COX-2 expression and cell cycle progression in human fibroblasts

Derek W. Gilroy1,2, Michael A. Saunders1, and Kenneth K. Wu1,2

1 Vascular Biology Research Center and Division of Hematology, University of Texas-Houston Medical School, Houston, Texas 77030; and 2 Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cyclooxygenase-2 (COX-2) is continuously expressed in most cancerous cells where it appears to modulate cellular proliferation and apoptosis. However, little is known about the contribution of transient COX-2 induction to cell cycle progression or programmed cell death in primary cells. In this study we determined whether COX-2 regulates proliferation or apoptosis in human fibroblasts. COX-2 mRNA, protein, and prostaglandin E2 (PGE2) were not detected in quiescent cells but were expressed during the G0/G1 phase of the cell cycle induced by serum. Inhibition of COX-2 did not alter G0/G1 to S phase transition or induce apoptosis at concentrations that diminished PGE2. Addition of interleukin-1beta to serum enhanced COX-2 expression and PGE2 synthesis over that by serum alone but had no effect on the progression of these cells into S phase. Furthermore, platelet-derived growth factor drove the G0 fibroblasts into the cell cycle without inducing detectable levels of COX-2 or PGE2. Collectively, these data show that transient COX-2 expression in primary human fibroblasts does not influence cell cycle progression.

serum; platelet-derived growth factor; interleukin-1beta ; proliferation; apoptosis; cyclooxygenase-2


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CYCLOOXYGENASE (COX) metabolizes arachidonic acid to prostaglandins (PGs) and thromboxane (20) and exists in two isoforms (23). Constitutively expressed COX-1 is present in most tissues where it synthesizes PGs continuously to maintain physiological functions (23, 29). In contrast, COX-2 is induced by proinflammatory stimuli, cytokines, and mitogens and synthesizes a large quantity of PGs (8, 10, 12). COX-2 is crucial to the inflammatory response (4, 28), and nonsteroidal anti-inflammatory drugs (NSAIDs) selective for its inhibition are now used clinically to treat inflammatory arthropathies. However, evidence is now emerging to show that COX-2 is also involved in tumorigenesis. For instance, COX-2 is constitutively expressed in various cancerous cells and tissues (13, 24), while in APCDelta 716 mice, a model of familial adenomatous polyposis bearing a COX-2 deletion, there was a dramatic reduction in intestinal polyp size and number (16). In the same model, mice treated with a selective COX-2 inhibitor had a reduced polyp formation (19). Indeed, epidemiological studies have demonstrated a 50% reduction in the rate of mortality from colorectal cancer in patients taking NSAIDs (6).

It has been shown that forced COX-2 overexpression arrests rat intestinal epithelial cells at G1 and confers resistance to apoptosis (5). COX-2 overexpression in ECV-304 cells is associated with a decrease in cells in S phase and an accumulation in G0/G1, while in COS-7 and human embryonic kidney 293 cells there is an increase in cells in G2/M (26). Additionally, H-ras-transfected rat intestinal epithelial cells constitutively overexpressing COX-2 traverse the cell cycle in the absence of serum and show significantly reduced proliferation after treatment with a selective COX-2 inhibitor (21). Collectively, these results suggest that continuous COX-2 expression causes phenotypic changes typical of cancerous cells by regulating, at least in part, cell cycle progression. However, despite the mounting evidence that pathological overexpression of COX-2 enhances proliferation and reduces apoptosis in cancerous or transformed cells, comparatively little is known about the role of transient COX-2 expression in primary cells. Therefore, in the present study we examined whether COX-2 regulates G0/G1 to S phase transition and programmed cell death in human foreskin fibroblasts (HFF), a well-characterized model for cell cycle studies. Our results show that COX-2 expression after serum stimulation was restricted to the G0/G1 phase of the cell cycle and that its inhibition did not alter G0/G1 to S transition or result in programmed cell death.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. HFF were obtained from American Type Culture Collection (Manassas, VA) and cultured on 10-cm plates in DMEM supplemented with 10% fetal bovine serum (FBS), 100 µg/ml streptomycin, and 100 U/ml penicillin. For all experiments, cells were washed twice with PBS and incubated in FBS-free medium for 24 h. FBS-free medium was replaced with medium containing 10% FBS to initiate the cell cycle. All tissue culture reagents were from Life Technologies. To evaluate the effects of COX inhibitors on cell cycle progression, NS-398 (Calbiochem), indomethacin, aspirin, or sodium salicylate (Sigma) was added with 10% FBS to G0 cells in culture.

