|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2672-2680
RAPID COMMUNICATION
Activated Endothelial Cells Induce Apoptosis in Leukemic Cells by
Endothelial Interleukin-8
By
Yasuhito Terui,
Masayuki Ikeda,
Hiroshi Tomizuka,
Tadashi Kasahara,
Tetsuya Ohtsuki,
Masaya Uwai,
Masaki Mori,
Takehito Itoh,
Miyuki Tanaka,
Muneo Yamada,
Seiichi Shimamura,
Yukihito Ishizaka,
Kazuma Ikeda,
Keiya Ozawa,
Yasusada Miura, and
Kiyohiko Hatake
From the Division of Hematology, Department of Internal Medicine,
Jichi Medical School, Kawachi-gun, Tochigi, Japan; the Biochemical
Research Laboratory, Morinaga Milk Industry Co Ltd, Zama City,
Kanagawa, Japan; and the Department of Biochemistry, Kyoritsu College
of Pharmåcy, Minato-ku, Tokyo, Japan.
 |
ABSTRACT |
Tumor cells are eradicated by several systems, including Fas
ligand-Fas and tumor necrosis factor (TNF)-tumor necrosis factor receptor (TNFR). In the previous study, we purified an
apoptosis-inducing factor (AIF) to homogeneity from a medium
conditioned by PDBu-treated HL-60 cells. N-terminal sequence analysis
showed that AIF is identical to endothelial interleukin-8 (IL-8). A
novel apoptosis system, in which endothelial cells participate via
endothelial IL-8 release, is identified here. Human umbilical vein
cells (VE cells) produce and secrete IL-8 by stimulation of IL-1 and
TNF-. Endothelial IL-8, which is secreted from VE cells by
stimulation of IL-1 and TNF- , induces apoptosis in myelogenous
leukemia cell line K562 cells. Monocyte-derived IL-8 could not induce
apoptosis in K562 cells. Moreover, interaction between VE cells and
K562 cells induces the release of endothelial IL-8 from VE cells, and
the attached K562 cells undergo apoptosis. Moreover, interactions between VE cell and other cell lines, such as HL-60, U937, Jurkat, and
Daudi, induce the secretion of endothelial IL-8 and the induction of
apoptosis in cell lines. Endothelial IL-8 significantly inhibits tumor
growth of intraperitoneal and subcutaneous tumor mass of K562 cells and
induces apoptosis in their cells in vivo. Endothelial IL-8 plays an
important role in apoptosis involving endothelial cells, which may
provide us with a new therapy for hematological malignancies.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
APOPTOSIS IS AN ACTIVE process in which
various types of cells are selectively deleted during embryonic
development and in the adult multicellular organism under certain
physiologic conditions.1 Cell death occurs during the
development and regulation of the immune system, leading to deletion of
self-reactive T and B lymphocytes, regulation of immunologic memory,
and lysis of target cells by cytotoxic T lymphocytes and natural killer
(NK) cells.2-4 Alteration of the genes that control
apoptosis may lead to a variety of diseases such as autoimmune,
malignant clonal growth, neurodegenerative diseases, as well as
prolonged survival of cells during latent viral infection.5
Cells with induced apoptosis appear shrunken, with condensed or
fragmented nuclei. Apoptotic cells have fragmented DNA, with DNA
ladders in electrophoresis.6,7
Tumor cells have systems to protect themselves from induction of
apoptosis in various physiologic conditions.8-10 Alteration of bcl-2 or bcl-x, well-known as antiapoptosis genes, is observed in
many tumor cells in leukemia, lymphoma, and other types of cancers.11,12 Overexpression of these genes in cancer cells leads to partial or complete resistance to several conditions such as
the presence of chemotherapeutic agents and
irradiation.13,14 Leukemic cells are known to undergo
apoptosis under several conditions, and the basic strategy of leukemia
therapy is the induction of apoptosis.15 Chemotherapeutic
agents such as etoposide and all-trans retinoic acid can induce
apoptosis, killing leukemic cells.15 Because cytosine
arabinoside can reduce expression of bcl-2 or bcl-x in leukemic cells,
their lifespan is shortened by induction of apoptosis.16,17
Some mechanisms induce apoptosis against tumor cells and leukemic
cells, removing them from the body. Fas ligand and TNF-, which
physiologically exist in the body, are also inducers of apoptosis in
leukemic cells and other cells.18,19 It has been reported
that signals for some cytokines might select induction of apoptosis or
proliferation in target cells,20 but no cytokine was able
to induce apoptosis in leukemic cells and other tumor cells. Moreover,
tumor cells are directly attacked by cytotoxic T cells, NK cells, and
macrophages, inducing apoptosis.21,22 For example,
cytotoxic T cells and NK cells attach to tumor cells and release
perforin. A serine protease, granzyme B, is transferred into the
cytosol of the target cells, and then apoptosis is
induced.23,24 It has been reported that endothelial cells
are also associated with inhibition of tumor cell
invasion,25 but the mechanism is not clear.
The human myelogenous leukemia cell line HL-6026 can be
induced to differentiate into monocyte/macrophage lineage by phorbol esters, undergoing apoptosis.27 In the previous study, we
have purified an apoptosis-inducing factor (AIF) to homogene ity from a
medium conditioned by PDBu-treated HL-60 cells.28
N-terminal sequence analysis showed that AIF is identical to
endothelial interleukin-8 (IL-8). Human recombinant endothelial IL-8
induces apoptosis in most leukemic cell lines, such as K562, HL-60,
Jurkat, KG-1, U937, and THP-1, but monocyte-derived IL-8 does not.
Endothelial IL-8, which has added five amino acids to the N terminus of
monocyte-derived IL-8, is active in the induction of apoptosis, but its
action against tumor cells in vivo has never been clarified.
To investigate the biological significance of endothelial IL-8, we
observed the antitumor effect of endothelial cells in vitro and the
antitumor effect of endothelial IL-8 in vivo.
| |
MATERIALS AND METHODS |
Reagents.
Lipopolysaccharide (LPS) was obtained from Sigma (St Louis,
MO). Recombinant human IL-1, recombinant human tumor
necrosis factor- (TNF-), recombinant human interferon-
(IFN-), and antihuman monoclonal IL-8 antibody were purchased from R
& D Systems Inc (Minneapolis, MN). Recombinant human endothelial and
monocyte-derived IL-8 were purchased from Genzyme (Cambridge, MA).
Cell lines and cell culture.
