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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 1100-1107
Expression of the Death Gene Bik/Nbk Promotes Sensitivity to
Drug-Induced Apoptosis in Corticosteroid-Resistant T-Cell Lymphoma and
Prevents Tumor Growth in Severe Combined Immunodeficient Mice
By
Peter T. Daniel,
Kwok-Tao Pun,
Silke Ritschel,
Isrid Sturm,
Jutta Holler,
Bernd Dörken, and
Robin Brown
From the Max Delbrück Center for Molecular Medicine; the
Department of Hematology, Oncology and Tumor Immunology,
Charité Campus Berlin-Buch, Humboldt University, Berlin-Buch,
Germany; and the Cell Biology Unit, Glaxo Wellcome Medicines Research
Centre, Stevenage, United Kingdom.
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ABSTRACT |
Members of the Bcl-2 gene family have been implicated in the
regulation of cell death induced by cytostatic drugs. In some malignancies such as B-cell lymphoma, there is evidence that high expression of Bcl-2 is an independent negative prognostic marker and
the overexpression of Bcl-2 has been shown to confer resistance to
cytotoxic drugs by preventing drug-induced apoptosis. This function of
Bcl-2 can be antagonized by apoptosis-promoting members of the Bcl-2
family. We previously showed that overexpression of Bax restores the
chemosensitivity of Bax-deficient breast cancer cell lines. Therefore,
we investigated whether the death-promoting Bcl-2 homologue Bik/Nbk can
enhance cytostatic drug-induced apoptosis. As a model, we used the
T-cell leukemia H9 (CD3+ and
CD4+CD8 ), which is resistant to
corticosteroid-induced cell death and does not express endogenous
Bik/Nbk. Sensitivity for drug-induced apoptosis was increased 10- to
39-fold in cells transfected with the full-length coding sequence of
Bik/Nbk. In addition, apoptosis induced via CD95/Fas or heat shock was
increased to a similar extent. These data show that Bik/Nbk, which,
unlike Bax, carries only a BH3 but no BH1 or BH2 domain may be a target
to enhance chemosensitivity. The complete suppression of tumor growth
in a severe combined immunodeficient mouse xenotransplant model
suggests that, in analogy to Bax, Bik/Nbk may function as a tumor
suppressor gene.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MEMBERS OF THE BCL-2 FAMILY are key
regulators of apoptosis and their deregulation has been implicated in
the development of malignancy1 and the resistance of tumor
cells to cytostatic drug-induced cell death.2,3 Previously,
we have shown that the proapoptotic gene Bax, if overexpressed in
breast cancer cells, which lack endogenous Bax,4 blocks the
ability of these cells to form tumors in severe combined
immunodeficient (SCID) mice.5 In addition, Bax confers to
these cells increased drug sensitivity.2 In metastatic
colorectal, cancer, we recently described that the lack of Bax is a
negative prognostic factor, especially in those patients carrying a
wild-type p53 gene.6
In this report, we have examined the effect of another member of the
Bcl-2 family on drug sensitivity in the mature T-ALL (acute
lymphoblastic/lymphocytic leukemia) cell line H9 (CD3+ and
CD4+CD8). Bik/Nbk7,8 is, like
Bax9 and Bak,10 a BH3 containing member of the
Bcl-2 family and is expressed only in a restricted subset of human
tissues, as we show in the present work. This suggests that Bik/Nbk may
play a role in the tissue specific regulation of apoptosis. To
investigate whether Bik/Nbk enhanced the sensitivity to apoptosis of H9
cells, we generated stable transfectants expressing Bik/Nbk. (The H9
parental cell line does not express Bik/Nbk). Interestingly, the H9
T-lymphoma cells are refractory to corticosteroid-induced cell death,
which appears to be a negative prognostic factor in childhood11,12 and possibly as well in adult13
acute lymphoblastic leukemia.
The transfectants showed an increased sensitivity to Fas-triggered
death and apoptosis after exposure to anticancer drugs or heat shock.
Moreover, their ability to form tumors in vivo was completely suppressed.
These experiments have, therefore, helped us to understand the role of
Bik/Nbk in the control of apoptosis and determine that Bik/Nbk
preferentially enhances cell death induced on DNA damage or
Fas-triggering. Therefore, this "minimal" death module, which, unlike other Bcl-2 homologues, expresses only the BH3 Bcl-2-homology domain, may be employed not only to further investigate the mechanisms of apoptosis induction but may also provide a target to enhance chemosensitivity of cancer cells.
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MATERIALS AND METHODS |
Cell culture and transfection.
All cells were maintained in 1640 RPMI (Seromed-Biochrom, Hamburg,
Germany), 10% heat-inactivated fetal calf serum (GIBCO-BRL, Karlsruhe,
Germany), 2 mmol/L L-Glutamine (GIBCO-BRL, Karlsruhe, Germany), and
penicilline-streptomycin (Seromed-Biochrom, Hamburg, Germany) as
described.14
Bik/Nbk cDNA was cloned as a 1 kilobase (kb) Bgl II fragment into the
mammalian expression vector pCIN4, a derivative of
pCIN1/pIRESneo15 (Clontech Lab, San Diego, CA). This
plasmid (30 µg) was transfected into H9 cells (CD3+,
CD4+CD8) by electroporation with a Bio-Rad
electroporator at 960 µF/250V. Stable clones were generated by
limiting dilution into 96-well flat-bottomed plates with selection in
normal medium containing 1 mg/mL G418-sulphate (GIBCO-BRL). Colonies
were diluted into larger well and flasks and expanded for analysis.
pCIN4 mock transfectants (H9 3D8) were generated in parallel and
displayed the same apoptotic behavior as the parental line.
Western blot analysis.
Cells in log phase were harvested and lysed in sodium dodecyl sulfate
(SDS) sample buffer containing 10%  -mercaptoethanol, then boiled
for 5 minutes and sonicated (4 × 5-second cycles at 10 µm
amplitude in an MSE soniprep 150 sonicator). Proteins were separated on Tris-glycine 4% to 20% gradient acrylamide gel and blotted onto Hybond enhanced chemoluminescence (ECL) nitrocellulose membrane (Amersham, Braunschweig, Germany). Immunodetection was performed at 4°C by using a polyclonal goat anti-Nbk antibody (Santa-Cruz, Santa Cruz, CA) at 1 µg/mL and visualized
by using the ECL detection system (Amersham), according to the
manufacturer's instructions. Glucocorticoid receptor (GR) protein was
detected as described above by the use of a polyclonal rabbit antiserum (diluted 1:1,000; Santa-Cruz), which recognized the N-terminus of both
GR-alpha (95 kD) and GR-beta (90 kD).
Northern blot analysis.
Human multiple tissue Northern blots (Clontech) were hybridized with a
32P- -dCTP (Amersham) random labeled 423bp
BamH1-Sma1 fragment of Bik/Nbk in 50% formamide buffer
at 43°C for 24 hours, followed by washes in 0.1% SDS 0.1 × standard salt concentration (SSC) buffer at 50°C. The
blots were exposed for 10 days with intensifying screens at  70°C.
RNA preparation, polymerase chain reaction (PCR), and Southern blot
analysis.
Total RNA was purified from 5 × 106 cells by using the
RNAzolB method.16 RNA (4 µg) was used for first strand
cDNA synthesis with the GeneAmp RNA PCR kit (Perkin Elmer, Weiterstadt,
Germany), according to the manufacturer's instructions.
PCR was performed by using a vector specific T7 forward primer:
5'-TAA-TAC-GAC-TCA-CTA-TAG-GG-3' and a Bik/Nbk specific reverse primer:
5'-TTC-CAA-AGA-ATC-GAA-GTC-CT-3' in PCR buffer containing 20 pmol of
each primer, 1.5 mmol/L MgCl2, 200 mmol/L dNTPs, and 2.5 units Taq polymerase. Cycling conditions were 30 cycles (94°C, 30 seconds; 60.5°C, 1 minute; 72°C, 1 minute). The PCR fragments were
analyzed on a 1.2% gel and identified as Bik/Nbk by Southern blotting
onto Hybond N+ membrane (Amersham) and high stringency hybridization
using a 32P- -ATP labeled oligonucleotide corresponding
to the 5' terminus of Bik/Nbk (4 to 28 bp inclusive).
Measurement of apoptosis.
After induction of apoptosis by cytotoxic drugs, anti-APO-1 IgG3,
anti-CD3, dexamethasone, or heat shock for 24 to 72 hours, the cells
were pelleted, washed with ice-cold phosphate-buffered saline in U-form
96-well plates and gently resuspended in 300 µL hypotonic
fluorochrome solution (propidium iodide 50 µg/mL, 0.1 mol/L sodium
citrate plus 0.1% Triton ×100) as previously described.14,17 After overnight incubation at 4°C in the
dark, the propidium-iodide content of the individual nuclei was
measured on a FACSort (Becton Dickinson, Heidelberg, Germany). Cell
debris was excluded by raising the forward-scatter threshold
adequately. Apoptotic nuclei displayed a decreased DNA content below
the G1 peak, paralleled by an increase of the side scatter. In
addition, apoptosis was measured in the dexamethasone-induced cultures
by assessing the decrease of cell size (forward light scatter signal, FSC) and the increase of membrane permeability (uptake of propidium iodide, 1 µg/mL) during late stage apoptosis as
described.18
Animal experiments.
