Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M503245200 on July 26, 2005

J. Biol. Chem., Vol. 280, Issue 37, 32379-32388, September 16, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
280/37/32379    most recent
M503245200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Marwaha, V.
Right arrow Articles by Goukassian, D. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Marwaha, V.
Right arrow Articles by Goukassian, D. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

T-oligo Treatment Decreases Constitutive and UVB-induced COX-2 Levels through p53- and NF{kappa}B-dependent Repression of the COX-2 Promoter*{boxs}

Vaneeta Marwaha{ddagger}1, Ya-Hui Chen{ddagger}12, Elizabeth Helms{ddagger}, Simin Arad{ddagger}, Hiroyasu Inoue§, Evelyn Bord¶, Raj Kishore¶, Raffi Der Sarkissian||, Barbara A. Gilchrest{ddagger}3, and David A. Goukassian{ddagger}4

From the {ddagger}Departments of Dermatology and ||Division of Facial Plastic and Reconstructive Surgery, Boston University School of Medicine, Boston, Massachusetts 02118, §Nara Women's University, Nara 630-8506, Japan and Division of Cardiovascular Research, St. Elizabeth's Medical Center, Boston, Massachusetts 02135

Received for publication, March 24, 2005 , and in revised form, July 14, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronically irradiated murine skin and UV light-induced squamous cell carcinomas overexpress the inducible isoform of cyclooxygenase (COX-2), and COX-2 inhibition reduces photocarcinogenesis in mice. We have reported previously that DNA oligonucleotides substantially homologous to the telomere 3'-overhang (T-oligos) induce DNA repair capacity and multiple other cancer prevention responses, in part through up-regulation and activation of p53. To determine whether T-oligos affect COX-2 expression, human newborn keratinocytes and fibroblasts were pretreated with T-oligos or diluent alone for 24 h, UV-irradiated, and processed for Western blotting. In both cell types, T-oligos transcriptionally down-regulated base-line and UV light-induced COX-2 expression, coincident with p53 activation. In fibroblasts with wild type versus dominant negative p53 (p53WT versus p53DN), T-oligos decreased constitutive expression of a COX-2 reporter plasmid by >50%. We then examined NF{kappa}B, a known positive regulator of COX-2 transcription. In p53WT but not in p53DN fibroblasts and in human keratinocytes, T-oligos decreased readout of an NF{kappa}B promoter-driven reporter plasmid and decreased NF{kappa}B binding to DNA. After T-oligo treatment and subsequent UV irradiation, binding of the transcriptional co-activator protein p300 to NF{kappa}B was decreased, whereas binding of p300 to p53 was increased. Human skin explants provided with T-oligos had markedly decreased COX-2 immunostaining both at base-line and post-UV light, coincident with increased p53 immunostaining. We conclude that T-oligos transcriptionally down-regulate COX-2 expression in human skin via activation and up-regulation of p53, at least in part by inhibiting NF{kappa}B transcriptional activation. Decreased COX-2 expression may contribute to the observed ability of T-oligos to reduce photocarcinogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nonmelanoma skin cancer accounts for well over 1 million cases of human malignancy annually in the United States, and the incidence continues to rise (1–3). The major initiator and promoter of skin cancer is UVB radiation (4, 5). Among the contributing effects of UVB radiation on skin are the formation of cyclobutane-pyrimidine dimers and pyrimidine (6-4) photoproducts (6, 7), which lead to mutations in key regulatory genes (8), epidermal hyperplasia (9, 10) allowing for expansion of mutated clones (11), immunosuppression (12, 13), and inflammation (14, 15).

One way inflammation in particular is thought to affect carcinogenesis is by promoting epidermal hyperplasia and proliferation through production of cytokines and various second messengers such as prostaglandin E2 (16). The major enzyme responsible for the UVB-induced prostaglandin synthesis is cyclooxygenase-2 (COX-2),5 the inducible isoform of the cyclooxygenase enzyme (17) that carries out the rate-limiting step of prostaglandin and thromboxane production (18–20). COX-2 has been shown to be overexpressed in numerous human malignancies, including colon, lung, and breast cancers (21–24). In relation to skin cancer, UVB irradiation increases both mRNA and protein levels of COX-2 in human keratinocytes (25). Recent studies have shown increased COX-2 expression in human skin in response to acute UVB exposure as well as increased COX-2 expression in human and murine tumors that were induced by chronic UVB exposure (26, 27). Furthermore, specific inhibitors of Cox-2 such as celecoxib have been shown not only to decrease tumorigenesis and increase tumor latency in hairless mice models (28) but also to decrease tumor growth in hairless mice with pre-existing UVB-induced tumors (29). In addition, Cox-2-overexpressing transgenic mice have shown dramatic increase in predisposition to tumor development in tumor promotion studies (30).

Given the evidence implicating COX-2 in tumorigenesis and tumor maintenance, methods to decrease COX-2 levels in response to UVB irradiation are currently being investigated as a promising means of cancer prevention. Known inhibitors of COX-2 include estrogens, antioxidants, and p53 (31–33). The presence of active p53 in particular has been shown to decrease both the mRNA and protein levels of Cox-2 in mouse embryo fibroblasts (33). In a study of head and neck squamous cell carcinomas (SCCs), tumors with mutated p53 showed higher COX-2 protein levels than tumors expressing wild type (WT) p53 (34). Evidence thus suggests that activating p53 and thereby reducing COX-2 expression in the absence of DNA damage might decrease photocarcinogenesis and inhibit growth of established tumors.

In mammalian cells, telomeres are tandem repeats of a short DNA sequence, 5'-TTAGGG-' and its complement that cap chromosome ends and form a large loop structure (35). The loop is held closed by an ~100–400-base single-stranded 3'-overhang that inserts into the proximal double-stranded telomere and is secured by binding proteins, particularly telomere repeat binding factor (TRF2) (36). Disruption of this loop structure by sequestration of the binding protein with a dominant negative construct (TRF2DN) leads to exposure of the 3'-overhang sequence (repeats of TTAGGG), digestion of the overhang, and signaling through ATM and p53 that induces DNA damage responses (37). We have found that providing cultured cells with DNA oligonucleotides substantially homologous to the telomere sequence, which we term T-oligos, mimics telomere loop disruption, leading to ATM/p53 signaling (38) and the DNA damage-like responses of senescence (39) or apoptosis (40) but apparently without disruption of the loop or other effects on genomic DNA (40). Because these 2–11-base T-oligos concentrate rapidly in the nucleus (40, 41), we hypothesize that T-oligos are interpreted by the cell as indicating telomere loop disruption, specifically exposure of the otherwise concealed 3'-overhang sequence (38–40), and hence initiate signaling for DNA damage-like responses without antecedent DNA damage.

T-oligos with 100% telomere homology, delivered at a sufficiently high dose, elicit predominantly "end point" cancer protective responses such as apoptosis or proliferative senescence that remove cells, particularly malignant cells, from the proliferative pool (40, 42, 43). However, it is also possible to provide cells with a less than maximal DNA damage signal by reducing the T-oligo dose and/or employing a shorter or less telomere-homologous oligonucleotide. Under these circumstances, it is possible to observe transient reversible cell cycle arrest (44), increased melanogenesis in pigment cells and intact skin (44–46), release of immunomodulatory cytokines from keratinocytes associated with abrogation of allergic contact sensitization or elicitation in intact skin (47, 48), enhanced rate and accuracy of DNA repair both in vitro and in vivo (49–51), and decreased mutation rate and tumor development in vivo (51). Moreover, malignant cells appear to undergo apoptosis or senescence more readily in response to a given T-oligo at a given dose than do their normal nontransformed cellular counterparts (42, 43).

In the present study, we show that treatment with either of two oligonucleotides with partial telomere homology decreases constitutive and UVB-induced COX-2 levels in cultured human fibroblasts, human skin explants, and intact murine skin. These responses are shown to occur at least in part through up-regulation and activation of p53, leading to transcriptional repression of COX-2 promoter activity. We propose that in addition to the previously reported protective DNA damage responses, treatment with T-oligos may also decrease the cutaneous inflammatory response through inhibition of COX-2 expression, a possible additional means of reducing photocarcinogenesis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Primary human neonatal fibroblast and keratinocytes cultures were established as described (49, 50). Cells were incubated at 37 °C in 5% CO2. Cell lines permanently (retroviral transfection) expressing WT p53 (R2FWT) or dominant negative p53 (R2FDD) (52–54) were the generous gift from Dr. Jim Rheinwald (Department of Dermatology, Harvard Skin Disease Research Center, Harvard Medical School, Boston) and were maintained in R2F medium containing 42.5% Dulbecco's modified Eagle's medium, 42.5% F-12, 15% calf serum, and 0.1% epidermal growth factor at 37 °C in 5% CO2.

Oligonucleotides—Previous experiments have shown that 100 µM of thymidine dinucleotide (pTT), representing one-third of the telomere repeat, and 40 µM of pGAGTATGAG (p9-mer), a 55% homologous sequence, are roughly bioequivalent concentrations for the elicitation of UV mimetic responses (49, 50, 55, 56), including p53 up-regulation and activation. Oligonucleotides (pTT and p9-mer) were synthesized with phosphodiester linkage by Midland Certified Reagent (Midland, TX) and diluted in H2O to form a 2 mM stock. This stock solution was then diluted in the appropriate culture medium to 100 or 40 µM, respectively, and added to culture dishes for use in experiments. Cells and skin explants were provided T-oligos only once at time 0 and then harvested at intervals, according to the design of the specific experiment. All experiments were conducted using both pTT and p9-mer and gave identical results with either T-oligo.