A human chronic myelogenous leukemia cell line, K562,29 was
obtained from ATCC (Rockville, MD) and was maintained in
GIT medium (Wako, Tokyo, Japan).30 Human myelogenous
leukemic cell line HL-60, human monocytic leukemia cell line U937,
human T-cell leukemia cell line Jurkat, and human myeloma cell line
Daudi were also obtained from ATCC. Human umbilical venous cells (VE
cells) were purchased from Cell Systems Co (Kirkland, WA) and were
maintained in CS-C serum-free medium. Normal human
monocytes were isolated in a nascent state from the peripheral blood of
healthy volunteers, as described previously.31 In brief,
collected mononuclear cells were resuspended in phosphate-buffered
saline (PBS) and then incubated onto an MSP-P plate (JIMRO, Gunma,
Japan) for 1 hour at 37°C. Adherent cells (monocyte; >90%
purity) were collected and were cultured in Dulbecco's modified
Eagle's medium (DMEM; Life Technologies Inc,
Gaithersburg, MD). Monocytes (1 × 106
cells/mL) and human umbilical venous cells (VE cells, confluent) were
cultured with or without LPS (500 ng/mL), IL-1 (10 U/mL), TNF-
(10 ng/mL), IFN- (200 U/mL), and macrophage colony-stimulating factor (M-CSF; 100 ng/mL) in 24-well plates. Monocytes or VE cells was
also cocultured with K562 cells with or without the factors listed
above in 24-well plates with culture chambers (Intercell (Kurabo, Osaka).
Detection of IL-8.
For detection of IL-8 in culture media, an enzyme-linked immunosorbent
assay (ELISA) system for human IL-8 (Amersham, Arlington Heights,
IL) was used.32 In brief, endothelial cells or
monocytes were cultured with various factors and the supernatants were
collected by centrifugation at 15,000 rpm after 2 days. Samples were
then applied to the wells of a microtiter plate coated with a specific monoclonal antibody for IL-8. After washing away any unbound sample proteins, an enzyme-linked polyclonal antibody specific for IL-8 was
added to the wells and allowed to bind to the IL-8 that was bound
during the incubation. After a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells
and color-developed in proportion to the amount of IL-8. Absorbance at
450 nm was determined by spectrophotometer (Bio-Rad, Hercules,
CA). For detection of intracytoplasmic IL-8 in endothelial cells cultured with or without K562 cells, immunofluorescent staining for intracellular IL-8 (CytoStain kit; PharMingen, San Diego, CA) was performed. In brief, endothelial cells cultured
with or without K562 cells for 3 days were fixed and permeabilized by Cytofix/Cytoperm solution and Perm/Wash solution. After washing, fixed/permeabilized cells were stained by fluorochrome-conjugated antihuman IL-8 antibody. After washing, flow cytometric analysis was
performed by FACScan (Becton Dickinson, Mountain View,
CA).
MTT assay.
K562 cells were seeded at 1 × 105 cells/mL onto
confluent human umbilical venous cells in CS-C medium. After 3 days,
K562 cells were collected and the ca pacity to reduce 3-[4,
5-Dimethylthiazol-2-yl]-2, 5-diphenylte trazolium bromide (MTT; Sigma)
was determined.33 After adding 10 µL of MTT solution (5 mg/mL MTT in PBS), the preparation was incubated at 37°C for 4 hours. Cells with MTT for mazan were dissolved in 0.04 N HCl in
2-propanol, and color absorbance was measured at 595 nm by microplate
reader (Bio-Rad).
TUNEL assay.
Additionally, residual cells were incubated with digoxigenin-dUTP
terminaldeoxynucleotidyl transferase mixture and subsequently were
stained with peroxidase-conjugated antibody to digoxigenin (Apop Tag
PLUS; Oncor, Gaithersburg, MD),34
counterstained with 1% methyl green in sodium acetate (pH 4.0), and
mounted. Specimens were examined and photographed with a microscope.
The percentage of apoptotic cells was determined by microscopically
counting more than 200 cells. Statistical analysis was performed using the Student's t-test.
In vivo experiments.
Male Balb/c nu/nu mice were purchased from Japan Charles River and were
age-matched (5 weeks of age) at the onset of each experiment. Nude mice
were inoculated with 106 K562 cells into the peritoneal
space. Intraperitoneal tumor masses of K562 cells were injected daily
with recombinant human endothelial IL-8 or monocyte-derived IL-8 for 2 days. As a control, saline was injected. Intraperitoneal cells were
collected daily by washing with 5 mL PBS. Cell counting, Wright-Giemsa
staining, and TUNEL assay were also performed. Mice were injected with
5 × 105 viable K562 cells by subcutaneous injection
in a midline ventral position in a total volume of 0.1 mL PBS. Test
mice bearing subcutaneously established K562 tumors (confirmed after 4 days of inoculation) were injected daily (for 11 days) into tumor with
endothelial IL-8 or monocyte-derived IL-8 in a total
volume of 0.1 mL saline. As a control, saline and TNF- were
injected. Tumor size was calculated using the formula described by
Kyriazis et al,35 as follows: tumor volume = width2 × length × 0.4.
Tumors were resected in toto, fixed in 10% neutral formalin solution
(Sigma), embedded in paraffin, sectioned at 4 mm, and stained with
hematoxylin and eosin or with TUNEL assay. The injected dose of IL-8
was 100 ng per mouse per day. The dose of TNF- was 200 U per mouse.
Statistical analysis was performed using the Student's t-test.
| |
RESULTS |
Secretion of IL-8 from monocytes and endothelial cells by various
factors.
IL-8 takes several forms of various lengths that are produced by
monocytes and endothelial cells. To examine by which factors monocytes
and endothelial cells are induced to produce IL-8, possible inducers
such as LPS, IL-1, TNF-, IFN-, and M-CSF were added to culture
media of monocytes and endothelial cells
(Fig 1). Concentrations of IL-8 in medium
were then measured by an ELISA system after 2 days. When monocytes were
stimulated with LPS, TNF-, IFN-, and M-CSF, concentrations of
IL-8 in culture medium significantly increased from 7.7 ± 1.2 ng/mL
to 56.7 ± 4.5 ng/mL, 30.8 ± 3.6 ng/mL, 26.9 ± 3.0 ng/mL,
and 19.4 ± 2.5 ng/mL, respectively. On the other hand, when LPS,
IL-1, and TNF- were added to culture media of endothelial cells,
concentrations of IL-8 in culture medium increased from 1.1 ± 1.0 ng/mL to 12.8 ± 2.3 ng/mL, 70.1 ± 10.6 ng/mL, and 61.5 ± 7.0 ng/mL, respectively.
View larger version (20K):
[in this window]
[in a new window]
| Fig 1.
Effect of various cytokines on production of IL-8 from
monocytes and endothelial cells. Monocytes (1 × 106
cells/mL) and human umbilical venous cells (VE cells, confluent) were
cultured with or without LPS (500 ng/mL), IL-1 (10 U/mL), TNF-
(10 ng/mL), IFN- (200 U/mL), and M-CSF (100 ng/mL) in 24-well
plates. After 2 days, supernatants were collected and an ELISA system
for IL-8 was performed. Columns represent the mean ± SD (bar) of
three independent experiments. Statistical analysis was performed using
the Student's t-test. *P < .01.
|
|
Secretion of IL-8 from endothelial cells by attachment to K562 cells.
To investigate the effect of interaction between monocytes or
endothelial cells and K562 cells on secretion of IL-8, monocytes or
human umbilical endothelial cells (VE cells) were cultured with or
without human leukemic cell line K562 cells for 2 days (Fig 2A). Concentrations of IL-8 in
conditioned media did not increase significantly when monocytes were
cultured with K562 cells. When VE cells were cultured with K562 cells,
the concentration of IL-8 in culture medium increased from 2.1 ± 1.1 ng/mL to 75.1 ± 5.8 ng/mL as compared with that in culture
medium of only VE cells. IL-8 in medium of K562 cells alone was 0.6 ± 0.3 ng/mL. Moreover, we observed a change in intracytoplasmic
IL-8 of VE cells during interaction between VE cells and K562 cells
using immunofluorescent staining by flow cytometric analysis to examine whether IL-8 is produced in VE cells (Fig 2B). The percentage of
intracellular IL-8-positive VE cells was 22.7% ± 5.3% during culture of VE cells alone. When VE cells were cocultured with K562
cells for 2 days, the percentage of intracellular IL-8-positive VE
cells increased to 55.5% ± 7.0%. These results suggest that VE
cells can produce and secrete IL-8 during interaction with K562 cells
but that monocytes cannot. Moreover, we examined concentrations of IL-8
when other cell lines such as HL-60, U937, Jurkat, and Daudi were
cocultured with VE cells. When VE cells were cultured with HL-60, U937,
Jurkat, or Daudi cells, concentration of IL-8 in culture medium
increased from 2.1 ± 1.1 ng/mL to 74.2 ± 2.0 ng/mL, 71.2 ± 2.4 ng/mL, 48.1 ± 3.3 ng/mL, or 44.6 ± 3.9 ng/mL as compared
with that in culture medium of only VE cells, respectively.
View larger version (24K):
[in this window]
[in a new window]
| Fig 2.
Effect of interaction between either monocytes or
endothelial cells and K562 cells on secretion of IL-8 from endothelial
cells. Monocytes (1 × 106 cells/mL) and human umbilical
venous cells (VE cells, confluent) were cultured with or without K562
cells (5 × 104 cells/mL) for 2 days, and supernatants
were then collected. (A) ELISA system for IL-8 was performed. Columns
show the means of three independent experiments. Statistical analysis
was performed using the Student's t-test. (B) When VE cells
were cocultured with (right) or without (left) K562 cells, the
remaining VE cells, after removal of K562 cells, were collected and
expression of intracytoplasmic IL-8 was examined as described in
Materials and Methods. Purified mouse Ig G1 was used as a control
antibody (CONTROL). The vertical axis indicates the frequency of
fluorescence-positive cells. Intracytoplasmic IL-8 density is depicted
on the horizontal axis. The representative data from three independent
experiments are shown.
|
|
Endothelial IL-8 secreted by various factors induces apoptosis in
K562 cells.
To examine whether IL-8 secreted from endothelial cells by several
factors induces apoptosis in K562 cells, K562 cells in Intercell-insert
were separately cultured with VE cells stimulated with various factors.
K562 cells in Intercell were collected after 2 days, and MTT and TUNEL
assays were performed (Fig 3). When K562
cells were cultured with various factors such as LPS, IL-1, TNF-,
IFN-, and M-CSF, growth of K562 cells was not suppressed as measured
by MTT assay (Fig 3A). However, when endothelial cells were cultured
with IL-1 or TNF-, growth of K562 cells was significantly suppressed to 74.2% or 74.6% as compared with control (no additives), respectively (Fig 3A). Moreover, when VE cells were cultured with IL-1 or TNF-, the percentage of apoptotic cells in K562 cells increased from 1.2% ± 0.1% to 25.6% ± 2.5% or 25.3% ± 1.7%, respectively (Fig 3B). This effect was blocked by monoclonal
anti-IL-8 antibody (data not shown). Our interpretation was that
interaction between endothelial cells and K562 cells during separate
culture by Intercell could neither inhibit cell growth nor induce
apoptosis against K562 cells (Fig 3A and B). This suggests that
endothelial IL-8, secreted from endothelial cells by various factors,
induces apoptosis in K562 cells.
View larger version (33K):
[in this window]
[in a new window]
| Fig 3.
Effect of IL-8 from endothelial cells on induction of
apoptosis in K562 cells. K562 cells alone (5 × 104
cells/mL) or human umbilical venous cells (VE cells, confluent) and
K562 cells (5 × 104 cells/mL) were cultured with or
without LPS (500 ng/mL), IL-1 (10 U/mL), TNF- (10 U/mL), IFN-
(200 U/mL), and M-CSF (100 ng/mL) in 24-well plates. Coculture of VE
cells and K562 cells was mediated by Intercell. After 2 days, MTT assay
(A) and TUNEL assay (B) were performed. Columns show the means of three
independent experiments. Statistical analysis was performed using the
Student's t-test. *P < .01.
|
|
Interaction between endothelial cells and K562 cells induces
apoptosis in K562 cells.
To identify the biological significance of endothelial IL-8, we
examined the effect of interaction between normal human venous endothelial cells (VE cells) and K562 cells on the inhibition of cell
growth and the induction of apoptosis (Fig
4). When K562 cells were attached onto VE cells, growth of K562 cells
was suppressed at 62.0% as compared with K562 cell culture alone (Fig
4A and B), and 32.0% ± 3.1% of K562 cells underwent apoptosis
(Fig 4A and C). Because the concentration of IL-8 in coculture medium of VE cells and K562 cells increased to 75.1 ng/mL from 2.1 ng/mL in
medium of VE cells alone (Fig 2), we examined whether the effects of
the interaction between VE cells and K562 cells on cell growth inhibition and apoptosis are blocked by anti-IL-8 antibody. When anti-IL-8 antibody was added to coculture media of VE cells and K562
cells, MTT reducing activity recovered to that of K562 cell culture
alone (Fig 4B). Moreover, the percentage of apoptotic cells decreased
to 7.8% ± 0.6% when anti-IL-8 antibody was added to
coculture media of VE cells and K562 cells (Fig 4C). This result suggests that the inhibition of K562 cell growth and the induction of
apoptosis were blocked by anti-IL-8 antibody. Western blot analysis
also showed that endothelial IL-8 was detected in the culture medium of
VE cells and K562 cells (data not shown). Moreover, when HL-60 cells,
U937 cells, Jurkat cells, or Daudi cells were attached onto VE cells,
34.7% ± 3.1%, 31.0% ± 3.6%, 24.3% ± 4.0%, or 20.7% ± 3.1% of cells underwent apoptosis, respectively. These findings
indicate that endothelial cells can induce apoptosis in the attached
K562 cells or other cell lines by releasing endothelial IL-8.
