(Received for publication, January 24, 1997, and in revised form, May 8, 1997)
From the Department of Physiology, University of Connecticut
School of Medicine, Farmington, Connecticut 06030, the
Department of Clinical Chemistry and Obstetrics and
Gynecology, Haartman Institute, Helsinki University, Helsinki, Finland,
and the ¶ Holland Laboratory, American Red Cross,
Rockville, Maryland 20855
The cyclooxygenase (Cox) enzyme catalyzes the
rate-limiting oxidative and peroxidative enzymatic steps in the
biosynthesis of prostanoids. Both Cox-1 and -2 genes encode the two
isoenzymes that carry out similar enzymatic steps. Enhanced Cox
activity is associated with proliferative diseases such as colon
cancer. To determine if a cause and effect relationship exists between Cox isoenzyme overexpression and tumorigenesis, the human Cox-1 and
Cox-2 isoenzymes were transfected into ECV immortalized endothelial cells. Although numerous clones of Cox-1 expressing cells were obtained, Cox-2 overexpression resulted in growth disadvantage and
increased cell death. In contrast, Cox-1 overexpressing cells expressed
high levels of the functional Cox-1 polypeptide in the endoplasmic
reticulum and the nucleus. In vitro proliferation of these
cells was reduced compared with vector-transfected ECV cells. Cox-1
overexpression also enhanced the tumor necrosis factor--induced apoptosis of ECV cells 2-fold. In contrast to the in vitro
behavior, ECV-Cox-1 cells proliferated aggressively and formed
tumors in athymic "nude" mice, whereas the vector-transfected
counterparts did not. The growth of Cox-1-induced tumors was not
inhibited by indomethacin, suggesting a nonprostanoid function of
Cox-1. ECV-Cox-1-derived tumors were angiosarcoma-like and contained numerous host-derived neovessels. These data suggest that Cox-1 overexpression in immortalized ECV endothelial cells results in nuclear
localization of the polypeptide and tumorigenesis.
Cyclooxygenase (Cox),1 also known as prostaglandin (PG) H synthase (EC 1.14.99.1), catalyzes the rate-limiting steps in the formation of prostaglandin endoperoxides (1). The Cox enzyme possesses oxygenase and peroxidase activities and thus catalyzes the formation of PGH2 from arachidonic acid (2). Two genes, designated as Cox-1 and -2 encode for the Cox isoenzymes (2). Both isoenzymes exhibit similar enzymatic properties; however, the Cox-1 enzyme is ubiquitously expressed, whereas the Cox-2 isoenzyme is expressed as an immediate-early gene after stimulation by a wide variety of extracellular stimuli (3). Gene knock-out studies have suggested that Cox-1 and -2 isoenzymes possess distinct functions (4-6). In addition, subcellular localization of the Cox isoenzymes exhibits differences. The Cox-2 enzyme is localized in the endoplasmic reticulum (ER) as well as the nuclear envelope, whereas the Cox-1 enzyme is localized primarily in the ER (7). These studies suggest that Cox-1 and -2 isoenzymes possess overlapping as well as distinct functions.
Prostanoids synthesized from both Cox-1 and -2 pathways can function as
extracellular or intranuclear messengers (1, 2). Classical prostanoids
such as PGF2 and PGE2 are secreted extracellularly and mediate their effects via the plasma membrane G-protein-coupled receptors (8). In contrast, PGJ2 and
PGA2 accumulate in the nucleus and activate the peroxisomal
proliferator activator receptor
family of transcription factors
(9). Differential subcellular localization may be important in
determining the nuclear versus extracellular action of
prostanoids. In addition, it is not known if the two isoenzymes can
suffice for each other for nuclear versus extracellular
actions.
The Cox-2 gene is overexpressed in several proliferative pathological
conditions such as rheumatoid arthritis and colon cancer (10-12).
Because chronic usage of nonsteroidal anti-inflammatory drugs such as
aspirin reduce the incidence of colon cancer (13) and because
nonsteroidal anti-inflammatory drugs such as sulindac block
tumorigenesis in experimental murine models of colon cancer (14), it is
assumed that the Cox isoenzymes may play causative roles in tumor
initiation and/or promotion. Prostanoid synthesis is generally enhanced
in tumors (15), and nonsteroidal anti-inflammatory drugs inhibit the
growth of certain well established tumor cell lines in vitro
and in athymic mice (16). However, evidence that the Cox enzymes are
necessary for tumor initiation and/or promotion is recently inferred
from studies in the murine system. For example, mating of tumor
suppressor APC gene knock-out mice, which develop spontaneous
intestinal polyps, with the Cox-2 knock-out mice resulted in the
suppression of polyp formation. In addition, a specific pharmacological
inhibitor of Cox-2 also inhibited polyp formation (17). These studies
strongly suggest that Cox-2 expression is required for the development
of colon cancer. However, even in Cox-2 /
, APC
/
animals,
significant polyp formation was observed, suggesting that Cox-1 is able
to substitute for the function of Cox-2 and participate in the
development of colon cancer. In a recent report, resveratrol, a natural
product found in grapes, was found to be a potent inhibitor of both the
peroxidase and cyclooxygease activities of Cox-1 preferentially (18).
Interestingly, resveratrol inhibited all three phases of
carcinogenesis, namely, initiation, promotion, and progression.
The mechanisms responsible for the functional role of Cox enzymes in tumorigenesis, however, is poorly understood. Overexpression of the Cox-2 enzyme in a gastrointestinal epithelial cell line is associated with the inhibition of apoptosis (19) and altered cell cycle kinetics (20). Because cells resistant to apoptosis may be prone to mutagenic events, inhibition of apoptosis is a potential mechanism for Cox-induced tumorigenesis. However, overexpression of Cox isoenzymes in other cell types promotes rather than inhibits apoptosis (see "Results and Discussion"). Exaggerated and dysregulated angiogenesis may be another mechanism via which Cox isoenzyme overexpression may regulate tumorigenesis. Dysregulated angiogenesis is a major hallmark of tumors and is thought to be necessary for the successful growth of many if not all solid tumors (21). PGE2, a major prostanoid produced by a variety of tumors (15), induces the production of angiogenic growth factors such as vascular endothelial cell growth factor (22) and induces angiogenesis in vivo (23). In this report, we investigated the relationship between the overexpression of the Cox-1 isoenzyme and tumorigenesis.
ECV304 cells (24) (CRL1994) were obtained from American Type Culture Collection (Rockville, MD) and were grown in medium 199 containing 10% fetal bovine serum (Hyclone) and antibiotic/antimycotic mixture (Life Technologies, Inc.). Cells were transfected with an expression vector pCDNA-I-Neo (Invitrogen) containing the 1.8-kilobase open reading frame of human Cox-1 or Cox-2 cDNAs (25). The characterization of Cox-1 and -2 cDNAs and expression of respective polypeptides have been described previously (25, 26). Transfection was achieved by a liposome-mediated method using LipofectamineTM (Life Technologies, Inc.). After transfection, cells were split and selected in growth medium containing 1 mg/ml of G418 (Life Technologies, Inc.). Expression of transgenes was assessed (see below) in pools of transfected cells (ECV-neo and ECV-Cox-1) after 3 weeks of selection. Two independently derived pools of transfected cells (for both Cox-1 and neo) were used in the studies described below.
Cell Growth and Apoptosis in VitroFor cell growth assays,
ECV-Cox-1 and ECV-neo cells (104 cells/well) were plated in
six-well tissue culture plates containing normal growth medium plus
G418. Cell numbers were obtained by trypsinization and counting the
cells in a Coulter counter. To induce apoptosis, subconfluent ECV cells
were treated for 16 h with 100 ng/ml of TNF- (R & D Systems)
and 10 µg/ml of cycloheximide (Sigma). This treatment was shown
previously to induce apoptosis of endothelial cells (27). After 16 h, apoptotic bodies were determined by staining with propidium iodide
and counting under a fluorescence microscope.
Total RNA from ECV-neo or ECV-Cox-1 cells were isolated by guanidinium isothiocyanate/phenol procedure (28). RNA was separated by 1% agarose gel electrophoresis and transferred onto a nylon membrane, and Northern blot analysis was conducted using the radiolabeled Cox-1 probe as described previously (29). Western blot analysis was conducted using the polyclonal Cox-1 antiserum as described before (12).
TLC-Autoradiography ProceduresECV cells were superfused with 12.5 µM [14C]arachidonic acid (NEN Life Science Products) in plain medium 199 for 15 min at 37 °C. Medium was then acidified and extracted, and TLC analysis of prostanoids was conduced using the solvent system Iw as described previously (25). Prostanoids were identified by comparison of Rf values of authentic standards (Cayman Chemical). The TLC plates were then autoradiographed.
Nude Mice StudiesEqual numbers (107) of
ECV-neo and ECV-Cox-1 cells were trypsinized, resuspended in 200 µl
of sterile phosphate-buffered saline, and injected intradermally into
athymic nu/nu
"nude" mice. Tumor volume
was measured at indicated time points (30). Upon completion of the
experiments, mice were inspected for the appearance of metastases in
major organs by gross as well as by histological procedures (see
below).
ECV-Cox-1 cells were grown on glass coverslips and fixed in 10% formalin for 24 h and 70% ethanol for at least 24 h. Cells were then subjected to immunostaining with 1:100 dilution of anti-Cox-1 antibody. This antiserum was determined to be selective for Cox-1 by Western blot procedures of expressed human Cox-1 and -2 polypeptides in Cos-7 cells (8, 10). For immunohistochemistry, antibody staining was visualized using a second antibody coupled to peroxidase (ABC kit, Vector Laboratories). Tumor tissues were dissected, fixed as above, and immunostained with anti-Cox-1 antibody (12) and anti-von Willebrand factor antibody (DAKO Inc.).
The ECV cell line is derived from spontaneous immortalization of human umbilical vein endothelial cells (HUVEC) (24). Although these cells retain several endothelial cell markers, such as induction of E-selectin and the formation of capillary-like tubular structures (24, 29, 31), the growth properties of these cells are significantly different from that of the parental HUVEC. For example, ECV cells proliferate maximally in serum containing medium alone, whereas HUVEC require growth factors such as fibroblast growth factor for survival (24, 27, 29). In addition, ECV cells are immortalized and do not exhibit the phenomenon of in vitro senescence, whereas HUVEC do (24, 27, 31). ECV cells do not express either Cox-1 or -2 genes under basal conditions (29). However, stimulation with phorbol myristic acetate or interleukin-1 results in the dramatic up-regulation of Cox-2 expression and the inhibition of cell growth (29). In contrast, no Cox-1 expression was detected.
Cox-1 and -2 cDNAs in the expression vector pCDNANeo were transfected into ECV cells, and G418-resistant cells were selected. Although numerous G418-resistant colonies (approximately 50 colonies/100-mm dish) were obtained from the Cox-1-transfected ECV cells, very few were obtained from Cox-2 transfection. When assayed for the expression of the transgene by Northern blot analysis, several slow growing ECV colonies expressed low but detectable levels of the Cox-2 transcript. These cells, however, exhibited poor survival in serum-containing medium, even in the presence of Cox inhibitors (10 µM indomethacin or NS-398). Numerous apoptotic cells were observed in these colonies. After several weeks of culture, the expression of Cox-2 was lost and rapid growth of these cells resumed. These studies suggested that Cox-2 overexpression in ECV cells conferred a growth disadvantage. In contrast to intestinal epithelial cells (19), Cox-2 overexpression may induce apoptosis in endothelial cells. These colonies were not further studied. In contrast, robust expression of Cox-1 cDNA was observed in stably transfected ECV cells, and the transfectants were further characterized.
These cells and the vector-transfected counterparts were grown in
medium containing 1 mg/ml of G418. The expression of the transfected
gene was assessed by Northern and Western blot analyses. As shown in
Fig. 1a, high levels of the
polypeptide and the mRNA for Cox-1 was detected in ECV-Cox-1 cells,
whereas the vector-transfected ECV-neo cells did not express detectable
levels of Cox-1 mRNA and protein. The functionality of the
transfected Cox-1 cDNA was assessed by measuring the production of
prostanoids after incubating the cells with
[14C]arachidonic acid. Prostanoids were extracted from
the medium, analyzed by thin layer chromatography, and visualized by
autoradiography as described (25). As shown in Fig. 1a,
significant production of 6-keto-PGF1, a stable
metabolite of prostacyclin and PGF2
and PGE2
as well as several unidentified arachidonic acid metabolites were
observed. Vector-transfected ECV-neo cells did not metabolize exogenous
[14C]arachidonic acid, which is consistent with the
nondetectable expression of the Cox-1 and -2 isoenzymes in these cells.
These data suggest that high level expression of the transfected Cox-1 gene was achieved in ECV cells.
Subcellular Localization of the Transfected Cox-1 Polypeptide
The Cox-1 and -2 polypeptides were shown to be localized in different subcellular compartments in NIH 3T3 cells, transfected Cos cells, and endothelial cells (7). Although the Cox-1 isoenzyme was localized primarily in the ER, the Cox-2 polypeptide was observed in both ER and nuclear compartments (1, 2, 7). Because products of the Cox isoenzymes may have functions extracellularly (8) as well as intranuclearly (9), we characterized the localization of the overexpressed Cox-1 polypeptide in stably transfected ECV cells. Immunohistochemical analysis with the polyclonal antiserum against Cox-1 on ECV-neo cells did not indicate appreciable staining, consistent with nondetectable expression of Cox-1 in these cells. However, high expression of Cox-1 immunoreactivity was seen in cobblestone-shaped ECV-Cox-1 cells (Fig. 1b). Strong immunoreactivity was observed in the nuclear membrane, within the nucleus and the ER. Confocal immunofluorescence microscopy confirmed these results (data not shown). These data suggest that overexpression of Cox-1 in ECV cells resulted in the localization in the ER and the nucleus. The nuclear localization of Cox-1 could be due to high intracellular concentration of this polypeptide, which is achieved by overexpression. Alternatively, it could be due to a characteristic unique to the immortalized nature of the ECV endothelial cells.
In Vitro Growth and Apoptosis of ECV-neo and ECV-Cox-1 CellsTo determine if elevated Cox-1 expression in ECV cells
resulted in the aberrant regulation of growth or apoptosis, we measured the growth rate of ECV-Cox-1 cells and compared it with that of ECV-neo
cells. As shown in Fig. 2a, both
ECV-Cox-1 and ECV-neo cells proliferated; however, the growth of
ECV-Cox-1 cells was reduced (approximately 45%) at day 7. The doubling
times of the ECV-Cox-1 and ECV-neo cells were 23.32 and 23.28 h,
respectively. The growth rates of both cell lines were not modulated
significantly by varying the serum concentration. These data suggest
that Cox-1 overexpression resulted in modest inhibition of in
vitro growth rate of ECV cells.
The effect of Cox-1 overexpression on the apoptosis of ECV cells was
assessed next. Treatment of ECV cells with TNF- and cycloheximide
potently induced apoptosis within 16 h. The rate of apoptosis of
ECV-Cox-1 cells was enhanced approximately 2-fold compared with the
ECV-neo cells (Fig. 2b). These data suggest that Cox-1
overexpression enhances the TNF-induced apoptosis of ECV cells in
vitro. Enhanced apoptosis in ECV-Cox-1 cells perhaps was the
reason why the growth rates of these cells were blunted in
vitro (Fig. 2a). Interestingly, inhibition of
prostaglandin synthesis with 2 µM indomethacin did not
block enhanced apoptosis in ECV-Cox-1 cells, suggesting that the
ability of Cox-1 to promote apoptosis is dissociable from its enzymatic
property of prostaglandin synthesis. Thus, overexpression of Cox-1 in
ECV cells does not dramatically alter the in vitro phenotype
of ECV cells. For example, the morphology of Cox-1 transfected cells
in vitro is indistinguishable from ECV-neo cells in that
both cell lines undergo contact inhibition, fail to grow under
anchorage-independent conditions, and exhibit a cobble-stone morphology
typical of endothelial cells. However, reduction in growth rate and
enhanced response to TNF-
-induced apoptosis was observed.
To test the
tumorigenic potential of Cox-1, ECV-neo and ECV-Cox-1 cells were
injected subcutaneously into the athymic nude mice. Although ECV cells
are immortalized, they are not transformed (24). ECV-neo cells behaved
similarly and did not grow as tumors at the site of injection or at
distant sites. In contrast, ECV-Cox-1 cells grew aggressively as tumors
(Fig. 3a). Large tumors that are
frequently red on the surface grew at the site of injection; however,
distant metastases were not observed. These data suggest that Cox-1
overexpression induces tumorigenic transformation of ECV cells in
vivo. To determine if secretion of prostanoids by the transfected
Cox-1 gene is involved in tumorigenesis, mice were administered
indomethacin in drinking water (14 µg/ml) for the duration of the
experiment. Because mice drink approximately 3-4 ml of water per day,
this translates to a dose of 1-1.4 mg/kg/day, which is effective in
blocking prostanoid synthesis under clinical situations (33). As shown
in Fig. 3b, indomethacin treatment did not inhibit
Cox-1-induced tumorigenesis. These data suggest that the ability of
Cox-1 to induce tumorigenesis is independent of prostanoid
biosynthesis.
The morphology of the tumor as well as expression of the transfected
Cox-1 polypeptide was analyzed by histopathological procedures. As
shown in Fig. 4a, the site of
injection of ECV-neo cells (panels A and B)
indicates the presence of a few ECV cells that are intermingled with
monocytic cells and host-derived fibroblastic capsule engulfing the
injected cells. Many of the ECV-neo cells appear necrotic, and mitotic
bodies were not observed. In contrast, ECV-Cox-1 cells appear highly
malignant (Fig. 4a, panels C and D).
The mass of the tumor is composed of ECV cells and vessel-like
structures. The nuclear morphology of ECV cells appears highly
abnormal, whereas the vessel-like structures contain flat nuclei and
thus may be host-derived. Numerous mitotic and apoptotic bodies were
observed in the ECV-Cox-1 tumors, suggesting a high turnover. In
addition, various degrees of differentiation of ECV-Cox-1 cells were
observed; some were highly mitotic and undifferentiated, whereas some
appeared more differentiated with an adenomatous phenotype. The
ECV-Cox-1 cells in the tumors still express the transfected Cox-1 gene, albeit in a heterogenous manner, as determined by immunohistochemical procedures (Fig. 4a, panel D). Such a pattern of
heterogenous yet exaggerated expression in tumor cells resembles the
pattern of Cox-2 expression in human colorectal cancer (12), a tumor of
epithelial origin.
The histology of the ECV-Cox-1 tumors with respect to the vasculature was characterized next. As shown in Fig. 4b, tumors derived by ECV-Cox-1 cells are highly angiogenic, as evident from the redness of the tumor. The tumor is composed of inflammatory and angiogenic periphery and a solid mass of rapidly proliferating ECV-Cox-1 cells (Fig. 4b). A necrotic center in the center of the tumor can be frequently seen (Fig. 4b). The vessels in the tumor appear to be host-derived because their nuclear architecture is normal and flat, in contrast to the proliferative ECV-Cox-1 cells that possess abnormally shaped nuclei. The vessel-like structures, especially those at the periphery of the tumor are positive for von Willebrand factor expression, a marker for vascular endothelial cells. There are several phenotypes of blood vessels within the ECV-Cox-1 tumor. Several of the angiogenic vessels appear injured and are occluded with thrombi composed primarily of platelets (Fig. 4b, panels D and F). The endothelial cells lining these vessels as well as the platelets stain for the von Willebrand factor antigen. Several parts of the tumor are composed of a solid mass of ECV-Cox-1 cells with an angiogenic periphery (Fig. 4b, panel E). Some of the vessels appear to be abnormally shaped and are sinusoidal in nature (Fig. 4b, panel F). These data suggest that ECV-Cox-1 cells induced enhanced angiogenesis of the host and thus allowed rapid proliferation of the tumor. In addition, secretion of high levels of vasoactive and thrombotic prostanoids may have induced thrombosis and vascular injury.
It is not known if extracellular or intranuclear action of Cox-1 is responsible for the enhanced angiogenesis and tumorigenesis. Indeed, PGE2, which is produced by the Cox-1 pathway is a potent inducer of angiogenesis (23). PGE2 is known to up-regulate vascular endothelial growth factor expression and thereby regulate angiogenesis (22). Alternatively, exaggerated expression of Cox-1 may allow a nuclear signaling event to take place in ECV cells that would express angiogenic factors and/or down-regulate angiogenic suppressors (32). However, because indomethacin treatment did not reverse the enhanced apoptosis in vitro (Fig. 2b and data not shown) and tumorigenesis in vivo (Fig. 3b), the observations in this study may relate to the nonprostanoid-mediated function of Cox-1. Further studies are needed to address such mechanisms.
In conclusion, we have shown that high level expression of Cox-1 in ECV cells, which normally do not express neither Cox-1 or -2 isoenzymes, results in nuclear and ER localization of functional Cox-1 polypeptide. Cox-1 overexpression induced tumorigenesis and exaggerated angiogenesis in vivo. Such mechanisms may be important in Cox-induced tumorigenesis in vivo. In addition, this system may be useful to further dissect the mechanisms involved.
We thank Carolyn Hue for expert technical assistance and Tom Maciag for support and encouragement.