UVB Irradiation—After 48 h of incubation in medium containing pTT, p9-mer, or diluent alone, cells or skin explants were placed in phosphate-buffered saline and irradiated through the plastic culture dish cover by using a solar simulator (Spectral Energy Corp., Westwood, NJ). The 1-kilowatt xenon arc lamp (XMN-1000-21; Optical Radiation Corp., Azuza, CA) irradiance was adjusted to 5 x 10-5 watts/cm2, and dishes were exposed to 15 mJ/cm2 as measured with a research radiometer fitted with a UV light probe at 285 ± 5 nm (model IL1700 A; International Light, Newburyport, MA) (56, 57), a protocol that exposes cells to a spectrum of light resembling terrestrial sunlight (58). Sham-irradiated cultures were handled identically, except that they were shielded with aluminum foil during irradiation. After irradiation, cells were given fresh medium lacking T-oligos.

Western Blot Analysis—Total cellular proteins were collected as described previously (46). Concentrations were determined by the BioRad method, and 50 µg of protein were run in each lane on a 10% denaturing SDS-polyacrylamide gel. Proteins were then transferred to a nitrocellulose membrane. Antibody reactions were performed with the following antibodies: phospho-p53Ser15 (Cell Signaling Technology, Beverly, MA), p53 DO-1, COX-2, NF{kappa}B/p65, and actin (all from Santa Cruz Biotechnology, Santa Cruz, CA). Western blot analysis was then performed as described (46).

Electrophoretic Mobility Shift Assays—Electrophoretic mobility shift assays (EMSAs) using consensus p53 and NF{kappa}B oligonucleotides (Santa Cruz Biotechnology) and 5 µg of nuclear protein from variously treated cells were carried out as described previously (59). Reactions were electrophoresed on 5% nondenaturing polyacrylamide gels, dried, and processed for autoradiography. For competition experiments, 50–100-fold excess of unlabeled DNA were added to the reaction 20 min before the addition of radiolabeled probe.

Transfection Studies—Constructs containing the full-length phPES2(-1432/+59) human Cox-2 promoter, a deletion construct of phPES2(-327/+59), and an NF{kappa}B binding region site-specific mutant of phPES2(-327/+59) attached to a luciferase reporter (60, 61) or an NF{kappa}B reporter plasmid (Promega Corp., Madison, WI) were employed. The pGL2 vector used for cloning the reporter construct was obtained from Promega (pGL2-Basic, Promega Corp., Madison, WI) and was used as an empty vector control. A plasmid containing Renilla luciferase (pRL-CMV, Promega Corp., Madison, WI) was co-transfected as a control for transfection efficiency. R2FWT and R2FDD cells were plated in 35-mm tissue culture dishes and incubated in R2F medium overnight to reach 50–60% confluence the next day. Cells were then transfected using the Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Two µg of plasmid DNA were co-transfected with 0.15 µg of the Renilla luciferase plasmid in each dish. After 4–6 h of transfection, cells were supplemented either with R2F medium alone (diluent) or R2F medium with 40 µM p9-mer or 100 µM pTT. Cells were then incubated for 24 and 48 h at 37 °C in 5% CO2 before harvesting for dual-luciferase assay. Promoter activity was then assayed using a dual-luciferase reporter assay system (Promega Corp., Madison, WI) according to the manufacturer's protocol. Firefly luciferase values were corrected for transfection efficiency according to Renilla luciferase values and revealed minimal differences in transfection efficiency among dishes. Luciferase activity was then expressed as percent of diluent value, setting diluent as 100%.



View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 1.
T-oligo pretreatment up-regulates and activates p53 and down-regulates constitutive and UV light-induced COX-2 expression. Human fibroblasts were pretreated once with diluent or T-oligo (p9-mer 40 µM; pTT data not shown) for 48 h. a, paired dishes were then sham-irradiated and harvested after 8 and 24 h and processed for Western blot analysis to evaluate the expression of COX-2, p53total, p53Ser15, and actin (loading control). Between 8 and 24 h, T-oligo pretreatment down-regulated constitutive COX-2 expression, which was inversely related to up-regulation of p53total and p53Ser15 levels. These experiments were repeated three times with similar results. b, paired dishes were then UVB-irradiated with 15 mJ/cm2, harvested, and then processed as for a. In diluent-treated UV light-irradiated samples, p53total and p53Ser15 levels increased from 8 to 24 h, whereas in T-oligo-treated UV light-irradiated cells, p53total and p53Ser15 expression was greater at 8 h. This earlier and higher up-regulation and activation of p53 in T-oligo-treated UV light-irradiated cells were accompanied by a striking decrease at 8 h and virtually complete inhibition of UV light-induced COX-2 expression in T-oligo-treated versus diluent-treated control cells. Similar results were obtained in three independent experiments with no difference between p9-mer and pTT results.

 
Immunoprecipitation and Immunoblotting—Cell lysates were pre-cleaned with protein G-Sepharose beads for 2 h at 4°C. Then 100 µg of total cell proteins were incubated with 2 µg of monoclonal p300 antibodies (GeneTex® Inc., San Antonio, TX), followed by the addition of protein G-Sepharose beads. The immunoprecipitated products were then subjected to SDS-PAGE and Western blot analysis as described (62). After transferring proteins to nitrocellulose membrane antibody reactions were performed with NF{kappa}B (p65) antibodies and p53 (DO-1) (both from Santa Cruz Biotechnology).

Human Skin Explant Studies—Human skin fragments from healthy donors (aged 56 ± 15 years, mean ± S.D.) were brought to the laboratory within 30 min after excision during plastic or facial reconstructive surgery. After removing subcutaneous fat and deep dermis, skin was cut into 5 x 5-mm squares and placed in 60-mm tissue culture dishes. Paired skin explants were then incubated in either medium alone or medium supplemented with 100 µM pTT or 40 µM p9-mer for 24 h. Medium consisted of Dulbecco's modified Eagle's medium with 10% calf serum plus KBM-2 with growth factors (50/50 v/v). The skin explants were then irradiated with a single dose of 30 mJ/cm2 UVB. One set was sham-irradiated as a negative control. For each treatment, one explant was harvested immediately after UVB irradiation. The dishes were then re-fed with fresh medium lacking T-oligos, and explants were harvested at 6, 18, and 24 h after UVB irradiation. Harvested skin was snap-frozen at -80 °C in OCT medium for later processing.

Immunohistochemistry and Immunofluorescence—Snap-frozen human skin explants were processed for staining by cutting 4–6-µm sections and fixing them in acetone for 10 min at -20 °C. COX-2 staining was performed using the Ultravision Detection System (TQ-015-HA, Labvision Corp., Fremont, CA) according to manufacturer's protocol. Primary antibodies used included anti-COX-2 (Santa Cruz Biotechnology), human anti-p53 DO-7 (DakoCytomation, Carpinteria, CA), and anti-phospho-p53Ser15 (Cell Signaling Technology, Beverly, MA). For the p53 DO-7 and p53Ser15 stainings, sections were blocked in 10% goat normal serum in Tris-buffered saline for 15 min at room temperature and then incubated with primary antibody overnight at 4 °C. Sections were then washed in Tris-buffered saline three times for 5 min each before being incubated with the appropriate fluorescein isothiocyanate-labeled secondary antibody at 37 °C for 45 min. Finally, sections were washed as before and mounted with Vectashield mounting medium containing 4',6-diamidino-2-phenylindole to visualize nuclei and were stored at -20 °C. We delineated {approx}10-µm x 1-mm areas using computer-assisted image analysis and counted p53total and p53Ser15 (+) nuclei in the epidermis. For each time point we analyzed an average of three randomly selected visual fields of p53total and p53Ser15-stained epidermis from three to five donors per treatment condition. To avoid bias all counts were done by a single investigator for whom all samples were blinded by another investigator.

Statistical Analysis—Difference in protein expression, Cox-2 promoter activity, and p53total and p53Ser15 (+) nuclei in T-oligo versus control-treated samples were analyzed by the analysis of variance post hoc analysis using the StatView statistical program (SAS Institute, Gary, NC). Groups were considered different when p < 0.05 (50).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
T-oligo Pretreatment Down-regulates Base-line and UVB-induced COX-2 Protein Levels That Coincide with Up-regulation and Activation of p53 Levels—The effect of T-oligo pretreatment on constitutive and UV light-induced levels of COX-2 and p53 was examined by Western blot analysis (Fig. 1), and results were confirmed by densitometric analysis of the blots (Fig. S1). In diluent-treated sham-irradiated cells, the constitutive p53total and p53Ser15 levels were negligible at 8 h and moderately increased 24 h later (Fig. 1a and Fig. S1, a and b), consistent with approaching confluence of the cultures.6 Reciprocally, COX-2 was constitutively expressed in diluent-treated sham-irradiated samples at 8 h and decreased by 24 h (Fig. 1a and Fig. S1c). In comparison, in T-oligo-treated sham-irradiated cells, COX-2 protein levels were strikingly lower at 8 h and virtually undetectable at 24 h, time points 56 and 72 h after T-oligo supplementation (Fig. 1a and Fig. S1c), suggesting that T-oligo treatment for 48 h down-regulates constitutive COX-2 levels in human fibroblasts. These decreases in COX-2 protein level were inversely related to the increases in both p53total and p53Ser15 in T-oligo-treated sham-irradiated cells (Fig. 1a and Fig. S1, a and b).

