Molecular Cancer Therapeutics
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Vol. 1, 1265-1271, December 2002     Molecular Cancer Therapeutics
© 2002 American Association for Cancer Research

Deregulated Cyclooxygenase-2 Expression in Oral Premalignant Tissues 1

Abhijit G. Banerjee, Velliyur K. Gopalakrishnan, Indraneel Bhattacharya and Jamboor K. Vishwanatha2

Departments of Biochemistry and Molecular Biology [A. G. B., V. K. G., J. K. V.] and Oral Biology [I. B.], University of Nebraska Medical Center, Omaha, Nebraska 68198


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Establishment of an early and reliable biomarker for oral carcinogenesis whose expression can be monitored through noninvasive techniques will enable early diagnosis of cancer. Cyclooxygenases (COXs) have been implicated previously in several human malignancies, and the therapeutic benefit of specific COX-2 inhibitors has been elucidated. The expression of COX-2 and subsequent markers of malignant progression was studied in archival human specimens representing premalignant and malignant stages of oral cancer. We find that changes in COX-2 gene expression precede changes in expression of biomarkers related to apoptosis and angiogenesis in oral premalignant tissues as a veritable phenotype. We also report for the first time COX-2 mRNA variants in dysplastic samples and in a human papillomavirus-transformed cell line HOK-16B, indicating a possible stabilization of COX-2 message by human papillomavirus infection as an early event in oral cancer. Expression of other markers of tumor progression related to apoptosis and angiogenesis pathway genes shows relatively low level of changes in oral premalignant tissue. However, a determinant shift toward decrease in antitumor immunity was observed by cytokine gene expression profile changes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Detection of oral cancer in early stages has remained an elusive goal for appropriate clinical management of the disease. Every year, >30,000 new cases are reported in America, of which ~9,000 die of the disease. The 5-year survival rate of this disease has been ~50%, despite several advancements in therapy regimens of this largely preventable disease (1, 2). The development of oral cancer, clinically a subgroup of head and neck cancer, has been linked to long-term use of tobacco and alcohol mostly aggravated attributable to poor diet in developing nations (3–5). Although smoking has declined, smokeless tobacco use by young males has increased, particularly in some regions of the United States. The 1986–1987 National Institute for Dental Research Survey of Oral Health in Schoolchildren found that 16% of males in grades 6–12 reported current or past use of smokeless tobacco products, and ~39% of current users of snuff products had detectable oral lesions (6, 7). The majority of histological types of oral malignancies (~90%) has been found to be squamous epithelial cell in origin. Precancerous oral lesions occurring in adult population are primarily designated as leukoplakias and erythroplakias, of which the latter has an increased propensity (70–90%) to develop into malignant phenotype (8, 9).

Molecular markers of oral cancer initiation involving HPV3 integration (10, 11) have been identified in several cell lines and tumors of oral origin. The progression and metastasis involve up-regulation of oncogenes (Ras, c-myc, and c-erbB2; Refs. 10 and 12–14), mutation, deletion or down-regulation of tumor suppressor genes (p53 and fragile histidine triad; Refs. 15–21), cell cycle-associated kinases and their inhibitors, growth factor receptors (epidermal growth factor receptor and insulin-like growth receptor), and down-regulation of nuclear receptors (retinoic acid receptor-{alpha} and retinoic acid receptor-ß; Refs. 22–26). In addition, altered expression levels of other cellular markers, such as proliferating cell nuclear antigen, cytokeratins, enzymes (COX-2), antiapoptotic genes (bcl family), proangiogenic genes (VEGF family), and immunomodulators (IL-10 and IL-12), have been implicated in squamous cell carcinoma progression (27–32). The need to identify a singular causative biological marker in progression of the disease as a diagnostic or prognostic tool, which correlates with biological behavior of tumors (also termed "biological staging") along with its clinical staging, has been imperative for a long time. Because of the limited amount of biopsy materials in primary clinics, sensitive but quantifiable detection techniques such as RT-PCR are currently being investigated to identify populations at risk of development of disease and also to determine residual disease (histopathologically undetectable) after tumor resection. An improved understanding of molecular mechanisms of disease initiation and progression in oral cancer would also help delineate further targets of therapeutic intervention or chemoprevention.

The present study aims to delineate molecular expression profile of such biological markers in premalignant and malignant lesions of oral cancer in relation to tumor progression and metastasis. Specifically, we have correlated the expression of inflammatory mediators like COX-2 in tissues from progressive clinical stages of oral carcinogenesis with the expression of known apoptosis and angiogenesis genes. The RT-PCR studies using RNA from representative premalignant and malignant stages were substantiated through immunohistochemical staining of clinical archival specimens. We also show that RT-PCR from buccal brush biopsies obtained from a primary dental clinic can be used to monitor gene expression and progression of the disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals.
All general-purpose chemicals and biochemical and molecular biology reagents were from Sigma Chemical Co., (St. Louis, MO). The cell culture reagents were purchased from Life Technologies, Inc. (Rockville, MD).

Cell Lines and Tissue Samples.
The immortalized human oral epithelial cell line HOK-16B (HPV immortalized) was cultured in keratinocyte growth medium supplemented with growth factors supplied in the bullet kit (Clonetics Corp., San Diego, CA; Ref. 32). The oral squamous cell carcinoma cell line SCC-66 and the immortalized human oral epithelial cell line OKF-4/E4E6 (gift of Dr. James G. Rheinwald, Harvard Institutes of Medicine, Boston, MA) were cultured in DMEM supplemented with 10% fetal bovine serum. The tissue samples were obtained as biopsy specimens from oral surgical units or from patients undergoing surgical resection of tumors after obtaining informed consent according to institutional guidelines. In total, 20 specimens of different progressive stages of oral tissues from matched or unmatched patient samples comprising of minimal epithelial dysplasia (n = 6), severe epithelial dysplasia (n = 5), moderately well-differentiated squamous cell carcinoma (n = 3), verrucopapillary dysplasia (n = 2), mild epithelial atypia (n = 1), atypical squamous hyperplasia (n = 1), verrucous dysplasia (n = 1), and verrucous carcinoma (n = 1) were studied by immunostaining, and representative samples of specific progressive stages were used for RT-PCR studies. No previous information regarding biomarker status of the specimens was known before use in our studies. The specimens for RNA isolation and subsequent RT-PCR were obtained from different oral epithelial cell lines as well as representative patient samples in the form of four buccal cytological brushings or tongue depressor swabs, one normal or dysplastic biopsy patient-matched sample, and one normal adjacent tissue with tumor samples from surgical resections. The tissue material was frozen in liquid nitrogen immediately for RT-PCR experiments or processed for paraffin embedding for histopathological and immunohistochemical analysis.

Immunohistochemistry.
Paraffin-embedded tissue sections were cut into 3-µm-thick sections. Sections were deparaffinized with tissue-deparaffinizing solution (Biogenex, Inc., San Ramon, CA) for 10 min, followed by rinsing in distilled water for 2 min. Tissue sections were washed in PBS and treated with 0.3% hydrogen peroxide (H2O2) in absolute methanol for 30 min at room temperature to inactivate endogenous peroxidase activity of the samples. The slides were washed with PBS three times for 5 min each and blocked with 2.5% normal horse serum for 30 min, followed by blocking with avidin and biotin blocking solution for 15 min at room temperature (Vector Labs, Inc., Burlingame, CA). Three intermittent rinses in PBS for 5 min each followed. The tissue sections were incubated with polyclonal goat antihuman COX-2 primary antibody (sc 1745; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 1:25 dilutions in parallel for overnight at 4°C. Sections were then rinsed with PBS and incubated for 30 min with biotinylated antigoat antibody (H + L) made in horse at room temperature. After rinsing in PBS, the sections were further incubated for 45 min in streptavidin-peroxidase complex (VectaStain kit; Vector Labs). Enzyme substrate color development was carried out with substrate solution containing 50 mM Tris (pH 7.5), 0.5% (w/v) diaminobenzidine hydrochloride, and 0.01% (volume for volume) hydrogen peroxide for 6 min at room temperature. Sections were counterstained with hematoxylin and mounted using aqueous mounting medium (VectaShield; Vector Labs). For negative controls, primary antibody was omitted from the samples. Specimens were scored positive for immunoreactivity to COX-2 when >5% of cells showed brown staining (Fig. 1).



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  		Fig. 1. COX-2 expression in oral specimen. H&E staining of oral biopsy specimens (a–d) and anti-COX-2 staining (e–h) at x100 magnification in various representative stages of normal (a and e), minimal epithelial dysplasia (b and f), severe dysplasia (c and g), and well-differentiated carcinoma specimens (d and h). Arrows, heavily stained dysplastic or malignant epithelial cells.

 
RNA Isolation and Reverse Transcription.
Total RNA was isolated from the cell monolayers or tissue samples using RNeasy mini kit (Qiagen, Inc., Valencia, CA) or by TRIzol method (Life Technologies, Inc.), and first-strand cDNA was synthesized using Superscript II (RNase H-) reverse transcriptase (Invitrogen, Inc., Carlsbad, CA) as per manufacturer’s protocol.

Multiplex PCR Analysis.
Direct RNA PCR experiments were performed with C. therm. polymerase RT-PCR system (Roche Molecular Biochemicals, Mannheim, Germany). Two inflammatory gene targets, COX-2- and iNOS-related primers, were designed initially to correlate expression in oral cell lines, using Primer3 software (an Internet-based application tool) for multiplex reaction conditions, and synthesized in house at the UNMC/Eppley molecular biology core facility at the UNMC. The primers designed for amplification of respective genes are as follows:

COX-2 forward: 5'-CCACCCGCAGTACAGAAAGT-3',
COX-2 reverse: 5'-CAGGATACAGCTCCACAGCA-3,
iNOS forward: 5'-TTGCGACAGAGACAGGAAAA-3',
iNOS reverse: 5'-GATTCTGCCGAGATTTGAGC-3'.

All primers have similar Tm and GC ratios to be compatible under single PCR amplification conditions. Each individual reaction condition for the multiplex PCR was optimized for different gene sets. RT-PCR amplification conditions for analysis of COX-2 and PGK (used as an internal control) in oral cell lines used multiplex primers at 1 pmol/µl per reaction. The thermal cycling conditions were one cycle of 94°C for 1 min, 64°C for 30 s, and 72°C for 1 min, followed by 29 cycles of 94°C for 30 s, 62°C for 30 s, and 72°C for 30 s. The final extension step was carried out for 3 min at 72°C followed by hold at 15°C. The multiplex PCR kits for genes involved in apoptosis pathway, angiogenesis pathway, and IL-10/IL-12p40 primers were purchased, and amplification conditions were optimized as per manufacturer’s guidelines (Maxim Biotech, Inc., South San Francisco, CA).

Cloning and Sequence Analysis.
The DNA sequence of PCR products was determined by cloning the PCR products and subsequent sequence analysis. Briefly, the PCR DNA fragment was purified from the agarose gels using Qiagen Gel extraction columns as per manufacturer’s recommendations and blunt end cloned into PCR Script Vector (Stratagene, La Jolla, CA) at the Srf I site. The resultant construct was transformed into CaCl2 competent Escherichia coli DH5{alpha} cells, and transformants were selected and analyzed for DNA inserts. Maxi prep plasmid DNA construct from appropriate clones was purified using Qiagen Maxi Kit column for automated sequence analysis using ABI Prism Fluorescent DNA sequencer (Applied Biosystems, Inc., Foster City, CA) as per manufacturer’s recommendation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Microscopic evaluation of the 20 specimens from different progressive stages of oral tissues from matched or unmatched patient samples was found to display immunoreactivity to human COX-2 as speckled cytoplasmic staining. COX-2 immunostaining was observed in minimal epithelial dysplasias, indicating up-regulation in early premalignant cells. The extent and intensity of staining were also found to vary between different stages (Fig. 1), and there was no definite correlation between site, grade, and/or COX-2 expression levels. The immunoreactivity was predominantly found to be cytoplasmic in localization in dysplastic and malignant epithelium rather than normal epithelium, often being concentrated around the perinuclear membrane area forming a discrete "halo." All (100%) dysplastic oral epithelium from various (14 of 14) independently and clinically graded dysplasia specimens stained positive for COX-2 expression (Fig. 2). The upper dead keratin layer, however, stained inadvertently. There was no previous information available on the COX-2 or other biological marker status of the oral tissue specimens used in our studies. Additionally, in some carcinoma samples, the invading malignant epithelium surrounded by mononuclear leukocytes also stained positive along with stromal fusiform fibroblasts, thus contributing to overall up-regulation of COX-2 expression in the tumor microenvironment. The high-grade squamous cell carcinoma specimen known to express COX-2 was used as positive control, and fibroma tissue specimens were used as negative control in each of our immunohistochemistry protocols.



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  		Fig. 2. Expression of COX-2 in a dysplastic specimen. The immuno-stained tissue section shows a magnified image (x200) of anti-COX-2-stained representative dysplasia biopsy specimen. The perinuclear cytoplasmic staining around the nucleus is strongly indicative of COX-2 overexpression in most of the dysplastic epithelial cells (arrows). Additionally, note the several mitotic atypical cells (arrowhead) observed in the specimen confirming the cytopathology.

 
COX-2 gene expression analysis was initially optimized by RT-PCR for three cell lines (Fig. 3) with specifically designed COX-2 primers either alone (Lanes 1–3) or together with PGK (alone: Lanes 4–6 or multiplexed: Lanes 7–9). The expected PCR product of ~200 bp was observed. However, an additional strong PCR product of ~700 bp was also observed in the HOK-16B cell line. The 700-bp product was gel purified, cloned into PCR script vector, and sequenced as described in "Materials and Methods." The DNA sequence information revealed it to be a primary transcript-derived PCR product containing nonspliced intron sequence between exons 9 and 10 (Fig. 4).



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  		Fig. 3. RT-PCR analysis of COX-2 and PGK in oral cell lines. The ethidium bromide-stained gel shows specific (~200 bp) and alternate splice variant band (~700 bp) with COX-2 primers in SCC-66 cell line (Lane 1) and OKF4 cell line (Lane 2) but only COX-2sv band (~700 bp) in HOK-16B cell line. A 444-bp band with PGK primers alone is seen in all of the three cell lines, respectively (Lanes 4–6). Multiplex PCR reactions containing both COX-2 and PGK primers together show the appropriate sized bands similar to single primer reactions (Lanes 7–9, respectively). The gene ruler 100-bp DNA ladder marker (MBI) was used in Lane 10.

 


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  		Fig. 4. DNA sequence of COX-2 splice variant PCR product. The alternate band was found to be a primary transcript-derived PCR product containing nonspliced intron sequence. The COX-2 upstream and downstream primers are italicized and underlined. The intron donor and acceptor signature sequences are in bold.

 
The COX-2 multiplex RT-PCR was carried out for buccal epithelial cytology samples with no known history or diagnosis for dysplasia or malignancy and representative normal adjacent, dysplastic, and tumor oral tissue-derived mRNA samples (Fig. 5). The dysplastic as well as buccal epithelial cell cytology samples showed the 700-bp COX-2 splice variant band in addition to increasing amounts of expected 200-bp PCR product derived from mature COX-2 mRNA. Buccal epithelial biopsies were included to assess sensitivity of the RT-PCR assays from minimal amount of clinical samples.



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  		Fig. 5. RT-PCR profile of COX-2 expression relative to PGK in different oral tissue samples. Lane 1, buccal swab left cheek brush; Lane 2, buccal swab left tongue depressor; Lane 3, buccal swab right cheek brush; Lane 4, buccal swab right tongue depressor; Lane 5, normal adjacent tissue; Lane 6, dysplastic biopsy; Lane 7, oral tumor; Lane 8, RT-PCR-positive control; Lane 9, RT-PCR-negative control. The gene ruler 100-bp DNA ladder marker (MBI) was used in Lane 10.

 
Presence of a second messenger, nitric oxide, has been associated with inflammation as well as tumor-related immunosupression when produced in relatively small amounts by tumor cells (33). The gene expression associated with nitric oxide production; i.e., iNOS is known to be an inducer of COX-2 (34). Therefore, this gene expression was studied in the three cell line samples by multiplexing additional iNOS primers in the same reaction (Fig. 6). The iNOS band of 600 bp was only seen in the SCC-66 cell line (Lane 4) but not in other cell lines and was not consistent in representative patient matched normal and tumor tissues (data not shown). Hence, it is thought that iNOS as a causative inflammatory mediator may not be significantly involved in oral carcinogenesis and was not pursued further.



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  		Fig. 6. Multiplex PCR analysis of PGK, COX-2, and iNOS. Multiplex reactions carried out show appropriate bands with all of the three primers together in a single reaction in HOK16 B cell line (Lane 2), OKF-4 cell line (Lane 3), and SCC-66 cell line (Lane 4). iNOS-specific band (~600 bp) could be only observed in SCC-66 sample. Lane 1, gene ruler 100-bp DNA ladder marker (MBI).

 
Multiplex PCR studies were carried out in the same samples to look for expression of Th1/Th2 cytokine profile, apoptosis pathway, and angiogenesis pathway-related genes. These studies were carried out to correlate expression of these genes as a consequence of up-regulated and constitutive COX-2 gene expression. Surprisingly, the cell mediated immune status in these samples (Fig. 7) as shown through Th1 (IL-12 p40) versus Th2 (IL-10) cytokine profiling; using both the primers in the same reaction revealed that immunosuppressive cytokine IL-10 was up-regulated in the dysplastic lesions with respect to IL-12 in resected tumor samples, as well as normal adjacent epithelial tissues irrespective of their stage.



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  		Fig. 7. Multiplex RT-PCR profile of IL-12 and IL-10 cytokine expression in different oral tissue samples. Lane 1, RT-PCR-negative control; Lane 2, 100-bp DNA marker; Lane 3, buccal swab left cheek brush; Lane 4, buccal swab left tongue depressor; Lane 5, buccal swab right cheek brush; Lane 6, buccal swab right tongue depressor; Lane 7, normal adjacent tissue; Lane 8, dysplastic biopsy; Lane 9, oral tumor; Lane 10, HOK 16 B cell line; Lane 11, RT-PCR-positive control cDNA.

 
Multiplex PCR analysis of apoptosis pathway-related genes shows that the antiapoptotic markers like Bcl2 and Bcl-XL expression emerge in the dysplastic samples and get up-regulated in tumors (Fig. 8). As a consequence, increased proliferation of neoplastic tissue is expected. Although Bax appears slightly up-regulated, it is known that its antiapoptotic activity depends on complex interaction with other Bcl2 family members and is subject to intricate post-translational regulation. These studies are further warranted in oral dysplasia, and mechanistic correlates were not envisaged for the present study. However, we intend to study the extensive mechanism of such regulation in the cell models currently being established in our laboratory.



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  		Fig. 8. Multiplex RT-PCR profile of Apoptosis gene set expression in different oral tissue samples. Lane 1, buccal swab left cheek brush; Lane 2, buccal swab left tongue depressor; Lane 3, buccal swab right cheek brush; Lane 4, buccal swab right tongue depressor; Lane 5, normal adjacent tissue; Lane 6, dysplastic biopsy; Lane 7, oral tumor; Lane 8, HOK 16 B cell line; Lane 9, 100-bp kit DNA marker; Lane 10, RT-PCR-negative control (without cDNA).

 
The angiogenesis pathway-related genes amplified by multiplex PCR show elevated levels of angiogenic factors like VEGFs with metastatic tumor samples only, though low level of expression is seen in dysplasia samples (Fig. 9). The appearance of the more angiogenic and tumorigenic VEGF isoform-related band (VEGF, 121 and 104 bp) in the tumor and HOK-16B samples confirms the acquired malignant progression capability through promotion of tumor mass neo-vascularization.



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  		Fig. 9. RT-PCR profile of angiogenesis gene set expression in oral tissue samples. Lane 1, 100-bp kit DNA marker; Lane 2, buccal swab left cheek brush; Lane 3, buccal swab left tongue depressor; Lane 4, buccal swab right cheek brush; Lane 5, buccal swab right tongue depressor; Lane 6, normal adjacent tissue; Lane 7, dysplastic biopsy; Lane 8, oral tumor; Lane 9, HOK 16 B cell line; Lane 10, RT-PCR-positive control cDNA; Lane 11, RT-PCR-negative control (without cDNA).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study was aimed at delineation of early gene expression changes in oral premalignant tissues, which can be used for follow-up of tumor growth promotion and their invasive potential and form the basis of chemoprevention studies. We have established through this study that deregulation of COX-2 gene expression occurs in early dysplastic oral tissues. This up-regulation of COX-2 in premalignant cells predisposes the cells to further deregulation of several progression-related gene expression markers and can be rapidly diagnosed by multiplex RT-PCR assays from minimal amounts of noninvasive samples (e.g., buccal brushing). It has been known through earlier studies that COX regulates expression of various genes in colon cancer (35, 36), non-small cell lung cancer (31), and prostate cancer (37). Recent evidence by Tsujii et al. (30) and Masunaga et al. (38) has shown that COX-2 augments the release of angiogenic peptides as a result of increased production of its metabolite PGE2, and a positive correlation of COX-2 versus microvessel density was observed. Our study confirms these earlier reports that constitutive COX-2 expression is an early causal event for tumor initiation through conversion of procarcinogens to carcinogens because of its peroxidase activity, as well as tumor progression because of its COX activity-related gene expression changes. Our results also confirm another earlier finding that COX-2 metabolite prostaglandin PGE2 is a potent inducer of IL-10 and predisposes to cytokine imbalance (31). The study by Huang et al. (31) demonstrated that PGE2 specifically caused up-regulation of IL-10 while down-regulating IL-12 (also known as natural killer cell activation factor). This causes disruption in immune surveillance against tumor cells through abrogation of natural killer cell activity, and the process of removal of neoplastic cells from the system is thus anergized, leading to growth advantage and tumor progression. VEGFs are the only factor that specifically regulates tumor angiogenesis by interacting with two endothelial receptor tyrosine kinases, Flt-1 and Flk-4, in vivo. VEGFs are known to be expressed in six variant isoforms of which VEGF-121 supposedly is more strongly tumorigenic, because it can readily diffuse from tumor cells (39). Therefore, our studies confirm the earlier finding that COX-2 promotes angiogenesis, and action of COX-2-specific inhibitors arrests tumor progression by specifically antagonizing tumor angiogenesis (36).

Our study also establishes that COX-2 gene expression changes precede other tumor progression-related biomarker expression, such as antiapoptotic- and angiogenesis-related genes in oral cancer. Hence, specific chemoprevention agents specific for blocking both the activities of COX-2 may be meaningful in decreasing the initiation and progression of oral cancer. We have also shown for the first time that HPV integration in oral tissues may have a role in deregulation of COX-2 primary transcript processing and stabilization of protein expression and activity. However, the exact mechanism of such deregulation is intent of study in the oral cancer progression cell models in our laboratory. Additionally, surrogate markers for HPV infection and integration in the oral tissues in the clinical samples need to be ascertained, although it has already been reported as a risk factor in oral epithelial carcinogenesis by several authors (15). We have established through these molecular studies that HOK-16B cell line could serve as a cell model system for additional molecular studies in oral cancer progression and integrated HPV-derived factors modulating COX-2 expression. Although our studies were in progress, another angiogenesis-correlated prognostic biomarker regulated by COX-2, the proto-oncogene eIF4E, has been reported in dysplasia of head and neck cancer (35). Munkarah et al. (40) have shown that PGE2 stimulates proliferation and reduces apoptosis in epithelial ovarian cancer cells. They also found an association of overexpression of COX-2 with dose-dependent increase in the ratio of Bcl-2:Bax mRNA. On the basis of our observations and that of other laboratories, we believe that chemoprevention trials using COX-2 as target and surrogate marker in oral dysplasia are warranted.


   Acknowledgments
 
We thank the Molecular Biology Core Facility at the Eppley Institute for Research in Cancer and Allied Diseases and the UNMC/Eppley Tumor Bank for assistance in our studies.


   Footnotes
 
1 Supported by Phillip Morris Incorporated (J. K. V.) and National Cancer Institute Grant T30CA36727. Back

2 To whom requests for reprints should be addressed, at Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 984525 Nebraska Medical Center, Omaha, NE 68198-4525. Phone: (402) 559-6663; Fax: (402) 559-6650; E-mail: jvishwan{at}unmc.edu Back

3 The abbreviations used are: HPV, human papillomavirus; COX, cyclooxygenase; IL, interleukin; VEGF, vascular endothelial growth factor; PGK, phosphoglycerate kinase; UNMC, University of Nebraska Medical Center; iNOS, inducible nitric oxide synthase; PGE2, prostaglandin E2; RT-PCR, reverse transcription-PCR. Back

Received 5/21/02; revised 8/15/02; accepted 10/29/02.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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