell surface DR4 significantly attenuated the sensitivity of parental HL60 cells to Apo2L/TRAIL, indicating that cell surface expression of DR4 plays a crucial role in regulating susceptibility of tumor cells to Apo2L/TRAIL. Taken together, our results demonstrate that malignant cells can develop resistance to Apo2L/TRAIL by several different mechanisms and multiple resistance mechanisms may develop in a single tumor cell (such as R3 cells). Understanding the basis of Apo2L/TRAIL resistance will help to predict sensitivity and to develop strategies to circumvent resistance.

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A stress inducible HSP72 cell surface expression is observed selectively on human tumor cells

European Journal of Cancer ◽

10.1016/0959-8049(95)99941-r ◽

1995 ◽

Vol 31 ◽

pp. S36

Author(s):

M. Wiesner ◽

G. Multhoff ◽

C. Botzler ◽

W. Wilmanns ◽

R.D. Issels

Keyword(s):

Cell Surface ◽

Tumor Cells ◽

Human Tumor ◽

Surface Expression ◽

Cell Surface Expression ◽

Human Tumor Cells

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Discrepancy between transcriptional products and cell surface expression of MHC class I antigens in metastatic and non-metastatic Lewis Lung tumor cells

Apmis ◽

10.1111/j.1699-0463.1990.tb04980.x ◽

1990 ◽

Vol 98 (7-12) ◽

pp. 624-636 ◽

Cited By ~ 2

Author(s):

H. R. Juul-Madsen ◽

L. Olsson

Keyword(s):

Cell Surface ◽

Tumor Cells ◽

Mhc Class I ◽

Lung Tumor ◽

Surface Expression ◽

Class I ◽

Cell Surface Expression ◽

Lewis Lung ◽

Mhc Class I Antigens

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Characterization of thrombospondin synthesis, secretion and cell surface expression by human tumor cells

Clinical & Experimental Metastasis ◽

10.1007/bf01753679 ◽

1989 ◽

Vol 7 (3) ◽

pp. 265-276 ◽

Cited By ~ 34

Author(s):

James Varani ◽

Bruce L. Riser ◽

Lisa A. Hughes ◽

Thomas E. Carey ◽

Suzanne E. G. Fligiel ◽

...

Keyword(s):

Cell Surface ◽

Tumor Cells ◽

Human Tumor ◽

Surface Expression ◽

Cell Surface Expression ◽

Human Tumor Cells

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Temporal aspects of O-glycosylation and cell surface expression of ascites sialoglycoprotein-1, the major cell surface sialomucin of 13762 mammary ascites tumor cells.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)75922-5 ◽

1987 ◽

Vol 262 (1) ◽

pp. 269-275

Author(s):

J Spielman ◽

N L Rockley ◽

K L Carraway

Keyword(s):

Cell Surface ◽

Tumor Cells ◽

Surface Expression ◽

Cell Surface Expression ◽

Ascites Tumor ◽

Temporal Aspects

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Concomitant induction of the cell surface expression of Ia determinants and accessory cell function by a murine macrophage tumor cell line.

Journal of Experimental Medicine ◽

10.1084/jem.155.2.629 ◽

1982 ◽

Vol 155 (2) ◽

pp. 629-634 ◽

Cited By ~ 49

Author(s):

E B Walker ◽

L L Lanier ◽

N L Warner

Keyword(s):

Cell Surface ◽

Tumor Cells ◽

Cell Function ◽

Tumor Cell Line ◽

Surface Expression ◽

Cell Surface Expression ◽

Accessory Cell ◽

Gene Products ◽

Region Gene ◽

Histocompatibility Complex

This study demonstrates that an uncharacterized soluble factor produced in concanavalin A-induced rat spleen cell suspensions has the capacity to induce the increased expression of cell surface H-2K and H-2D molecules and the expression of I-region gene products on murine monocyte-macrophage lineage tumors that are not Ia positive in the absence of the factor. In parallel with induction of serologically defined Ia specificities, Ia-induced WEHI-3 macrophage tumor cells are capable of providing accessory cell function in stimulating IL-2 production by T-T hybridomas that are activated in a major histocompatibility complex-restricted, antigen-dependent fashion. The uninduced Ia-negative WEHI-3 tumor cells do not trigger a comparable response in this assay system.

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Natural Killer Cell Death Mediated By NKG2D-Trogocytosis

Blood ◽

10.1182/blood.v122.21.18.18 ◽

2013 ◽

Vol 122 (21) ◽

pp. 18-18

Author(s):

Kyohei Nakamura ◽

Masafumi Nakayama ◽

Mitsuko Kawano ◽

Tomonori Ishii ◽

Hideo Harigae ◽

...

Keyword(s):

Cell Death ◽

Confocal Microscopy ◽

Cell Surface ◽

Nk Cells ◽

Tumor Cells ◽

Nk Cell ◽

Surface Expression ◽

Cell Surface Expression ◽

Target Cells ◽

Dependent Manner

Abstract Introduction The activating receptor, NKG2D, plays crucial role in natural killer (NK) cell-mediated effector function. NKG2D is involved not only in host defense against tumor and viral infection, but also in autoimmune diseases by recognizing stress-induced self-ligands (NKG2DLs). However, the negative feedback regulation of NKG2D has not been fully understood. It has been reported that NK cells undergo rapid apoptosis upon interaction with NK-sensitive tumor cells, suggesting that activation-induced NK cell death can be triggered in certain situations. In this study, we aimed to elucidate underlying mechanism of NK cell death, especially focused on NKG2D-NKG2DLs interaction. Methods NK cells were purified from splenocytes of C57BL/6, perforin-/-, and DAP10-/-/12-/- mice, and cultured with rhIL-2 (1000 U/ml) for 5 days. We used these IL-2-activated NK cells as effector cells and three target cell lines: mouse T cell lymphoma RMA cells (RMA), RMA lacking MHCI expression (RMA-S), and RMA stably expressing an NKG2DL, Rae-1δ (RMA/Rae-1δ). CFSE-labeled NK cells were co-cultured with target cells for 2 hours, and stained with anti-NK1.1 mAb propidium iodide (PI). The percentage of PI-positive cells within CFSE+ NK1.1+ population was measured by flowcytometry. The cell surface expression of Rae-1 on NK cells after co-culture with target cells was evaluated by flowcytometry and confocal microscopy. Results NK cells from WT mice rapidly underwent cell death when co-cultured with Rae-1δ, but not with RMA or RMA-S, suggesting that NKG2D-Rae-1 interaction is involved in NK cell death. NK cells from perforin-/-, and DAP10-/-/12-/- mice did not undergo cell death, highlighting the importance of the NKG2D pathway for NK cell death. However, cross-linking of NKG2D receptor failed to induce NK cell death. To understand underlying the mechanism of NK cell death, we evaluated the cell surface expression of NKG2DLs on NK cells after co-culture with tumor cells. We found that cell surface expression of Rae-1 on NK cells was remarkably induced after co-culture with RMA/Rae-1δ, but no with RMA or RMA-S, implying that these Rae-1-positive NK cells may be lysed by NK cells through NKG2D-induced perforin pathway. The cell surface induction of Rae-1 on NK cells was very rapid (within 5min), and it occurred cell-cell contact dependent manner. Interestingly, NK cells from C57/BL6 mice rapidly became BALB/c allele Rae-1γ-positive after co-culture with RMA/Rae-1γ, indicating that NK cells acquire tumor-derived Rae-1. Consistently, acquisition of Rae-1 by NK cells was confirmed by confocal microscopy. Therefore, NK cells rapidly dress tumor-derived Rae-1 after interaction with tumor cells through intercellular membrane transfer, namely trogocytosis. Trogocytosis of Rae-1 was significantly inhibited in NK cells from DAP10-/-/12-/- mice and by chemical inhibitors of PI3K and Syk, indicating that it requires NKG2D-signaling. To confirm whether Rae-1-dressed NK cells can be recognized and lysed by other NK cells, we used sort-purified Rae-1-dressed NK cells as target cells. Rae-1-dressed NK cells were lysed by WT NK cell in an E/T-ratio dependent manner through NKG2D-induced perforin pathway. Furthermore, adoptively transferred Rae-1-dressed NK cells were significantly eliminated in Rag-1-deficient mice, indicating that Rae-1-dressed NK cells are also recognized and lysed in vivo. Conclusion Upon interaction with NKG2DLs-expressing tumor cells NK cells rapidly dress tumor-derived NKG2DLs, and subsequently undergo fratricide. Our results provide novel insights into activation-induced NK cell death via dynamic intercellular communications. Disclosures: No relevant conflicts of interest to declare.

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