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Regular Articles |
§
From the Ottawa Regional Cancer Centre,*
Ottawa; the
Department of Biochemistry, Microbiology and
Immunology,
University of Ottawa, Ottawa; the
Department of Pathology and Laboratory
Medicine,
Ottawa Hospital, General Campus,
Ottawa; and the Department of Pathology and Laboratory
Medicine,§
University of Ottawa, Ottawa,
Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The linkage between inflammation and genotoxicity is strengthened by recent findings that some chronic inflammatory diseases are associated with an increase in mutant frequency (MF) at the hypoxanthine phosphoribosyltransferase (Hprt) locus. This locus is widely used as a marker of genotoxic insult because it is a non-essential gene in which mutational events can be readily scored on the basis of resistance to 6-thioguanine.13 T lymphocytes from the peripheral blood of patients with autoimmune diseases such as systemic lupus erythematosus, multiple sclerosis, scleroderma and rheumatoid arthritis have an elevated MF.14-17 T lymphocytes from the diseased synovium of rheumatoid arthritis patients exhibited an even higher MF.17 In a lacZ transgenic mouse injected with lymphoma cells to produce inflammation in the spleen and lymphatic tissues, an increase in MF in host cells was seen.18 Thus, the association between chronic inflammation and cancer may be explained by the presence of mutagenic factors in inflammatory milieu.
Genomic instability of cancer cells is well documented, and is a feature of tumor progression. Instability may be manifest as aneuploidy, structural chromosome abnormalities, loss of heterozygosity, homogeneously staining regions (amplification) and accumulation of mutations.19 The contribution to genetic instability of factors in the tumor microenvironment, such as products of inflammatory cells, has been addressed in only a few experimental studies.20 An increase in mutagenicity in cells grown in vivo as subcutaneous tumors compared to the same cells grown in culture has been reported. C3H/10T1/2 cells grown in C3H mice had a very high frequency of minisatellite sequence instability compared to in vitro.21 Mutatect cells22 grown in C57BL/6 mice had a fourfold higher MF at the Hprt locus.23 Murine LN12 cells grown in athymic nude mice exhibited a similar increase in MF.24 The nature of the mutagenic species in the tumor microenvironment is unknown, but is suspected of being reactive oxygen species (ROS) or nitrogen species (RNS).10,18,23,25-31 Because ROS and RNS are known to be mutagenic, it is possible that they are responsible for the genotoxicity associated with both the inflammatory and the tumor microenvironments.
Solid tumors frequently contain inflammatory cells, whose presence may
be explained by chemotactic factors. For example, interleukin-8 (IL-8),
a potent chemoattractant for neutrophils of the C-X-C
type,32
has been shown in glioblastoma.33
IL-8 has also been identified in human gastric, colorectal and
bronchioloalveolar carcinomas34-36
and in B-cell chronic
lymphocytic leukemia.37
High expression of several C-C
chemokines, in particular, monocyte chemoattractant protein-1, was
identified in epithelial ovarian cancers where CD68+ macrophages and
CD8+/CD45RO+ T cells were the primary infiltrating
cells.38
Recruited neutrophils and hypoxic vascular
endothelial cells may be another source of IL-8 and other
chemotaxins.32,39
Since thrombotic occlusion of blood
vessels is common in tumors, clotting-related factors can also
contribute to the recruitment of inflammatory cells.38
Activated platelets can secrete chemokines such as monocyte
chemoattractant protein-1 and IL-8.38,40
Human
-thrombin is a chemoattractant for monocytes.41
Fibrin-derived polypeptides are chemoattractants for
neutrophils.42
It would thus appear that many chemotactic
factors may be present in tumors; the particular mix of factors present
in any individual tumor may dictate the type of leukocytes that
infiltrate.
To better understand the contribution of inflammatory cells and their products to genotoxicity in solid tumors, we have used the Mutatect mouse tumor model.22 This is a murine fibrosarcoma that can be propagated as a subcutaneous tumor in syngeneic C57BL/6 mice as well as in culture. This model is unique in that it permits the ready detection of mutations at the Hprt locus that may arise in vivo or in vitro, either spontaneously or induced by agents such as ionizing radiation.22,23 In the present study, we show that the number of Hprt mutations in individual tumors is a direct function of the number of infiltrating neutrophils and amount of nitric oxide synthase.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 female mice, 8 to 10 weeks of age (Charles River Laboratories, St.-Constant, QC, or Taconic, Germantown, NY) were injected subcutaneously (s.c.) in the right flank with 5 x105 Mutatect MN-11 cells in 0.1 ml PBS (140 mmol/L NaCl, 2.7 mmol/L KCl, 8 mmol/L Na2HPO4, 1.5 mmol/L KH2PO4, pH 7.4). Tumors were first detectable at day 7, measured about 0.5 cm in diameter at day 11 and were harvested when they were about 1 cm in diameter (day 14–15). Other details and culture conditions are described elsewhere.23 Experiments were carried out at the Animal Care and Veterinary Service of the University of Ottawa in accordance with guidelines of the Canadian Council on Animal Care.
Detection of S-Phase Cells in Tumors by 5-Bromo-2'-deoxyuridine (BrdU) Labeling
To detect S-phase cell in tumors, BrdU (30 mg/kg) was injected 2 hours before sacrifice. BrdU incorporation into DNA was detected immunohistochemically using a mouse monoclonal anti-BrdU antibody (Sigma Chemical Co., St. Louis, MO), biotinylated horse anti-mouse IgG (Dimension Lab, Inc., Mississauga, ON), avidin-peroxidase (Dimension Lab) and 0.02% diaminobenzidine tetrahydrochloride (DAB).
Determination of in Vivo MF
Tumors were allowed to form following s.c. injections as described above. When tumors reached 1 to 1.5 cm in diameter (14–16 days), animals were sacrificed by cervical dislocation. Tumors were removed under aseptic conditions and gently homogenized by passage several times through a syringe. The suspension was allowed to settle for 5 minutes and the supernatant containing predominantly single cells was removed and incubated for 2 to 4 days to establish ex vivo cultures. The MF was estimated by culturing 1 x 105 viable cells (determined by trypan blue exclusion) per 10-cm dish in the presence of 50 µmol/L 6-thioguanine. MF is expressed as mutants per 1 x 105 viable G418R cells (MN-11 cells harbor a neo gene that allows them to be readily distinguished from host cells), as described earlier.23
Histochemical Assays for Detecting Tumor-Infiltrating Host Cells
Tumor tissue was fixed for 24 hours in 10% neutral buffered formalin, embedded in paraffin, sectioned at 3 to 5 µm, and processed for histochemical analysis. Paraffin was removed with xylene and sections were rehydrated with a series of decreasing concentrations of ethanol. Frozen sections were used where indicated. Fresh tumor tissue was fixed with modified Zamboni’s fixative43 for 2 hours. The fixative was removed with ice-cold 100 mmol/L sodium phosphate buffer, pH 7.2 (PB), and the tumor fragment was stored in a 1:1 mixture of 10% sucrose, 3% Triton X-100 in PB for a minimum of 24 hours. Tumor tissue was embedded in Tissue-Tek OCT Compound (VWR-Canlab, Mississauga, ON), frozen immediately in liquid nitrogen, and stored at -80°C until sectioned. Hematoxylin and eosin (H&E) stained sections were examined for infiltrating host cells. Other stains were used as indicated. Neutrophils were identified in frozen sections of mouse tissue by staining for myeloperoxidase activity; this enzyme appears to be inactive in paraffin-embedded sections of mouse and human tissues. Five-micron cryosections were stained using 0.02% DAB, 0.6% H2O2 in TBS (50 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.6) for 10 minutes at room temperature. Sections were washed in running water and counterstained with Mayer’s hematoxylin. Brown-stained multinucleated neutrophils were readily identifiable. Macrophages were identified by staining for nonspecific esterase, using frozen sections incubated in the presence of 2% sodium nitrite, 2% pararosanilin (Sigma-Aldrich) for 20 minutes at 37°C.44 Mast cells were identified in fixed, paraffin-embedded sections after staining with 1% toluidine blue in 50% isopropanol for 10 minutes at room temperature and destaining in absolute isopropanol for 1 minute.
iNOS Immunohistochemistry
Formalin-fixed tumor tissues was deparaffinized and incubated with TBS containing 1% H2O2 and 0.25% Triton-X 100 for 15 minutes at room temperature to destroy endogenous peroxidase activity. Nonspecific immunoglobulins were blocked with 1% normal swine serum at room temperature for 30 minutes. Excess liquid was drained and sections were incubated in a humid chamber with 1:100 dilution (2.5 µg/ml) of rabbit polyclonal antibody to mouse macrophage iNOS (Transduction Laboratories, Lexington, KY). Sections were incubated overnight at 4°C followed by an incubation at room temperature for 30 minutes with 1:200 (7.5 µg/ml) goat anti-rabbit biotinylated IgG (Vector Laboratories). The biotinylated conjugate was detected by incubating sections with avidin-peroxidase 1:1000 dilution (5 µg/ml, Vector Laboratories, Burlingame, CA) at room temperature for 30 minutes. Immunolabeling was detected using DAB as the chromogen, washed, and counterstained with Mayer’s hematoxylin.
Nitrotyrosine Immunohistochemistry
Formalin-fixed tumor tissue sections were deparaffinized and heated at 90–100°C for 12 minutes in 0.01 mol/L sodium citrate, pH 6.0. Sections were then incubated with TBS containing 3% H2O2 for 10 minutes at room temperature. Nonspecific immunoglobulins were blocked with 1% normal swine serum at room temperature for 30 minutes. Excess liquid was drained and sections were incubated in a humid chamber with 2.5 µg/ml of a rabbit polyclonal antibody to nitrated KLH (Upstate Biotechnology, Lake Placid, NY). Sections were incubated for 1 hour at room temperature followed by incubation for 30 minutes with DAKO Envision peroxidase conjugated to goat anti-rabbit/anti-mouse Ig antibody (DAKO Corp., Carpinteria, CA). Immunolabeling was detected using DAB as the chromogen, washed and counterstained with Mayer’s hematoxylin. Three tests of the specificity of the antibody for nitrotyrosine were carried out. Before incubation with tissue sections, the primary antibody was preincubated for 1 hour at room temperature with either 10 mmol/L nitrotyrosine (Sigma) or 50 µg/ml nitrated bovine serum albumin (prepared by incubation of albumin (6 mg/ml) for 18 hours at room temperature with 10 mmol/L NaNO2, 0.3% H2O2, 9 µmol/L FeCl3 and subsequent precipitation with 4 vol ethanol). A third control involved reduction of tissue nitrotyrosine to aminotyrosine with sodium hydrosulfite.45
Nitric Oxide Synthase (NOS) Activity in Tumor Extracts
Fragments of the same tumors collected for MF analysis (vide supra) were frozen in liquid nitrogen and stored at -80°C. For assay of NOS activity, a previously described method was followed.46 Tumor fragments (100 mg) were thawed and homogenized (Polytron PT-1200, Kinematica, Lucerne, Switzerland) in 250 µl of an ice-cold solution containing 320 mmol/L sucrose, 10 mmol/L HEPES, 1 mmol/L dithiothreitol, 1 mmol/L EDTA, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml leupeptin, and 2 µg/ml aprotinin, pH 7.4. The homogenates were centrifuged at 13,000 x g at 4°C for 30 minutes. The supernatants were removed and assayed for NOS activity by following the conversion of L-[14C]arginine (Amersham, Arlington Heights, IL) to [14C]citrulline.46 Forty microliters of supernatant were mixed with 100 µl of assay buffer containing 30 mmol/L KH2PO4, 1 mmol/L MgCl2, 0.2 mmol/L CaCl2, 1 mmol/L ß-NADPH, 6 mmol/L L-valine, 18 µmol/L L-arginine, and 1 µCi14 C-L-arginine, pH 7.4. Reactions were incubated at 37°C for 20 minutes and terminated by dilution in 1 ml of 1 mmol/L cyclohexanediamine tetraacetate (CDTA, Sigma-Aldrich) and immediately applied to columns containing 0.4 ml Spectra/Gel cation exchanger 50W-X8 to remove [14C]arginine. [14C]Citrulline was recovered by elution with 3 ml of 1 mmol/L CDTA. Fractions were mixed with scintillation fluid (Aquasol-2) and counted in a Packard model 1600 TR liquid scintillation counter. We have not ruled out that arginase may have contributed in part to the observed activity, since inhibition by 1 mmol/L NG-monomethyl-L-arginine was incomplete (average of 50%). Protein content of tumor homogenates was determined spectrophotometrically with Bradford reagent,47 using bovine serum albumin as standard. NOS activity was expressed as pmole of citrulline generated per minute per milligram of protein.
IL-8 Injections
On day 11 after inoculation, tumors were injected directly with IL-8 or N-formyl-met-leu-phe (fMLP) in combination with prostaglandin E2 (PGE2). Control mice received endotoxin-free sterile saline. Amounts injected were 1 x 10-12 mol IL-8 (Endogen, Woburn, MA), 3 x 10-10 mol PGE2 (Sigma-Aldrich), and 1 x 10-10 mol fMLP (Pierce, Rockford, IL) in a total volume of 6 µl using an ethanol-sterilized glass Hamilton syringe and semiautomatic dispenser. On day 15 after tumor inoculation (4 days after IL-8 + PGE2 injections), animals were sacrificed by cervical dislocation and tumors were excised aseptically. A section of the tumor taken from the middle was washed in PB and fixed in a modified Zamboni’s fixative, and myeloperoxidase activity was detected histochemically. The remaining tumor tissue was used for analysis of MF as described.23
Histological Grading of Neutrophil Infiltration and Necrosis
Neutrophil and necrosis counts were performed by two observers on coded tumor sections. A 2-mm grid was drawn on the coverslip over the tumor section. The four corners of each 2-mm square within the grid were scored for neutrophil and necrosis counts using a 40x objective with a 10x eyepiece. Between 5 and 14 squares (depending on tumor size) were chosen at random and the same fields were scored for both neutrophil and necrosis counts. The extent of tumor necrosis was determined by the stereological point counting method48 and expressed as percentage of tumor necrosis of total area counted. Results were expressed as the mean number of neutrophils and mean percentage necrosis per field. Neutrophils found in blood vessels were not included in the counts.
Statistical Analysis
The MF of individual tumors exhibited considerable variation that
appeared to be non-Gaussian. Data were therefore analyzed using
nonparametric tests. Comparison between two groups was made using the
Mann-Whitney test and correlations using Spearman’s rank test.
Calculations were done using GraphPad Instat version 3.0. A value of
P 
0.05 was considered to be statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MN-11 cells readily form subcutaneous tumors in syngeneic C57BL/6
mice. At day 14 after injection of 5 x104, 1
x105, 2.5 x105, or 5
x105
cells, the percentages of animals (6 per
group) bearing a tumor were 17, 67, 67, and 100%, respectively. This
percentage did not increase by day 18. The tumor volume was estimated
from its length, width, and height according to Isaacs and
Coffey.49
The tumor volume increased most rapidly between
days 10 and 15; tumor volume doubling time following injection of
5x105
cells was estimated to be 2.7 days. For
example, the average volume was 40 ± 3.5
mm3
(n = 6) at day 10 and
256 ± 10.6 mm3
at day 15. The growth
fraction in the tumors was estimated using the BrdU labeling technique,
in which newly synthesized DNA is detected using an antibody to BrdU.
Tumors 15, 19, and 21 days old were examined 2 hours after injection of
BrdU. The percentage of nuclei into which BrdU was incorporated was
11.0 ± 1.1, 8.0 ± 1.2, and 5.3 ± 0.3%, respectively,
at the periphery of the tumors and 3.9 ± 0.8, 2.8 ± 1.1,
and 1.6 ± 0.7%, respectively, in the core of the tumors.
Statistically significant decreases (from day 15) are day 19 and 21 at
the periphery and day 21 in the core (analysis of variance,
n = 4 to 8 tumors in each group, P <
0.01) Tumors that were 
2 cm in diameter (usually older than 17 days)
tended to have necrotic cores. A low magnification micrograph of a
15-day-old MN-11 tumor is shown in Figure 1A
. Tumors were typically surrounded by a
capsule and rarely showed evidence of invasion. Histological
examination following H&E staining revealed a poorly differentiated
fibrosarcoma with considerable variation in cell and nuclear size. Most
tumors were very vascular. In some, a heavy neutrophil infiltrate was
seen in and around blood vessels (Figure 1B)
and in necrotic areas
(Figure 1C)
. Fibrin deposits were also seen in necrotic areas (Figure 1C)
. Neutrophils were present to a lesser extent in non-necrotic areas
and at the periphery of the tumor. Less frequently seen were
infiltrating lymphocytes, mainly at the tumor periphery (Figure 1D)
.
Nonspecific esterase staining was used to identify macrophages; these
were infrequent and found only at the periphery of tumors (Figure 1E)
.
Toluidine blue staining was used to identify mast cells; these were
also infrequent and at the periphery of tumors (Figure 1F)
. On the
basis of the examination of 20 Mutatect MN-11 tumors, we concluded that
central necrosis was more frequently seen in 2 cm tumors and that
neutrophils were the predominant infiltrating host cell type.
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To study the relationship between infiltrating neutrophils and MF,
a neutrophil count was performed on MN-11 tumor sections, using
myeloperoxidase staining to facilitate counting. Thirty-four MN-11
tumors were examined both for MF and for infiltrating neutrophils, as
described in Materials and Methods (Figure 2A)
. There was a statistically
significant correlation between the number of infiltrating neutrophils
and the MF (r = 0.63, P <
0.0001, Spearman’s rank correlation). The same tumors were examined
for necrosis. Again, there was a statistically significant correlation
between the number of infiltrating neutrophils and the extent of tumor
necrosis (r = 0.73, P <
0.0001). These findings demonstrate a clear positive correlation
between the neutrophils and MF in Mutatect tumors.
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It is possible to increase neutrophils by using chemoattractants such as IL-8 + PGE2 or fMLP.50 The neutrophil content of treated and control 11-day tumors was first examined in a pilot experiment involving 7 tumors per treatment group. Although both fMLP and IL-8 + PGE2 injected tumors were more heavily infiltrated with neutrophils than control tumors, IL-8 + PGE2 appeared to be more effective (data not shown). No increase in the number of infiltrating macrophages or lymphocytes was seen.
To confirm and extend these observations, an experiment involving 64
animals was carried out. Thirty-six animals were injected
intratumorally with IL-8 + PGE2 and the remainder
with saline on day 11, when tumors were about 0.5 cm in
diameter. On day 15, the tumors were excised and some were examined
quantitatively for neutrophil infiltrate (Figure 2B)
. There was a
statistically significant increase in neutrophil content between the
IL-8 + PGE2-injected (n =
16) group and the control group (n = 12;
Mann-Whitney test, P = 0.0007). The percentage of tumor
necrosis was also significantly increased (P =
0.01). Tumors were also analyzed for MF. The IL-8 +
PGE2 injected group (n =
36) had a highly statistically significant elevation in MF compared to
the control group (n = 28) (Mann-Whitney test,
P = 0.0002; Figure 2C
). These data indicate clearly
that IL-8 + PGE2 was effective at recruiting
neutrophils to MN-11 tumors and that this was associated with an
increase in MF.
For 20 tumors injected with IL-8 + PGE2, both MF
and neutrophil data were available. The relationship between neutrophil
content and MF was explored (Figure 2D)
. As in untreated tumors, there
was a strong correlation between the MF and the neutrophil content in
this group of IL-8 + PGE2-treated tumors
(Spearman’s rank correlation, r = 0.63,
P = 0.003). Percentage of tumor necrosis and MF were
also strongly correlated (r = 0.70,
P = 0.0006).
Because neutrophil count was correlated with percentage of necrosis in the mouse tumors, a similar study of human adenocarcinoma of the lung was carried out. A statistically significant correlation between the number of neutrophils and the extent of necrosis was seen (n = 30; r = 0.70; P < 0.0001). The presence of IL-8 was assessed immunohistochemically in 12 specimens; most tumors showed at least some IL-8-staining areas, localized to the cytoplasm of tumor cells with occasional staining of macrophages and neutrophils (data not shown).
Correlations between NOS Activity and MF in MN-11 Tumors
The Mutatect model was developed to study whether factors such as
nitric oxide and/or related species in the tumor microenvironment might
be genotoxic and mutagenic. Nitric oxide is produced from arginine by a
family of enzymes, NOS. To explore whether NOS activity was present in
MN-11 tumors, extracts of 56 individual tumors (15–22 days of in
vivo growth) were examined. Figure 3
shows that NOS activity varied appreciably among tumors (range,
1.1–33.7 pmol/minute/mg protein). The level of NOS activity was
compared to the MF in the corresponding tumor. The data indicate a
positive correlation between NOS activity and MF
(r = 0.77, P < 0.0001) in these
tumors.
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Nitric oxide is a potentially genotoxic reactive nitrogen species
formed in vivo by nitric oxide synthases. Inducible nitric
oxide synthase (iNOS) is an enzyme associated primarily with
macrophages. Because MN-11 tumors contain very few macrophages but many
neutrophils, it was of interest to determine whether tumor-infiltrating
neutrophils expressed iNOS. Tumor sections probed with an antibody to
mouse macrophage iNOS demonstrated strong staining of neutrophils.
Immunostaining was seen in both non-necrotic (Figure 4A)
and necrotic (Figure 4B)
areas of the
tumor. Macrophages and mast cells at the periphery of the tumor were
also stained, but were fewer in number (data not shown). No staining of
tumor cells, lymphocytes, or vascular endothelial cells was seen.
Specimens of human lung adenocarcinoma were also examined
immunohistochemically for the presence of iNOS. In an analysis of 6
cases, iNOS was seen in macrophages and eosinophils but not in
neutrophils or tumor cells (data not shown). Thus, it appears that iNOS
is present in mouse neutrophils but not in human tumor-infiltrating
neutrophils.
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It is difficult to provide direct evidence for the formation of
reactive oxygen or nitrogen species in vivo and therefore
indirect evidence of oxidative damage to tissues is often used. The
availability of antibodies to protein nitrotyrosine has facilitated
such analyses.51
MN-11 tumors probed with an
anti-nitrotyrosine antibody revealed diffuse staining throughout much
of the tumor (Figure 4C)
. Both diffuse cytoplasmic staining and nuclear
staining was seen. No staining was seen in negative controls in which
nitrated serum albumin (Figure 4D)
, nitrotyrosine amino acid or sodium
hydrosulfite was used. These observations supports the notion that
peroxynitrite or other reactive nitrogen species capable of nitrating
proteins was present in MN-11 tumors.
Comparison of Interanimal and Intra-Animal Tumor MF Variation
A possible source of the interanimal variability in spontaneous tumor MF was investigated in an experiment in which two subcutaneous tumors were grown in each of 14 mice. The MF of tumors grown on the left flank was compared to the MF of tumors grown on the right flank in this series. There was no significant correlation between the MF of the 2 tumors grown in the same animal (Spearman’s rank correlation test, P = 0.48) (data not shown). Thus, it does not appear that the variability can be explained by systemic factors and therefore may be due to stochastic events occurring within individual tumors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neutrophils are a potential source of ROS (such as superoxide and hydrogen peroxide) and RNS (such as nitric oxide and peroxynitrite), which are potentially genotoxic.10,18,23,25-31 The enzyme responsible for the production of nitric oxide from arginine in tissues is NOS. Immunohistochemical analysis of MN-11 tumors demonstrated iNOS in infiltrating neutrophils and biochemical measurements of tissue extracts showed NOS activity. The presence of iNOS in mouse neutrophils52,53 and the presence of high NOS activity in some human cancers has been reported.54 Further evidence of the presence of RNS in MN-11 tumors was provided by immunohistochemical analysis for nitrotyrosine; immunoreactivity was observed throughout the tumor, both in the cytoplasm and the nucleus of tumor cells. Nuclear staining has also been observed in human atherosclerotic lesions.45 Specificity of staining was proven by blocking with nitrated bovine serum albumin or nitrotyrosine and reduction of nitrotyrosine to aminotyrosine using sodium hydrosulfite. Neutrophils can produce superoxide, nitric oxide and myeloperoxidase, all of which can indirectly nitrate tyrosine.55 We have recently shown that a nitric oxide-donating drug, glyceryl trinitrate, can induce mutations in Mutatect tumors and that both spontaneous and drug-induced mutations can be inhibited by dietary vitamin E56 (Sandhu JK, Haqqani AS, and Birnboim HC, submitted for publication). Taken together, these data provide strong evidence that RNS/ROS are generated in situ in the MN-11 tumor microenvironment, where they are likely to be mutagenic. Because of the complexity of interactions between these reactive species,31 the pathways responsible for mutations cannot be identified with certainty.
Neutrophils are commonly observed in human malignancies, but are not
thought to be a source of RNS.57
In our limited study, we
noted a correlation between the number of neutrophils and necrosis in
adenocarcinoma of the lung. When these same specimens were stained for
IL-8, most showed reactivity in tumor cells but there was no obvious
correlation of IL-8 with neutrophil content. In bronchioloalveolar
carcinoma,35
IL-8 was found in most specimens but again
its presence was not correlated with neutrophil content. In an study of
human glioblastoma, Desbaillets et al have observed that IL-8
expression was found in hypoxic tumor cells surrounding areas of
necrosis.33
In gastric carcinomas, the presence of IL-8
was correlated with microvessel count.34
A model to depict
some of the events that may be occurring in the complex tumor
microenironment is presented in Figure 5
.
Because tumors typically have very poorly developed blood vessels and
have regions of hypoxia,24
small areas of necrosis may
arise and produce chemotactic factors for neutrophils, which can in
turn generate IL-8.32
Our IL-8 experiments using MN-11
tumors suggest that an influx of neutrophils is associated with
necrosis. Thus, both events can occur: necrosis may recruit neutrophils
and neutrophils may produce additional chemoattractants. We propose
that the poor microvasculature of rapidly growing tumors fosters
hypoxia, necrosis, and microthrombi. These can be a source of
chemotactic factors, promoting the infiltration of neutrophils and
other inflammatory cells. Neutrophil products may in turn cause damage
to the tumor microvasculature and promote this cycle of events. Because
neutrophils are the only known source of ROS and RNS, they appear to be
responsible for the observed high frequency of mutations seen in
Mutatect MN-11 tumors.
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Acknowledgements |
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Footnotes |
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Supported by grant MT-8728 from the Medical Research Council of Canada (to H. C. B.). H. C. B. is a Senior Career Scientist of Cancer Care Ontario.
Accepted for publication October 26, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ala active site mutant recombinant -thrombin. Exp Cell Res 1995, 219:650-656[Medline]
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Md. A. Rahman, D. K. Dhar, E. Yamaguchi, S. Maruyama, T. Sato, H. Hayashi, T. Ono, A. Yamanoi, H. Kohno, and N. Nagasue Coexpression of Inducible Nitric Oxide Synthase and COX-2 in Hepatocellular Carcinoma and Surrounding Liver: Possible Involvement of COX-2 in the Angiogenesis of Hepatitis C Virus-positive Cases Clin. Cancer Res., May 1, 2001; 7(5): 1325 - 1332. [Abstract] [Full Text]
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 A. S. Haqqani, J. K. Sandhu, and H.C. Birnboim Constitutive expression of interleukin-8 by Mutatect cells markedly affects their tumor biology Carcinogenesis, February 1, 2001; 22(2): 243 - 250. [Abstract] [Full Text] [PDF]
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A. Murakami, K. Kawabata, T. Koshiba, G. Gao, Y. Nakamura, K. Koshimizu, and H. Ohigashi Nitric Oxide Synthase Is Induced in Tumor Promoter-sensitive, but not Tumor Promoter-resistant, JB6 Mouse Epidermal Cells Cocultured with Interferon- {{gamma}}-stimulated RAW 264.7 Cells: The Role of Tumor Necrosis Factor-{{alpha}} Cancer Res., November 1, 2000; 60(22): 6326 - 6331. [Abstract] [Full Text]
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J. K. Sandhu, A. S. Haqqani, and H. C. Birnboim Effect of Dietary Vitamin E on Spontaneous or Nitric Oxide Donor-Induced Mutations in a Mouse Tumor Model J Natl Cancer Inst, September 6, 2000; 92(17): 1429 - 1433. [Abstract] [Full Text] [PDF]
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 A. S. Haqqani, J. F. Kelly, and H. C. Birnboim Selective Nitration of Histone Tyrosine Residues in Vivo in Mutatect Tumors J. Biol. Chem., January 25, 2002; 277(5): 3614 - 3621. [Abstract] [Full Text] [PDF]
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