View larger version (39K):
[in this window]
[in a new window]
| Fig 4.
Effect of interaction between human venous endothelial
cells (VE cells) and K562 cells on secretion of endothelial
IL-8 and apoptosis. K562 cells (5 × 104
cells) were seeded with or without confluent VE cells in 24-well
plates. Cell culture was boosted with or without anti-IL-8 antibody (5 µg/mL) every 24 hours. After 2 days, Wright-Giemsa staining was
performed in 24-well plates (A). K562 cells and supernatants were
collected, and MTT assay (B), TUNEL assay (C), and ELISA for IL-8 were
performed. For (A), original magnification × 60. Data shown come from
three independent experiments. Statistical analysis was performed using
the Student's t-test.
|
|
Endothelial IL-8 suppresses cell growth of K562 cells and induces
apoptosis in vivo.
Therefore, we investigated whether endothelial IL-8 can induce
apoptosis or suppress cell growth in leukemic cells in vivo. Endothelial IL-8 was in jected daily for 2 days into intraperitoneal tumor masses of K562 cells in nude mice
(Fig 5). Apoptosis and suppression of cell
growth were observed after 2 days in intraperitoneal K562 cells (Fig 5B
and C), and apoptotic cells were phagocytosed by macrophages (Fig 5A).
After 2 days, apoptotic cells increased to 12.8% ± 2.2%.
Moreover, numbers of K562 cells decreased to 33.3% of control.
Monocyte-derived IL-8 did not significantly suppress cell growth or
induce apoptosis in intraperitoneal K562 cells. In addition, we
investigated whether endothelial IL-8 could suppress cell growth or
induce apoptosis against subcutaneous K562 cells
(Fig 6). Endothelial IL-8 was then injected
daily from day 4 to day 11 into subcutaneous K562 cell tumors
established in nude mice, and the antitumor and the apoptosis-inducible
effects of endothelial IL-8 were examined. Mice showed a visible
response to endothelial IL-8 intratumor inoculations characterized by
apoptosis (Fig 6A). Therefore, tumor size of endothelial IL-8 treatment decreased in 52.2% of control (saline; Fig 6B). Tumor size of TNF-
treatment decreased in 41.3% of control (Fig 6B). On the other hand,
monocyte-derived IL-8 treatment did not either induce apoptosis or
suppress cell growth against subcutaneous K562 cells (Fig 6).
View larger version (38K):
[in this window]
[in a new window]
| Fig 5.
Inhibition of cell growth and induction of apoptosis on
treatment of endothelial IL-8 in nude mice. The peritoneal space of
nude mice (5 examined) were inoculated with K562 cells, and agents such
as saline, endothelial IL-8 (IL-8 [E]), and monocyte-derived IL-8
(IL-8 [M]) were injected daily as described in Materials and Methods.
(A) Morphology (Wright-Giemsa staining) and apoptotic cells (TUNEL
assay) of intraperitoneal cells were collected. Arrows indicate the
apoptotic cells. Original magnification × 160 and × 80. (B)
Inhibition of cell growth of K562 cells by endothelial IL-8. Horizontal
bars show the means. (C) The percentage of apoptotic cells was
determined microscopically by counting more than 200 cells in situ on
staining slides. Columns represent the means ± SD (bar) of three
independent experiments. Statistical analysis was performed using the
Student's t-test.
|
|
View larger version (33K):
[in this window]
[in a new window]
| Fig 6.
Endothelial IL-8 suppressed growth of subcutaneous K562
cell tumors. The subepiderms of nude mice (10 examined per group) were
inoculated K562 cells, and agents were injected with endothelial IL-8
(IL-8 [E]), monocyte-derived IL-8 (IL-8 [M]), saline, and TNF-
daily as described in Materials and Methods. As controls,
monocyte-derived IL-8, saline, and TNF- were used. Data shown come
from 10 nude mice. Statistical analysis was performed using the
Student's t-test. In (B), *P < .05 and **P < .005. (A) Photograph of suppression of subcutaneous K562 tumor by
endothelial IL-8 (IL-8 [E]). As controls, saline, monocyte-derived
IL-8 (IL-8 [M]), and TNF- were used.
|
|
Antitumor effect of endothelial IL-8 is due to induction of apoptosis
in K562 cells.
To investigate whether the antitumor effect of endothelial IL-8 is due
to induction of apoptosis, pathological examination was performed by
hematoxylin-eosin staining and TUNEL staining (Fig 7). Histologically, subcutaneous K562
tumors that responded to either endothelial IL-8 or TNF- (10 mice of
each were examined) generally displayed homogenous central necrosis
with intratumor bleeding (Fig 7H and K). Within the viable tumor
tissue, many tumor cells became smaller than control cells and showed
either condensation or fragmentation of nuclei (Fig 7B and H).
Neutrophil and lymphocyte infiltrations were unremarkable in both
groups. Control K562 tumors (saline- and monocyte-derived IL-8 groups) displayed little or no tumor necrosis (Fig 7A and D), and their cells
had no change in cell size or nuclei (Fig 7B and E). Tumor sections
were stained with TUNEL assay specific for apoptotic cells. TUNEL assay
showed that in 45.2% or 47.8% of K562 tumor cells inoculated with
either endothelial IL-8 or TNF-, apoptosis was induced (Fig 7I and
L). However, control tumor cells showed little apoptosis (Fig 7C and
F).
View larger version (160K):
[in this window]
[in a new window]
| Fig 7.