Cell cycle analysis. The cell cycle of the 24-h serum-starved HFF was initiated by adding 10% FBS to fresh medium. At indicated time points, cells were trypsinized, washed in ice-cold PBS, and fixed using ice-cold acetone-free methanol (methanol:PBS ratio was 2:1). Cellular DNA was stained with 0.01% propidium iodide (Calbiochem) in PBS containing 0.1% Triton X-100 and 0.037% EDTA followed by the addition of 100 U/ml RNase (Worthington Biochemical) to remove RNA. Samples were filtered through a 35-µm nylon mesh, and the percentage of cells in sub-G0/G1, G0/G1, S, and G2/M phase was assessed using flow-assisted cell sorting (FACS) analysis (Becton Dickinson). Data were analyzed using ModFit software (Verity software).

Northern blotting. RNA was isolated from cultured cells by using RNA-STAT 60 (TEL-TEST; Friendswood, TX). RNA (25-30 µg) was fractionated on 1% agarose and was transferred to a positively charged nylon membrane. As a COX-2 probe, agarose gel-purified, full-length, 1.9-kb COX-2 cDNA was used (31). Hybridization and detection by autoradiography were performed according to a procedure previously reported (31).

Western blotting. Cell pellets were lysed with PBS (pH 7.4) containing 0.1% Triton X-100, 0.01% EDTA, 1 mM phenylmethylsulfonyl fluoride, 1.5 mM pepstatin A, and 0.2 mM leupeptin. Lysates were centrifuged at 13,000 rpm for 10 min. The supernatants were boiled for 5 min with equal volumes of 2× gel-loading buffer (100 mM Tris, 10% mercaptoethanol, 20% glycerol, 4% SDS, 2 mg/ml bromphenyl blue). The protein concentration of the supernatant was determined by the bicinchoninic acid assay method (Pierce Chemical). Protein (5 µg) was applied to and separated on 10% SDS-polyacrylamide minigels (Hoefer Scientific Instruments) using the Laemmli buffer system and transferred to polyvinylidine difluoride membranes (Amersham Pharmacia Biotech). Nonspecific IgGs were blocked with 5% nonfat dried milk containing 1 mg/ml globulin-free bovine serum albumin and incubated with specific antibodies to COX-1 and COX-2 (both 1:1,000; Santa Cruz Biotechnology). Protein bands were detected using enhanced chemiluminescence (Amersham Pharmacia Biotech).

COX activity. Because PGE2 is the major metabolite of COX enzyme catalysis in fibroblasts, we measured its levels in the culture medium by an enzyme immunoassay detection kit (Amersham Pharmacia Biotech).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

COX-2 and COX-1 expression during serum-driven cell cycle progression. We have recently shown by flow cytometry that, after FBS deprivation for 24 h, 90% HFF were in the G0/G1 phase of the cell cycle, which entered into S phase 16 h after the addition of 10% FBS, and, by 24 h, >50% of cells were in S phase (7). These results are consistent with previous reports that the vast majority of HFF cultured in medium deprived of serum are quiescent (G0) (17). COX-2 mRNA was undetectable in quiescent cells, and, after addition of FBS, low levels were measurable at 1 and 2 h, and the level peaked at 4 h (Fig. 1A). COX-2 mRNA levels declined thereafter, becoming barely detectable at 12 h. The COX-2 protein was also expressed in a time-dependent manner after serum addition. It peaked at 6-8 h after serum treatment and declined thereafter (Fig. 1B). In contrast, COX-1 protein was constitutively expressed in quiescent cells, increased slightly after the addition of FBS, peaked at 12 h, and remained at the same level up to 32 h (Fig. 1C). Thus COX-1 is involved in a housekeeping function during cell cycle progression, while COX-2 expression, which coincides with G1 phase of the cell cycle, could play a role in cell cycle regulation.


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Fig. 1.   Time course of cyclooxygenase (COX) isoform expression after serum stimulation of quiescent human foreskin fibroblasts (HFF). A: COX-2 mRNA. B: COX-2 protein. C: COX-1 protein. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA blotting was used as an internal control. Representative blots from 4 experiments are shown.

PGE2, the main metabolite of COX in fibroblasts, was undetectable in serum-starved cells. After addition of FBS, it became detectable at 4 h and reached a plateau at 12 h (Fig. 2A). The accumulated PGE2 levels in the media changed very little thereafter. To determine the relative contributions of COX-2 and COX-1 to PGE2 synthesis by serum stimulation, quiescent cells were treated with a selective COX-2 inhibitor, NS-398, concomitant with FBS stimulation. At 10-7 M, NS-398 inhibited PGE2 synthesis by >95% up to 24 h (Fig. 2A). Indomethacin (10-6 M), a dual COX-1/COX-2 inhibitor, also caused a marked inhibition of PGE2 synthesis (Fig. 2B). These results indicate that, after initiation of the cell cycle by FBS, COX-2 was responsible for synthesizing large quantities of PGE2 during the G1 phase of the cell cycle.


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Fig. 2.   Effects of COX inhibitors on PGE2 release from serum-stimulated quiescent HFF. Cells were arrested in G0 by serum deprivation for 24 h followed by stimulation with 10% fetal bovine serum (FBS). Effects of NS-398 (A; 10-7 M) and indomethacin (B; 10-6 M) on COX activity were assessed by measuring PGE2 levels in the culture medium. Both drugs were added to the medium at the same time as FBS and were dissolved in DMSO, which, when used as a control, had no effect on PGE2 synthesis (data not shown). Data representing means ± SE from 4 experiments are shown. Open and filled bars denote absence and presence of an inhibitor, respectively.

Effect of COX inhibitors on cell cycle progression and apoptosis. Expression of COX-2 and PGE2 synthesis in the G0/G1 phase of the cell cycle implicates a role for COX-2 in regulating G0/G1 to S transition. To discern this, we treated quiescent cells with NS-398 at the same time as FBS stimulation. Despite an almost complete obliteration of PGE2 synthesis by NS-398 (Fig. 2A), proportions of cells that progressed through G1 and entered S phase were unaltered by this selective COX-2 inhibitor compared with the controls (Fig. 3A). Because indomethacin was previously reported to arrest cells in G1 (1), we also treated quiescent cells with this dual COX inhibitor in the presence of FBS but observed no change in the percentages of cells in G0/G1 compared with controls (Fig. 3B). Western blot analysis revealed that neither NS-398 nor indomethacin inhibited COX-2 protein expression (data not shown). We also evaluated the effect of aspirin, which was shown previously to inhibit COX-2 mRNA and protein levels in these cells (31), on cell cycle progression. Aspirin pretreatment had no significant effect on altering percentages of cells entering the S phase. Similarly, sodium salicylate (10-4 M) did not change cell cycle progression (data not shown).


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Fig. 3.   Effects of COX inhibitors on the progression of cells through G0/G1 and into S phase. HFF cells were arrested in G0 by serum deprivation for 24 h followed by stimulation with 10% FBS. Effects of NS-398 (A; 10-7 M) and indomethacin (B; 10-6 M) on the percentage of cells in G1 were determined by flow-assisted cell sorting and compared with controls (open circle ). Both drugs were added to the medium at the same time as FBS and were dissolved in DMSO, which, when used as a control, had no effect on cell cycle progression (data not shown). Data representing means ± SE from 4 experiments are shown. open circle , No treatment; , NS-398 treatment; , indomethacin treatment.

Evidence obtained from various transformed and cancerous cell lines suggests that COX-2 can arrest cells in either the S or G2/M phases of the cell cycle (26) in addition to protecting against apoptosis (27). Therefore, we carried out experiments with both NS-398 (10-7 to 10-9 M) and aspirin (10-4 to 10-7 M) and found that COX-2 inhibition did not arrest HFF at any phase of the cell cycle or induce programmed cell death. Data representing the highest doses of each drug are shown in Fig. 4. Apoptosis was determined by the presence/absence of a sub-G0/G1 peak using propidium iodide staining by FACS analysis. For these experiments, apoptosis and cell cycle analysis were carried out at a number of critical time points after serum stimulation representing mid-G1 (8 h), initiation of S phase (16 h), maximal S phase (24 h), and completing a cell cycle (32 h), when cells had traversed the cell cycle and begun to reenter G1 again. Neither drug exerted a significant effect on apoptosis or cell cycle arrest (Fig. 4). Taken together, these results indicate that the transient expression of COX-2 and resultant PGE2 synthesis at the G1 phase of the cell cycle did not regulate G1 to S transition, apoptosis, or progression of cells through either S or G2/M phases.


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Fig. 4.   Effects of NS-398 (10-7M) or aspirin (10-4M) on apoptosis (sub-G0/G1) and cell cycle progression in serum-starved HFF stimulated with 10% FBS. Cells at sub-G0/G1, G0/G1, S, and G2/M at selected time points (8, 16, 24, and 32 h) after addition of FBS were determined by flow cytometry. NS-398, aspirin, and vehicle control (DMSO) were added 30 min before addition of serum. Data representing means ± SE for 3 experiments are shown. *Sub-G0/G1 that were undetectable at all time periods. Open bars, G0/G1 phase; solid bars, S phase; hatched bars, G2/M phase.

Effects of interleukin-1beta on COX-2 induction, PGE2 synthesis, and cell cycle progression induced by serum. To examine further the role of COX-2 in fibroblast proliferation, we treated quiescent HFF with a combination of serum and the proinflammatory cytokine interleukin-1beta (IL-1beta ; 1 nM), a well-known inducer of COX-2 and PGE2 synthesis. It was found that, while this combination of stimuli caused a sustained increase in COX-2 protein expression (Fig. 5A) and PGE2 synthesis (Fig. 5B) over and above that of serum alone during G0/G1, there was no corresponding alteration in cell cycle progression into S phase (Fig. 5C).


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Fig. 5.   Effects of serum and interleukin-1beta (IL-1beta ) on COX-2 expression, PGE2 synthesis, and the transition of HFF into S phase. Cells were arrested in G0 by serum starvation for 24 h. Thereafter, IL-1beta (1 nM) or vehicle was added with 10% FBS to G0 cells. A: time course of COX-2 protein stimulated by IL-1beta  + 10% FBS. COX-2 proteins determined by Western blots are shown on top. Densitometry of 4 experiments is shown on bottom. Each bar is mean ± SE. B: PGE2 levels in the cultured medium produced by cells treated with 10% FBS + IL-1beta () vs. 10% FBS alone (open circle ). C: percentage of G0/G1 cells after treatment with 10% FBS plus IL-1beta () vs. 10% FBS alone (open circle ).

Platelet-derived growth factor drove the cell cycle without inducing COX-2 expression. Platelet-derived growth factor (PDGF) is known to drive quiescent cells into the cell cycle. We determined whether PDGF-driven cell cycle progression was similarly accompanied by COX-2 expression as serum. To our surprise, despite a similar time course of cell cycle progression as serum (Fig. 6A), PDGF at a submaximal concentration (1 ng/ml) did not induce COX-2 protein expression during the 24-h period (Fig. 6B) or increase PGE2 synthesis (Fig. 6C), in contrast to the reported data of COX-2 induction by PDGF at a higher concentration (10 ng/ml) (30). Our results with submaximal concentrations of PDGF further support the notion that COX-2 expression is not essential for cell cycle progression.


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Fig. 6.   Submaximal concentration (1 ng/ml) of platelet-derived growth factor (PDGF) drove the cell cycle (A) but did not induce COX-2 protein expression (B) or PGE2 production (C). black-triangle, PDGF-treated cells; open circle , serum-treated cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we determined COX-2 expression during synchronized cell cycle progression by using a fibroblast model that has been widely used to characterize the cellular and molecular events and control mechanisms of the cell cycle (17). We used FBS and PDGF to drive quiescent cells into the cell cycle, and the results show a striking difference in COX-2 expression. COX-2 is expressed in the G1 phase of FBS-driven cells, whereas, in PDGF-driven cell cycle progression, COX-2 protein was undetectable up to 32 h after PDGF treatment. These results suggest that COX-2 expression is not an integral part of cell cycle progression but rather is induced in G0 cells by FBS, which is coincidental with the G0 phase of the cell cycle. It is possible that cell cycle progression and COX-2 expression are induced by different factors in FBS. IL-1beta greatly increases COX-2 expression and PGE2 production induced by serum but has no effect on serum-driven cell cycle progression, despite the production of large quantities of PGE2. We have recently shown that addition of IL-1beta alone without FBS to quiescent fibroblasts does not drive the quiescent cells into the cell cycle, and yet it induces COX-2 expression (7). These results support the notion that cells in G0 phase are highly responsive to exogenous stimuli in COX-2 expression. Taken together, these results suggest that COX-2 expression is not inherently tied to cell cycle progression but is an important property of G0 cells in response to exogenous stimuli including serum, IL-1beta , and phorbol esters.

