Professional Documents
Culture Documents
DOI 10.1007/s10495-009-0349-3
ORIGINAL PAPER
123
Introduction
Tumor necrosis factor-related apoptosis-inducing ligand
(TRAIL), also called apoptosis-inducing ligand 2 (Apo2L),
triggers programmed cell death in various types of cancer
cells but not in most normal cells [1]. TRAIL is a homotrimeric protein that interacts with five receptors. Binding
with DR4 and DR5 receptors induces death signals to the
intracellular apoptotic machinery [2]. DcR1 and DcR2 are
designated as decoy receptors that compete with DR4 and
DR5 for TRAIL binding and antagonize TRAIL-induced
apoptosis [3]. Osteoprotegerin (OPG) is a soluble TRAILinteracting molecule which may have a more prominent
role in bone and myeloid cell development than in regulating TRAIL-induced apoptosis [4].
Many cancer cell lines express both DR4 and DR5, and
each of these receptors can initiate apoptosis independently
of the other. Recent evidence indicates that both tumor and
normal cells can acquire resistance to TRAIL-induced killing by upregulating either of the two antagonistic receptors,
DcR1 and DcR2 so the four TRAIL receptors are involved in
regulation of TRAIL-induced apoptosis [59]. DcR1 and
DcR2 are unable to recruit FADD and form active deathinducing signaling complex (DISC). DcR2 inhibits apoptosis by sequestering TRAIL or by forming heteromeric
receptor complex with DR5 that is unable to activate signaling [6, 7]. Inhibition of TRAIL induced apoptosis by
DcR2 critically depends on its association with DR5 via the
NH2-terminal preligand assembly domain overlapping the
first partial cysteine-rich domain of both receptors. Ligand
binding by DcR2 is dispensable for its apoptosis inhibitory
function, thereby excluding the possibility that DcR2 is a
decoy to inhibit apoptosis by binding of TRAIL. It have
been proposed that DcR2 is a regulatory rather than
decoy receptor that inhibits TRAIL-induced apoptosis
signaling through ligand-independent mechanism [6, 8].
To investigate the relative contribution of DR4 and DR5
to ligand-induced apoptosis several receptor-selective
TRAIL variants were generated recently using a novel
approach that enabled phage display of mutated trimeric
proteins. Selective binding to DR4 or DR5 was achieved
with three to six amino acid substitutions [9]. The TRAIL
variants with optimal selectivity and activity were marked
as DR5-8 and DR4-8. The DR4-8 variant showed a
markedly reduced ability to trigger apoptosis, whereas the
DR5-8 variant had minimally decreased or slightly
increased apoptosis-inducing activity in cell lines where
induction of apoptosis was mediated mainly through DR5
receptor. Another DR5-selective TRAIL variant (D269H)
was generated using the automatic design algorithm
FOLD-X. This variant did not induce apoptosis in DR4responsive cell lines but show a significant increase of
biological activity in DR5-responsive cancer cell lines [10].
Here we have generated new DR5-selective variants of
TRAIL (designated as DR5-A and DR5-B) by combining
DR5-8 and D269H variants. DR5-A and DR5-B demonstrated high selectivity to DR5 receptor and practically did
not bind to DR4 or DcR1 receptors. Surface plasmon resonance (SPR) binding experiments demonstrated that
DR5-B variant had also very low affinity to DcR2 and OPG
receptors. DR5-A and DR5-B were significantly more
effective than wild type TRAIL in induction of apoptosis in
different cell lines were the cell death was mediated mainly
through DR5 receptor. The new selective mutants of
TRAIL will serve as a good tool to study the TRAILinduced apoptotic signaling in cell lines with different
expression of death and decoy receptors.
779
Analytical ultracentrifugation was carried out with Beckman Model E centrifuge connected to PC. TRAIL solution
(0.5 mg/ml in PBS) was analyzed at 60,000 rpm in standard 0.4 ml cells. Sedimentation coefficient s20,w was calculated using software SEDFIT [14] (available at NIH site
123
780
http://www.analyticalcentrifugation.com). Sedimentation
analysis data demonstrated that 98% of recombinant wild
type and mutant TRAIL formed trimers with a relative
molecular mass of 5860 kDa (Fig. 1).