Animals were kept and treated in accordance with German animal
protection laws. C.B-17 scid/scid mice (SCID mice) were from our own breeding colony. The mice were kept in isolators under gnotobiotic conditions. Food and water were autoclaved. The mice were
not subjected to antibiotic drug treatment. No mouse pathogens were
detected. For serology, sterile sentinel mice were added to the colony.
These mice were serologically negative for Sendai, PVM, MVM, Reo3, MHV
(Corona), Theiler's GD VII, Polyoma, K-Adeno, and m.-Adeno virus.
Leakiness was determined by measuring mouse Igmol/L and mouse IgG, and
mice expressing serum titers above 50 µg/mL IgM or IgG were excluded
from the study. H9 lymphoma cells were injected s.c. into the inguinal
region (107 per animal). Tumor diameters were measured with
calipers in two dimensions and the tumor volume was calculated as
described.19
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RESULTS |
Resistance to cytostatic drug-induced apoptosis is the major obstacle
for the cure of lymphoid malignancies. In this regard, the resistance
to corticosteroid-induced death appears to be a prominent negative
clinical prognostic factor in acute lymphoblastic leukemia.11,12 In this line, bcl-2 and bcl-xL have been
shown to prevent cytostatic drug-induced cell death.3
Therefore, we were interested in testing whether the expression of the
death promoting Bcl-2 homologue Bik/Nbk may enhance sensitivity for drug-induced apoptosis in the T-lymphoma cell line H9, which is refractory to corticosteroid-triggered cell death.
Northern blot analysis showed that Bik/Nbk has a restricted tissue
distribution with expression being detected mainly in epithelial cells
(Fig 1). High levels of expression are seen
in the kidneys and pancreas (Fig 1A) with lower levels in placenta,
lungs, liver (Fig 1A), prostate, and testis (Fig 1B). No hybridization
was seen in the heart, brain, skeletal muscle, spleen, thymus, ovary, small intestine, colon, and peripheral blood leukocytes (Fig 1A and B).
The cell lines Raji (EBV-positive type III Burkitt lymphoma) and SW480
(colon adenocarcinoma) had the highest levels of mRNA expression with
lower levels in HL-60 and MOLT-4 cells (Fig 1C). Thus, in contrast to
the lack of Bik/Nbk expression in nonmalignant lymphoid or colon
tissue, Bik/Nbk mRNA appears to be expressed ectopically in some
tumor-derived cell lines.
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| Fig 1.
Multiple tissue Northern blot analysis for Bik/Nbk
expression. PolyA+ RNA (2 µg) were loaded per lane. Arrows indicate
the position of the 1.1 kb Bik/Nbk transcript. (A) Multiple human
tissue Northern blot analysis. Lane 1, heart; 2, brain; 3, placenta; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; 8, pancreas. (B) Lane 1, spleen; 2, thymus; 3, prostate; 4, testis; 5, ovary; 6, small
intestine; 7, colon; 8, peripheral blood leukocytes. (C) Human cell
lines Northern blot analysis. Lane 1, promyelocytic leukemia HL60; 2, cervix carcinoma HeLa S3; 3, erythroid leukemia K562; 4, T-cell
leukemia MOLT4; 5, type III Burkitt lymphoma Raji; 6, colorectal
adenocarcinoma SW480; 7, lung carcinoma A549; 8, melanoma G361.
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With regard to the above expression pattern where Bik/Nbk was detected
preferentially in the epithelial but not the lymphoid compartment
(disregarding the ectopic expression in Raji cells), a lymphoid cell
system appeared suitable for the functional analysis of Bik/Nbk. The
ability of Bik/Nbk to enhance sensitivity for drug-induced apoptosis
was, therefore, assessed in the corticosteroid-resistant H9 T-ALL cell
line, which does not express detectable levels of endogenous Bik/Nbk as
shown by Western blot analysis (Fig 2B).
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| Fig 2.
Analysis for Bik/Nbk expression in transfected H9
T-lymphoma cells. (A) RT-PCR and Southern blot: The exogenous mRNA
transcript was detected by RT-PCR with primers designed to vector
specific forward sequence and Bik/Nbk specific reverse sequence,
yielding a 292-bp fragment. DNA was electrophoresed, Southern blotted
and hybridized by the use of a third, radiolabeled oligonucleotide
specific for the 5' terminus of Bik/Nbk. Positive and negative controls
were pCIN.Nbk plasmid (lane 1) and water (lane 2). Lane 3, H9 3D8
control transfected cells; lane 4, clone no. 2; lane 5, clone no. 7;
lane 6, clone no. 10; lane 7, clone no. 13; lane 8, clone no. 16. (B)
Western blot analysis for Bik/Nbk expression: Protein extracts from
SW480 colon carcinoma cells (lane 1), H9 3D8 control cells (lane 2) or
Bik/Nbk transfected clones no. 2 (lanes 3 and 4) and 10 (lane 5) were
separated by SDS-polyacrylamide gel electrophoresis and Western blot
analysis with a goat antiserum against Bik/Nbk. Yeast expressed Bik/Nbk
Gal4-Nbk fusion protein served as a positive control (lane 6). Bands
were visualized by means of ECL.
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Therefore, the full-length cDNA of Bik/Nbk was cloned into the pCIN4
vector (pCIN4.Nbk) and stably overexpressed in the H9 cells. Clones
selected for resistance to G418 and isolated by limiting dilution were
screened for transgene expression by reverse transcription (RT)-PCR
followed by Southern blot analysis. In Fig 2A, no exogenous Bik/Nbk is
detected in the mock transfectants (H9 3D8), whereas a signal is
detected in the pCIN4.Nbk transfectants. The clones no. 2, 10, and 16 showed a strong Bik/Nbk hybridization signal and were subjected to
further functional assays as described below.
Protein expression was determined by Western blot analysis. In the H9
3D8 control cells no Bik/Nbk could be detected (Fig 2B). In contrast,
the clones nos. 2 and 10 show overexpression of the 22.5 kD Bik/Nbk
protein. A Gal4-Nbk fusion protein expressed in yeast served as a
positive control. However, the colon adenocarcinoma cell line SW480,
which shows strong expression of Bik/Nbk RNA (Fig 1C), showed only
weak, but detectable Bik/Nbk endogenous protein expression (Fig 2B).
To test for the effect of Bik/Nbk on apoptosis sensitivity, the
transfectants were exposed to the cytostatic drugs etoposide, epirubicin, and taxol. After a 72-hour culture, apoptosis was assessed
on the single cell level by flow cytometric measurement of the nuclear
DNA content. All three clones showed an increased susceptibility for
drug-induced apoptosis. Sensitivity for all three drugs was strongly
enhanced as compared with the control cells (Fig 3A through
C). Comparison of the ED50
concentrations for apoptosis induction shows a 10.1- to 39.3-fold
sensitization for drug-induced apoptosis in the Bik/Nbk transfectants
as compared with the H9 mock transfectants (Table
1).
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| Fig 3.
Sensitization of H9 cells for drug-induced cell death.
Cells were exposed to cytostatic drugs for 72 hours. Apoptosis was
determined on the single cell level by measuring the DNA content of
individual nuclei by flow cytometry. Hypodiploid nuclei were considered
as apoptotic. (A) Epirubicin, (B) etoposide, (C) taxol. Cell culture in
the presence of the drugs ( ). Medium controls (only in [A]) ( ).
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In T cells, the CD95 death receptor and its ligand have been implicated
in the control of apoptosis.14,17,20,21 In addition, drug-induced apoptosis was suggested to depend, in part, on activation of the Fas ligand and subsequent CD95/Fas ligation. Therefore, we
assessed the effect of Bik/Nbk on CD95 triggered death of the H9 T
cells. CD95/Fas-triggered death was augmented in all three clones (Fig
4B), whereas activation-induced death on
crosslinking of the CD3 chain of the T-cell receptor was only
marginally enhanced (Fig 4A). The CD95 or CD3 -chain receptor
densities in the transfectants were measured by flow cytometry and
showed no significant difference (data not shown). As in the case of
drug-mediated apoptosis, the Bik/Nbk transfectants showed an increased
apoptosis susceptibility on ligation of the CD95/Fas death receptor by
agonistic anti-CD95 mab (anti-APO-1 IgG319) as compared
with cultures exposed to an isotype matched control mab
(FII23c19).
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| Fig 4.
Sensitization of H9 cells for cell death induced by
Fas/CD95, CD3 crosslinking, or dexamethasone. (A)
Activation-induced cell death on CD3 cross-linking by immobilized
anti-CD3 monoclonal antibody (MoAb) (clone OKT3). Plates were coated
with OKT3 (coating concentration 0.1 to 100 µg/mL) as
described.14,17 (B) Induction of cell death by CD95/Fas
triggering by (soluble) anti-CD95 MoAb (clone anti-APO-1 IgG3). (C)
Cell death induction by dexamethasone, which was added to the cultures
at concentrations from 108 mol/L to 105
mol/L. Cell death was determined on the single-cell level by measuring
the DNA content of individual nuclei by flow cytometry. Data represent
the mean of triplicates ± SD. (A through C) H9 control cells ( ),
Bik/Nbk clone no. 2 ( ), Bik/Nbk clone no. 10 ( ), Bik/Nbk clone
no. 16 ( ). Medium control in (A) or cultures incubated with FII23c
isotype-matched control antibody (0.12 µg/mL19) in (B):
H9 control cells ( ), Bik/Nbk clone no. 2 ( ), Bik/Nbk clone no. 10 ( ), Bik/Nbk clone no. 16 ( ).
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In contrast to the effect seen with the DNA damaging agents and taxol
or heat shock (see below), we observed no increase in cell death
susceptibility to dexamethasone (Fig 4C). Addition of dexamethasone in
concentrations ranging from 108 to 105
mol/L did not elevate the death rate above percentages seen in the
medium controls of the H9 3D8 control cells or the Bik/Nbk transfectants. To address the question of whether corticosteroid occurs
in the absence of endonuclease activation in the H9 cells, we performed
a flow cytometric analysis of H9 cells in which we assessed cell size
and cell membrane permeability of H9 cells during apoptosis induction
as additional parameters. Apoptotic cells are known to shrink and this
leads to a decrease of the forward light scatter (FSC) as measured by
flow cytometry. In addition, late stage apoptotic cells show an
increased membrane permeability, which was determined by addition of
propidium iodide (PI). Thus, cells with a decrease in FSC and an
increased uptake of PI can be considered as apoptotic.18 In
comparison and in clear contrast to anti-CD3-induced H9 cells, neither
H9 3D8 cells nor Bik/Nbk H9 transfectants showed such signs of
apoptosis after induction with dexamethasone, thereby showing that
dexamethasone does not induce apoptosis in these cells (not shown). We
were also unable to observe induction of a DNA ladder after induction with dexamethasone, unlike CD3 or CD95/Fas triggering which induce activation of genomic DNA fragmentation.22
To further exclude that dexamethasone does not induce apoptosis in the
H9 T cells because of defects in GR expression and function we
performed a Western blot analysis for GR expression in H9 3D8 and the
Bik/Nbk transfectants (Fig 5A). There was
no difference in GR-alpha (95 kD) or GR-beta (90 kD) expression levels. In addition, the GR expression is known to be under control of steroid
responsive elements in T cells.23 Thus, exposure of H9 T
cells to dexamethasone led to an induction of GR expression in both the
H9 3D8 control cells and the Bik/Nbk transfectants (Fig 5B and C).
Additional evidence for the presence of a functional GR receptor and
signaling pathway come from the observation that glucocorticoids induce
promoter activation in H9 T cells transfected with retroviral long
terminal repeat promoter constructs containing GR-response
elements.24 Therefore, the GR receptor and signaling pathway appear to be intact in the H9 T cells and the Bik/Nbk clones.
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| Fig 5.
Expression and induction of the GR in H9 cells. (A)
Western blot analysis for GR- (95 kD, upper band) and GR- (90 kD,
lower band) expression. Lane 1, H9 3D8 cells; lane 2, H9 Bik/Nbk clone
no. 16, lane 3, clone no. 10; lane 4, clone no. 2. (B) Western blot
analysis for induction of GR expression by dexamethasone in H9 3D8
cells. Lane 1, medium control; lane 2, 24-hour culture in the presence
of dexamethasone (106 mol/L); lane 3, 48 hours; lane 4, 72-hour induction. (C) Induction of GR expression by dexamethasone in
H9 Bik/Nbk clone no. 16. Lane 1, medium control; lane 2, 24-hour
culture in the presence of dexamethasone (106 mol/L);
lane 3, 48 hours; lane 4, 72 hours.
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Thus, Bik/Nbk could promote sensitivity toward drug-induced apoptosis
and CD95/Fas-mediated death, but could not render the steroid
refractory H9 cells sensitive for steroid-induced apoptosis.
To assess the effect of Bik/Nbk on another physiologic apoptotic
stimulus, and in addition to the death induced by cytostatic drugs, we
investigated the response to heat shock (Table
2). The cells were incubated for 4 hours at
temperatures ranging from 37°C to 45°C. After heat shock, the cells
were cultured for a further 24 to 48 hours at 37°C. Cell death by
apoptosis was induced at temperatures of 39°C and above. Apoptosis
was detectable in the H9 3D8 mock transfectants after 24 hours (Table
2) and reached a maximum after 48 hours (Table 2). No further increase
was seen after 72 to 96 hours (not shown). At higher temperatures, the death rate increased but cell death was necrotic as evidenced by uptake
of trypan blue (data not shown) and decreased DNA fragmentation as
compared with the lower temperatures. Overexpression of Bik/Nbk increased the response for heat shock triggered apoptosis. Whereas, 15% of the H9 control cells underwent apoptosis after 48 hours, apoptosis induction was observed in 28% to 38% of the Bik/Nbk transfectants (Table 2).
In previous experiments, we observed a decrease of tumorigenicity of
breast cancer cells when transfectants overexpressing the apoptosis
promoter Bax were xenotransplanted into SCID mice. Therefore, we tested
whether the ectopic overexpression of Bik/Nbk decreases tumorigenicity
of H9 T lymphoma cells in such a xenotransplantation model in SCID
mice. Cells (107) were injected subcutaneously (s.c.)
into the inguinal region. Tumor growth was observed starting at week 6 after transplantation (Table 3). The H9 3D8
mock transfected cells grew to large local s.c. tumors. Some mice
developed macroscopic lymph node dissemination. Hind limb paralysis was
observed as previously encountered in the case of Nalm-6 pre-B-ALL
xenotransplantation.25 Mice were, therefore, sacrificed 9 weeks after transplantation. In contrast, no tumors developed in the
mice transplanted with Bik/Nbk transfectants (Table 3). These mice
remained tumor free for a further 3 months after the mice transplanted
with H9 control cells had to be sacrificed.
| |
DISCUSSION |
Members of the Bcl-2 family are key regulators of apoptosis.
Overexpression of Bcl-2 in the B-cell compartment of transgenic mice
leads to B-cell hyperplasia. Subsequent dysregulation of genes such as
c-myc can lead to the development of B-cell lymphoma.26 The
ability of Bcl-2 to prevent apoptosis is antagonized by the proapoptotic members of the Bcl-2 family.9,27 Tissue
hyperplasia and tumor promotion can be achieved by the inactivation of
such proapoptotic genes as demonstrated by the phenotype of Bax knock out mice28 and transgenic Bax k.o. mice carrying a
truncated SV40 large T antigen.29 The mechanism of action
of the proapoptotic genes is nevertheless unclear, although recent
evidence has implicated the APAF-1 gene, a homologue of the
Caenorhabditis elegans ced-4 gene.30
In this model, Bcl-xL, caspase-9, and APAF-1 form a ternary
complex.31,32 The role of Bax in this complex could be to
compete for binding to the caspase activating protein APAF-1 and effect
cell death. Additional data show that Bcl-2 prevents mitochondrial
permeability shift transition33 and that Bax may directly
activate mitochondria resulting in the induction of the mitochondrial
permeability transition and the release of cytochrome C.34
In previous experiments, we observed that breast cancer cells have a
defect in Bax expression.4 The reconstitution of Bax in the
breast cancer cells restored apoptosis sensitivity for serum starvation
or Fas-triggered death.5 Additional experiments showed that
Bax, as well, increased the sensitivity of the breast cancer cells to
apoptosis induced by exposure to DNA damaging agents such as
anthracyclin drugs.2 In metastatic colorectal cancer, we
recently described that the lack of Bax is a negative prognostic
factor, especially in those patients carrying a wild-type p53
gene.6 In contrast to the broad expression of Bax and Bak, the expression of Bik/Nbk seems to be restricted to epithelial tissues.
Given the proapoptotic properties, Bik/Nbk could be involved in tissue
specific maintenance of homeostasis in these adult tissues as suggested
for Bcl-2 or Bax.35 In this context, it was surprising to
find that some of the cell lines tested showed constitutive overexpression of Bik/Nbk. In the case of Raji, this overexpression shows a good correlation with a high sensitivity for Fas-triggered apoptosis.14 Mutations in the Bik/Nbk gene like in the case of Bax,36 p53,37 or deregulated activation of
the Bik/Nbk gene by EBV-encoded factors (in the case of Raji), may also
be the cause for the ectopic expression. Nevertheless, we cannot exclude that this overexpression is paralleled by defects in the downstream death signaling cascade and concomittant deregulation of the
upstream death effector Bik/Nbk. In this line, the SW480 cells, which
show elevated Bik/Nbk mRNA, express only low levels of endogenous
Bik/Nbk protein.
H9 T cells do not express detectable levels of endogenous Bik/Nbk and
are resistant to glucocorticoid-induced apoptosis. Therefore, we
investigated in this model whether the overexpression of Bik/Nbk in
stably transfected cells increased the sensitivity to cytostatic drug-induced apoptosis and rendered them sensitive to steroid-induced cell death. This question is of interest in light of data that suggest
that primary resistance to steroid-induced cell death is an important
negative prognostic factor that may predict treatment failure in acute
lymphocytic leukaemia.11,12
In H9 cells stably transfected with Bik/Nbk, we observed sensitization
of the cells to cytostatic drug-induced cell death as compared with the
parental line and the mock transfectants. This increase of drug
sensitivity amounted to a 10- to 39-fold reduction in the ED50 for the
topoisomerase inhibitors epirubicin and etoposide as well as for the
microtubule disrupting drug paclitaxel (taxol). These results show and
confirm that the induction of apoptosis by these agents in H9 cells is
controlled, in part, by a Bcl-2-dependent pathway, which is enhanced by
the overexpression of Bik/Nbk. In contrast, we were unable to reverse
the steroid resistance in these cells. Previous reports showed that
Bcl-2 prevents steroid-induced death in normal and malignant lymphoid cells.38,39 The glucocorticoid receptor expression levels
and signaling both appear to be functional in the H9 cells and the transfectants. Thus, the resistance to dexamethasone-induced apoptosis may be because of a defective response upstream of Bcl-2, Bax, or
Bik/Nbk. Nevertheless, recent evidence suggests that Bcl-2 and Bax or
Bik/Nbk independently regulate cell death.1,40 In this
regard, it is unclear whether Bik/Nbk (and Bax) regulate a
corticosteroid-independent pathway of apoptosis as opposed to the
antagonization of corticosteroid-triggered apoptosis by Bcl-2.
In a xenotransplantation SCID mouse model, Bik/Nbk overexpressing cells
were unable to form tumors. The data obtained from these experiments
are in line with our previous observations that reconstitution of Bax
expression in xenotransplanted breast cancer cells decreases tumor
formation in SCID mice. The clonogenic potential of H9 T cells can,
thus, be abrogated by the overexpression of Bik/Nbk. This is in line
with the decreased clonogenicity (data not shown) and elevated
spontaneous (background) apoptosis of the Bik/Nbk transfectants in
vitro. In this context, it would be interesting to examine whether
Bik/Nbk expression is lost or downregulated during tumorigenesis as we
observed for Bax in breast cancer4,5 and high-grade
metastatic colorectal cancer.6
In the Bik/Nbk transfectants, we observed an increase in sensitivity to
CD95 triggered cell death. Thus, Bik/Nbk not only sensitizes cells to
drug-induced apoptosis, but also to CD95/Fas-triggered apoptosis, ie,
one of the major physiologic programmed cell death pathways in lymphoid
cells. In malignant disease, recent observations have suggested that
drug-induced cell death by a variety of compounds, including
antimetabolites, leads to induced expression of CD95/Fas and
FasL.41 The Bik/Nbk data are consistent with this model and
suggest that Bik/Nbk acts on a pathway that sensitizes the cells to
apoptosis that is common to both stimuli. The Bik/Nbk enhancement of
cell death under these circumstances is also in line with results that
suggest CD95/Fas triggered death can be inhibited by overexpression of
Bcl-2 and can be enhanced by Bax expression.5
The effect of Bik/Nbk overexpression was examined in another
physiologic model of cell death. We observed an enhancement of cell
killing after heat shock, in contrast to treatment with anti-CD3 and
dexamethasone in which killing was unaffected. This was surprising in
the case of the anti-CD3-mediated death, because this is considered to
be mediated by the CD95/Fas ligand. Nevertheless, there is evidence
that additional, Fas-independent pathways participate in the
activation-induced cell death on CD3-crosslinking, which could be
Fas-independent, such as other members of the death ligand and receptor
superfamilies, eg, tumor necrosis factor receptor-mediated signals.42 In addition we were not able, as previously
shown,14,17,20 to completely abrogate CD3-triggered
apoptosis by addition of high-affinity blocking anti-CD95/Fas
antibodies19 to the culture. These and many other findings
from other groups show that the activation-induced cell death of T
cells is not mediated exclusively by the CD95/Fas receptor/ligand
interaction. This is also in line with the fact that antigen
receptor-triggered apoptosis in B cells is clearly independent from
CD95/Fas,14 which further corroborates the fact that
additional, CD95/Fas-independent signals participate in antigen
receptor-mediated cell death in lymphoid cells.
Thus, Bik/Nbk differentially sensitized the T-lymphoma cells to a
variety of apoptotic stimuli. This might be related to structural properties of Bik/Nbk.
The Bcl-2 family protein Bik/Nbk7,8 contains only one of
the signature domains of the Bcl-2-family, the BH3 domain. The BH3
domain is conserved both in the proapoptotic and the antiapoptotic Bcl-2 family proteins. The BH3 domain of the proapoptotic proteins may
serve a dual function. It appears to be essential for their cell death
activity and for mediating homodimerization with antiapoptosis proteins.8,43,44 Because BH3-alone containing proapoptotic proteins (Bik/Nbk, Bid, Hrk, Bad, Mtd) share only the BH3 domain in
common, it has been discussed whether the BH3 domain is a death effector module and is postulated to elicit its cell death activity by
inactivating the antiapoptotic proteins through heterodimerization. This is supported by the observation that the BH3 domain is an apoptosis effector in a cell-free system.44 However,
mutational analysis of the BH3 domain in Bik40 and a novel
proapoptotic Bcl-2 homologue, Mtd,45 suggests that
heterodimerization via the BH3 domain with survival proteins alone is
insufficient to explain their cell death inducing activity.
Nevertheless, Bik/Nbk may also function as a naturally dominant
negative antagonist whose role in the cell is to bind to and inactivate
antiapoptotic genes, eg, Bcl-2 and Bcl-xL, like in the case of Bad.
Given the above described capability of members of the Bcl-2 family to
induce apoptosis even in the absence of a functional BH3 domain we
would favor the direct induction of the mitochondrial apoptotic
signaling cascade and subsequent activation of caspases. Such a view of a direct action of Bik/Nbk, independent from dimerization to Bcl-2 or
Bcl-xL, is supported by data where the BH3 domain in Bax or Bak was
deleted.27,46 This prevents homodimerization of Bax but the
BH3-deleted mutants retain their capability to enhance apoptosis.
The Bik/Nbk data presented here establish that Bik/Nbk differentially
controls the cellular apoptotic response, depending on the type of
induction stimulus. In our previous work, we showed that overexpression
of Bax may enhance chemosensitivity.2 Recent data show that
such an effect of Bax may be caused by the direct activation of the
mitochondrial death cascade.34 Such an effect appears to be
independent from the interaction with Bcl-2/Bcl-x via the BH1/BH2
domains, which would be in line with our findings that the proapoptotic
Bcl-2 homologue Bik/Nbk, which lacks the BH1 and BH2 domain, enhances
drug and Fas-mediated apoptosis. Taken together, these data support the
role of Bik/Nbk as a direct death effector not only in propagation of
cell death on DNA damage, but also for CD95/Fas crosslinking. Such a
differential control of different apoptotic pathways could be mediated
by functional domains apart from the BH3 signature domain.
Finally, the fact that Bik/Nbk can sensitize cells to drug-induced
apoptosis suggests that upregulation of such tissue-specific dominant
negative Bcl-2-like protein might yield a therapeutic strategy to
overcome drug resistance and tumors refractory to cytotoxic therapies.
| |
ACKNOWLEDGMENT |
The authors thank Clarissa von Haefen for expert technical assistance
and P.H. Krammer (German Cancer Research Center, Heidelberg) for the
generous gift of anti-CD95/Fas MoAb clone anti-APO-1 IgG3 and B. Champion and S. Rees (Glaxo Wellcome, Stevenage) for providing H9 cells
and the pCIN4 vector, respectively.
| |
FOOTNOTES |
Submitted November 2, 1998; accepted April 7, 1999.
P.T.D. and K.-T.P. equally contributed to this work and share first authorship.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Peter T. Daniel, MD,
Department of Hematology, Oncology and Tumor Immunology,
CharitéCampus Berlin-Buch, Humboldt-University, Lindenberger
Weg 80, 13125 Berlin-Buch, Germany; e-mail: pdaniel{at}mdc-berlin.de.
| |
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ABSTRACT
INTRODUCTION