Our results further suggest that COX-2 expression is not essential for cell cycle progression. Selective inhibition of COX-2 with NS-398 obliterates almost the entire PGE2 synthesis without changing the duration of G1 and the proportion of cells that enter the S phase of the cell cycle. Aspirin, which inhibits COX-1/COX-2 activities and suppresses COX-2 expression (31), also has no effect on the cell cycle. This is further supported by cell cycle progression induced by PDGF despite a lack of COX-2 induction or PGE2 synthesis. Thus our results are in striking contrast to those from persistent transfection of COX-2, which causes cell cycle arrest. One possible explanation for the difference is that persistent COX-2 overexpression during the S and G2/M phases of the cell cycle causes DNA damage, which signals cell cycle arrest. It has been reported that proliferative cells are more susceptible to DNA damage and mutation (2). COX-2 overexpression is accompanied by generation of metabolites, including malondialdehyde, which causes oxidative stress to DNA (18), and inhibition of COX-2 by nebutame in asynchronized cells, which reduces the level of DNA oxidation (25). We have recently observed that that COX-2 expression in response to phorbol 12-myristate 13-acetate and IL-1beta is subdued in proliferative fibroblasts compared with quiescent cells (7). These results suggest that COX-2 expression is controlled in a cell cycle-dependent manner in normal cells. Under the control mechanism, COX-2 is transiently expressed predominantly in cells at quiescent or early G1 of the cell cycle. Work is in progress to elucidate the control mechanism.

It was reported that indomethacin induces G1 arrest and causes apoptosis in both COX-2-positive and COX-2-negative cell lines (22), as well as induces apoptosis in transformed murine fibroblasts in the absence of both COX isoforms (32). Indomethacin has been shown to bind and activate peroxisome proliferator-activated receptor-gamma (PPARgamma ) (14), a nuclear receptor that, when activated in human colorectal cancer cell lines, causes G1 arrest and apoptosis (3). However, in the present study, indomethacin had no effect on either cell cycle progression or apoptosis in nontransformed primary HFF. It may be speculated that activation of the PPARgamma is also without effect on cellular proliferation and programmed cell death in HFF. Thus there are striking differences in response to indomethacin between normal and transformed cells. These differences may reflect derangements of the endogenous control of COX-2-dependent and COX-2-independent cell cycle arrest, apoptosis resistance, and cell proliferation in transformed and cancer cells, in contrast to a tight control of COX-2 expression in normal cells. Transient expression of COX-2 during G0/G1 phase of the cell cycle by serum is likely to play a physiological role such as wound healing. It has been shown by microarray that several genes including COX-2, transforming growth factor-beta (TGF-beta ), and metalloproteinase-1 (MMP-1) are expressed in mid G0/G1 in serum-stimulated quiescent human dermal diploid fibroblasts (9). COX-2 and PGE2 have been reported to modulate the expression of TGF-beta (11) and MMP-1 (15) in asynchronous cells. However, we did not observe such modulation in our synchronized fibroblast model, since NS-398 at doses reducing PGE2 synthesis by >95% had no effect on TGF-beta or MMP-1 levels stimulated by serum (data not shown). Our results suggest that serum stimulates quiescent cells to express COX-2 and several other genes that act in concert to promote wound healing and other pathophysiological processes.


    ACKNOWLEDGEMENTS

We thank Dr. Jeffrey J. Yen for assistance in cell cycle analysis and in the preparation of this manuscript.


    FOOTNOTES

This work was supported by National Institutes of Health Grants P50 NS-23327 and R01 HL-50675 (to K. K. Wu) and by Taiwan Academia Sinica.

Address for reprint requests and other correspondence: K. K. Wu, Vascular Biology Research Center and Division of Hematology, Univ. of Texas-Houston Medical School, 6431 Fannin St., MSB 5.016, Houston, TX 77030 (E-mail: Kenneth.K.Wu{at}uth.tmc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 27 November 2000; accepted in final form 21 February 2001.


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RESULTS
DISCUSSION
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