Assay of apoptosis induction
Human acute T leukemia Jurkat, monoblastic leukemia
U937 and Philadelphia chromosome-positive acute myeloid leukemia K562 cell lines were maintained in RPMI
1640 medium supplemented with 10% (v/v) fetal calf
serum, 2 mM glutamine, 100 U/ml streptomycin and
100 U/ml penicillin at 37C and 5% CO2. A concentration
series of the TRAIL variants were made in cell culture
medium. For viability tests, cells were seeded in 96-well
plates (1 9 105 cells/well) and incubated with serial dilutions of TRAIL mutants for 24 h at 37C and 5% CO2.
Subsequently, MTT reagent was added to final concentration 0.5 mg/ml. Cell viability was determined after 3 h of
incubation by measuring the absorption at 570 nm with
Multiscan plate reader. HeLa-Bcl-2 cells were generated
using retrovirus vector [15] in the Institute of Poliomyelitis,
Russian Academy of Medical Sciences and kindly provided
by Professor VI Agol. Human cervical carcinoma HeLa
and HeLa-Bcl-2 cell lines were grown in Dulbeccos
modified Eagles medium (DMEM) supplemented with
10% (v/v) fetal calf serum, 2 mM glutamine, 100 U/ml
streptomycin and 100 U/ml penicillin at 37C and 5% CO2.
In some experiments the inhibitor of protein synthesis
123
781
Results
Generation of new DR5-selective TRAIL mutant
variants
The new DR5-selective TRAIL variants DR5-A and DR5-B
were designed based on previously described variants
DR5-8 [9] generated using a novel phage display technology and D269H [10] generated using the automatic
design algorithm FOLD-X (Table 1). DR5-8 variant demonstrated higher selectivity than D269H but the affinity of
D269H binding to DR5 receptor was several fold higher
than for DR5-8. To improve the selectivity without loosing
affinity to DR5 receptor we have generated DR5-A mutant
by insertion D269H mutation in amino acid sequence of
DR5-8 variant which contained six other amino acid substitutions. Analysis of the original data indicated that D267
residue in TRAIL amino acid sequence did not play a
References
Wt TRAIL
DR4-8
Y189A
Q193S
N199 V
K201R
Y213 W
S215D
DR5-8
Y189N
R191K
Q193R
H264R
I266L
D267Q
[9]
D269H
D269H
[10]
DR5-A
Y189N
R191K
Q193R
H264R
I266L
D267Q
D269H
Present report
DR5-B
Y189N
R191K
Q193R
H264R
I266L
D269H
Present report
[9]
123
782
DR5
DR4
DcR2
OPG
Wild type
0.51 0.028
0.46 0.014
1.09 0.069
0.36 0.009
0.99 0.017
D269H
0.13 0.003
8.47 0.335
8.69 0.628
0.73 0.014
5.26 0.980
DR5-8
1.18 0.017
NB
NB
3.32 0.048
10.9 0.448
DR5-A
0.33 0.005
NB
NB
4.18 0.082
12.2 0.138
DR5-B
0.71 0.013
NB
NB
101 7.0
221 19a
143 13
DR4-8
NB
3.26 0.010
2.49 0.950
2.10 0.075
21.0 0.140
Standard deviation was calculated by fitting of sensorgrams using ProteOn Manager Version 2.0.1 software (Bio-Rad) as Chi-square criterion
NB no detectable binding
a
123
783
with maximum 60% cell death. This was a saturated percent for wild type and DR4-8 variant when D269H variant
at higher concentrations of ligand (up to 400 ng/ml)
appeared even higher effectivity (up to 85% cell death)
(Fig. 3).
In U937 monoblastic leukemia cell line cytotoxic
activity of DR5-8, DR5-A and DR5-B variants was significantly higher than of wild type TRAIL showing both an
increased maximum response (almost twice) and 1.8, 3.0
and 3.4 decreased EC50 values, respectively. D269H variants activity was similar to the wild type in these cells.
In our previous studies we have demonstrated that
expression of Bcl-2 blocks TRAIL-induced release of
cytochrome c from mitochondria into cytosol and apoptosis
in Hela cells [20]. No one of the DR5-selective TRAIL
variants could overcome this block of apoptosis (Fig. 4).
We have shown earlier that the inhibitor of microtubules,
123
784
Table 3 ED50 values of TRAIL variants induced apoptosis on different cell lines
TRAIL variants HeLa (?0.05 lg/ml emetine) K562
Jurkat
EC50 (ng/ml) Cell death (%) EC50 (ng/ml) Cell death (%) EC50 (ng/ml) Cell death (%) EC50 (ng/ml) Cell death (%)
Wild type
6.2 0.5
93 6
0.22 0.02
60 4
0.28 0.03
37 3
0.27 0.03
D269H
2.1 0.2
91 6
0.23 0.03a 85 7
0.17 0.02
43 3
0.27 0.03
25 2
DR5-8
6.0 0.6
45 3
0.27 0.02
0.15 0.02
48 4
0.15 0.02
43 3
27 3
22 2
DR5-A
2.2 0.2
86 7
0.73 0.06
34 3
0.07 0.01
56 5
0.09 0.01
43 4
DR5-B
1.5 0.1
78 6
1.11 0.07
35 3
0.07 0.01
53 5
0.08 0.01
50 3
DR4-8
1.6 0.2
25 3
0.23 0.02
62 7
0.71 0.06
26 3
0.26 0.02
14 3
taxol and the inhibitor of actin microfilaments, cytochalasin D enhanced efficiency of TRAIL and allow it to
overcome antiapoptotic effect of Bcl-2 [20]. All the DR5selective TRAIL variants did not lose the capacity to break
Bcl-2 block in combination with the inhibitors of cytoskeleton (Fig. 4). It should be mentioned that TRAILinduced apoptosis in HeLa cells was strongly enhanced by
inhibitor of the protein synthesis, emetine. The same effect
in the case of TNF-induced apoptosis was traditionally
ascribed to inhibition of NF-jB-dependent expression of
antiapoptotic proteins. As one can see from Fig. 4, in
combination with all TRAIL variants taxol but not cytochalasin D completely substitute emetine. Moreover, the
effect of taxol was not summated with emetine. The nature
of this effect is currently under investigation.
All our results clearly demonstrated that DR5-A and
DR5-B variants are more specific to DR5 receptor and
demonstrate higher biological activity in comparison to
DR5-8 and D269H TRAIL variants.
Discussion
123
785
123
786
References
1. Ashkenazi A, Pai RC, Fong S, Leung S, Lawrence DA, Marsters
SA et al (1999) Safety and anti-tumor activity of recombinant
soluble Apo2 ligand. J Clin Invest 104:155162. doi:10.1172/
JCI6926
2. LeBlanc HN, Ashkenazi A (2003) Apo2L/TRAIL and its death
and decoy receptors. Cell Death Differ 10:6675. doi:10.1038/
sj.cdd.4401187
123
787
26. Ashkenazi A, Herbst RS (2008) To kill a tumor cell: the potential
of proapoptotic receptor agonists. J Clin Invest 118:19791990.
doi:10.1172/JCI34359
27. MacFarlane M, Inoue S, Kohlhaas SL, Majid A, Harper N,
Kennedy DBJ et al (2005) Chronic lymphocytic leukemic cells
exhibit apoptotic signaling via TRAIL-R1. Cell Death Differ
12:773782. doi:10.1038/sj.cdd.4401649
28. Inoue S, MacFarlane M, Harper N, Wheat LMC, Dyer MJS,
Cohen GM (2004) Histone deacetylase inhibitors potentiate TNFrelated apoptosis-inducing ligand (TRAIL)-induced apoptosis in
lymphoid malignancies. Cell Death Differ 11(Suppl 2):193206.
doi:10.1038/sj.cdd.4401535
29. Pietro R, Secchiero P, Rana R, Gibellini D, Visani G, Bemis K
et al (2001) Ionizing radiation sensitizes erythroleukemic cells
but not normal erythroblasts to tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity by
selective up-regulation of TRAIL-R1. Blood 97:25962603.
doi:10.1182/blood.V97.9.2596
30. Perez-Cruz I, Carcamo JM, David W, Golde DW (2007) Caspase8 dependent trail-induced apoptosis in cancer cell lines is inhibited by vitamin C and catalase. Apoptosis 12:225234.
doi:10.1007/s10495-006-0475-0
31. Gregorini A, Tomasetti M, Cinti C, Colomba D, Colomba S
(2006) CD38 expression enhances sensitivity of lymphoma T and
B cell lines to biochemical and receptor-mediated apoptosis. Cell
Biol Int 30:727732. doi:10.1016/j.cellbi.2006.05.004
32. Kim K, Fisher MJ, Xu SQ, El-Deiry WS (2000) Molecular
determinants of response to TRAIL in killing of normal and
cancer cells. Clin Cancer Res 6:335346
123