UV irradiation up-regulated p53total and p53Ser15 levels by 8 h and through 24 h and up-regulated COX-2 within 24 h, as reported previously (25, 50, 55), in diluent-treated cells (Fig. 1b and Fig. S1, d–f). T-oligo-treated cells also showed UV light-induced increases in p53total and p53Ser15, with both sham- and UV light-induced levels markedly higher than in diluent-treated controls, as expected (55). As also expected if active p53 negatively regulates COX-2 levels, in T-oligo-treated cells COX-2 was virtually undetectable by 24 h after UVB (Fig. 1b and Fig. S1f), the time of maximal UV light-induced COX-2 protein expression reported by others (27, 63). After UV irradiation, maximal p53 induction and activation are reported to occur at ~2–24 h (depending on UV light dose) (64). Because T-oligo pretreatment appeared to accelerate the time of peak, UV light induction and activation of p53 as well as to increase the magnitude of p53 induction and activation (Fig. 1), we examined a more detailed time course, harvesting fibroblasts pretreated with T-oligo or diluent alone for 48 h immediately after UV irradiation and after 4, 6, 8, 16, and 24 h (Fig. S2). Consistent with our interpretation of the experiment shown in Fig. 1, diluent-pretreated cells showed peak phospho-p53Ser15 induction at 6–8 h, with a return to base-line by 24 h, whereas T-oligo-pretreated cells had ~85% higher phospho-p53Ser15 levels immediately post-irradiation and had tripled this induction within 4 h of an ~3-fold increase that gradually declined through 24 h (Fig. S2). Total p53, in contrast to activated p53, showed similar patterns in both T-oligo and diluent pretreated fibroblasts with substantial inductions by 4 h of post-UV light that declined only slightly by 24 h. However, in diluent-pretreated cells, p53total levels were far lower (~4-fold) of p53total levels in T-oligo-pretreated cells (Fig. S2). In combination, these data are consistent with a delayed direct effect of T-oligos on COX-2 expression, such as altered transcription rate subsequently reflected in protein levels. Alternatively or in addition, this may indicate involvement of an subsequent event downstream of p53 activation, such as p53-mediated inhibition of a known positive COX-2 transcriptional regulator, such as NF{kappa}B, NF-IL6, or AP1 (65–67).

T-oligo Pretreatment Down-regulates COX-2 through p53-dependent Repression of COX-2 Promoter—To evaluate further the involvement of p53 in T-oligo-induced down-regulation of COX-2 expression, we performed transient transfection studies using two isogenic fibroblast cell lines, one that expresses WT p53 (R2FWT) and one that is permanently transfected with dominant negative p53 (R2FDD) (52–54).

As shown by Western blot analysis and quantification by densitometry, R2FDD cells expressed higher constitutive levels of p53total and p53Ser15 than R2FWT cells (~6- and or 40-fold, respectively), as expected (54), and showed virtually no T-oligo-induced up-regulation of either p53total or p53Ser15 (Fig. 2a and Fig. S3a), whereas in R2FWT cells by 24 h T-oligo induced a more than 5-fold increase in p53Ser15 and a 37% increase in p53total levels (Fig. 2a and Fig. S3b).

To verify that the p53 status of the cell lines had not changed over time in culture, we next evaluated T-oligo-induced p53 DNA binding activity. EMSA showed that in T-oligo-treated R2FDD cells, consensus sequence binding was minimal and did not increase over time, whereas in contrast binding in R2FWT cells was far higher as early as 8 h and was maximal by 24 h (Fig. 2b). These data confirm the reported p53 status in the R2FWT versus R2FDD cells.

The transcription factor NF{kappa}B is a known positive regulator of COX-2 gene expression (68). Because recent reports indicate that activation of p53 by various stimuli inhibits NF{kappa}B activity (69, 70), we next evaluated NF{kappa}B DNA binding to its consensus sequence after T-oligo treatment. In both R2FWT and R2FDD cells that received T-oligos in fresh medium, NF{kappa}B binding decreased between 1.5 and 8 h and then increased again by 16 h (Fig. 2c), consistent with the known serum-mediated bi-phasic increase in NF{kappa}B activity (71–73). However, by 24 h, the time of maximal p53 activation in R2FWT cells (Fig. 2b), there was a marked decrease in NF{kappa}B binding activity compared with R2FDD cells (Fig. 2c), suggesting that p53 activation inhibited NF{kappa}B DNA binding activity after T-oligo treatment.

We then evaluated the effect of T-oligos on NF{kappa}B-driven transcription by transfecting R2FWT and R2FDD cells with an NF{kappa}B reporter plasmid. In cells expressing p53WT, within 24 h T-oligo treatment decreased NF{kappa}B-driven transcription by ~35%, whereas in p53DN cells T-oligo treatment had no inhibitory effect on NF{kappa}B-driven transcription (Fig. 2d). We next transfected these cells with the COX-2 reporter plasmid. In R2FWT cells, treatment with T-oligos decreased COX-2 transcription by more than 50%, whereas in R2FDD cells T-oligo treatment had virtually no effect (Fig. 2e). In combination, these results demonstrate that functional p53 is required for T-oligo-induced repression of the COX-2 promoter and suggest that the effect may be mediated at least in part through NF{kappa}B.

To examine if T-oligo-induced p53-mediated repression of the COX-2 promoter depends on inhibition of NF{kappa}B transcriptional activity, we evaluated the effect of T-oligo treatment on NF{kappa}B-driven transcription by transfecting R2FWT and R2FDD cells with the COX-2 gene promoter with a site-specific mutation of the NF{kappa}B binding region (see diagram in Fig. S4a). In R2FWT cells transfected with WT COX-2 promoter, treatment with T-oligos decreased COX-2 transcription by more than 70%, whereas in R2FWT cells transfected with the mutated COX-2 promoter, T-oligo treatment had no inhibitory effect (Fig. S4b). In R2FDD cells, transfected with either WT or NF{kappa}B mutant COX-2 plasmid, T-oligo treatment had no effect (data not shown). These data demonstrate direct involvement of NF{kappa}B in T-oligo-induced p53-mediated repression of COX-2 promoter.

T-oligo Pretreatment Down-regulates Base-line and UVB-induced COX-2 Protein Levels in Human Keratinocytes, Coinciding with Activation of p53, p53-dependent Inhibition of NF{kappa}B Activity, and NF{kappa}B-dependent Transactivation—Because keratinocytes are a primary target for UVB damage and are the cells that give rise to UV light-induced actinic keratoses and SCC, we also examined the effect of T-oligos on primary human keratinocytes. Keratinocytes were pretreated with either T-oligos or diluent alone for 48 h and then sham or UV light-irradiated. Cells were then placed in medium without T-oligos and harvested for analysis of COX-2 protein levels and p53 DNA binding activity 8 and 24 h after irradiation. As shown by Western blot analysis (Fig. 3a) and quantified by densitometry (Fig. S5) in sham-irradiated keratinocytes, constitutive COX-2 expression was low at 8 and 24 h and similar in diluent-treated versus T-oligo-treated cells.

As reported previously (27, 74), UV irradiation markedly up-regulated COX-2 within 24 h in diluent-treated control cells (Fig. 3b and Fig. S5). However, as expected if T-oligos negatively regulate COX-2 in keratinocytes as well as in fibroblasts, COX-2 levels in T-oligo-treated keratinocytes were reduced by >50% at both 8 and 24 h (Fig. 3b and Fig. S5), the time of maximal UV light-induced COX-2 protein expression reported by others in this cell type (27, 74).

To substantiate further the involvement of p53 in T-oligo-induced down-regulation of COX-2 expression in human keratinocytes, we next evaluated T-oligo-mediated changes in p53 DNA binding activity using the protein from the same samples that was used to evaluate COX-2 protein levels in keratinocytes. At 8 and 24 h, EMSA showed minimal p53 DNA binding activity in T-oligo-treated or diluent-treated sham-irradiated cells, similar to the negative control (Fig. 3c, lanes 7–10 versus 2). As expected, UV irradiation increased p53 binding activity in diluent-treated keratinocytes between 8 and 24 h (Fig. 3c, lanes 3 versus 5). However, in T-oligo-treated UV light-irradiated keratinocytes, p53 binding activity was increased 4-fold as early as 8 h after UVB compared with the levels in diluent-treated UV light-irradiated cells (Fig. 3c, lanes 4 versus 3), although by 24 h p53 DNA binding activity was less and was comparable in diluent- versus T-oligo-treated UV light-irradiated samples (Fig. 3c, lanes 6 versus 5). These data confirm an inverse relationship between p53 activity and COX-2 levels in human keratinocytes, as well as in fibroblasts.



