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Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3394-3404
Identification of Four Genes in Endothelial Cells Whose
Expression Is Affected by Tumor Cells and Host Immune Status A
Study in Ex Vivo-Isolated Endothelial Cells
By
Birgit Liliensiek,
Marian Rocha,
Victor Umansky,
Axel Benner,
Jie Lin,
Reinhard Ziegler,
Peter P. Nawroth, and
Volker Schirrmacher
From the Department of Internal Medicine I, University of Heidelberg,
Heidelberg; and the German Cancer Research Center, Tumor Immunology
Program and the Division of Biostatistics, Heidelberg, Germany.
 |
ABSTRACT |
A spontaneously metastasizing, well-defined mouse lymphoma was
chosen as an in vivo model to study the effect of tumor-host interaction on gene expression in liver sinusoidal endothelial cells.
Forty-nine bovine aortic endothelial cell (BAEC) genes, recently
isolated by a differential screening approach of a cDNA library
enriched for tumor necrosis factor- (TNF-) suppressed genes, were
investigated. Four of these genes were finally selected because they were affected differentially by host immuno-competence, TNF-, and tumor cells. Sequence analysis showed them to encode the
bovine polyubiquitin (A4), elongation factor 1 (B2), the acidic
ribosomal phosphoprotein PO (C3), and the ribosomal protein S2 (E10).
Gene expression was analyzed by dot-blot or Northern blot analysis.
TNF- and tumor cell conditioned supernatant suppressed the genes
additive in BAEC but not in other endothelial cells except for bovine
capillary endothelial cells. Ex vivo-isolated liver endothelial cells
of tumor-bearing syngeneic DBA/2 mice showed strong downregulation of
these four genes in comparison to normal control values. In contrast,
endothelial cells of tumor-bearing immuno-incompetent Balb/c (nu/nu)
mice showed no downregulation but upregulation of these genes.
Consistently, all four genes were also downregulated when BAEC were
incubated with supernatants derived from ex vivo-isolated liver
metastases from immuno-competent but not from -incompetent mice. Thus,
the expression of a group of genes involved in protein translation and
processing was more profoundly altered in endothelial cells in vivo
than in vitro, suggesting that microenviromental factors and cell-cell
and cell-matrix interactions play an important role.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
VASCULAR ENDOTHELIAL CELLS exert a
variety of functions which go far beyond the original hypothesis of an
"inert barrier."1-3 They can participate in immune
reactions by affecting adherence and other functions of
immuno-competent cells. Endothelial cell products such as
platelet-derived growth factor induce T-cell synthesis of interleukin-2
(IL-2), while suppressing synthesis of IL-4, -6, and
interferon- .4 Vascular endothelial cell growth factor
(VEGF) enhances leukocyte functional antigen-1 and very late
antigen-4-dependent natural killer (NK) cell adhesion to intercellular
adhesion molecule-1 and vascular adhesion molecule-1 on vascular
endothelial cells.5 Basic fibroblast growth factor in
contrast inhibits NK cell adhesion through the regulation of these
adhesion molecules on tumor vasculature.5 Hence, some angiogenic factors facilitate lymphocyte recognition of angiogenic vessels, while others protect vessels from cytotoxic
lymphocytes.5 These findings suggest that the interaction
of endothelial cells with other cells such as lymphocytes or tumor
cells can not only be viewed under the aspect of gene induction, but
also gene suppression. Gene suppression by mediators of the immune
response has received only little attention.
Tumor necrosis factor- (TNF-), known to be angiogenic in vivo, is
one of the cytokines most extensively studied in cultured endothelial
cells. Many genes induced by TNF- have been described, including
vasoconstrictors, leukocyte adhesion molecules, procoagulant receptors,
and others.6-15 We have recently used a differential screening approach to isolate cDNAs suppressed by TNF- in cultured bovine aortic endothelial cells (BAEC).16 About 0.25% of
all mRNA species present in cultured endothelial cells were found to be
suppressed by TNF-, indicating that gene suppression may be an
important feature of cytokine-endothelial cell interaction.
Very few data are available with respect to the in vivo response of
endothelial cells to a metastasizing tumor cell line and the role of
the host immune status in regulating endothelial cell properties. For
the present study a model of ex vivo-isolated liver sinusoidal
endothelial cells was established. A previously well-defined mouse
tumor was chosen which metastasizes spontaneously to the
liver.17 Liver sinusoidal endothelial cells were isolated ex vivo from normal or tumor-bearing immuno-competent (DBA/2) or
-incompetent [Balb/c (nu/nu)] mice. In preliminary experiments, 49 of
the above-mentioned TNF- suppressed genes were tested. Of these,
four genes were chosen for further analysis because they were affected
by tumor cells and host immune status. The expression of these four
genes was strongly affected in vivo by microenvironmental factors like
tumor-derived factors and by cell-cell interactions like tumor-host
interactions. According to the analysis, the liver endothelial cell
response (gene expression phenotype) of late-stage tumor-bearing nude
mice differs strikingly from that of immuno-competent mice which
survive about twice as long. In vitro, a less prominent change in gene
expression of the selected genes was detected after incubation of BAEC
with supernatants derived from ex vivo-isolated late-stage liver
metastases of immuno-competent DBA/2 and immuno-incompetent Balb/c
(nu/nu) mice. Downregulation of the four selected genes by TNF- and
conditioned ESbl-lacZ tumor cell supernatant was detected in
vitro in BAEC. Gene expression after incubation with TNF-
and/or tumor cell supernatant was also investigated in other
cultured endothelial cells. The sequences of the cDNAs which will be
shown revealed that the genes are involved in protein translation and
processing.
| |
MATERIALS AND METHODS |
Cell lines and in vitro assays.
BAEC and human umbilical vein endothelial cells (HUVEC; kindly provided
by Dr P. Quehenberger) cultured as described
previously.18,19 Bovine adrenal cortex endothelial cells
(ACE) were isolated and characterized as described.20
Experiments were performed with quiescent cells that had been confluent
for 2 days.
The mouse endothelial cells TC10 were cultured as
described.21 Mouse endothelioma cells (FN1) were kindly
provided by Dr M. Clauss and cultured in Dulbecco's
modified Eagle's medium (GIBCO-BRL, Eggenstein, Germany) supplemented
with 10% fetal calf serum (FCS; Boehringer, Ingelheim, Germany), 1%
nonessential amino acids (Bio Whittaker, Ingelheim, Germany), 1%
sodium pyruvate (Bio Whittaker, Ingelheim, Germany), 0.4%
mercaptoethanol, and 1% penicillin/streptomycin.
DBA/2 derived and with the  -galactosidase gene (lacZ)
transduced ESbL lymphoma cells (clone L-CI.5s) (ESbL-lacZ) were
cultured as described.17 After 24 hours of incubation,
tumor cell supernatant (TS) was collected and incubated with the
different endothelial cells for 8 hours. Supernatants derived from
tumor and sinusoidal cells of metastatic livers at the late stage of
tumor growth were incubated with BAEC for 8 hours.
Human recombinant TNF- was added at a saturating dose of 1 nmol/L (1 × 108 U/mg; Knoll AG, Ludwigshafen,
Germany) for 8 hours.
Mice and tumor cell inoculation.
Euthymic DBA/2 mice and athymic nude mice [Balb/c (nu/nu)] were
obtained from Iffa Credo (Lyon, France) and used at 6 to 12 weeks of
age. ESbL-lacZ cells were washed in phosphate-buffered saline
(PBS) (137 mmol/L NaCl, 2.7 mmol/L KCl, 1.5 mmol/L
KH2PO4, 8.1 mmol/L
Na2HPO4) and adjusted to the appropriate
concentration. For standard injection, 104 cells were
injected intradermally at the shaved flank of anesthesized (Rompun
[0.1%]: Ketanest [0.25%]: PBS diluted 1:1:3 [volume]) animals.
Isolation of tumor and sinusoidal cells from metastatic livers.
Cell isolation was performed as described.22 Briefly,
livers from anesthetized tumor-bearing mice were washed in situ by perfusion through the portal vein at 37°C with 10 mL -modified Eagle's medium (-MEM) containing 15 mmol/L
N-(2-hydroxyethyl)-piperazino-N'-2-ethanol sulfonic acid (HEPES) at a
flow rate of 3 mL/min. Tissue digestion was performed during perfusion
with 10 mL of -MEM/HEPES containing 0.05% pronase E (Boehringer
Mannheim, Mannheim, Germany) at 1 mL/min and then with 15 mL
of the same medium containing 0.03% pronase E, 0.05% collagenase A
(from Clostridium histolyticum; Boehringer Mannheim). After
perfusion, livers were minced and stirred in 13 mL -MEM/HEPES
containing 0.04% pronase E, 0.04% collagenase A, and 0.0004% DNase
(Sigma Chemical Co, St Louis, MO) at 37°C for 10 minutes. The cell
suspension was then filtered through a nylon gauze and centrifuged at
300g for 10 minutes. To remove cell debris and erythrocytes,
the cell pellet was centrifuged at 1,400g for 15 minutes in
-MEM/HEPES containing 17.5% (wt/vol) metrizamide (Sigma), followed
by washing of the top layer with -MEM/HEPES at 300g for 10 minutes.
In some experiments, tumor and sinusoidal cells were isolated from
metastatic livers at a late stage of tumor growth and metastasis (ie,
day 28 in immuno-competent DBA/2 mice and day 12 in immuno-incompetent Balb/c [nu/nu] mice) and cultured at 37°C for 1 and 5 days in complete RPMI. At these time points, supernatants were collected for
the incubation with BAEC.
FDG-staining and flow cytometric analysis of tumor cells.
Quantitation of liver metastasis was performed at the single-cell level
by staining with fluorescein -galactosidase (FDG; Molecular Probes,
Eugene, OR) as described.17 Isolated cells, 1 × 106, were washed in PBS supplemented with 5% FCS and
incubated in 100 µL 5% FCS at 37°C for 10 minutes. One hundred
microliters of prewarmed FDG in H2O was added to the cell
suspension, and the mix was briefly vortexed and then incubated for 1 to 4 minutes at 37°C (hypotonic shock). Ice-cold 5% FCS/PBS, 1.8 mL, was then added, and the cells were kept for 10 minutes on ice and
then stained with 1.5 µmol/L propidium iodide. Flow cytometry was
performed using a FACScan (Becton Dickinson, Heidelberg, Germany) with
30,000 cells/sample. Cells were simultaneously measured for forward
angle light scatter (FSC) and integrated side scatter (SSC), as well as
green (FL1) and red (FL3) fluorescences (expressed as logarithm of the
integrated fluorescence light). Recordings were made only on propidium
iodide-negative (viable) cells. Data were expressed as percentage of
positive cells.
Isolation of liver endothelial cells.
After isolation of liver sinusoidal endothelial and tumor cells, they
were cultured on type 1 collagen-coated plastic Petri dishes in -MEM
supplemented with 10% FCS at 37°C in a 5% CO2 incubator. Two hours later supernatants were collected and adherent cells (sinusoidal endothelial cells) were scraped off with a rubber spatula, counted, pelleted, snap frozen in liquid nitrogen, and kept at
 70°C for RNA studies. The same procedure was used for isolation of endothelial cells from normal (non-tumor-bearing) mice.
Purity of endothelial cell populations was evaluated by phase contrast
microscopy, mannose-receptor expression, and measurement of the
mitochondrial activity, as described.23,24
RNA preparation.
Cultured BAEC were washed three times with 0.9% sodium chloride (NaCl)
solution at 4°C before the guanidine isocyanate
solution was added. Subsequently total RNA was extracted using a
procedure of guanidinium isocyanate lysis followed by
ultracentrifugation through cesium chloride, as
described.25
Pellets of ex vivo-isolated liver sinusoidal endothelial cells were
homogenized with 0.2 mL RNA-Clean TM (Angewandte Gentechnologie Systeme
GmbH, Heidelberg, Germany) per 2 × 106 cells. Total
RNA extraction was performed by the chloroform/phenol technique. The
RNA was precipitated with isopropanol, the pellet washed in ethanol,
dried under vacuum, and resuspended in diethyl pyrocarbonate-treated
water. The quantity of RNA was measured by absorbance at 260 nm. Poly
(A) + RNA was isolated using an mRNA purification kit (Qiagen GmbH,
Hilden, Germany).
Dot-blot analysis.
The filters for dot hybridization were prepared essentially as
described by Kafatos et al.26 Plasmid pBluescript SK
containing cDNA inserts of 49 independent BAEC genes, suppressed after
incubation with 1 nmol/L TNF- for 6 hours in BAEC, and several
control genes6,10,27,28 were used to prepare the filters.
The cDNA for the housekeeping gene glyceraldehyde-3-phosphate
dehydrogenase (GADPH; American Type Culture Collection [ATCC],
Rockville, MD) was used to standardize the hybridized cDNAs. The vector
pBluescript SK served as a control for background signals. One
microgram of linearized plasmid was denatured in 0.4 mol/L NaOH at room
temperature for 30 minutes and chilled on ice. The solution was then
brought to 10× SSC (1.5 mol/L sodium chloride, 0.15 mol/L sodium
citrate, pH 7.0) (4°C) and blotted on Hybond N membranes (Amersham
Buchler, Braunschweig, Germany), using a 96-well manifold apparatus
(Bio-Rad Laboratories GmbH, München, Germany).
Poly(A) + RNA was used to synthesize radioactive-labeled cDNA probes
using Moloney-murine leukemia virus reverse transcriptase (GIBCO-BRL)
and [-32P]dCTP (Redivue; Amersham Buchler,
Braunschweig, Germany). Prehybridization was performed in 5× SSPE
(1× SSPE contains: 180 mmol/L NaCl, 10 mmol/L
Na3PO4, 1 mmol/L EDTA, pH 7.7), 0.5% sodium
dodecyl sulfate (SDS), 5× Denhardt's solution,25
0.02 mg/mL salmon sperm DNA (Sigma-Aldrich, Deisenhofen, Germany), and
50% formamide at 42°C for 2 hours. The filters were hybridized at
42°C for 24 hours with 1 × 106 cpm/mL for
experiments with ex vivo-isolated liver sinusoidal endothelial
cells. Washing was performed once in 1× SSC (150 mmol/L NaCl, 15 mmol/L sodium citrate)/0.1% SDS at room temperature for 15 minutes, in
0.1× SSC/0.5% SDS at 65°C for 20 minutes, and the filters
were subsequently exposed to AGFA Curix 1.000G x-ray films (Sigma, München, Germany) with an intensifying screen at
80°C for 1 to 4 days.
Northern blot analysis.
Total RNA per lane, 12.5 µg, was separated on 1.1% agarose gels
containing 6.4% formaldehyde. The integrity of RNA was checked by
ethidium bromide staining of the 18S and 28S ribosomal RNA. After
electrophoresis, RNA was transferred overnight by capillary blotting in
20× SSC to Hybond N nylon membrane (Amersham Buchler). Hybridization (using 3 to 4 × 106 cpm labeled probe
per mL hybridization solution) and washing were performed as described
for dot-blot analysis. Selected cDNAs were labeled with the random
primer labeling system (Promega, Heidelberg, Germany). The filters were
stripped and rehybridized against human GAPDH to standardize the amount
of RNA loaded.
Densitometric quantitation.
Gene expression levels were quantified by densitometry of
autoradiograms using the Adobe Photoshop Program and the SCAN analysis program from Macintosh (München, Germany). Each
relative expression value represents the ratio between the densities of
specific mRNA transcripts to GAPDH transcripts. Measurable background
signals were not detected for the vector pBluescript SK for dot blots. Therefore, the background signals were not included in the calculation.
Sequencing.
The four selected genes were used for dideoxy sequencing with the
Sequenase Version 2.0 Kit (Amersham Buchler) and
[-35S]dATP (Redivue; Amersham Buchler). The clones
were partially sequenced from both ends, using 40 and reverse
primers for the pBluescript SK vector (Stratagene, Heidelberg,
Germany). At least 200 bp from both ends were used to search for
similarities to previously published genes with BLASTN of the HUSAR
sequence analysis program package (Version 4.0; Genetics Computer
Group, Inc, Heidelberg, Germany). Full-length cDNA
sequences were obtained for three unpublished bovine sequences.
Automatic sequencing was performed commercially by MWG-Biotech
(Ebersberg, Germany). The bovine full-length sequences were analyzed
with the HUSAR sequence analysis program package (Version 4.0). The
applications BLASTN, MAP, and FASTA29 were used.
Statistical analysis.
The relative densitometry units resulting from autoradiograms of
Northern blots or dot blots were statistically analyzed using the
paired t-test (see Figs 3 and 4) and the Wilcoxon
rank sum test (see Figs 2, 5, and 6). For the statistical analysis
untreated controls of one experiment representing the relative
densitometry units of the selected four genes were considered to be one
group and were compared with the corresponding treated group. A
probability P < .05 was considered statistically significant.
| |
RESULTS |
Effect of tumor growth on expression of multiple genes in liver
sinusoidal endothelial cells.
Forty-nine genes isolated from a subtractive cDNA library of
BAEC,16 representing genes suppressed by TNF-, were used
to identify their potential relevance in vivo upon tumor growth and liver metastasis. Gene expression was tested in four independent experiments of carefully ex vivo-isolated endothelial cells. The relative densitometry units of the 49 genes and of several control genes6,10,27,28 expressed in endothelial cells of
tumor-bearing mice (y-axis) were compared with those of normal control
mice (x-axis). Representative data of the mRNA expression levels
(relative densitometry units) are shown for immuno-competent DBA/2 mice in Fig 1A and for immuno-incompetent Balb/c
(nu/nu) mice in Fig 1B. Dots around the diagonal represent genes
without expression changes. In late-stage metastases of
immuno-competent DBA/2 mice (A) a number of genes were downregulated
(below the diagonal), whereas in respective immuno-incompetent nude
mice (B) expression levels of several genes were upregulated (above the
diagonal). Four genes that were reproducibly affected by tumor growth
and host immuno-competence in repeated experiments were selected and they are labeled as A4, B2, C3, and E10 in Fig 1. Many other genes (those along the diagonal line) were not significantly affected by
tumor growth in vivo, indicating selectivity as to the kinds of genes
affected.
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| Fig 1.
Analysis of the effect of tumor growth on the expression
of multiple genes in liver sinusoidal endothelial cells in vivo. Gene
expression was analyzed by dot-blot mRNA analysis in cells from
tumor-bearing immuno-competent DBA/2 (A) and from immuno-incompetent
Balb/c (nu/nu) (B) mice. The relative densitometry units were
calculated from the autoradiograms such as shown in Figs 3 and 4. They
represent the ratio between the density of the specific mRNA transcript
to the GAPDH transcript as described in Materials and Methods. The
expression levels at day 0 (control, x-axis) were plotted versus those
obtained at the indicated days after tumor inoculation
(ESbl-lacZ, y-axis). Dots around the diagonal represent genes
without expression changes, whereas dots below (suppression) or above
(induction) the diagonal represent genes with altered expression within
the experiments. Representative data from two in vivo experiments for
immuno-competent DBA/2 and immuno-incompetent Balb/c (nu/nu) mice are
shown. For further analysis selected genes are labeled (A4, B2, C3, and
E10).
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TNF- and tumor cell supernatant exert a suppressive
effect in vitro on gene expression by BAEC.
Confluent bovine aortic endothelial cell monolayers were incubated for
8 hours with either 1 nmol/L TNF- (TNF, a saturating dose as shown
in previous experiments) or tumor cell supernatant (TS) of the
well-characterized highly metastatic ESbL-lacZ lymphoma cells17 or with both factors together (TS + TNF).
Expression of the genes A4, B2, C3, and E10 was investigated by
Northern blot analysis.
The size of the detected mRNAs in cultured BAEC was determined based on
18S and 28S ribosomal RNA. The molecular weights of the bands
recognized by the cDNA probe A4 are 4.2 kb (minor transcript) and 1.2 kb (major transcript). The weakly detectable 4.2-kb mRNA of the clone
A4 is not shown in Fig 2. The mRNAs
recognized by the other cDNAs have a size of 1.8 kb for B2, 1.05 kb for
C3, and 0.9 kb for E10.
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| Fig 2.
TNF- and tumor cell supernatant exert suppressive
effects in vitro on the expression of selected genes by BAEC. Confluent
BAEC were cultured for 8 hours either without (C) or with 1 nmol/L
TNF- (TNF) and/or with lymphoma cell conditioned supernatant
(TS). The mRNA expression was detected by Northern blot analysis for
the genes A4, B2, C3, and E10. The autoradiograms depicted below the
histograms were quantitated by densitometry as described in the legend
to Fig 1 and in Materials and Methods. Histograms for the calculated
relative densitometry units (y-axis) for the single genes are shown.
The bar graphs represent different treatments. The differences seen
between C and the TNF + TS group were significant (P = .015, Wilcoxon rank sum test).
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As shown in the histograms of Fig 2, gene A4 was downregulated from
6.29 (C) to 4.35 (TNF), 4.65 (TS), and 3.1 (TNF + TS) relative
densitometry units. Gene B2 was downregulated from 0.94 (C) to 0.83 (TNF), 0.8 (TS), and 0.72 (TNF + TS) relative densitometry units. The
calculated relative densitometry units for C3 are 1.85 (C), 1.3 (TNF),
1.39 (TS), and 0.9 (TNF + TS). Gene E10 was downregulated from 2 (C) to
1.39 (TNF), 1.52 (TS), and 1.01 (TNF + TS) relative densitometry units.
The mRNAs of the four selected genes were similarly suppressed by TNF
and TS. A stronger suppression was detected for TNF and TS together.
The mean of the densitometry units of the four selected genes was 2.77 (C), 1.97 (TS), 2.09 (TNF), and 1.43 (TS + TNF). The differences in the
TNF- and TS-treated groups in comparison with the controls only
indicated trends (P = .14 for TNF and P = .23 for TS), while those between the C and the TNF + TS group were significant (P = .015) when using the Wilcoxon rank sum test. The same results were obtained when performing dot-blot analysis. Thus,
dot-blot analysis had a similar sensitivity as Northern blots and could
therefore be used in further experiments with ex vivo-isolated
endothelial cells when amounts of isolated RNA were small.
The results represented in Fig 2 show that metastatic tumor cells
secrete factors modulating gene expression in BAEC. The finding that TS
when given together with a saturating dose of TNF- had a stronger
suppressive effect on the expression of the four genes suggests that TS
and TNF are not identical and that there is more than one factor that
can negatively affect gene expression. Addition of VEGF and TNF- did
not have an effect like TS (data not shown), suggesting that it is not
VEGF in TS causing the observed effect.
Endothelial cells ex vivo from livers of tumor-bearing syngeneic
DBA/2 mice show downregulation of gene expression.
To investigate the functional significance of the observed suppression
of gene expression in BAEC after treatment with TNF- and/or
with lymphoma cell conditioned supernatant, we checked the expression
of the same genes in liver sinusoidal endothelial cells isolated
directly ex vivo from either normal mice (control) or from mice 28 days
after intradermal injection of ESbL-lacZ lymphoma cells. At day
28 the mice carry large primary tumors and have macroscopic liver
metastases.17 The isolated endothelial cells had a purity
of 95% and contained less than 5% of other sinusoidal
cells.23,24
As shown in Fig 3 by dot-blot analysis, the
mRNA expression of A4, B2, C3, and E10 was reduced in liver sinusoidal
endothelial cells from tumor-bearing animals in comparison with the
control group. The mRNA expression of these genes in control
(untreated) mice remained unchanged during the 28 days of the
experiment (data not shown). Gene A4 was downregulated from 9.3 (C) to
3.6 (day 28) relative densitometry units, gene B2 from 7.2 (C) to 3.4 (day 28) units, gene C3 from 4.6 (C) to 2.9 (day 28), and C3 from 3.8 (C) to 2.1 (day 28) units. The difference in the expression of all four
genes, shown as relative densitometry units in Fig 3, between the mean
of the controls (6.23) and the mean of tumor-bearing hosts (3.0) was
significant at P = .044 (paired t-test).
Thus, there was, in immuno-competent mice, a prominent suppressive
effect in vivo on the four endothelial genes during massive invasion of
the liver with metastatic syngeneic lymphoma cells.
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| Fig 3.
Downregulation of gene expression in ex vivo-isolated
liver endothelial cells from tumor-bearing immuno-competent mice.
ESbL-lacZ lymphoma cells were injected intradermally into
syngeneic DBA/2 mice. Twenty-eight days later, endothelial cells were
isolated using liver perfusion and differential adhesion to collagen
pretreated Petri dishes. Then mRNA was extracted from control (C) and
tumor-bearing mice (day 28), and dot-blot analysis performed and
evaluated as described in the legend to Fig 1 and in Materials and
Methods. The pBluescript SK DNA which was used as vector gave no
densitometric signal. The difference in the expression of all four
genes between C and the day 28 tumor-bearing immuno-competent host was
significant (P = .044, paired t-test).
|
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Endothelial cells ex vivo isolated from livers of tumor-bearing
Balb/c (nu/nu) mice show upregulation of gene expression.
To investigate to what extent the reduced gene expression observed in
sinusoidal endothelial cells from metastatic livers of syngeneic DBA/2
mice was a direct effect from the tumor as shown in vitro (Fig 2) or
was dependent on host immuno-competence, we performed an analogous
experiment in nude mice lacking mature T lymphocytes. Gene expression
in liver sinusoidal endothelial cells was tested again in cells ex vivo
isolated from the final phase of tumor growth and metastasis (day 7 after tumor cell inoculation). It was previously shown that in this
final phase the load of liver metastases is comparable to that of DBA/2
immuno-competent mice at day 28 after tumor cell
inoculation.17
Data presented in Fig 4 show an increased
mRNA expression of the four analyzed genes in tumor-bearing nude
compared with the control mice. The mRNA expression of these genes in
control (untreated) mice remained unchanged during the 7 days of the
experiment (data not shown). Gene A4 was upregulated from 2.3 (C) to
3.4 (day 7) relative densitometry units, the gene B2 from 1.9 (C) to
3.0 (day 7) units, C3 from 1.6 (C) to 2.6 (day 7), and E10 from 1.2 (C) to 3.0 (day 7) units. The difference in the expression of all four
genes, shown as relative densitometry units in Fig 4, between the mean
of the controls (1.73) and the mean of the tumor-bearing hosts (2.94)
was significant (P = .003, paired
t-test). A similar upregulation of the expression
of these genes was found in endothelial cells isolated from nude mice
at day 12 after tumor inoculation. Thus, expression of the investigated
genes in ex vivo-isolated liver sinusoidal endothelial cells from
tumor-bearing nude mice was significantly higher than in
non-tumor-bearing mice. This suggests an inductive effect in vivo
during massive invasion of the liver with metastatic lymphoma cells in
the absence of mature T lymphocytes.
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| Fig 4.
Upregulation of gene expression in ex vivo-isolated
liver endothelial cells from tumor-bearing immuno-incompetent mice.
ESbL-lacZ lymphoma cells were injected intradermally into
BALB/c (nu/nu) mice. Seven days later, endothelial cells were isolated
from control (C) and tumor-bearing mice (day 7) and analyzed as
described in the legend to Fig 3 and in Materials and Methods. The
pBluescript SK DNA (vector), which was used as a control, gave no
nonspecific background signals. The difference in the expression of all
four genes between C and the day 7 tumor-bearing immuno-incompetent
host was significant (P = .003, paired t-test).
|
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Supernatants of tumor and sinusoidal cells from late-stage metastatic
livers alter gene expression in BAEC.
To determine whether the microenvironment of the tumor (sinusoidal
cells) or the tumor cells were responsible for the upregulation or
downregulation of the above genes, sinusoidal and tumor cells from
metastatic livers were reisolated at day 28 from DBA/2 and at day 12 from Balb/c (nu/nu) mice after tumor injection. FDG staining and FACS
analysis showed that the number of tumor cells 24 hours after isolation
was 73% in DBA/2 mice and 76% in Balb/c (nu/nu) mice. Five days later
in culture, 97.7% and 98%, respectively, of the cells were tumor
cells. Supernatants derived from the reisolated cells were incubated
for 8 hours with BAEC (Fig 5). Afterwards, total RNA was isolated, and Northern blots were performed and analyzed
by densitometry. As shown in Fig 5, the mRNA expression of A4, B2, C3,
and E10 was reduced in comparison to the control group in BAEC when
incubated with supernatants derived from ex vivo-isolated liver
metastasis from immuno-competent DBA/2 mice. Gene A4 was downregulated
from 1.49 to 1.13 and 1.3 (day 1 and 5, respectively) relative
densitometry units, gene B2 from 0.57 to 0.4 and 0.44, gene C3 from
0.33 to 0.19 and 0.20, and gene E10 from 0.26 to 0.17 and 0.15. The
mean of the relative densitometry units of all four genes for control
(C) was 0.66, for treatment with DBA/2-derived supernatants from day 1 (S1) 0.47 and from day 5 (S5) 0.52. Differences were statistically
significant (P < .001, Wilcoxon rank sum test). In contrast,
when BAEC were incubated with supernatants derived from ex
vivo-isolated metastases from immuno-incompetent nude mice, there was
only a slight downregulation of the expression of these genes. These
results suggest that the microenvironment of the tumor (sinusoidal
cells) is responsible for the alteration of gene expression in BAEC and
that within the sinusoidal cell population, T cells seem to be
important, because supernatants derived from isolated cells from nude
mice were inactive.
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| Fig 5.
Supernatants of tumor and sinusoidal cells isolated from
metastatic livers of immuno-competent DBA/2 and immuno-incompetent
Balb/c (nu/nu) mice at a late stage of tumor growth alter gene
expression in BAEC. Supernatants of the re-isolated and cultured cells
were collected at day 1 (S1) and day 5 (S5). BAEC were incubated with
the supernatants for 8 hours and total RNA was extracted. BAEC
incubated in the culture medium of the tumor cells served as control
(C). The mRNA expression of the genes A4, B2, C3, and E10 was analyzed
by Northern blot hybridization as described in the legend to Fig 2. The
bar graphs represent different treatments, with the corresponding
autoradiograms of the hybridization signals depicted below the
histograms. The differences in the expression of all four genes between
C and the DBA/2-derived supernatants were significant (P < .001, Wilcoxon rank sum test).
|
|
Gene expression response to TNF- and TS in
endothelial cells of other vessel origin and species.
We also investigated endothelial cells of other vessel origin and
species with regard to expression of the four genes after incubation
with TNF and/or tumor cell supernatant (TS). No significant changes were observed when fetal human endothelial cells (HUVEC), mouse
endothelial cells (TC10), and murine endothelioma cells (FN1) were
incubated for 8 hours with TNF and/or TS.
Figure 6 shows the results obtained from
Northern blot analysis of bovine capillary endothelial cells (ACE).
Although the expression level of A4 was not affected, B2 was
downregulated from 1.37 (C) to 1.06 (TS) and 0.98 (TNF + TS) relative
densitometry units, and C3 from 1.94 (C) to 1.64 (TS) and 1.56 (TNF + TS) relative densitometry units. Gene expression was not affected by
TNF- alone. E10 was only slightly downregulated. The mean of the
densitometry units of the three genes B2, C3, and E10 was 2.11 (C), 1.8 (TS), and 1.74 (TNF + TS). The difference between C and the TNF + TS
group was significant (P = .027, Wilcoxon rank sum test).
Nevertheless, the effects seen are minor.
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| Fig 6.
Gene expression of A4, B2, C3, and E10 in cultured bovine
capillary endothelial cells after TNF- and tumor cell supernatant is
different from BAEC. Confluent ACE were cultured for 8 hours either
without (C) or with 1 nmol/L TNF- (TNF) and/or with lymphoma
cell conditioned supernatant (TS). The mRNA expression was analyzed by
Northern blots described in the legend to Fig 2 and in Materials and
Methods. The differences in the expression of B2, C3, and E10 between C
and the TNF + TS group were significant (P = .027, Wilcoxon
rank sum test).
|
|
Sequence analysis of the four studied bovine genes.
The four genes investigated in detail show differential regulation
during the process of metastasis in liver sinusoidal endothelial cells
in immuno-competent and immuno-incompetent animals. With regard to the
direction of regulation (upregulation or downregulation), the four
genes behaved similarly, suggesting common regulatory response
elements. The four investigated genes were then sequenced. The DNA and
protein sequences of the genes B2, C3, and E10 are new and are
presented in Fig 7A through
C. The gene A4 was identified as an already published sequence of the
bovine polyubiquitin (accession number BTPOLYUB) and, therefore, is not
shown. The sequence analysis with the HUSAR program package showed that
B2 codes for the bovine elongation factor 1, C3 for the bovine
acidic ribosomal phosphoprotein PO, and E10 for the bovine ribosomal
protein S2 (LLRep3 protein). The bovine elongation factor 1 (B2)
cDNA shares 91% homology with the human DNA sequence and 100%
homology with the protein sequence.30 The predicted bovine
protein sequence lacks 149 amino acids at the amino terminus in
comparison with the human protein sequence. The acidic ribosomal
phosphoprotein P0 (C3) shows 93% identity with the human cDNA
sequence, 98.3% identity at the protein level,31 and the
predicted bovine protein lacks 16 amino acids at the amino terminus in
comparison with the human protein sequence. The ribosomal protein S2
(E10) shows 88% homology with the rat cDNA sequence and 98.3%
homology with the rat protein sequence,32 and the bovine
protein sequence lacks 7 amino acids at the amino terminus. The DNA and
protein sequence of the human LLRep3 gene,33 another member
of this gene family, which codes for the ribosomal protein
S2,32 is completely included within the identified bovine
sequence of E10.
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| Fig 7.
DNA and protein sequence of the cDNAs B2, C3, and E10.
The clones B2, C3, and E10 were commercially sequenced and compared to
published sequences using the application BLASTN of the HUSAR program
package (Version 4.0). The bovine cDNAs represent partial sequences of
the elongation factor 1 (B2, A), the acidic ribosomal phosphoprotein
PO (C3, B) and the ribosomal protein S2 (E10, C). Within the sequence
of the ribosomal protein S2 the sequence of LLRep3 is included. The
start codon (ATG) and the first Methionin (M) of the LLRep3 sequence
are underlined and enlarged. The cDNA sequences were submitted to
GenBank and the accession numbers are AF013213 (B2), AF013214 (C3),
AF013215 (E10). The open reading frames of the cDNA sequences were
translated into the deduced amino acid sequences and placed under the
corresponding DNA sequence. The numbering starts with the first
nucleotide or amino acid. The potential polyadenylation signal
sequences are underlined.
|
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| |
DISCUSSION |
In cultured BAEC we previously identified 49 genes that were suppressed
by TNF-.16 To test the possible relevance of these genes
in vivo in pathological or physiological situations, we decided to
investigate the influence of a tumor and its metastases on the gene
expression of liver sinusoidal endothelium. For this purpose we
established a technique of direct ex vivo isolation of liver sinusoidal
endothelial cells of high purity from normal and tumor-bearing mice.
After in situ enzyme perfusion, dissociation and selective in vitro
adhesion to collagen-coated Petri dishes, maximally 1 to 2 × 106 endothelial cells could be isolated per organ allowing
only semiquantitative dot-blot hybridization. The isolated endothelial
cells had a purity over 95% according to the parameters described in
Materials and Methods. From cell-culture experiments, which allow us to
isolate relatively large amounts of RNA, we performed Northern blot and dot-blot analysis and obtained comparable results. For experiments with
ex vivo-isolated cells with limiting amounts of RNA, the methodology
had to be restricted to dot blot. The recently developed ex vivo cell
isolation technology allowed us to study the endothelial response of
the liver as an important target organ of metastases.
Of the previously isolated 49 TNF- suppressed genes, four genes (A4,
B2, C3, and E10) were selected because of their altered expression in
livers of tumor-bearing immuno-competent and immuno-incompetent hosts
in comparison to normal mice (Fig 1). In cultured BAEC the four genes
were not only suppressed by saturating amounts of TNF- but also by
tumor-cell-conditioned supernatant (Fig 2). Both factors together had
a stronger suppressive effect, indicating that in tumor supernatant
factors other than TNF- exist, which can affect endothelial gene
expression in vitro. Thus, tumor cell products as well as
mediators of the host immune response such as TNF- were found to
affect the expression of the four genes studied in BAEC. Expression of
these genes was also investigated in other cultured endothelial cells.
The genes B2 and C3, but not A4, were also downregulated by tumor cell
supernatant in bovine capillary endothelial cells (ACE) (Fig 6).
Expression of A4, B2, C3, and E10 was not affected by TNF-
and/or tumor cell supernatant in human HUVEC cells or murine
TC10 and FN1 endothelial cells. The results obtained in endothelial
cells of different vessel origin or from different species thus
indicate that the observed downregulation in BAEC is not a general
phenomenon in all endothelial type cells.
In vivo in mice we found an even more prominent suppression of these
four genes (Fig 3) than in cultured BAEC (Fig 2). However, this
observation required an immuno-competent host. In immuno-incompetent Balb/c (nu/nu) mice, expression of these genes was upregulated in liver
endothelial cells of tumor-bearing compared with non-tumor-bearing animals (Fig 4). The results corroborate our previous observations: (1)
that the pattern and load of spontaneous liver metastasis depends on
the host immune status,17 (2) that liver endothelial cells
participate in T-cell-dependent host resistance to lymphoma metastasis
by production of nitric oxide in vivo,23 and (3) that
dynamic expression changes in vivo of adhesion and costimulatory molecules determine load and pattern of lymphoma liver
metastasis.22 Thus, gene expression of liver endothelial
cells in tumor-bearing animals with liver metastasis is under control
of tumor cells and tumor-mediated immune reactions.
Incubation of cultured BAEC with supernatants derived from reisolated
and cultured sinusoidal and tumor cells from DBA/2 late-stage metastatic livers resulted in partial suppression of the four genes A4,
B2, C3, and E10 (Fig 5). The strongest downregulation was observed
using supernatant conditioned by 73% tumor cells and 27% sinusoidal
cells from immuno-competent mice. Gene expression was not significantly
altered using supernatants from immuno-incompetent mice. Therefore, the
tumor and its microenvironment, in particular T cells, seem to be
responsible for the observed downregulation of the four genes in murine
liver endothelial cells (Fig 3) and in cultured BAEC (Fig 5).
Alteration in the expression of the genes A4, B2, C3, and E10 was more
prominent in vivo, shown in ex vivo-isolated liver endothelial cells
(Figs 3 and 4), than in cultured BAEC and other endothelial cells using
TNF- and different conditioned supernatants (Figs 2, 5, and 6).
Therefore, not only the secreted mediators of the tumor and its
microenvironment seem to be responsible for the changes in gene
expression; other conditions of the in vivo situations such as
cell-cell contacts and cell contacts with the extracellular matrix may
also be important for expression changes of the four investigated
genes.
There is not much known about the molecular regulation of gene
expression of the four described genes. Within the murine elongation factor 1 promotor three p53-responsive elements were
found,34 whereas five Sp1 sites and one Ap-1 site were
found within the first intron of the human gene.35 Further
studies are required to understand the molecular mechanism of
downregulation of the genes.
Sequence analysis of the four genes revealed their identity (Fig 7).
They encode bovine polyubiquitin (A4), elongation factor 1 (B2),
acidic ribosomal phosphoprotein P0 (C3), and ribosomal protein S2
(E10). Database analysis showed the bovine genes to be newly cloned
homologues to previously isolated human or rat genes. The homology to
the human DNA sequence is 91% for the elongation factor 1 (B2) and
93% for acidic ribosomal phosphoprotein P0 (C3). The ribosomal protein
S2 (E10) shows 88% homology with the rat cDNA sequence. The cloned
bovine polyubiquitin sequence (A4, only partially sequenced) is
identical with an already published bovine sequence.
Interestingly, all four genes are involved in protein translation and
processing. Polyubiquitin (A4) is an important ubiquitous molecule
involved in a variety of cellular processes including cell cycle,
stress response, protein folding and translocation, protein tagging for
nonlysosomal degradation, DNA repair, transcription, and
apoptosis.36-39 Elongation factor 1 (B2) is a guanosine
triphosphate (GTP)-binding protein that catalyzes the binding of
aminoacyl-transfer RNAs to the ribosome in the protein synthesis
process40 and was also identified as microtubule-severing
protein.34 Multiple phosphorylated forms of the acidic
ribosomal phosphoprotein PO (C3) were found to be located within the
large subunit (60S) of eukaryotic ribosomes. They form a pentameric
complex with the ribosomal proteins P1 and P2 in the ribosome, which
interacts with the elongation factors 1 and 2.41 The
ribosomal protein S2 (E10) belongs to a highly conserved repetitive
mammalian gene family designated LLRep3.33 It participates
in aminoacyl-transfer RNA binding to the ribosome and this potentially
affects the fidelity of the mRNA translation.32
In addition, ubiquitin,42 elongation factor
1,43-45 the acidic ribosomal phosphoprotein
PO,46,47 and the ribosomal protein S248,49 have
been shown to be upregulated in tumor cells. This appears to be the
first report on upregulation and downregulation of these genes in
normal cells, namely liver sinusoidal endothelial cells. If one assumes
that upregulation of these genes in different cultured tumor cells and
in vivo in tumor models are of importance for the survival of tumor
cells, then it is intriguing to speculate on our observation that the
same genes are differentially regulated in ex vivo-isolated liver
sinusoidal endothelial cells of liver tumor-bearing mice, depending on
their immune status. The four genes were upregulated in liver
endothelial cells of Balb/c (nu/nu) mice (Fig 4), but downregulated in
the respective cells from immuno-competent mice, which survived twice
as long (Fig 3). Thus, upregulation of these genes seems to correlate
with tumor cell growth, both in vitro and now also in vivo, in this
liver metastasis model.17 In the context of immune response
influences it is noteworthy that ubiquitin has been shown to be
involved in antigen presentation.50 Further studies are
required to provide evidence for a causal relationship between
upregulation of these genes in endothelial cells and facilitation of
tumor metastasis on one hand and downregulation of these genes and
retardation of liver metastasis formation.
While using the ex vivo isolation technique of liver endothelial cells,
it was possible to study the influence of microenvironmental factors,
immune response, and cell-cell and cell-matrix interactions on
endothelial gene expression in vivo. The effects observed in freshly ex
vivo-isolated cells were more profound than those obtained in tissue
culture experiments. This study exemplifies how the combination of
techniques of molecular biology, cell biology, immunology, and cancer
research allows for the unraveling of complex in vivo phenomena, such
as metastasis formation, at the molecular level.
| |
FOOTNOTES |
Submitted November 19, 1997;
accepted June 25, 1998.
B.L. and M.R. contributed equally to this work.
Supported by a grant from the Dr Mildred Scheel Stiftung (no.
10-0980-Schi2, M.R. and V.U.), the Schilling Stiftung (P.P.N.), a grant
of the Deutsche Forschungsgemeinschaft (to P.P.N.) and the
Sonderforschungsbereich 405 supported by the Deutsche
Forschungsgemeinschaft (P.P.N.).
Address reprint requests to Volker Schirrmacher, PhD, Tumor Immunology
Program, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany; e-mail: V.Schirrmacher{at}dkfz-heidelberg.de.
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.
| |
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Abstract
Introduction
Abstract