Microscopic morphology of progressive and regressing
subcutaneous K562 tumors. Balb/c nu/nu mice were injected
subcutaneously with K562 cells and were subsequently injected with
saline (A through C), monocyte-derived IL-8 (D through F), endothelial
IL-8 (G through I), and TNF- (J through L). Tumors were removed in
toto, and hematoxylin and eosin staining (A, B, D, E, G, H, J, and K)
or TUNEL assay (C, F, I, and L) was performed after 8 days. Original
magnification × 5 for (A), (D), (G), and (J); × 20 for (B), (C),
(E), (F), (H), (I), and (K).
|
|
| |
DISCUSSION |
In this study, we observed that endothelial cells were able to secrete
endothelial IL-8 after stimulation with IL-1 and TNF- and that
endothelial IL-8 was able to induce apoptosis in leukemic cells.
Moreover, endothelial cells that attached to leukemic cells secreted
IL-8, and endothelial IL-8 induced apoptosis in leukemic cells. In the
previous study, we purified an apoptosis-inducing factor derived from
differentiated HL-60 cells.28 This apoptosis-inducing factor is identical to endothelial IL-8. Human recombinant endothelial IL-8 is able to induce apoptosis in most leukemic cell lines, but
monocyte-derived IL-8 is not.
IL-8 was originally isolated from culture supernatants of stimulated
human monocytes and identified as a protein of 72 amino acids.36,37 The open reading frame of the IL-8 cDNA encodes for 99 amino acids,38 and the mature form is processed
further at the N terminus, yielding several biologically active
truncation analogs.39-41 The occurrence of N-terminal
variants depends on cell type and culture conditions. Of the two major
forms, the 72-amino acid form (monocyte-derived IL-8; SAKELRC...)
predominates in cultures of monocytes and macrophages,40
and the 77-amino acid form (endothelial IL-8;
AVLPRSAKELRC...) prodominates in cultures of tissue cells
such as endothelial cells42 and fibroblasts.43 Endothelial IL-8 has five extra N-terminal amino acids lacking in
monocyte-derived IL-8. Because endothelial IL-8 is converted to
monocyte-derived IL-8 by serine proteases such as
thrombin,44 monocyte-derived IL-8 exists mainly in
plasma.45 Many previous reports have demonstrated that
monocyte-derived IL-8 is an inflammatory chemoattractant for
neutrophils and that several types of cells, such as monocytes,
lymphocytes, and fibroblasts, produce IL-8 after stimulation with
various agents.46 On the other hand, the chemotactic
activity of endothelial IL-8 is one twentieth as strong as that of
monocyte-derived IL-8. The difference of whether IL-8 has 5 extra
N-terminal amino acids may decide the induction of chemotaxis or
apoptosis. Moreover, it has been reported that leukemic cell
dissemination to extravascular space is mediated by interaction between
leukemic cells and endothelial cells.47 Our study
demonstrated that endothelial IL-8 plays an important role in the
antitumor action of endothelial cells.
Recently, it has been demonstrated that vascular cells are important
participants in antitumor host defense. Vascular cells, which express
nitric oxide synthase in response to IFN- and TNF-, can kill
leukemic cells.48 However, the mechanism of the antitumor host defense system associated with vascular cells has not been clarified. Many researchers have discussed angiogenesis for tumor vascularization and tumor metastasis.49 Tumor cells secrete metalloproteinase to destroy matrix proteins and to damage endothelial cells, and then they invade extravascular space.50 We
demonstrated that endothelial IL-8 can protect against tumor invasion
in this system. Because endothelial IL-8 is known to be secreted mainly by endothelial cells and fibroblasts,51 endothelial cells
secreting IL-8 have the antitumor property of inducing apoptosis in
contacting leukemic cells like other antitumor cells, such as
macrophages and NK cells. This is a novel function of endothelial cells
involving tumor cell eradication in the body, mainly via endothelial
IL-8. Endothelial IL-8 may have an important role in induction of
apoptosis in tumor cells in the blood stream when they anchor to
endothelial cells.
Surprisingly, in our in vivo experiment, endothelial IL-8 inhibited
growth of K562 cell tumors in the same manner as TNF-. We
demonstrated that TNF- did not directly suppress cell growth or
induce apoptosis in K562 cells, but that TNF- allows endothelial cells to secrete IL-8 and can indirectly kill leukemic cells. It has
been reported that TNF- modulates expression of various biological
molecules in endothelial cells.52 The in vivo effect of
TNF- on killing tumor cells may be explained by both a direct death
signal mediated through its receptor and the indirect release of
biological modulators such as endothelial IL-8 from endothelial cells.
Therefore, it is possible that the antitumor effect of TNF- in vivo
may be mediated by secretion of endothelial IL-8 from intratumor
endothelial cells. However, angiogenesis for tumor vascularization will
aid tumor progression, and tumor cells produce and secrete some
endogenous regulators such as endostatin and angiostatin.53,54 In this study, injected TNF- acted on
endothelial cells growing in tumors, and endothelial IL-8 might then be
released from endothelial cells. There are two possible roles of IL-8
from endothelial cells in host defense against tumor cells. First, endothelial cells will secrete endothelial IL-8 when tumor cells in the
vessels contact endothelial cells, and then the anchored tumor cells
may be directly killed by endothelial IL-8. Second, when the growing
endothelial cells in tumors are stimulated with some cytokines such as
TNF-, they will secrete endothelial IL-8 and induce apoptosis.
If we know how to stimulate release or production of endothelial IL-8,
we may be able to develop new methods of treatment. In 10 of 14 clinical cases, we observed that apoptosis was significantly induced in
fresh leukemic cells by endothelial IL-8 in vitro. Endothelial IL-8,
with five extra N-terminal amino acids, can induce apoptosis, but
monocyte-derived IL-8, which lacks them, cannot do so. Because most
IL-8 in blood plasma exists in the form of monocyte-derived
IL-845 and does not induce apoptosis in leukemic
cells,28 this phenomenon will be important in leukemia therapy.
VP-16 is well-known to be an anticancer agent and an inducer of
apoptosis in leukemic cells. When leukemic cells were treated with both
0.1 µmol/L VP-16 and 20 ng/mL endothelial IL-8 for 48 hours,
apoptotic cells increased as compared with the treatment of only 0.1 µmol/L VP-16 (data not shown). This result suggests that endothelial
IL-8 can enhance the effect of VP-16 on the induction of apoptosis and
may therefore be clinically promising in combination with VP-16.
Understanding of the receptor for induction of apoptosis will be
important to further research on chemokines, and we are currently investigating receptor(s) and apoptosis-signaling for them in our
system. Anti-IL-8 receptor antibody or IL-8 receptor antagonist, or
specific binding protein(s) for endothelial IL-8, should be characterized. About 20% of target cells (K562 cells) undergo apoptosis by endothelial IL-8; therefore, we need to have more susceptible cells as targets, using subclones of the cells. In a
preliminary experiment, we separated whole K562 cells into three fractions of cell cycle-phases, ie, G0/G1-phase, S-phase, and G2/M-phase fractions by counterflow centrifugal elutriation
system,30 and then examined the susceptibility of K562
cells in each cell-cycle phase to apoptosis induced by endothelial
IL-8. This experiment demonstrated that susceptibility to apoptosis is
higher in the G0/G1 phase of cell cycle than in the S and G2/M phases
in K562 cells (data not shown). In our experiment, proliferation can be less changed but apoptosis markedly increased (Fig 3A). We showed bcl-2-overexpressing cells in any cell cycle phases lost
susceptibility to apoptosis.30 Moreover, thymocyte
apoptosis by methylpredonisolone and etoposide is independent of
proliferation.55 This indicates that the antiapoptotic
genes-expressing cells escaped from endothelial IL-8-induced apoptosis
can proliferate.
In conclusion, endothelial cells involving a novel apoptotic system
have been identified. In the mechanism of this system, endothelial IL-8
release plays an important role. It is not only an inflammatory
mediator but also an apoptosis-inducing factor in leukemic cells.
| |
FOOTNOTES |
Submitted May 18, 1998;
accepted July 28, 1998.
Supported by a grant-in-aid from the Ministry of Education, Science and
Culture of Japan; the Research on Advanced Medical Technology; the
Japanese Foundation for Multidisciplinary Treatment of Cancer; and
Jichi Medical School Young Investigator Award.
Address reprint requests to Kiyohiko Hatake, MD, PhD, Division of
Hematology, Department of Internal Medicine, Jichi Medical School,
3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi 329-04, Japan; e-mail: kiyohiko{at}jichi.ac.jp.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank S. Kurokawa, H. Ishikawa, and T. Ishida for technical
assistance.
| |
REFERENCES |
1.
Raff MC:
Social controls on cell survival and cell death.
Nature
356:397,
1992[Medline]
[Order article via Infotrieve]
2.
Cohen JJ,
Duke RC,
Fadok VA,
Sellins KS:
Apoptosis and programmed cell death in immunity.
Annu Rev Immnol
10:267,
1992[Medline]
[Order article via Infotrieve]
3.
Green DR,
Scott DW:
Activation-induced apoptosis in lymphocytes.
Curr Opin Immunol
6:476,
1994[Medline]
[Order article via Infotrieve]
4.
Griffith TS,
Ferguson TA:
The role of FasL-induced apoptosis in immune privilege.
Immunol Today
18:240,
1997[Medline]
[Order article via Infotrieve]
5.
Thompson CB:
Apoptosis in pathogenesis and treatment of disease.
Science
267:1456,
1995[Abstract/Free Full Text]
6.
Kerr JFR,
Wyllie AH,
Currie AR:
Apoptosis: A basic biological phenomenon with wild-ranging implications in tissuekinetics.
Br J Cancer
26:239,
1972[Medline]
[Order article via Infotrieve]
7.
Wyllie AH,
Kerr JFR,
Currie AR:
Cell death: The significance of apoptosis.
Int Rev Cytol
68:251,
1990
8.
Mannick JB,
Miao XQ,
Stamler JS:
Nitric oxide inhibits Fas-induced apoptosis.
J Biol Chem
272:24125,
1997[Abstract/Free Full Text]
9.
Rokhlin OW,
Hostager BS,
Bishop GA,
Sidorenko SP,
Glover RA,
Gudkov VA,
Cohen MB:
Dominant nature of the resistance to Fas-and tumor necrosis factor-alpha-mediated apoptosis in human prostatic carcinoma cell lines.
Cancer Res
57:3941,
1997[Abstract/Free Full Text]
10.
Ohtsu M,
Sakai N,
Fujita N,
Kashiwagi M,
Gasa S,
Shimizu S,
Eguchi S,
Tsujimoto Y,
Sakiyama Y,
Kobayashi K,
Kuzumaki K:
Inhibition of apoptosis by the actin-regulatory protein gelsolin.
EMBO J
16:4650,
1997[Medline]
[Order article via Infotrieve]
11.
Krajewski S,
Krajewska M,
Ehrmann J,
Sikorska M,
Lach B,
Chatten J,
Reed JC:
Immunohistochemical analysis of Bcl-2, Bcl-X, Mcl-1, and Bax in tumors of central and peripheral nervous system origin.
Am J Pathol
150:805,
1997[Abstract]
12.
Pallis M,
Zhu YM,
Russell NH:
Bcl-x(L) is heterogenously expressed by acute myeloblastic leukemia cells and is associated with autonomous growth in vitro and with P-glycoprotein expression.
Leukemia
11:945,
1997[Medline]
[Order article via Infotrieve]
13.
Kim CN,
Wang X,
Huang Y,
Ibrado AM,
Liu L,
Fang G,
Bhalla K:
Overexpression of Bcl-X(L) inhibits Ara-C-induced mitochondrial loss of cytochrome c and other perturbations that activate the molecular cascade of apoptosis.
Cancer Res
57:3115,
1997[Abstract/Free Full Text]
14.
Simonian PL,
Grillot DA,
Nunez G:
Bcl-2 and Bcl-XL can differentially block chemotherapy-induced cell death.
Blood
90:1208,
1997[Abstract/Free Full Text]
15.
Fisher DE:
Apoptosis in cancer therapy: Crossing the threshold.
Cell
78:539,
1994[Medline]
[Order article via Infotrieve]
16.
Decaudin D,
Geley S,
Hirsch T,
Castedo M,
Marchetti P,
Macho A,
Kofler R,
Kroemer G:
Bcl-2 and Bcl-XL antagonize the mitochondrial dysfunction preceding nuclear apoptosis induced by chemotherapeutic agents.
Cancer Res
57:62,
1997[Abstract/Free Full Text]
17.
Benito A,
Silva M,
Grillot D,
Nunez G,
Fernandez LJL:
Apoptosis induced by erythroid differentiation of human leukemia cell lines is inhibited by Bcl-XL.
Blood
87:3837,
1994[Abstract/Free Full Text]
18.
Takahashi T,
Tanaka M,
Brannan CI,
Jenkins NA,
Copeland NG,
Suda T,
Nagata S:
Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand.
Cell
76:969,
1994[Medline]
[Order article via Infotrieve]
19.
Heller RA,
Kronke M:
Tumor necrosis factor receptor-mediated signaling pathways.
J Cell Biol
126:5,
1994[Free Full Text]
20.
Miyazaki T,
Liu ZJ,
Kawahara A,
Minami Y,
Yamada K,
Tsujimoto Y,
Barsoumian EL,
Permutter RM,
Taniguchi T:
Three distinct IL-2 signaling pathways mediated by bcl-2, c-myc, and lck cooperate in hematopoietic cell proliferation.
Cell
81:223,
1995[Medline]
[Order article via Infotrieve]
21.
Walker PR,
Saas P,
Dietrich PY:
Role of Fas ligand (CD95L) in immune escape: The tumor cell strikes back.
J Immunol
158:4521,
1997[Abstract]
22.
Whiteside TL,
Chikamatsu K,
Nagashima S,
Okada K:
Antitumor effects of cytolytic T lymphocytes (CTL) and natural killer (NK) cells in head and neck cancer.
Anticancer Res
16:2357,
1996[Medline]
[Order article via Infotrieve]
23.
Heusel JW,
Wesselschmidt RL,
Shresta S,
Russell JH,
Ley TJ:
Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells.
Cell
76:977,
1994[Medline]
[Order article via Infotrieve]
24.
Sarin A,
Williams MS,
Alexander MMA,
Berzofsky JA,
Zacharchuk CM,
Henkart PA:
Target cell lysis by CTL granule exocytosis is independent of ICE/Ced-3 family proteases.
Immunity
6:209,
1997[Medline]
[Order article via Infotrieve]
25.
Sternlicht MD,
Barsky SH:
The myoepithelial defense: A host defense against cancer.
Med Hypotheses
48:37,
1997[Medline]
[Order article via Infotrieve]
26.
Collins SJ,
Gallo RC,
Gallagher RE:
Continuous growth and differentiation of human myeloid leukemic cells in suspension culture.
Nature
270:347,
1977[Medline]
[Order article via Infotrieve]
27.
Terui Y,
Furukawa Y,
Sakai T,
Kikuchi J,
Sugahara H,
Kanakura Y,
Kitagawa S,
Miura Y:
Up-regulation of VLA-5 expression during monocytic differentiation and its role in negative control of the survival of peripheral blood monocytes.
J Immunol
156:1981,
1996[Abstract]
28.
Terui Y,
Ikeda M,
Tomizuka H,
Kasahara T,
Ohtsuki T,
Uwai M,
Mori M,
Itoh T,
Tanaka M,
Yamada M,
Shimamura S,
Miura Y,
Hatake K:
Identification of a novel apoptosis-inducing factor derived from leukemic cells: Endothelial interleukin-8, but not monocyte-derived, induces apoptosis in leukemic cells.
Biochem Biophys Res Commun
243:407,
1998[Medline]
[Order article via Infotrieve]
29.
Lozzio CB,
Lozzio BB:
Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome.
Blood
45:321,
1975[Abstract/Free Full Text]
30.
Terui Y,
Furukawa Y,
Kikuchi J,
Saito M:
Apoptosis during HL-60 cell differentiation is closely related to a G0/G1 cell cycle arrest.
J Cell Physiol
164:74,
1995[Medline]
[Order article via Infotrieve]
31.
Hashimoto S,
Yoda M,
Yamada M,
Yanai N,
Kawashima T,
Motoyoshi K:
Macrophage colony-stimulating factor induces interleukin-8 production in human monocytes.
Exp Hematol
24:123,
1996[Medline]
[Order article via Infotrieve]
32.
Ko Y,
Mukaida N,
Panyutich A,
Voitenok NN,
Matsushima K,
Kawai T,
Kasahara T:
A sensitive enzyme-linked immunosorbent assay for human interleukin-8.
J Immunol Methods
149:227,
1992[Medline]
[Order article via Infotrieve]
33.
Efferth T,
Fabry U,
Osieka R:
Apoptosis and resistance to daunorubicin in human leukemic cells.
Leukemia
11:1180,
1997[Medline]
[Order article via Infotrieve]
34.
Williams GT:
Programmed cell death: Apoptosis and oncogenesis.
Cell
65:1097,
1991[Medline]
[Order article via Infotrieve]
35.
Kyriazis AP,
Kyriazis AA,
Scarpelli DG,
Fogh J,
Rao MS,
Lepera R:
Human pancreatic adenocarcinoma line Capan-1 in tissue culture and the nude mouse: Morphologic, biologic, and biochemical characteristics.
Am J Pathol
106:250,
1982[Abstract]
36.
Walz A,
Peveri P,
Aschauer H,
Baggiolini M:
Purification and amino acid sequence of NAF, a novel neutrophil-activating factor produced by monocytes.
Biochem Biophys Res Commun
149:755,
1987[Medline]
[Order article via Infotrieve]
37.
Yoshimura T,
Matsushima K,
Tanaka S,
Robinson EA,
Appella E,
Oppenheim JJ,
Leonard EJ:
Purification of a human monocyte-derived neutrophil chemotactic factor that has peptide sequence similarity to other host defense cytokines.
Proc Natl Acad Sci USA
84:9233,
1987[Abstract/Free Full Text]
38.
Schmid J,
Weissmann C:
Induction of mRNA for a serine protease and a  -thromboglobulin-like protein in mitogen-stimulated human leukocytes.
J Immunol
139:250,
1987[Abstract]
39.
Yoshimura T,
Robinson EA,
Appella E,
Matsushima K,
Showalter SD,
Skeel A,
Leonard EJ:
Three forms of monocyte-derived neutrophil chemotactic factor (MDNCF) distinguished by different length of the amino-terminal sequence.
Mol Immunol
26:87,
1989[Medline]
[Order article via Infotrieve]
40.
Lindley I,
Aschauer H,
Seifert JM,
Lam C,
Brunowsky W,
Kownatzki E,
Thelen M,
Peveri P,
Dewald B,
von Tscharner V,
Walz A,
Baggiolini M:
Synthesis and expression in Escherichia coli of the gene encoding monocyte-derived neutrophil-activating factor: Biological equivalence between natural and recombinant neutrophil-activating factor.
Proc Natl Acad Sci USA
85:9199,
1988[Abstract/Free Full Text]
41.
Van Damme J,
Van Beeumen J,
Conings R,
Decock B,
Billiau A:
Purification of granulocyte chemotactic peptide/interleukin-8 reveals N-terminal sequence heterogeneity similar to that of beta-thromboglobulin.
Eur J Biochem
181:337,
1989[Medline]
[Order article via Infotrieve]
42.
Gimbrone MA Jr,
Obin MS,
Brock AF,
Luis EA,
Hass PE,
Hébert CA,
Yip YK,
Leung DW,
Lowe DG,
Kohr WJ,
Darbonne WC,
Bechtol KB,
Baker JB:
Endothelial interleukin-8: A novel inhibitor of leukocyte-endothelial interactions.
Science
246:1601,
1989[Abstract/Free Full Text]
43.
Schröder J-M,
Sticherling M,
Henneicke HH,
Preissner WC,
Christophers E:
IL-1 or tumor necrosis factor- stimulate release of three NAP-1/IL-8-related neutrophil chemotactic proteins in human dermal fibroblasts.
J Immunol
144:2223,
1990[Abstract]
44.
Leavell KJ,
Peterson MW,
Gross TJ:
Human neutrophil elastase abolishes interleukin-8 chemotactic activity.
J Leukoc Biol
61:361,
1997[Abstract]
45.
Baggiolini M,
Dewald B,
Moser B:
Interleukin-8 and related chemokines-CXC and CC chemokines.
Adv Immunol
55:97,
1993
46.
Van Damme J:
Interleukin-8
, in Thomson A
(ed):
The Cytokine Handbook.
London, UK, Academic
, 1994
, p 185
47.
Azzarelli B,
Easterling K,
Norton JA:
Leukemic cell-endothelial cell interactions in leukemic cell dissemination.
Lab Invest
60:45,
1989[Medline]
[Order article via Infotrieve]
48.
Geng Y-J,
Hellstrand K,
Wennmalm A,
Hansson GK:
Apoptotic death of human leukemic cells induced by vascular expressing nitric oxide synthesis in response to -interferon and tumor necrosis factor-.
Cancer Res
56:866,
1996[Abstract/Free Full Text]
49.
Folkman J,
Ingber D:
Angiogenesis.
J Biol Chem
267:10931,
1992[Free Full Text]
50.
Thorgeirsson UP,
Lindsay CK,
Cottam DW,
Gomez DE:
Tumor invasion, proteolysis, and angiogenesis.
J Neurooncol
18:89,
1993
51.
Baggiolini M,
Walz A,
Kunkel SL:
Neutrophil-activating peptide-1/interleukin 8, a novel cytokine that activates neutrophils.
J Clin Invest
84:1045,
1989
52.
Mantovani A,
Bussolino F,
Introna M:
Cytokine regulation of endothelial cell function: From molecular level to the bedside.
Immunol Today
18:231,
1997[Medline]
[Order article via Infotrieve]
53.
O'Reilly MS,
Boehm T,
Shing Y,
Fukai N,
Vasios G,
Lane WS,
Flynn E,
Birkhead JR,
Olsen BR,
Folkman J:
Endostatin: An endogenous inhibitor of angiogenesis and tumor growth.
Cell
88:277,
1997[Medline]
[Order article via Infotrieve]
54.
Dong Z,
Kumar R,
Yang X,
Fidler IJ:
Macrophage-derived metalloelastase is responsible for the generation of angiostatin in Lewis lung carcinoma.
Cell
88:801,
1997[Medline]
[Order article via Infotrieve]
55.
Evans DL,
Tiby M,
Dive C:
Differential sensitivity to the induction of apoptosis by cisplatin in proliferating and quiescent immature rat thymocyte is independent of the levels of drug accumulation and DNA adduct formation.
Cancer Res
54:1596,
1994[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
G. Spinetti, G. Bernardini, G. Camarda, A. Mangoni, A. Santoni, M. C. Capogrossi, and M. Napolitano
The chemokine receptor CCR8 mediates rescue from dexamethasone-induced apoptosis via an ERK-dependent pathway
J. Leukoc. Biol.,
January 1, 2003;
73(1):
201 - 207.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Mishima, Y. Matsumoto-Mishima, Y. Terui, M. Katsuyama, M. Yamada, M. Mori, Y. Ishizaka, K. Ikeda, J.-i. Watanabe, N. Mizunuma, et al.
Leukemic Cell-Surface CD13/Aminopeptidase N and Resistance to Apoptosis Mediated by Endothelial Cells
J Natl Cancer Inst,
July 3, 2002;
94(13):
1020 - 1028.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Kurita-Ochiai, K. Ochiai, N. Suzuki, K. Otsuka, and K. Fukushima
Human Gingival Fibroblasts Rescue Butyric Acid-Induced T-Cell Apoptosis
Infect. Immun.,
May 1, 2002;
70(5):
2361 - 2367.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Jakubowski, B. Browning, M. Lukashev, I. Sizing, J. S. Thompson, C. D. Benjamin, Y.-M. Hsu, C. Ambrose, T. S. Zheng, and L. C. Burkly
Dual role for TWEAK in angiogenic regulation
J. Cell Sci.,
January 15, 2002;
115(2):
267 - 274.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Mori, Y. Terui, M. Tanaka, H. Tomizuka, Y. Mishima, M. Ikeda, T. Kasahara, M. Uwai, M. Ueda, R. Inoue, et al.
Antitumor Effect of {beta}2-Microglobulin in Leukemic Cell-bearing Mice via Apoptosis-inducing Activity: Activation of Caspase-3 and Nuclear Factor-{{kappa}}B
Cancer Res.,
June 1, 2001;
61(11):
4414 - 4417.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. J. Merkle, I. M. Moore, B. S. Penton, B. J. Torres, R. K. Cueny, R. C. Schaeffer Jr., and D. W. Montgomery
Methotrexate Causes Apoptosis in Postmitotic Endothelial Cells
Biol Res Nurs,
July 1, 2000;
2(1):
5 - 14.
[Abstract]
[PDF]
|
|
|
|
|
|
|
Y. Terui, H. Tomizuka, Y. Mishima, M. Ikeda, T. Kasahara, M. Uwai, M. Mori, T. Itoh, M. Tanaka, M. Yamada, et al.
NH2-Terminal Pentapeptide of Endothelial Interleukin 8 Is Responsible for the Induction of Apoptosis in Leukemic Cells and Has an Antitumor Effect in Vivo
Cancer Res.,
November 1, 1999;
59(22):
5651 - 5655.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Mori, Y. Terui, M. Ikeda, H. Tomizuka, M. Uwai, T. Kasahara, N. Kubota, T. Itoh, Y. Mishima, M. Douzono-Tanaka, et al.
beta 2-Microglobulin Identified as an Apoptosis-Inducing Factor and Its Characterization
Blood,
October 15, 1999;
94(8):
2744 - 2753.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Kimura, Y. Mizukami, T. Miura, K. Fujimoto, S. Kobayashi, and M. Matsuzaki
Orphan G Protein-coupled Receptor, GPR41, Induces Apoptosis via a p53/Bax Pathway during Ischemic Hypoxia and Reoxygenation
J. Biol. Chem.,
July 6, 2001;
276(28):
26453 - 26460.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract