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Experimental Therapeutics |
Auckland Cancer Society Research Centre, University of Auckland School of Medicine, Auckland, New Zealand 1000
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and for interferon-inducible protein 10 (IP-10) were
strongly induced in both spleen and Colon 38 tumor tissue after DMXAA
treatment, whereas that for IFN-
was induced in spleen but not in
tumor. Expression of mRNA for IFN-ß and for the p35 or the p40
subunits of interleukin 12 was not observed in either tissue. Splenic
IP-10 mRNA induction was not a result of IFN- production
induced with DMXAA because spleen tissue from DMXAA-treated mice that
lacked functional IFN- receptors expressed similar amounts of
IP-10 mRNA as those from wild-type mice. A single i.p. injection of
DMXAA (20 mg/kg) was sufficient to reduce fibroblast growth
factor-induced endothelial cell invasion of Matrigel implants in
athymic nude mice by nearly 100%. The inactive analogue
8-methylxanthenone-4-acetic acid did not up-regulate the genes for
IP-10 or IFNs and did not inhibit endothelial cell invasion. Antibodies
to IP-10 reversed the inhibition of DMXAA of endothelial cell invasion
by 58%; antibodies to tumor necrosis factor-, IFN-, and IFN-
reversed inhibition by 7%, 5%, and 0%, respectively. The data
support the hypothesis that DMXAA, in addition to antivascular effects
mediated by tumor necrosis factor-, may have an antiangiogenic
effect mediated largely by the induction of IP-10. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, a new anticancer agent synthesized in this laboratory
(1)
, is currently in Phase I clinical trial and is
particularly effective against transplantable murine tumors with an
established vasculature (1)
. DMXAA induces irreversible
tumor vascular collapse within min of administration (2
, 3)
, and the ensuing tumor ischemia and hemorrhagic necrosis
account for a significant amount of tumor cell death. Although these
effects are a prerequisite for DMXAA activity, they appear to be
insufficient for complete tumor regression, and other factors may
contribute to the overall antitumor action (4)
.
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(11)
and
IL-12 (14)
, is thought to mediate the antiangiogenic
action of these cytokines (14
, 15)
. Thirdly, DMXAA induces
TNF (16)
, which in addition to its antivascular action
(17)
alters endothelial cell permeability
(18)
, stimulates angiogenesis at low doses, and inhibits
angiogenesis at high doses (19)
.
In this report, we examine the in vivo induction of mRNA for
IP-10, IFN-, IFN-ß, IFN-, IL-12, and TNF- in the spleen and
tumor of mice after DMXAA administration. We also measure up-regulation
of mRNA for MIP-1. Although not antiangiogenic, MIP-1 shares with
IP-10 the chemotactic properties of enhancing lymphocyte migration and
diapedesis through the vascular endothelium (20)
. As a
measure of antiangiogenic activity, the ability of DMXAA to inhibit
bFGF-induced endothelial cell infiltration into Matrigel plugs in
vivo has been examined.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The anti-IP-10 antibody 5171 was made available through the generosity
of Dr. Joshua Farber (Laboratory of Clinical Investigation, National
Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD). It is
a rabbit-neutralizing antiserum raised against IP-10 purified
from insect cells infected with recombinant baculovirus
(22)
. The other antibodies used were antimouse TNF-
(R&D Systems, Inc.; catalogue number AB-410-NA), antimouse IFN- (R&D
Systems, Inc.; catalogue number AF-485-NA), and antimouse IFN-
(Serotec Ltd.; catalogue number MCA1431).
Mice.
Athymic BALB/c-nu/nu mice were from the Animal
Laboratories, University of Auckland School of Medicine. Female C57Bl/6
were from the Department of Laboratory Animal Sciences, Otago Medical
School, Dunedin, New Zealand. IFN-R0/0 mice
and their wild-type counterparts were a generous gift from Dr. James D.
Watson (Genesis Research and Development Corporation, Auckland, New
Zealand) and were the offspring from the interbreeding of homozygous
wild-type or IFN-R0/0 (129/Sv/Ev x C57Bl/6)F1 mice (23)
. All of
the mice were maintained under constant temperature and humidity
according to institutional ethical guidelines and used between 8–12
weeks of age.
Matrigel Assay for Endothelial Cell Activity.
This assay was performed as described by Passaniti et al.
(24)
. Matrigel, either alone or mixed with bFGF (final
concentration 150 ng/ml) in a total volume of 0.5 ml at 4°C, was
injected s.c. into the mid-abdominal region of athymic nude mice. DMXAA
(20 mg/kg) was administered as a single i.p. injection (0.01 ml/g body
weight) after Matrigel inoculation. Matrigel polymerizes at body
temperature to form a solid plug. After 7 days, the Matrigel plug,
together with the underlying epidermis and dermis, was removed, fixed
in 10% neutral buffered formalin for at least 24 h, dehydrated
through ascending concentrations of alcohol, and embedded in paraffin
under vacuum. Sections through all three layers were stained with
GMasson trichrome. Counterstaining for factor VIII
(24)
established that >95% of cells stained with Masson
trichrome within the Matrigel plug were endothelial cells. The area
occupied by infiltrating endothelial cells in histological sections was
quantified in four nonoverlapping Matrigel sections/plug (two to four
mice/group).
Control rabbit serum was prepared from blood obtained from untreated
rabbit and clotted overnight on ice. Undiluted, polyclonal rabbit
antimouse IP-10 antibody or normal rabbit serum (50 µl) was mixed
with 500 µl of Matrigel plus bFGF/implant and also administered
3 h and 24 h after DMXAA treatment (300 µl/injection i.p.).
Neutralizing antibodies to TNF- were reconstituted at 1 mg/ml, and
neutralizing antibodies to IFN- and IFN- were reconstituted at
100 and 200 µg/ml, respectively, according to the manufacturer’s
instructions. Antibody (50 µl) was added to the Matrigel implant, and
each antibody was diluted 3-fold and injected (300 µl) 1 h and
24 h after implantation.
Tumor Implantation.
Colon 38 tumor fragments (1 mm3
) were implanted
s.c. in the left flank of anesthetized (sodium pentobarbitone; 86
mg/kg) C57Bl/6 and IFN-R0/0 mice. Tumors were
used when they had reached approximately 6 mm in diameter, generally
9–10 days after implantation. The implantation rate of Colon 38 tumors
was 100% in C57Bl/6 mice and approximately 70% in the
IFN-R0/0 mice.
Northern Blot Analysis.
Mice were sacrificed by cervical dislocation. Spleens and tumors were
removed aseptically, and the tissues were minced using a pair of
scalpels. Total cellular RNA was extracted using RNAzol (Life
Technologies, Inc.) according to manufacturer’s instructions. RNA (10
µg) was denatured and electrophoresed in 1% agarose-formaldehyde
gels as described previously (25)
. RNA was then
transferred by capillary action onto nylon membranes
(Hybond-N+; Amersham), which were UV cross-linked
(120 mjoule; UV-Stratalinker; Stratagene, San Diego, CA) and baked
(80°C for 30 min). Each membrane was prehybridized (2 h; 42°C) in 7
ml of hybridization mix containing 50% formamide, 0.075 M
sodium chloride, 0.05 M sodium dihydrogen phosphate, 5
mM EDTA, 0.001% polyvinyl pyrrolidine, 0.001% BSA,
0.001% Ficoll, 0.01 mg/ml herring sperm DNA, and 0.5% SDS. The cDNA
to the cytokine gene of interest was labeled with
[32P]dCTP (Amersham) using a random priming
kit (RTS Radprime DNA labeling system; Life Technologies, Inc.). Excess
radioactivity was removed by elution through a G-50 Sephadex column,
and labeled probe (106 cpm/ml hybridization mix)
was then added to the membrane and hybridized for 36 h at 42°C.
The blots were washed twice in 2 x SSC with 0.1% SDS
for 10 min at 42°C and finally in 0.2 x SSC with
0.1% SDS for 10 min at 65°C. Blots were exposed to X-ray film for
1–3 days at -70°C. After hybridization with one probe, membranes
were stripped (two washes in 300 ml of 0.1 x SSC with
1% SDS for 15 min at 80°C) and rehybridized with another probe.
Intensity of signals was quantitated by laser densitometric scanning.
Loading of lanes was determined from the intensity of bands hybridized
with the probe for human ß-actin or glyceraldehyde-3-phosphate
dehydrogenase.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, IFN-, and
IFN- was maximal at 2 h and then rapidly declined (Fig. 2)
. IP-10 and MIP-1 mRNA expression peaked at 4 h and was
maintained for up to 12 h after DMXAA administration.
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, IP-10, and MIP-1 and lesser amounts of IFN-
than splenic tissue (Fig. 3)
. Surprisingly, expression of mRNA for IFN- was reproducibly not
observed in the tumor, although it was induced in the spleen (Fig. 3)
.
Furthermore, up-regulation of mRNA for the p35 or p40 subunits of IL-12
and for IFN-ß mRNA was not detected in either tissue (Figs. 2
and 3)
.
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Production by
DMXAA., which in turn
is induced by IL-12 (14
, 15)
. The results shown in Figs. 2
and 3
suggested that IP-10 induction in response to DMXAA did not
involve IL-12 or IFN-. IL-12 mRNA could not be detected (Fig. 2)
,
and IP-10 mRNA expression in the tumor was independent of IFN- mRNA
induction (Fig. 3)
. To further clarify whether IFN- mediated IP-10
induction by DMXAA, we examined the induction of IP-10 mRNA in mice
lacking functional receptors for IFN-. DMXAA strongly induced IP-10
mRNA in splenic and tumor tissues of IFN-R0/0
mice to a level that was comparable with that induced in their
wild-type counterparts (Fig. 4)
. Although IFN- mRNA was induced in the spleens of both the
wild-type and IFN-R0/0 mice, no induction was
observed in the Colon 38 tumor tissues implanted in either host (Fig. 4)
.
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. The results of multiple sections were analyzed (Fig. 5E)
.
Matrigel plugs without bFGF contained few invading endothelial cells
compared with plugs of Matrigel plus bFGF. DMXAA treatment reduced the
amount of bFGF-induced endothelial cell invasion into the Matrigel by
88% when administered on the same day as the Matrigel implantation.
Timing of DMXAA administration after Matrigel implantation was
critical, because no inhibition was obtained when DMXAA was given 3
days after Matrigel implantation and only 20% inhibition when given
after 24 h.
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. After administration of 8-MeXAA, no induction of
mRNA for IP-10, IFN-, IFN-, and TNF- was observed in spleens
of nude mice used for Matrigel implants (data not shown).
Antibodies to IP-10 Neutralize the Inhibition of DMXAA of
Endothelial Cell Invasion.
To determine whether cytokines were involved in the inhibition of
endothelial cell invasion obtained after DMXAA administration, normal
rabbit serum, anti-IP-10, anti-TNF-, anti-IFN-, or anti-IFN-
antibodies were mixed with the Matrigel implant. Mice were also treated
with antibodies 1 or 3 h and 24 h after DMXAA treatment. The
number of endothelial cells in the Matrigel was quantitated after 7
days. Addition of anti-IP-10 antibodies reversed DMXAA-induced
inhibition of endothelial cells in the Matrigel plug by 58% (Table 1)
. In contrast, antibodies to IFN- and normal rabbit serum had no
effect, whereas anti-TNF- and anti-IFN- antibodies reversed the
inhibition by 7% and 5%, respectively.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The potential of DMXAA to inhibit the
development of new tumor blood vessels must therefore be considered in
addition to its known immune effects (27
, 28)
and
antivascular effects (2
, 3)
.
DMXAA might exert antiangiogenic activity either directly,
e.g., by affecting vascular endothelial cells, or indirectly
by inducing angiogenesis-modulating factors. We have extended a
previous observation using cultures of primary macrophages
(7)
by demonstrating that DMXAA up-regulates IP-10 mRNA
expression in spleen and tumor tissues (Figs. 2
and 3)
. At least in
spleen tissue, the induction of IP-10 after DMXAA administration was
not a consequence of IFN- production, because it was induced in the
spleens of both the wild-type and IFN-R0/0
mice (Fig. 4)
. The result is not completely unequivocal in tumor tissue
because tumor cells could have IFN- receptors and respond to the
high levels of circulating IFN- present in
IFN-R0/0 mice (8)
. It is possible
that DMXAA activates the gene for IP-10 directly, although maximal
expression of mRNA for IP-10 occurred later than that of TNF- or
IFN- mRNA expression.
Antibodies to IP-10 provided a 58% reversal of the inhibitory effects
of DMXAA, suggesting that IP-10 is the main cytokine responsible for
endothelial cell invasion. Antibodies to IFN- had no effect, whereas
anti-TNF- and anti-IFN- antibodies reversed the inhibition only
marginally. The lack of significant effect with anti-IFN- antibodies
is consistent with the observation that the induction of IP-10 by DMXAA
is independent of IFN- production. There is a surprisingly narrow
time window for the effect of DMXAA on the Matrigel assay, with
administration of DMXAA 3 days after implantation having no effect
(Fig. 5)
. It is likely that the competition between the opposing
effects of bFGF and IP-10 determines the outcome of the Matrigel assay,
and because both may be relatively short-lived, this competition can be
observed only over a short time.
After DMXAA treatment, greater expression of the genes for IP-10 and
MIP-1 was seen in tumor tissue than in the spleen (Fig. 3)
. This
behavior is different from that observed in viral infections or after
IFN- administration, where more IP-10 is produced in the spleen than
in the liver (29)
. The high induction of IP-10 and
MIP-1 in tumor tissues raises the question of whether these
chemokines contribute to the antitumor response through their
chemotactic and antiangiogenic properties. Chemokines produced in an
inflammatory response serve to attract leukocytes to the site of
inflammation (20)
, and the production of IP-10 or MIP-1
might mediate an influx of host leukocytes into the tumor. The primary
target for both IP-10 and MIP-1 appears to be natural killer cells
and activated T lymphocytes (10
, 30)
, and both these cell
types have been implicated in the action of DMXAA (28)
.
Inoculation of tumor cells engineered to express high amounts of IP-10
in mice resulted in regression of the tumors, as well as coinoculated
tumors that did not produce IP-10 (11)
. Antitumor activity
has been found to depend on the recruitment of T lymphocytes
(11)
, and it has been shown that activated T lymphocytes
selectively express the CXCR3 receptor, which is specific for IP-10 and
the closely related chemokine Mig (31)
.
One striking result emerging from these investigations is that,
although DMXAA induces mRNA for IFN- in spleen, it does not in the
Colon 38 tumor (Fig. 3)
. One possible explanation is that the
Th1 subset of T-helper lymphocytes that produce IFN- does not
infiltrate the Colon 38 tumor. We have yet to examine if expression of
mRNA for other cytokines that are produced by the Th1 subset, such as
IL-2 and lymphotoxin, is similarly absent in the tumor. Alternatively,
Th1 cells could be present, but the production of IFN- has been
suppressed within the tumor microenvironment. This latter explanation
would be compatible with other studies (32)
reporting
that, although the ability of tumor-associated T cells to produce IL-2
and IFN- decreased with tumor progression, transforming growth
factor ß and IL-6 activity increased. These authors suggested that
transforming growth factor ß inhibited the production of the T-cell
cytokines and that the tumor-bearing state induced an abnormal cytokine
network under which T-cell cytokines are negatively regulated. However,
we saw no significant difference in IFN- mRNA expression between
spleens of normal and tumor-bearing mice.
In summary, we have shown here that DMXAA induces the antiangiogenic chemokine IP-10 and that a single administration of DMXAA inhibits bFGF-induced neovascularization of Matrigel. Thus, DMXAA may exert significant antiangiogenic activity through IP-10 production, as well as antivascular action through production of TNF and other cytokines (33 , 34) . The balance between these two types of antitumor activity may well depend on the schedule of drug administration, with single high doses favoring antivascular effects and repeated low doses favoring antiangiogenesis. Drug combination studies may well facilitate such distinctions, and we are currently studying the effects of administration schedule on the activity of DMXAA in combination with thalidomide (35) .
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FOOTNOTES |
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1 Supported by the Cancer Society of New Zealand
(Auckland Division) and the Health Research Council of New Zealand. The
research was conducted during the tenure (by Z. C.) of a Postgraduate
Scholarship of the Health Research Council of New Zealand. 
2 To whom requests for reprints should be
addressed, at the Auckland Cancer Society Research Centre, University
of Auckland School of Medicine, Private Bag 92019, Auckland, New
Zealand 1000. Phone (64-9) 3737-999; Fax (64-9) 3737-502; E-mail: l.ching{at}auckland.ac.nz
3 The abbreviations used are: DMXAA,
5,6-dimethylxanthenone-4-acetic acid; IP-10, interferon-inducible
protein 10; IL, interleukin; bFGF, basic fibroblast growth factor; TNF,
tumor necrosis factor; MIP-1, macrophage inhibitory protein-1;
8-MeXAA, 8-methylxanthenone-4-acetic acid; IFN-R0/0,
IFN- receptor knock-out.
Received 6/13/00. Accepted 12/15/00.
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