View larger version (49K):
[in this window]
[in a new window]
  		FIGURE 2.
T-oligo down-regulates COX-2 promoter activity through activation of p53. R2FWT and R2FDD were additionally transiently transfected with a full-length COX-2 promoter-firefly luciferase reporter construct. a, cells treated once 48 h after transfection with either diluent or T-oligo (p9-mer 40 µM) and then processed for Western blot analysis after 24 h. In R2WT cells, T-oligo increased p53Ser15 levels and minimally increased p53total levels compared with diluent treatment. However, in R2FDD cells, there was a high base-line expression of p53total and p53Ser15 with no up-regulation by T-oligo. b, nuclear protein isolated from paired cultures 8, 16, and 24 h after addition of T-oligo (p9-mer 40 µM) shows substantial p53 DNA binding activity only in R2FWT cells. Specificity of binding is confirmed by the disappearance of the band in the control lane of R2FWT cell lysate treated with T-oligos for 24 h, reacted with a 50x excess of cold probe. c, nuclear protein isolated from paired cultures after T-oligo (p9-mer 40 µM) addition in fresh medium showed a bi-phasic pattern of NF{kappa}B DNA binding activity in R2FDD as well as R2FWT cells, but at 24 h, the time of increased p53 activity in R2FWT cells, NF{kappa}B binding activity was markedly decreased. Specificity of binding is confirmed by lack of a band in the control lane 3 reacted with a 100x excess of cold probe compared with the strong positive band in lane 2 containing a nuclear extract of lipopolysaccharide (LPS)-treated U937 cells. d, all cells were co-transfected with a Renilla luciferase reporter, to normalize for transfection efficiency of the NF{kappa}B reporter plasmid or the empty vector pGL2, a negative control. Transfectants were treated once with either diluent or T-oligo for 24 h before harvesting for the dual luciferase assay. A set of cells was transfected with the empty vector used to create the NF{kappa}B reporter construct. Results shown are the average of three separate experiments in duplicate for relative luciferase activity (RLA) for the firefly versus Renilla luciferase in the same dishes. In R2FWT cells, T-oligo treatment (p9-mer 40 µM) decreased NF{kappa}B-driven transcription by ~34% (100 ± 3 versus 66 ± 4, p < 0.008, diluent versus T-oligo), but in R2FDD cells, treatment with T-oligo had no effect (100 ± 11 versus 102 ± 16, p = 0.8, diluent versus T-oligo). e, all cells were processed as described in d but were transfected with a COX-2 promoter construct. Results shown are the average of three separate experiments in triplicate with firefly luciferase values normalized against Renilla luciferase values in the same dishes. In R2FWT cells, T-oligo treatment (p9-mer 40 µM) decreased COX-2 promoter activity by ~50% (125 ± 30 versus 58 ± 8, p < 0.04, diluent versus T-oligo), but in the R2FDD cells, T-oligo treatment had no effect (100 ± 6 versus 94 ± 30, p = not significant (NS), diluent versus T-oligo).

 
Because NF{kappa}B is a known positive regulator of COX-2 transcription and we found that T-oligo treatment inhibits NF{kappa}B DNA binding activity in fibroblasts, experiments were performed to confirm this presumptive mechanism for indirect T-oligo-mediated p53-dependent inhibition of COX-2 transcription in keratinocytes. We reasoned that a possible limiting factor for the transcriptional activity of both p53 and NF{kappa}B may be the binding to the transcriptional co-activator protein p300 (70, 75). To address this possibility, we pretreated human keratinocytes with diluent or T-oligo for 24 h. Cells were then UV light-irradiated with 15 mJ/cm2 followed by incubation in medium without T-oligos for 8 and 24 h and harvested for immunoprecipitation with anti-p300 antibodies followed by Western blot analysis for NF{kappa}B/p65 and p53 (Fig. 3d). In diluent-treated UV light-irradiated keratinocytes, NF{kappa}B/p65 binding to p300 increased markedly between 8 and 24 h after UV irradiation (Fig. 3d, lanes 1 versus 3). In contrast, in T-oligo-pretreated UV light-irradiated keratinocytes, NF{kappa}B binding to p300 was decreased relative to control as early as 8 h, and by 24 h NF{kappa}B binding to p300 was virtually undetectable (Fig. 3d lanes 2 versus 4). These data strongly suggest that T-oligo treatment decreases NF{kappa}B transcriptional activity via reduction of NF{kappa}B binding to its transcriptional co-activator p300 protein. Consistent with this interpretation, the amount of p53 binding to p300 was strikingly increased in T-oligo-treated versus control cells at both 8 and 24 h (Fig. 3d, lanes 1 and 3 versus 2 and 4, respectively). These data are consistent with an indirect mechanism of T-oligo-mediated p53-dependent inhibition of NF{kappa}B transcriptional activity in which activated p53 successfully competes with NF{kappa}B for binding to their common co-activator, p300.



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 3.
T-oligo pretreatment activates p53 and down-regulates constitutive and UV light-induced COX-2 expression in keratinocytes. Human newborn keratinocytes were treated as in Fig. 1. a, paired dishes were then sham-irradiated and harvested after 8 and 24 h for evaluation of COX-2 and actin (loading control) protein levels. These experiments were repeated two times with similar results. b, paired dishes were then UVB-irradiated with 15 mJ/cm2, harvested, and processed as for a. At 8 h COX-2 expression was comparable in diluent-treated and T-oligo-treated samples. In UV light-irradiated control samples, COX-2 expression increased by 8 h with a further increase at 24 h, whereas in T-oligo-treated UV light-irradiated cells, COX-2 expression was significantly down-regulated throughout the 24 h. Similar results were obtained in three independent experiments. c, cells lysates from the same samples that were used to evaluate COX-2 protein levels in keratinocytes in a were used to evaluate T-oligo-mediated changes in p53 DNA binding activity in EMSA. Specificity of binding is confirmed by significant reduction of the band in the control lane using 50x excess of cold probe and protein lysate of keratinocytes treated with T-oligos for 8 h (lane 2 versus lane 4). d, cell lysates from the same samples that were used to evaluate COX-2 protein levels and p53 binding activity in keratinocytes in b and c (only T-oligo-treated UV light-irradiated) were used for immunoprecipitation (IP) with anti-p300 antibodies followed by Western blot analysis for NF{kappa}B/p65 and p53. T-oligo treatment decreased binding of NF{kappa}B to p300 by 8 h and was virtually undetectable by 24 h, whereas in T-oligo-treated UB-irradiated keratinocytes p53 binding to p300 was increased already by 8 h and was even greater by 24 h.

 



View larger version (93K):
[in this window]
[in a new window]
  		FIGURE 4.
T-oligo treatment decreases constitutive and UVB-induced COX-2 levels in human skin explants. Human skin explants (10 per donor, 5 for each T-oligo) were prepared from otherwise discarded normal adult skin and treated immediately as described under "Experimental Procedures." Untreated nonirradiated skin showed constant low COX-2 expression over the 48-h experiment. a, in paired explants harvested after 24 h, T-oligos (pTT 100 µM; p9-mer data not shown) modestly decreased low base-line levels of COX-2 expression in the suprabasal epidermis. b, in paired explants harvested after T-oligo treatment and subsequent UVB irradiation, T-oligos strikingly decreased UVB-induced levels of COX-2 expression in the suprabasal epidermis as early as 18 h and continued through 24 h after irradiation. Specificity of COX-2 antibody staining was confirmed by staining sections of nontreated and diluent/T-oligo-treated and UVB-irradiated samples with mixed keratin human monoclonal mouse anti-human antibodies (DakoCytomation, Carpinteria, CA). No difference in epidermal staining pattern between nontreated and treated and then UV light-irradiated samples was detected, suggesting specificity of COX-2 antibody staining (Fig. S6, a and b). Dermal epidermal junction is indicated by the dashed line; x200 magnification, all panels.

 
T-oligo Pretreatment Down-regulates Both Base-line and UVB-induced COX-2 Expression in Human Explants—The effect of T-oligos on COX-2 levels in human skin was studied using adult skin explants. Explants were treated as described under "Experimental Procedures" and then processed for immunostaining for COX-2, p53total, and phospho-p53Ser15 levels. COX-2 was constitutively expressed throughout the epidermis, particularly in the suprabasal layers, as reported previously (76). Pretreatment with T-oligos modestly down-regulated constitutive COX-2 levels (Fig. 4a) and strongly inhibited UV light-induced COX-2 levels (Fig. 4b). By 18 and 24 h after a single 30 mJ/cm2 dose of UVB, there was a significant decrease in COX-2 immunostaining in T-oligo-treated skin treated once 42 or 48 h previously with T-oligo when compared with sham-irradiated untreated control skin. In contrast, in vehicle-treated skin there was a dramatic increase in COX-2 immunostaining by 16 h that diminished slightly but persisted through 24 h (Fig. 4b).



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 5.
T-oligo treatment increases constitutive and UVB-induced p53total levels in human skin. Adjacent sections of the same explants shown in Fig. 4 were reacted with a fluorescently tagged antibody to total p53 and examined under a fluorescent microscope. There is no change in low p53 expression in untreated nonirradiated explants over the 48-h experiment. a, T-oligo (pTT 100 µM; p9-mer data not shown) increased constitutive levels of p53total expression, as shown by the number of positive (+) nuclei in the epidermis. b, T-oligo treatment increases UV light-induced p53total expression 6 h after UVB, but the number of (+) nuclei in T-oligo versus vehicle-treated explants is comparable after 24 h and far greater than in sham-irradiated control explants. c and d, quantitative analysis of p53total (+) nuclei as determined by blindly examining at least 3–5 fields (x200) and averaging. Values are expressed per 10-4 µm2 of epidermal area and represent explants prepared from three donors. In addition, to confirm the validity of quantifying p53total (+) nuclei per 10-4 µm2 of epidermal area, we also quantified p53total (+) nuclei per number of nuclei (4',6-diamidino-2-phenylindole + cells). No difference in the relative expression of p53total (+) nuclei was found using either quantification method (Fig. S7, a and b). Note that after 24 h nonirradiated T-oligo-treated explants have p53 positivity comparable with that of UV light-irradiated explants. C, control; V, vehicle; T, T-oligo; NS, not significant. x200 magnification in all panels.

 
T-oligo-induced Inhibition of COX-2 Expression in Human Skin Is Associated with Up-regulation and Activation of p53 Protein—To confirm our in vitro findings of an inverse relationship between T-oligo-induced decreases in constitutive and UV light-induced COX-2 levels and increases in p53 level and activity, adjacent sections of the same human tissue that were used for COX-2 immunostaining were processed for both p53total and p53Ser15 immunofluorescent staining. In T-oligo-treated versus vehicle-treated skin after 24 h, there was a >100% increase in the number of constitutively p53total positive (+) nuclei (26 ± 4 versus 12 ± 2, p < 0.09) (Fig. 5, a and c). In UV light-irradiated samples by 6 h the numbers of p53total (+) nuclei in T-oligo-treated samples were increased more than 3-fold above vehicle controls (38 ± 5 versus 12 ± 6, p < 0.004), and more than 10-fold above sham-irradiated samples, whereas the modest and variable increase in UV light-irradiated control samples remained insignificant (Fig. 5, b and d). By 24 h, the numbers of p53total (+) nuclei were similar in T-oligo-versus diluent-treated UV light-irradiated skin samples (29 ± 4 versus 26 ± 5, p = 0.6) and more than 20-fold higher in both cases than in the sham-irradiated controls (Fig. 5, b and d).

T-oligo treatment minimally increased the number of nuclei with detectable constitutive p53Ser15 levels (0.3 ± 0.2 versus 0.5 ± 0.2, vehicle versus T-oligo, p = 0.09) (Fig. 6, a and c). However, compared with vehicle-treated UV light-irradiated skin, there was a modest but statistically significant increase in the number of p53Ser15 (+) nuclei in T-oligo-treated UV light-irradiated skin samples at 6 and 24 h (6 h, 10.3 ± 0.8 versus 14 ± 0.6, p < 0.001; and 24 h, 8.5 ± 0.9 versus 13 ± 0.6, vehicle versus T-oligo, p < 0.001) as well as striking and highly significant increases above sham-irradiated untreated samples in both cases (Fig. 5, b and d). Taken together with the data of COX-2 immunostaining (Fig. 4b), these results establish an in vivo relevance of the cause-and-effect inverse relationship between increased p53 levels and activity and decreased COX-2 levels in T-oligo-treated human fibroblasts and keratinocytes (Fig. 1, a and b, and Fig. 3, a–c).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
UV irradiation is the major environmental carcinogen for human skin (77, 78), initiating and promoting development of both melanoma and nonmelanoma skin cancers (79–81). Mechanisms that contribute to UV light-induced mutagenesis and carcinogenesis include inactivation of tumor suppressor genes and/or activation of oncogenes (82–84), events that may also lead to clonal expansion of affected cells (85–89). However, in recent years a great deal of evidence has emerged suggesting that UV light-induced inflammation also plays an important role in tumor promotion and progression (26, 90–93). In murine models of skin carcinogenesis, it has been shown that administration of nonsteroidal anti-inflammatory drugs, especially selective COX-2 inhibitors, reduces the prevalence and multiplicity of UV light-induced neoplasms (74, 92–95), strongly implying direct involvement of COX-2 in cutaneous carcinogenesis.

The present study demonstrates that topical application of telomere 3'-overhang homolog DNA oligonucleotides, collectively termed T-oligos, inhibits UV light-induced up-regulation of COX-2 in vitro and ex vivo in human skin. These data expand our earlier reports that T-oligos elicit UV light-protective and cancer-preventative responses in skin cells (40, 45, 46, 50, 51). Most if not all of these responses are mediated at least in part through up-regulation and activation of p53, with subsequent effects on p53-regulated downstream target genes (49–51, 55, 56). Our current data show that T-oligo-dependent UV light-protective responses can be mediated specifically by transcriptional repression of gene products such as the inducible isoform of cyclooxygenase, COX-2 (17), that catalyzes the rate-limiting step of prostaglandin and thromboxane production (18–20). Western blot analysis of normal human fibroblasts and keratinocytes showed an inverse relationship between p53 up-regulation and activation and down-regulation of constitutive and UV light-induced COX-2 levels in both cell types. Studies in paired isogenic fibroblast lines with wild type versus dominant negative p53 confirmed that p53 transcriptional activity is required for this effect on COX-2. In addition, the present study also demonstrates that T-oligo addition to human skin explants up-regulates and activates constitutive p53 and, after subsequent UV light exposure, blocks COX-2 overexpression.



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 6.
T-oligos treatment increases constitutive and UVB-induced p53Ser15 levels in human skin. Adjacent sections of the same explants shown in Figs. 4 and 5 were reacted with a fluorescently tagged antibody to phospho-p53Ser15 and then analyzed as described for Fig. 5. T-oligo (pTT 100 µM; p9-mer data not shown) treatment variably and minimally increased constitutive levels of p53Ser15 expression (a) with the number of p53Ser15 (+) nuclei in the epidermis consistently below 0.7 per µm2x 10-4 in all groups (p = not significant (NS)). c, nonspecific retention of label in the stratum corneum was also observed in most specimens. After UV irradiation, the number of p53Ser15 (+) nuclei increased dramatically compared with sham-irradiated controls within 6 h and through 24 h for both T-oligo- and vehicle-treated explants. b, quantitative analysis of p53Ser15 (+) nuclei performed as for Fig. 5 revealed a roughly 20–30-fold increase in p53Ser15 above preirradiation levels for both vehicle-treated and T-oligo-treated explants, with T-oligo-treated explants showing ~40 and 53% higher numbers of (+) nuclei at 6 and 24 h, respectively, a significant difference (p < 0.001) at both times (d). x200 magnification, a and b, upper panel; x400 magnification, b, lower panel.

 
We demonstrate that T-oligos decrease COX-2 levels at least in part by decreasing COX-2 transcription, an effect that is dependent on p53 activity, not on total p53 levels. It is known that p53 suppresses a variety of promoters that contain one or more TATA boxes (96, 97). It was suggested that p53 suppresses gene expression by interfering with formation of the transcription preinitiation complex with TATA-binding proteins (TBPs) by preventing binding of TBPs to the TATA motif (98). Many genes are reported to be negatively regulated by p53. These include c-Fos, c-Jun, c-Myc, IL-6, heat shock protein 70 gene (HSP70), multidrug resistance gene (MDR1), and B-cell lymphoma gene-2 (Bcl2) (98–103).

Relevant to p53-dependent regulation of COX-2, it was shown that p53 inhibits the formation of complexes between TBPs and the murine and human COX-2 promoters in a cell-free system (33). The same authors also reported that wild type but not temperature-sensitive mutant p53 competed with TATA-binding proteins for binding to the mouse and human COX-2 promoters over a 100-bp segment surrounding the transcription initiation start on COX-2 promoter (33). Accordingly, to test the hypothesis that T-oligo-mediated p53-dependent suppression of COX-2 depends on repression of COX-2 promoter activity by activated p53, we tested T-oligo effect in p53WT versus p53DN cells. In cells expressing p53WT, treatment with T-oligos decreased constitutive COX-2 promoter-driven transcription of a reporter plasmid by 54%, whereas in p53DN cells T-oligo treatment had virtually no effect on COX-2 promoter activity, demonstrating that functional p53 is required for T-oligo-induced repression of the COX-2 promoter. Our findings may also resolve apparently conflicting data in the literature concerning regulation of COX-2 gene expression by p53, as investigators reporting positive p53 regulation of COX-2 in various mutant and tumor-derived cell lines correlated only the level of expression of the two proteins and did not assess p53 activity (104).

Cellular responses to DNA-damaging stimuli, including UV irradiation, are usually complex and often regulated by more than one transcription factor, for example by both p53 and NF{kappa}B (105). Furthermore, several studies have established a reciprocal inhibitory regulation of the transcription factors NF{kappa}B and p53 in various cell lines treated with DNA-damaging agents, mediated through competitive binding of p53 and NF{kappa}B to the transcriptional co-activator p300 protein (69, 70, 106). We found that in human fibroblasts and keratinocytes, T-oligo treatment up-regulates and activates p53, coinciding with decreased NF{kappa}B DNA binding activity and inhibition of transcription from NF{kappa}B-driven promoter constructs. Moreover, in human keratinocytes we also showed that treatment with T-oligo prior to UV irradiation inhibited p300 binding to NF{kappa}B and increased p300 binding to p53, as shown by immunoprecipitation of p300, followed by Western blot for NF{kappa}B/p65 and p53 proteins, coinciding with p53 activation and decreased COX-2 expression. This second and indirect mechanism of inhibition of COX-2 expression may explain the somewhat delayed T-oligo-mediated inhibition of COX-2 expression observed in our studies. Alternatively or in addition, the detailed regulation of COX-2 may also depend on the stimulus, cell type, and tissue environment, leading to predominant/selective regulation of the COX-2 promoter at different times by such known positive transcription factors as AP1, NF-IL6, NF{kappa}B, NFAT, and PEA3 (19). In fact, in our previous publications we have shown that T-oligos appear to exert their protective effects by activating ATM kinase (38) and its downstream effectors, including p53 and p95/Nbs1 (38, 40, 49), as well as by up-regulating a variety of other genes including E2F1, p16INK4a, and the p53 homologue p73 not known to be regulated by ATM (38–40, 49, 55).

E2F1 may be of particular relevance to our findings, as there is evidence suggesting E2F1 mediated inhibition of NF{kappa}B in various cell types (107, 108). Indeed, Tanaka et al. (107) showed that endogenous E2F1 competes with NF{kappa}B/p50 for binding to the p65 subunit of NF{kappa}B and that this physical interaction of E2F1/p65 inhibits NF{kappa}B transcriptional activities. Additionally, Phillips et al. (109) have shown in Saos2 cells lacking p53 that E2F1-induced inhibition of NF{kappa}B nuclear translocation and activity is mediated by the abrogation of TRAF-2 protein and inhibition of I{kappa}B kinase phosphorylation. It will be of interest to further investigate, specifically in cells with nonfunctional p53, such as the majority of UV light-induced skin neoplasms, whether T-oligo-mediated E2F1-dependent inhibition of NF{kappa}B in the absence of functional p53 may also inhibit COX-2 expression.

To date, attention has been directed toward inhibition of COX-2 enzyme activity, and much less attention has been given to modulation of COX-2 protein levels that are constitutively increased in many tumor cells (110). However, compared with young skin, there is an age-associated increase in constitutive and UV light-induced prostaglandin E2 production and COX-2 expression in human skin (26). The resulting chronic low grade inflammation may have crucial pathophysiologic implications for many aging processes, such as loss of collagen, and may contribute to the development and progression of age-associated diseases, including malignancies. Indeed, experimental COX-2 overexpression has been also reported to increase tumorigenesis in a variety of organ systems, including colon, lung, prostate, breast cancer, urinary bladder, pancreas, and liver (76, 111–115), in addition to skin (26–29). COX-2 overexpression is also increasingly implicated in the process of tumor angiogenesis (neovascularization) through increasing vascular endothelial growth factor levels as well as producing prostaglandins and thromboxanes that promote endothelial cell migration (18).

We conclude that T-oligo treatment beneficially affects multiple contributors to carcinogenesis; T-oligos transiently inhibit cell proliferation (38–41, 44–51, 55, 56), enhance DNA repair capacity (49–51), and decrease mutagenesis and photocarcinogenesis (51), as well as induce apoptosis and senescence of malignant cells (38, 40, 42, 43). In the present report we demonstrate that T-oligos also decrease constitutive and UV light-induced levels of COX-2, an inflammatory mediator strongly implicated in the processes of photocarcinogenesis, chemical and spontaneous carcinogenesis, and tumor angiogenesis (116–118). Furthermore, we suggest that T-oligos may also combat the tendency for chronic low grade inflammation that accompanies aging (26).


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant RO-1 CA105156-01 and by the Herzog Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Figs. S1–S7. Back

1 Both authors contributed equally to this work. Back

2 Supported by the Veterans General Hospital, Kaohsiung, Taiwan. Present address: Dept. of Dermatology, Veterans General Hospital, Kaohsiung, Taiwan. Back

3 To whom correspondence may be addressed: Dept. of Dermatology, Boston University School of Medicine, 609 Albany St., Boston, MA 02118. Tel.: 617-638-5541; Fax: 617-638-5515; E-mail: bgilchre{at}bu.edu. 4 To whom correspondence may be addressed: Dept. of Dermatology, Boston University School of Medicine, 609 Albany St., Boston, MA 02118, Tel.: 617-638-5541; Fax: 617-638-5515; E-mail: dgoukass{at}bu.edu.

5 The abbreviations used are: COX-2, cyclooxygenase-2; pTT, thymidine dinucleotide; WT, wild type; DN, dominant negative; SCC, squamous cell carcinoma; T-oligos, telomere homolog oligonucleotide (repeats of TTAGGG); EMSAs, electrophoretic mobility shift assays; TBPs, TATA-binding proteins; ATM, ataxia telangiectasia mutated. Back

6 V. Marwaha, B. A. Gilchrest, and D. A. Goukassian, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Christina Wu and Cynthia Curry for their valuable assistance in promoter regulation studies and to Kathleen Huard and Daniella Adrien for assistance in preparation of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Jemal, A., Murray, T., Ward, E., Samuels, A., Tiwari, R. C., Ghafoor, A., Feuer, E. J., and Thun, M. J. (2005) CA–Cancer J. Clin. 55, 10-30[Abstract/Free Full Text]
  2. Albert, M. R., and Weinstock, M. A. (2003) CA–Cancer J. Clin. 53, 292-302[Abstract/Free Full Text]
  3. Geller, A. C., Zhang, Z., Sober, A. J., Halpern, A. C., Weinstock, M. A., Daniels, S., Miller, D. R., Demierre, M. F., Brooks, D. R., and Gilchrest, B. A. (2003) J. Am. Acad. Dermatol. 48, 34-41[CrossRef][Medline] [Order article via Infotrieve]
  4. Brash, D. E., and Ponten, J. (1998) Cancer Surv. 32, 69-113[Medline] [Order article via Infotrieve]
  5. Zoumpourlis, V., Solakidi, S., Papathoma, A., and Papaevangeliou, D. (2003) Carcinogenesis 24, 1159-1165[Abstract/Free Full Text]
  6. Patrick, M. H. (1977) Photochem. Photobiol. 25, 357-372[Medline] [Order article via Infotrieve]
  7. Wood, R. D. (1996) Annu. Rev. Biochem. 65, 135-167[CrossRef][Medline] [Order article via Infotrieve]
  8. de Gruijl, F. R., van Kranen, H. J., and Mullenders, L. H. (2001) J. Photochem. Photobiol. B Biol. 63, 19-27[CrossRef][Medline] [Order article via Infotrieve]
  9. Sterenborg, H. J., de Gruijl, F. R., and van der Leun, J. C. (1986) Photodermatol. 3, 206-214[Medline] [Order article via Infotrieve]
  10. Lavker, R. M., Gerberick, G. F., Veres, D., Irwin, C. J., and Kaidbey, K. H. (1995) J. Am. Acad. Dermatol. 32, 53-62[CrossRef][Medline] [Order article via Infotrieve]
  11. Remenyik, E., Wikonkal, N. M., Zhang, W., Paliwal, V., and Brash, D. E. (2003) Oncogene 22, 6369-6376[CrossRef][Medline] [Order article via Infotrieve]
  12. Aubin, F. (2003) Eur. J. Dermatol. 13, 515-523[Medline] [Order article via Infotrieve]
  13. Schwarz, T. (2002) Photodermatol. Photoimmunol. Photomed. 18, 141-145[CrossRef][Medline] [Order article via Infotrieve]
  14. Bielenberg, D. R., Bucana, C. D., Sanchez, R., Donawho, C. K., Kripke, M. L., and Fidler, I. J. (1998) J. Invest. Dermatol. 111, 864-872[CrossRef][Medline] [Order article via Infotrieve]
  15. Hruza, L. L., and Pentland, A. P. (1993) J. Invest. Dermatol. 100, 35S-41S[CrossRef][Medline] [Order article via Infotrieve]
  16. Furstenberger, G., Gross, M., and Marks, F. (1989) Carcinogenesis 10, 91-96[Abstract/Free Full Text]
  17. Brecher, A. R. (2002) J. Drugs Dermatol. 1, 44-47[Medline] [Order article via Infotrieve]
  18. Turini, M. E., and DuBois, R. N. (2002) Annu. Rev. Med. 53, 35-57[CrossRef][Medline] [Order article via Infotrieve]
  19. Dannenberg, A. J., and Subbaramaiah, K. (2003) Cancer Cell 4, 431-436[CrossRef][Medline] [Order article via Infotrieve]
  20. Parente, L., and Perretti, M. (2003) Biochem. Pharmacol. 65, 153-159[CrossRef][Medline] [Order article via Infotrieve]
  21. Howe, L. R., Subbaramaiah, K., Brown, A. M., and Dannenberg, A. J. (2001) Endocr. Relat. Cancer 8, 97-114[Abstract]
  22. Castelao, J. E., Bart, R. D., 3rd, DiPerna, C. A., Sievers, E. M., and Bremner, R. M. (2003) Ann. Thorac. Surg. 76, 1327-1335[Abstract/Free Full Text]
  23. Marnett, L. J., and DuBois, R. N. (2002) Annu. Rev. Pharmacol. Toxicol. 42, 55-80[CrossRef][Medline] [Order article via Infotrieve]
  24. Ferrandez, A., Prescott, S., and Burt, R. W. (2003) Curr. Pharm. Des. 9, 2229-2251[CrossRef][Medline] [Order article via Infotrieve]
  25. Chen, W., Tang, Q., Gonzales, M. S., and Bowden, G. T. (2001) Oncogene 20, 3921-3926[CrossRef][Medline] [Order article via Infotrieve]
  26. Seo, J. Y., Kim, E. K., Lee, S. H., Park, K. C., Kim, K. H., Eun, H. C., and Chung, J. H. (2003) Mech. Ageing Dev. 124, 903-910[CrossRef][Medline] [Order article via Infotrieve]
  27. An, K. P., Athar, M., Tang, X., Katiyar, S. K., Russo, J., Beech, J., Aszterbaum, M., Kopelovich, L., Epstein, E. H., Jr., Mukhtar, H., and Bickers, D. R. (2002) J. Photochem. Photobiol. B Biol. 76, 73-80
  28. Orengo, I. F., Gerguis, J., Phillips, R., Guevara, A., Lewis, A. T., and Black, H. S. (2002) Arch. Dermatol. 138, 751-755[Abstract/Free Full Text]
  29. Fischer, S. M., Conti, C. J., Viner, J., Aldaz, C. M., and Lubet, R. A. (2003) Carcinogenesis 24, 945-952[Abstract/Free Full Text]
  30. Muller-Decker, K., Neufang, G., Berger, I., Neumann, M., Marks, F., and Furstenberger, G. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 12483-12488[Abstract/Free Full Text]
  31. Ahmed, S., Rahman, A., Hasnain, A., Lalonde, M., Goldberg, V. M., and Haqqi, T. M. (2002) Free Radic. Biol. Med. 33, 1097-1105[CrossRef][Medline] [Order article via Infotrieve]
  32. Ospina, J. A., Brevig, H. N., Krause, D. N., and Duckles, S. P. (2004) Am. J. Physiol. 286, H2010-H2019
  33. Subbaramaiah, K., Altorki, N., Chung, W. J., Mestre, J. R., Sampat, A., and Dannenberg, A. J. (1999) J. Biol. Chem. 274, 10911-10915[Abstract/Free Full Text]
  34. Gallo, O., Schiavone, N., Papucci, L., Sardi, I., Magnelli, L., Franchi, A., Masini, E., and Capaccioli, S. (2003) Am. J. Pathol. 163, 723-732[Abstract/Free Full Text]
  35. Griffith, J. D., Comeau, L., Rosenfield, S., Stansel, R. M., Bianchi, A., Moss, H., and de Lange, T. (1999) Cell 97, 503-514[CrossRef][Medline] [Order article via Infotrieve]
  36. Stansel, R. M., de Lange, T., and Griffith, J. D. (2001) EMBO J. 20, 5532-5540[CrossRef][Medline] [Order article via Infotrieve]
  37. Karlseder, J., Broccoli, D., Dai, Y., Hardy, S., and de Lange, T. (1999) Science 283, 1321-1325[Abstract/Free Full Text]
  38. Eller, M. S., Li, G. Z., Firoozabadi, R., Puri, N., and Gilchrest, B. A. (2003) FASEB J. 17, 152-162[Abstract/Free Full Text]
  39. Li, G. Z., Eller, M. S., Firoozabadi, R., and Gilchrest, B. A. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 527-531[Abstract/Free Full Text]
  40. Eller, M. S., Puri, N., Hadshiew, I. M., Venna, S. S., and Gilchrest, B. A. (2002) Exp. Cell Res. 276, 185-193[CrossRef][Medline] [Order article via Infotrieve]
  41. Hadshiew, I. M., Eller, M. S., Gasparro, F. P., and Gilchrest, B. A. (2001) J. Dermatol. Sci. 25, 127-138[CrossRef][Medline] [Order article via Infotrieve]
  42. Puri, N., Eller, M. S., Byers, H. R., Dykstra, S., Kubera, J., and Gilchrest, B. A. (2004) FASEB J. 18, 1373-1381[Abstract/Free Full Text]
  43. Li, G. Z., Eller, M. S., Hanna, K., and Gilchrest, B. A. (2004) Exp. Cell Res. 301, 189-200[CrossRef][Medline] [Order article via Infotrieve]
  44. Pedeux, R., Al-Irani, N., Marteau, C., Pellicier, F., Branche, R., Ozturk, M., Ranchi, J., and Dore, J. F. (1998) J. Invest. Dermatol. 111, 472-477[CrossRef][Medline] [Order article via Infotrieve]
  45. Eller, M. S., Yaar, M., and Gilchrest, B. A. (1994) Nature 372, 413-414[CrossRef][Medline] [Order article via Infotrieve]
  46. Eller, M. S., Ostrom, K., and Gilchrest, B. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1087-1092[Abstract/Free Full Text]
  47. Curiel-Lewandrowski, C., Venna, S. S., Eller, M. S., Cruikshank, W., Dougherty, I., Cruz, P. D., and Gilchrest, B. A. (2003) Exp. Dermatol. 12, 145-152[CrossRef][Medline] [Order article via Infotrieve]
  48. Cruz, P. D., Jr., Leverkus, M., Dougherty, I., Gleason, M. J., Eller, M., Yaar, M., and Gilchrest, B. A. (2000) J. Invest. Dermatol. 114, 253-258[CrossRef][Medline] [Order article via Infotrieve]
  49. Eller, M. S., Maeda, T., Magnoni, C., Atwal, D., and Gilchrest, B. A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12627-12632[Abstract/Free Full Text]
  50. Goukassian, D. A., Bagheri, S., el-Keeb, L., Eller, M. S., and Gilchrest, B. A. (2002) FASEB J. 16, 754-756[Abstract/Free Full Text]
  51. Goukassian, D. A., Helms, E., van Steeg, H., van Oostrom, C., Bhawan, J., and Gilchrest, B. A. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 3933-3938[Abstract/Free Full Text]
  52. Tubo, R. A., and Rheinwald, J. G. (1987) Oncogene Res. 1, 407-421[Medline] [Order article via Infotrieve]
  53. Shaulian, E., Zauberman, A., Ginsberg, D., and Oren, M. (1992) Mol. Cell. Biol. 12, 5581-5592[Abstract/Free Full Text]
  54. Rheinwald, J. G., Hahn, W. C., Ramsey, M. R., Wu, J. Y., Guo, Z., Tsao, H., De Luca, M., Catricala, C., and O'Toole, K. M. (2002) Mol. Cell. Biol. 22, 5157-5172[Abstract/Free Full Text]
  55. Goukassian, D. A., Eller, M. S., Yaar, M., and Gilchrest, B. A. (1999) J. Invest. Dermatol. 112, 25-31[CrossRef][Medline] [Order article via Infotrieve]
  56. Maeda, T., Eller, M. S., Hedayati, M., Grossman, L., and Gilchrest, B. A. (1999) Mutat. Res. 433, 137-145[Medline] [Order article via Infotrieve]
  57. Gilchrest, B. A., Zhai, S., Eller, M. S., Yarosh, D. B., and Yaar, M. (1993) J. Invest. Dermatol. 101, 666-672[CrossRef][Medline] [Order article via Infotrieve]
  58. Werninghaus, K., Handjani, R. M., and Gilchrest, B. A. (1991) Photodermatol. Photomed. 8, 236-242
  59. Kishore, R., Spyridopoulos, I., Luedemann, C., and Losordo, D. W. (2002) Circ. Res. 91, 307-314[Abstract/Free Full Text]
  60. Inoue, H., Yokoyama, C., Hara, S., Tone, Y., and Tanabe, T. (1995) J. Biol. Chem. 270, 24965-24971[Abstract/Free Full Text]
  61. Inoue, H., Taba, Y., Miwa, Y., Yokota, C., Miyagi, M., and Sasaguri, T. (2002) Arterioscler. Thromb. Vasc. Biol. 22, 1415-1420[Abstract/Free Full Text]
  62. Kishore, R., Luedemann, C., Bord, E., Goukassian, D., and Losordo, D. W. (2003) Circ. Res. 93, 932-940[Abstract/Free Full Text]
  63. Athar, M., An, K. P., Morel, K. D., Kim, A. L., Aszterbaum, M., Longley, J., Epstein, E. H., Jr., and Bickers, D. R. (2001) Biochem. Biophys. Res. Commun. 280, 1042-1047[CrossRef][Medline] [Order article via Infotrieve]
  64. Hall, P. A., McKee, P. H., Menage, H. D., Dover, R., and Lane, D. P. (1993) Oncogene 8, 203-207[Medline] [Order article via Infotrieve]
  65. Ramsay, R. G., Ciznadija, D., Vanevski, M., and Mantamadiotis, T. (2003) Int. J. Immunopathol. Pharmacol. 16, 59-67[Medline] [Order article via Infotrieve]
  66. Perfettini, J. L., Roumier, T., Castedo, M., Larochette, N., Boya, P., Raynal, B., Lazar, V., Ciccosanti, F., Nardacci, R., Penninger, J., Piacentini, M., and Kroemer, G. (2004) J. Exp. Med. 199, 629-640[Abstract/Free Full Text]
  67. Margulies, L., and Sehgal, P. B. (1993) J. Biol. Chem. 268, 15096-15100[Abstract/Free Full Text]
  68. Hung, J. H., Su, I. J., Lei, H. Y., Wang, H. C., Lin, W. C., Chang, W. T., Huang, W., Chang, W. C., Chang, Y. S., Chen, C. C., and Lai, M. D. (2004) J. Biol. Chem. 279, 46384-46392[Abstract/Free Full Text]
  69. Webster, G. A., and Perkins, N. D. (1999) Mol. Cell. Biol. 19, 3485-3495[Abstract/Free Full Text]
  70. Culmsee, C., Siewe, J., Junker, V., Retiounskaia, M., Schwarz, S., Camandola, S., El-Metainy, S., Behnke, H., Mattson, M. P., and Krieglstein, J. (2003) J. Neurosci. 23, 8586-8595[Abstract/Free Full Text]
  71. Rodel, F., Hantschel, M., Hildebrandt, G., Schultze-Mosgau, S., Rodel, C., Herrmann, M., Sauer, R., and Voll, R. E. (2004) Int. J. Radiat. Biol. 80, 115-123[CrossRef][Medline] [Order article via Infotrieve]
  72. Han, S. J., Ko, H. M., Choi, J. H., Seo, K. H., Lee, H. S., Choi, E. K., Choi, I. W., Lee, H. K., and Im, S. Y. (2002) J. Biol. Chem. 277, 44715-44721[Abstract/Free Full Text]
  73. Caba, E., and Bahr, B. A. (2004) Acta Neuropathol. 108, 173-182[Medline] [Order article via Infotrieve]
  74. Pentland, A. P., Scott, G., VanBuskirk, J., Tanck, C., LaRossa, G., and Brouxhon, S. (2004) Cancer Res. 64, 5587-5591[Abstract/Free Full Text]
  75. Ikeda, A., Sun, X., Li, Y., Zhang, Y., Eckner, R., Doi, T. S., Takahashi, T., Obata, Y., Yoshioka, K., and Yamamoto, K. (2000) Biochem. Biophys. Res. Commun. 272, 375-379[CrossRef][Medline] [Order article via Infotrieve]
  76. Buckman, S. Y., Gresham, A., Hale, P., Hruza, G., Anast, J., Masferrer, J., and Pentland, A. P. (1998) Carcinogenesis 19, 723-729[Abstract/Free Full Text]
  77. Miller, D. L., and Weinstock, M. A. (1994) J. Am. Acad. Dermatol. 30, 774-778[Medline] [Order article via Infotrieve]
  78. Scotto, J., and Fears, T. R. (1987) Cancer Investig. 5, 275-283[Medline] [Order article via Infotrieve]
  79. Giles, G. G., and Thursfield, V. J. (1996) Cancer Forum 20, 188-191
  80. Gilchrest, B. A., Eller, M. S., Geller, A. C., and Yaar, M. (1999) N. Engl. J. Med. 340, 1341-1348[Free Full Text]
  81. Sachs, D. L., Marghoob, A. A., and Halpern, A. (2001) in Clinics in Geriatric Medicine (Gilchrest, B. A., ed) Vol. 17, pp. 715-738, W. B. Saunders Co., Philadelphia[CrossRef][Medline] [Order article via Infotrieve]
  82. Osada, H., and Takahashi, T. (2002) Oncogene 21, 7421-7434[CrossRef][Medline] [Order article via Infotrieve]
  83. Kuper, H., Adami, H. O., and Trichopoulos, D. (2000) J. Intern. Med. 248, 171-183[CrossRef][Medline] [Order article via Infotrieve]
  84. Kopnin, B. P. (2000) Biochemistry (Moscow) 65, 2-27[Medline] [Order article via Infotrieve]
  85. Zhang, W., Remenyik, E., Zelterman, D., Brash, D. E., and Wikonkal, N. M. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 13948-13953[Abstract/Free Full Text]
  86. Brash, D. E., Ziegler, A., Jonason, A. S., Simon, J. A., Kunala, S., and Leffell, D. J. (1996) J. Investig. Dermatol. Symp. Proc. 1, 136-142[Medline] [Order article via Infotrieve]
  87. Aszterbaum, M., Epstein, J., Oro, A., Douglas, V., LeBoit, P. E., Scott, M. P., and Epstein, E. H., Jr. (1999) Nat. Med. 5, 1285-1291[CrossRef][Medline] [Order article via Infotrieve]
  88. Oro, A. E., Higgins, K. M., Hu, Z., Bonifas, J. M., Epstein, E. H., Jr., and Scott, M. P. (1997) Science 276, 817-821[Abstract/Free Full Text]
  89. McCormick, F. (1999) Trends Cell Biol. 9, 53-56
  90. Vanderveen, E. E., Grekin, R. C., Swanson, N. A., and Kragballe, K. (1986) Arch. Dermatol. 122, 407-412[Abstract/Free Full Text]
  91. Cerutti, P. A., and Trump, B. F. (1991) Cancer Cells 3, 1-7[Medline] [Order article via Infotrieve]
  92. Fischer, S. M. (2002) J. Environ. Pathol. Toxicol. Oncol. 21, 183-191[Medline] [Order article via Infotrieve]
  93. Fischer, S. M., Lo, H. H., Gordon, G. B., Seibert, K., Kelloff, G., Lubet, R. A., and Conti, C. J. (1999) Mol. Carcinog. 25, 231-240[CrossRef][Medline] [Order article via Infotrieve]
  94. Pentland, A. P., Schoggins, J. W., Scott, G. A., Khan, K. N., and Han, R. (1999) Carcinogenesis 20, 1939-1944[Abstract/Free Full Text]
  95. Pentland, A. P. (2002) Arch. Dermatol. 138, 823-824[Free Full Text]
  96. Mack, D. H., Vartikar, J., Pipas, J. M., and Laimins, L. A. (1993) Nature 363, 281-283[CrossRef][Medline] [Order article via Infotrieve]
  97. Ginsberg, D., Mechta, F., Yaniv, M., and Oren, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9979-9983[Abstract/Free Full Text]
  98. Ragimov, N., Krauskopf, A., Navot, N., Rotter, V., Oren, M., and Aloni, Y. (1993) Oncogene 8, 1183-1193[Medline] [Order article via Infotrieve]
  99. Santhanam, U., Ray, A., and Sehgal, P. B. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7605-7609[Abstract/Free Full Text]
  100. Wang, Q., and Beck, W. T. (1998) Cancer Res. 58, 5762-5769[Abstract/Free Full Text]
  101. Agoff, S. N., Hou, J., Linzer, D. I., and Wu, B. (1993) Science 259, 84-87[Abstract/Free Full Text]
  102. Miyashita, T., Harigai, M., Hanada, M., and Reed, J. C. (1994) Cancer Res. 54, 3131-3135[Abstract/Free Full Text]
  103. Kley, N., Chung, R. Y., Fay, S., Loeffler, J. P., and Seizinger, B. R. (1992) Nucleic Acids Res. 20, 4083-4087[Abstract/Free Full Text]
  104. Han, J. A., Kim, J. I., Ongusaha, P. P., Hwang, D. H., Ballou, L. R., Mahale, A., Aaronson, S. A., and Lee, S. W. (2002) EMBO J. 21, 5635-5644[CrossRef][Medline] [Order article via Infotrieve]
  105. Campbell, K. J., Chapman, N. R., and Perkins, N. D. (2001) Biochem. Soc. Trans. 29, 688-691[CrossRef][Medline] [Order article via Infotrieve]
  106. Ravi, R., Mookerjee, B., van Hensbergen, Y., Bedi, G. C., Giordano, A., El-Deiry, W. S., Fuchs, E. J., and Bedi, A. (1998) Cancer Res. 58, 4531-4536[Abstract/Free Full Text]
  107. Tanaka, H., Matsumura, I., Ezoe, S., Satoh, Y., Sakamaki, T., Albanese, C., Machii, T., Pestell, R. G., and Kanakura, Y. (2002) Mol. Cell 9, 1017-1029[CrossRef][Medline] [Order article via Infotrieve]
  108. Kundu, M., Guermah, M., Roeder, R. G., Amini, S., and Khalili, K. (1997) J. Biol. Chem. 272, 29468-29474[Abstract/Free Full Text]
  109. Phillips, A. C., Ernst, M. K., Bates, S., Rice, N. R., and Vousden, K. H. (1999) Mol. Cell 4, 771-781[CrossRef][Medline] [Order article via Infotrieve]
  110. Koki, A. T., and Masferrer, J. L. (2002) Cancer Control 9, Suppl. 2, 28-35[Medline] [Order article via Infotrieve]
  111. Soslow, R. A., Dannenberg, A. J., Rush, D., Woerner, B. M., Khan, K. N., Masferrer, J., and Koki, A. T. (2000) Cancer 89, 2637-2645[CrossRef][Medline] [Order article via Infotrieve]
  112. Yoshimura, R., Sano, H., Masuda, C., Kawamura, M., Tsubouchi, Y., Chargui, J., Yoshimura, N., Hla, T., and Wada, S. (2000) Cancer 89, 589-596[CrossRef][Medline] [Order article via Infotrieve]
  113. Shiota, G., Okubo, M., Noumi, T., Noguchi, N., Oyama, K., Takano, Y., Yashima, K., Kishimoto, Y., and Kawasaki, H. (1999) Hepatogastroenterology 46, 407-412[Medline] [Order article via Infotrieve]
  114. Kulkarni, S., Rader, J. S., Zhang, F., Liapis, H., Koki, A. T., Masferrer, J. L., Subbaramaiah, K., and Dannenberg, A. J. (2001) Clin. Cancer Res. 7, 429-434[Abstract/Free Full Text]
  115. Tucker, O. N., Dannenberg, A. J., Yang, E. K., Zhang, F., Teng, L., Daly, J. M., Soslow, R. A., Masferrer, J. L., Woerner, B. M., Koki, A. T., and Fahey, T. J., 3rd (1999) Cancer Res. 59, 987-990[Abstract/Free Full Text]
  116. Gately, S., and Li, W. W. (2004) Semin. Oncol. 31, 2-11[Medline] [Order article via Infotrieve]
  117. Dormond, O., Foletti, A., Paroz, C., and Ruegg, C. (2001) Nat. Med. 7, 1041-1047[CrossRef][Medline] [Order article via Infotrieve]
  118. Fosslien, E. (2001) Ann. Clin. Lab. Sci. 31, 325-348[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Am. J. Pathol.Home page
S. Arad, E. Zattra, J. Hebert, E. H. Epstein Jr., D. A. Goukassian, and B. A. Gilchrest
Topical Thymidine Dinucleotide Treatment Reduces Development of Ultraviolet-Induced Basal Cell Carcinoma in Ptch-1+/- Mice
Am. J. Pathol., May 1, 2008; 172(5): 1248 - 1255.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
H. Aoki, E. Iwado, M. S. Eller, Y. Kondo, K. Fujiwara, G.-Z. Li, K. R. Hess, D. R. Siwak, R. Sawaya, G. B. Mills, et al.
Telomere 3' overhang-specific DNA oligonucleotides induce autophagy in malignant glioma cells
FASEB J, September 1, 2007; 21(11): 2918 - 2930.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. Gu, R. P. Singh, S. Dhanalakshmi, C. Agarwal, and R. Agarwal
Silibinin Inhibits Inflammatory and Angiogenic Attributes in Photocarcinogenesis in SKH-1 Hairless Mice
Cancer Res., April 1, 2007; 67(7): 3483 - 3491.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
K.-S. Chun, J. K. Akunda, and R. Langenbach
Cyclooxygenase-2 Inhibits UVB-Induced Apoptosis in Mouse Skin by Activating the Prostaglandin E2 Receptors, EP2 and EP4
Cancer Res., March 1, 2007; 67(5): 2015 - 2021.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
E. Lopez-Knowles, S. Hernandez, M. Kogevinas, J. Lloreta, A. Amoros, A. Tardon, A. Carrato, S. Kishore, C. Serra, N. Malats, et al.
The p53 Pathway and Outcome among Patients with T1G3 Bladder Tumors.
Clin. Cancer Res., October 15, 2006; 12(20): 6029 - 6036.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
280/37/32379    most recent
M503245200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Marwaha, V.
Right arrow Articles by Goukassian, D. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Marwaha, V.
Right arrow Articles by Goukassian, D. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement