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Clinical Sciences |

p53 Expression and Relation to Human Papillomavirus Infection in Pingueculae, Pterygia, and Limbal Tumors FREE

Nicholas Dushku, MD; Sandra L. S. Hatcher, MSc; Daniel M. Albert, MD; Ted W. Reid, PhD
[+] Author Affiliations

From the Department of Ophthalmology, Kaiser Permanente Medical Center (Dr Dushku), and the Department of Pathology, University of California–Davis Medical Center (Ms Hatcher), Sacramento; the Department of Ophthalmology, University of Wisconsin Medical Center (Dr Albert), Madison; and the Departments of Ophthalmology and Visual Sciences and Cell Biology and Biochemistry, Texas Tech University Health Sciences Center and South West Cancer Center (Dr Reid), Lubbock.


Arch Ophthalmol. 1999;117(12):1593-1599. doi:10.1001/archopht.117.12.1593.
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Published online

Background  The tumor suppressor gene p53 is expressed without apoptosis in the limbal basal stem cells of all pterygia and limbal tumors and most pingueculae from which these growths seem to originate. Oncogenic human papillomaviruses (HPVs) have been found in pterygia and limbal tumors, and HPV and p53 overexpression commonly coexist in oropharyngeal and penile carcinomas.

Objective  To search for HPV DNA as a cofactor in the development of pingueculae, pterygia, and limbal tumors.

Methods  We examined specimens—1 of pinguecula, 13 of pterygia (7 primary, 1 recurrent, 1 with dysplasia, and 4 primary not tested for p53), and 10 of limbal tumors (2 with actinic keratosis dysplasia, 1 with conjunctival intraepithelial neoplasia, 3 with carcinoma in situ, and 4 with squamous cell carcinoma)—expressing p53. Specimens were tested for the presence of HPV DNA by the polymerase chain reaction using degenerate consensus primers for the highly conserved portion of the L1 region that encodes a capsid protein of the virus. This assay has a wide spectrum with capability of detecting essentially all known HPV types. Nested polymerase chain reaction was performed on all specimens. Primers of the cystic fibrosis gene were used to confirm the presence of genomic DNA and to rule out inhibitors. Purified HPV DNA type 11 was the positive control, and HPV-negative genomic DNA was the negative control.

Results  Using consensus primers for the highly conserved portion of the L1 region, all specimens of pingueculae, pterygia, and limbal tumors studied were negative for HPV DNA by nested polymerase chain reaction.

Conclusions  Human papillomavirus DNA is not required as a cofactor in the development of pterygia and limbal tumors. These data support the theory that increased p53 expression in the limbal epithelia of pingueculae, pterygia, and limbal tumors indicates the probable existence of p53 mutations in these cells as an early event in their development, which is consistent with UV irradiation causation. Thus, due to a damaged p53-dependent programmed cell death mechanism, mutations in other genes may be progressively acquired. This would allow for the multistep development of pterygia and limbal tumor cells from p53-mutated limbal epithelial basal stem cells overlying pingueculae.

Figures in this Article

BASED ON the intermediate-filament immunohistochemical analysis of surgical primary, and recurrent pterygia specimens, we discovered that the pathogenesis of pterygia was a result of an invasion of the cornea by vimentin-expressing altered limbal epithelial basal cells, the pterygium cells, followed by conjunctival epithelium.1,2

Because epidemiologically, UV-B radiation correlates as the etiologic agent for pterygia3,4 and limbal tumors58 and because UV-B is known to be mutagenic for the p53 tumor suppressor gene,912 abnormal p53 expression was searched for immunohistochemically in the altered limbal basal cells of pterygia and limbal tumors and in pingueculae from which they seem to originate.13 Increased nuclear p53 expression without apoptosis was found in the limbal epithelium of pterygia, limbal tumors, and most pingueculae, indicating the probable existence of p53 mutations in these cells as an early event in their development, which is consistent with their causation by UV irradiation. Because of this damaged p53-dependent programmed cell death mechanism,14 mutations in other genes are progressively acquired that allow the multistep15 development of pterygia and limbal tumor cells from p53-expressing cells overlying a pinguecula.

Another known mechanism for the reduction of the functional effectiveness of the wild-type p53 protein, other than point mutations, is by infection with cer tain oncogenic human papillomaviruses (HPVs).16,17 Human papillomavirus is the only DNA tumor virus that has been shown18 to be involved in human cancer. The binding to the wild-type p53 proteins of the E6 oncoproteins encoded by HPV types 16 and 18 results in the rapid degeneration of p53 through the ubiquitin-mediated pathway. Low levels of nuclear p53 protein lead to a damaged p53-dependent programmed cell death mechanism, which is similar to that caused by p53 mutations.19,20 This HPV mechanism would also permit mutations in other genes to accumulate and allow the multistep development of pterygia and limbal tumors from HPV-infected limbal stem cells.

Human papillomavirus and p53 overexpression commonly coexist in oropharyngeal carcinomas,21 penile carcinomas,22 grade III cervical intraepithelial neoplasia,23 and invasive squamous cell carcinomas of the cervix,23 but show a negative correlation rate with esophageal squamous cell carcinomas24 and certain cervical cancers.25,26 Because other investigators have found certain strains of HPV in pterygia27 and limbal tumors,2831 we used the polymerase chain reaction (PCR) to search for HPV DNA as a cofactor in the development of these abnormal growths.32

TISSUE SPECIMENS

Specimens of pinguecula (n = 1), pterygia (n = 13: 7 with primary, 1 with recurrent, 1 with dysplasia, and 4 primary not tested for p53), and limbal tumors (n = 10: 2 with actinic keratosis dysplasia, 1 with conjunctival intraepithelial neoplasia, 3 with carcinoma in situ, and 4 with squamous cell carcinoma) were examined. For immunohistochemical analysis, fresh normal human surgical specimens of superior (4 specimens) and lateral (1 specimen) limbal epithelia and 1 cadaver specimen of normal human medial corneal, limbal, and conjunctival tissue were used for negative controls. Positive tissue controls included specimens of colon carcinoma and breast carcinoma (BioGenex Laboratories, San Ramon, Calif).

IMMUNOHISTOCHEMICAL ANALYSIS

The primary antibody p53, Ab-6, clone DO-133 (Oncogene Science Inc, Uniondale, NY), was used to demonstrate p53 protein expression in limbal basal cells in pingueculae, pterygia, and limbal tumors. An avidin-biotin complex–immunoperoxidase technique (Vectastain Elite ABC Kit; Vector Laboratories, Burlingame, Calif) was selected for staining with the primary antibody.34,35 Hematoxylin was used for counterstaining.

APOPTOSIS TUNEL ASSAY

We performed the apoptosis TUNEL (terminal deoxynucleotidyltransferase–mediated deoxyuridine 5-triphosphate and biotin nick end labeling) assay on specimens of pingueculae, pterygia, and limbal tumors that had also been stained for p53 expression. This assay was performed according to the procedure of Lovelace et al36 using the TACS 2 TdT-DAB kit from Trevigen, Gaithersburg, Md.

PCR ASSAY
Quality Control

Control specimens were run with each patient specimen. The positive control was purified HPV type 11 isolated from HPV viral culture, and the negative control was DNA that was known to be negative for HPV. Each of these was amplified in the same manner as the patient specimen.37 It was presumed that viral and genomic DNA were isolated simultaneously. As a control for DNA isolation and amplification, each patient specimen that did not amplify for HPV was amplified with a set of primers that amplify a fragment of a human gene.

DNA Analysis of HPV

The DNA was extracted from frozen tissue using DNA isolation reagents (PureGene; Gentra Systems Inc, Research Triangle Park, NC). Total DNA (500 ng) was amplified with primers that amplify a 425-base pair (bp) region of the cystic fibrosis conductance regulator gene. These primers verified the successful isolation of genomic DNA and ruled out the presence of inhibitors. Specimens that did not demonstrate a 425-bp fragment were amplified using nested primers that yield a 295-bp fragment.

A sample of DNA (500 ng) from each specimen was enzymatically amplified to generate "consensus" oligonucleotide primers that amplify a highly conserved portion of the L1 region of the HPV. This assay is capable of detecting essentially all known HPV types. Fragments were amplified in 100 µL containing My09 primer (5′-CGTCCMARRGGAWACTGATC-3′ [R = A or G, W = A or T, Y = C or T, and M = A or C]), 0.5 µmol/L; My11 primer (5′-GCMCAGGGWCATAAYAATGG-3′), 0.5 µmol/L; deoxynucleoside 5′-triphosphate, 0.2 mmol/L of each; Taq DNA polymerase, 2 units; magnesium chloride, 1.5 mmol/L; and 1X Taq buffer. To prevent evaporation, the specimens were overlaid with mineral oil, 100 µL. They were then placed in a programmable heating block with an initial denaturation step of 94°C for 2 minutes, followed by 35 cycles at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 55 seconds. The specimens were electrophoresed on 1% agarose, 25 mL, and 2% NuSieve genetic technology grade agarose (FMC Corporation Bioproducts, Rockland, Me) in 0.5X buffer (TRIS base, boric acid, and EDTA at pH 8.3 [TBE]) in a mini-sub cell apparatus (Bio Rad Laboratories, Hercules, Calif). Gels were electrophoresed at 10 V/cm for 1 hour. Immediately after electrophoresis, gels were stained in 0.5X TBE containing ethidium bromide, 0.5 µg/mL, for 10 minutes, destained in 0.5X TBE for 10 minutes, and photographed. This fragment is approximately 450 bp. The size of the fragment varies depending on the HPV type present.

The presence of the 450-bp fragment indicates that the specimen contains HPV. If the 450-bp fragment was not present, 5 µL of the PCR product from the My09/My11 reaction was amplified in a nested PCR with a set of primers located internal to the My09/My11 primers. Fragments were amplified in 100 µL containing HPV 1 primer (5′-TTTGTTACTGIGGTAGATAC-3′), 0.5 µM; HPV 2 primer (5′-GAAAAATAAACTGTAAATCA-3′), 0.5 µmol/L; deoxynucleoside 5′-triphosphate, 0.2 mmol/L of each; Taq DNA polymerase, 2 units; magnesium chloride, 1.5 mmol/L; and 1X Taq buffer. To prevent evaporation, the specimens were overlaid with mineral oil, 100 µL. They were then placed in a programmable heating block for 35 cycles at 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 55 seconds. The specimens were electrophoresed on 1% agarose and 2% NuSieve genetic technology grade agarose, 25 mL, in 0.5X TBE buffer. Gels were electrophoresed at 10 V/cm for 1 hour. Immediately after electrophoresis, gels were stained in 0.5X TBE containing ethidium bromide, 0.5 µg/mL, for 10 minutes, destained in 0.5X TBE for 10 minutes, and photographed. This fragment is approximately 139 bp. The size of the fragment varies depending on the HPV type present.

PHOTOGRAPHY

Ethidium bromide–stained gels were photographed under UV light. Sections that immunostained for p53 were photographed with a photoscope (Zeiss Ultra Phot; Carl Zeiss Inc, Thornwood, NY).

p53 EXPRESSION

In normal control studies, p53 immunostaining was negative in 5 fresh surgical specimens of superior (n = 4) and lateral (n = 1) limbal epithelia and in the medial interpalpebral limbal-corneal epithelia of 1 cadaver eye. All tested specimens of pingueculae (1/1), pterygia (9/9), and limbal tumors (10/10) expressed nuclear p53 in limbal epithelial cells but not in the fibroblasts (Figure 1).

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Figure 1.

Nuclear p53 immunostaining of altered limbal epithelial cells of pterygia, pingueculae, and limbal tumors with p53 antibodies. A, A specimen of primary medial pterygium of the left eye shows nuclear immunostaining of altered limbal epithelial basal cells (pterygium cells) in the migrating limbus (to the left of the arrowhead pointing to Bowman's layer). Pterygium cells are also migrating onto the corneal basement membrane overlying Bowman's layer. Fibroblasts that have accumulated beneath the migrating limbus do not immunostain for p53 and are making collagenlike material. Fibroblasts immediately next to Bowman's layer appear to be dissolving Bowman's layer (original magnification, ×250). B, In a specimen of recurrent medial pterygium of the left eye, hyperproliferative corneal epithelium overlies Bowman's layer located at the leading edge of the pterygium. An island of fibroblasts (arrowhead) between Bowman's layer and corneal basement membrane does not express p53 and has dissolved Bowman's layer (original magnification, ×325). Directions of basal cell migration: left to right for A and B. C, A specimen of raised pinguecula shows altered limbal cells in limbal epithelium (original magnification, ×425). D, A specimen of conjunctival intraepithelial neoplasia shows dysplastic limbal cells (original magnification, ×250). E, A specimen of carcinoma in situ shows p53-expressing limbal tumor cells with no stromal invasion (original magnification, ×250). F, The p53-expressing limbal tumor cells invade the stroma of a specimen of squamous cell carcinoma (original magnification, ×250).

Graphic Jump Location
TUNEL ASSAY

For the specimens of pinguecula and pterygia, we found 0% to less than 1% apoptosis, and for the specimens of limbal tumors, we found 0% to less than 5% apoptosis.

PCR ASSAY

Amplification of the 425-bp or nested 295-bp fragment by the cystic fibrosis gene primers in all patient specimens confirmed that genomic DNA had been isolated from all specimens and that all patient specimens were free of PCR inhibitors (Figure 2 and Figure 3). We were unable to detect the 450-bp or the nested 139-bp L1 region HPV DNA fragments in all specimens of pingueculae (1/1), pterygia (13/13), and limbal tumors (10/10) tested (Figure 4 and Figure 5).

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Figure 2.

Polymerase chain reaction amplification of genomic DNA (CFTR gene) in specimens of pterygia, pinguecula, and limbal tumors. Lane 1 indicates positive control; lane 2, primary pterygium (A); lane 3, recurrent pterygium (B); lane 4, pinguecula (C); lane 5, conjunctival intraepithelial neoplasia (D); lane 6, carcinoma in situ (E); lane 7, squamous cell carcinoma (F); lane 8, negative control; and lanes M, 123–base pair (bp) DNA ladder. Letters refer to designators in Figure 1.

Graphic Jump Location
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Figure 3.

Nested polymerase chain reaction for genomic DNA (CFTR gene) in specimens of pterygia, pinguecula, and limbal tumors. Lane 1 indicates positive control; lane 2, primary pterygium (A); lane 3, pinguecula (C); lane 4, S92-18318; lane 5, S94-18485; lane 6, negative control; and lanes M, 123–base pair (bp) DNA ladder. Letters refer to designators in Figure 1.

Graphic Jump Location
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Figure 4.

Polymerase chain reaction amplification for human papillomavirus (HPV) DNA in specimens of pterygia, pinguecula, and limbal tumors. Lane 1 indicates positive control (HPV type 11); lane 2, primary pterygium (A); lane 3, recurrent pterygium (B); lane 4, pinguecula (C); lane 5, conjunctival intraepithelial neoplasia (D); lane 6, carcinoma in situ (E); lane 7, squamous cell carcinoma (F); lane 8, negative control; and lanes M, 123–base pair (bp) DNA ladder. Letters refer to designators in Figure 1.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 5.

Nested polymerase chain reaction for human papillomavirus (HPV) DNA in specimens of pterygia, pingvecula, and limbal tumors. Lane 1 indicates positive control (HPV type 11); lane 2, primary pterygium (A); lane 3, recurrent pterygium (B); lane 4, pinguecula (C); lane 5, conjunctival intraepithelial neoplasia (D); lane 6, carcinoma in situ (E); lane 7, squamous cell carcinoma (F); lane 8, negative control; and lanes M, 123–base pair (bp) DNA ladder. Letters refer to designators in Figure 1.

Graphic Jump Location
UV LIGHT, NOT HPV

Epidemiologically3,4,6 and histopathologically,38(p304) UV light correlates as the etiologic agent for pingueculae, pterygia, and limbal tumors. Because oncogenic HPVs have been found by other researchers in pterygia27 and limbal tumors,2831 we used PCR32 to search for HPV DNA in p53-expressing pingueculae, pterygia, and limbal tumors.

We found no HPV DNA in any of these growths and conclude that HPV DNA is not required as a cofactor for the etiology of these lesions, either through the control of the action of p53 or through any other mechanism.

BASEMENT MEMBRANES AND STEM CELLS IN LIMBAL EPITHELIUM

Important in the pathogenesis of pterygia and limbal tumors is the histological evidence that the limbal, corneal, and conjunctival epithelia have adjacent and joined basement membranes 42,43 that allow possible migration of any of their basal cells onto each other's basement membrane.

Although macroscopically the limbal epithelium looks like conjunctival epithelium and previously was considered to be composed of conjunctival epithelial cells, current evidence4244 indicates that the limbal epithelium instead contains corneal stem cells.

Because tumors are thought to arise from stem cells,45 knowledge of the existence of stem cells in the limbal epithelium, but not in the adjacent differentiated corneal and conjunctival epithelia, is also important in the understanding of the pathogenesis of pterygia and limbal tumors from stem cells. Normally, the parental stem cells in the limbal epithelium next to the conjunctival cells maintain a constant posterior border and do not migrate across that border onto conjunctival basement membrane. Instead, they produce daughter cells that migrate circumferentially onto limbal epithelial basement membranes and centripetally onto corneal basement membrane, where they differentiate into corneal basal cells. The adjacent differentiated conjunctival basal cells originally migrate from conjunctival stem cells43 located in the palpebral fornices and differentiate into mature conjunctival basal cells as they move onto bulbar conjunctival basement membranes.

LIMBAL STEM CELLS IN PINGUECULAE, PTERYGIA, AND LIMBAL TUMORS

The limbal epithelium over a pinguecula contains stem cells and can be the site of the development of pterygia and limbal tumors. Pterygia originate from a migration of an entire segment of altered limbal epithelial basal stem cells onto corneal basement membrane, followed by conjunctival epithelial cells.1,2 Before migration of the limbal epithelial segment, altered limbal basal stem cells (the pterygium cells) infiltrate centrifugally onto adjacent corneal, limbal, and conjunctival epithelial basement membranes. Consistent with these findings2,13 is our proposal that pterygia recur because of the incomplete removal of altered limbal basal stem cells that have invaded basal corneal, conjunctival, and circumferential limbal epithelia, and that recurrent pterygia are not a separate process of an invasion of vascular scar tissue in response to surgical injury.

The unusual developmental pattern of pterygia is consistent with the peculiar growth patterns of limbal tumors, such as corneal intraepithelial neoplasia or epithelial dysmaturation of Jakobiec.46 Stem cell migrations in the postnatal period are not a new biological phenomenon and may occur in such diseases as Barret esophagus47(pp832-834) and ectropion of the uterine cervix.48 Other limbal tumors that originate from limbal basal stem cells do not migrate as a segment of the limbal epithelium onto corneal basement membrane but have stationary limbal epithelia whose altered cells grow centrifugally and usually infiltrate locally.49,50

PATHOGENESIS OF PTERYGIA AND LIMBAL TUMORS FROM LIMBAL EPITHELIAL CELLS

Based on our findings of p53 expression without apoptosis in the limbal epithelia of pingueculae, pterygia, and limbal tumors13 and the absence of HPV, we propose a theory for the pathogenesis of these growths from 2 p53 pathways (Figure 6).

Place holder to copy figure label and caption
Figure 6.

Possible pathways for the formation of pterygia and limbal tumors. Rb indicates retinoblastoma protein; TGF-β, transforming growth factor β; question mark, it is not known if these mutations are in the fibroblast cells; and up arrow, increase.

Graphic Jump Location
Damaged p53-Dependent Programmed Cell Death Mechanism Pathway

Albedo UV light51 causes an early event mutation in the UV-sensitive p53 tumor suppressor genes in the parental limbal basal stem cells. Because of a damaged p53-dependent programmed cell death mechanism, mutations in other genes are progressively acquired that allow the multistep development of pterygia and limbal tumor cells from p53-expressing cells in the limbal epithelium overlying a pinguecula.

p53-Rb–TGFβ Pathway

In pterygia, p53 mutations in the limbal stem cells result in a damaged p53-dependent programmed cell death mechanism and the overproduction by the pterygium cells of transforming growth factor β (TGF-β) by the p53-Rb-TGF-β pathway.49,52 Many of the tissue changes seen in pterygia can be explained by this mechanism.13,49,5265 First, in the migrating limbus of pterygium cells, TGF-β production occurs, resulting in a decreased number of cell layers (average, 3-4 layers).49,5255 Second, among the TGF-β–expressing pterygium cells of the migrating limbus epithelium and in the TGF-β–soaked subepithelial stroma, increased capillaries and vessels occur, indicating angiogenesis.13,49,5456,58,59 Third, also within the epithelial cell layers (personal observations) and stroma under the pterygium cells, monocytes can accumulate.49 Fourth, a group of normal-appearing fibroblasts (Figure 1, A) gathers beneath the TGF-β–expressing pterygium cells, producing normal-appearing collagen.13,49,57,60 Fifth, some of these fibroblasts (Figure 1, A) next to the basic fibroblast growth factor–rich Bowman's layer appear to dissolve Bowman's layer, probably through the up-regulation of collagenase.13,49,61 Sixth, another group of these collagenase-producing fibroblasts (Figure 1, B) migrates anteriorly to the leading edges of the pterygia between Bowman's layer and the basement membrane of pterygium cells to form little islands of fibroblasts that locally dissolve Bowman's layer.13,49,61 Seventh, pterygium cells are mobile locally as they move on top of their basement membranes. The parental basal stem cells leave their permanent locations next to conjunctival basal cells and migrate onto corneal basement membrane, followed by conjunctival cells. Excess TGF-β production by the parental stem cells makes these cells more mobile by a mechanism whereby it induces increased production of collagenase that more readily dissolves hemidesmosome attachments.57,6265

PINGUECULAE

In the interpalpebral sun-exposed bulbar area of the eye, UV irradiation causes stromal fibroblast changes to occur beneath both limbal and conjunctival epithelia, leading to atrophic flat scars or raised vascular areas of abnormal elastic material production similar to that in the stroma of sun-damaged skin.13,38 However, in the skin, these stromal changes cannot be seen underneath the opaque epidermis, whereas in the interpalpebral area of the eye, the epithelium is transparent and the stromal changes, called pingueculae, are visible.

The limbal epithelium over the limbal portion of a pinguecula has stem cells and can also have UV-induced mutations in the p53 genes (Figure 1, C), leading to vascularized raised pingueculae, limbal tumors, or pterygia. Thus, both the limbal epithelium of pingueculae and the altered fibroblasts in the stroma of pingueculae become storage centers for damaged genes that can lead to growths in the limbal area.

LIMBAL TUMORS INITIATED BY HPV INFECTION

Limbal stem cells infected with oncogenic HPV might produce some limbal tumors. The present results indicate, however, that HPV plays a minor role, if any, in the development of these tumors.

The mechanism of HPV DNA limbal tumorigenesis could be related to the rapid degradation of p53 by the ubiquitin pathway.16,17 Low levels of nuclear p53 protein lead to a damaged p53-dependent programmed cell death mechanism, similar to what occurs when p53 is mutated. This would lead to the accumulation of damaged DNA and the multistep development of limbal tumors.

Such a situation probably occurs in viral papillomas at the limbus and limbal dysplasias that arise outside the UV-exposed interpalpebral region. Human papillomavirus infections in these growths could explain why topical antiviral treatment with interferon alfa-2b can lead to the regression of these tumors6669 by a restoration of the p53-dependent programmed cell death mechanism, which can cause these HPV-infected tumor cells to commit suicide.

Even though we did not find HPV DNA in any of our specimens, HPV DNA may still be a cofactor in certain pterygia and limbal tumor development. Because of HPV DNA's effect on p53 degradation, HPV infection can be a first or early event in tumor development, especially in limbal tumors such as those growths found in immunocompromised patients with pathogenic HPV 16 and 18 ocular infections or in growths found in parts of the world where ocular HPV is endemic.

Accepted for publication July 13, 1999.

This study was supported by grant 161-9895 from the Kaiser Foundation Research Institute, Oakland, Calif (Dr Dushku).

We thank Samuel Woo for photography assistance, Jane Bruner, MT, for polymerase chain reaction assays, and Judy Walls for immunohistochemical analyses, all affiliated with the University of California–Davis Medical Center, Sacramento.

Reprints: Nicholas Dushku, MD, Department of Ophthalmology, Kaiser Permanente Medical Offices, Point West, 1650 Response Rd, Sacramento, CA 95815.

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Tabrizi  SNMcCurrach  FEDrewe  RHBorg  AJGarland  SMTaylor  HR Human papillomavirus in corneal and conjunctival carcinoma. Aust N Z J Ophthalmol. 1997;25211- 215
Link to Article
Dushku  NAlbert  DMReid  TW The use of PCR to test for human papilloma virus DNA in p53 expressing limbal stem cells of pinguecula, pterygia, and limbal tumors. Invest Ophthalmol Vis Sci. 1998;39S543[abstract]
Vojtesek  BBartek  JMidgley  CALane  DP An immunochemical analysis of the human nuclear phosphoprotein p53. J Immunol Methods. 1992;151237- 244
Link to Article
Man  YGTavassoli  FA A simple epitope retrieval method without the use of microwave oven or enzyme digestion. Appl Immunohistochem. 1996;4139- 141
Taylor  CRShi  SRChaiwun  SYoung  LImam  SACote  RJ Strategies for improving the immunohistochemical staining of various intranuclear prognostic markers in formalin-paraffin sections: androgen receptor, estrogen receptor, progesterone receptor, p53 protein, proliferating cell nuclear antigen, and Ki-67 antigen revealed by antigen retrieval techniques. Hum Pathol. 1994;25263- 270
Link to Article
Lovelace  CZhang  JVanek  PCollier  G Detecting apoptotic cells in situ. Biomed Prod. 1996;2176- 77
Ting  YManos  MM Detection and typing of genital human papillomavirus. PCR Protocols A Guide to Methods and Applications. Orlando, Fla Academic Press Inc1990;356- 367
Spencer  WH Ophthalmic Pathology: An Atlas and Textbook. 3rd ed. Philadelphia, Pa WB Saunders Co1985;
Blum  HF Carcinogenesis by Ultraviolet Light.  Princeton, NJ Princeton University Press1959;
Buschke  WFriedenwald  JSMoses  SG Effects of ultraviolet irradiation on corneal epithelium: mitosis, nuclear fragmentation, post-traumatic cell movements, loss of tissue cohesion. J Cell Compr Physiol. 1945;26147- 164
Link to Article
Coroneo  MTFilipic  M Focal elastotic degeneration in the angle and ciliary body of eyes with pterygia. Invest Ophthalmol Vis Sci. 1997;39S1082[abstract]
Schermer  AGalvin  SSun  TT Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol. 1986;10349- 62
Link to Article
Wei  ZGWu  RLLavker  RMSun  TT In vitro growth and differentiation of rabbit bulbar, fornix, and palpebral conjunctival epithelia: implications on conjunctival epithelial transdifferentiation and stem cells. Invest Ophthalmol Vis Sci. 1993;341814- 1828
Lavker  RMDong  GCheng  SZKudoh  KCostarelis  GSun  TT Relative proliferative rates of limbal and corneal epithelia: implications of corneal epithelial migration, circadian rhythm, and suprabasally located DNA-synthesizing keratinocytes. Invest Ophthalmol Vis Sci. 1991;321864- 1875
Miller  SJLavker  RMSun  TT Keratinocyte stem cells of cornea, skin and hair follicle: common and distinguishing features. Dev Biol. 1993;4217- 240
Link to Article
Jakobiec  SA Corneal tumors. Kaufman  HBarron  BAMcDonald  MBWaltman  SReds.The Cornea. London, England Churchill Livingstone Inc1988;563- 598
Cotran  RSKumar  VRobbins  SL Robbins Pathologic Basis of Disease. 4th ed. Philadelphia, Pa WB Saunders Co1989;832- 834
Gigi-Leitner  OGeiger  BLevy  RCzemobilsky  B Cytokeratin expression in squamous metaplasia of the human uterine cervix. Differentiation. 1986;31191- 205
Link to Article
Reid  TWDushku  N Pterygia and limbal epithelial cells: relationship and molecular mechanisms. Prog Retin Eye Res. 1996;15297- 329
Link to Article
Dushku  NAlbert  DMReid  TW Classification of p53 expression in limbal-cells of pingueculae, pterygia, and premalignant and malignant limbal tumors. Invest Ophthalmol Vis Sci. 1997;38S1082[abstract]
Coroneo  MT Pterygium as an early indicator of ultraviolet insolation: a hypothesis. Br J Ophthalmol. 1993;77734- 739
Link to Article
Dushku  NReid  TW Immunohistochemical evidence that pterygia originate from Rb and TGFβ-expressing, p53-transformed, limbal basal stem cells. Invest Ophthalmol Vis Sci. 1995;36S1027
Kria  LOhira  AAmemiya  T Immunohistochemical localization of basic fibroblast growth factor, platelet derived growth factor, transforming growth factor-β and tumor necrosis factor-α in the pterygium. Acta Histochem. 1996;98195- 201
Link to Article
Ren  XLiu  YPTan  DTHSchultz  GS Elevated expression of TGFβ and EGF system in pterygia tissues and matched superior conjunctiva . Invest Ophthalmol Vis Sci. 1998;395509[abstract]
Pasquale  LRDorman-Pease  MELutty  GAQuigley  HAJampel  HD Immunolocalization of TGF-β1, TGF-β2, and TGF-β3 in the anterior segment of the human eye. Invest Ophthalmol Vis Sci. 1993;3423- 30
Roberts  ABSporn  MBAssoian  RKSmith  JMRoche  NSWakefield  LM Transforming growth factor type β: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci U S A. 1986;83416- 417
Link to Article
Liotta  LAStetler-Stevenson  WSteeg  PS Metastasis suppressor genes. DeVita  VTHellman  SRosenberg  SAeds.Oncology. Philadelphia, Pa JB Lippincott1991;85- 100
Seiffert  PSekundo  W Capillaries in the epithelium of pterygium. Br J Ophthalmol. 1998;8277- 81
Link to Article
Dameron  KMVolpert  OVTainsky  ABouck  N Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1. Science. 1994;2651582- 1584
Link to Article
Kay  EPLee  HKPark  KSLee  SC Indirect mitogenic effect of transforming growth factor-β on cell proliferation of subconjunctival fibroblasts. Invest Ophthalmol Vis Sci. 1998;39481- 486
Van der Zee  EEverts  VBeertsen  W Cytokines modulate routes of collagen breakdown. J Clin Periodontol. 1997;24297- 305
Link to Article
Salo  TLyons  JGRahemtulla  FBirkedal-Hansen  H Transforming growth factor-β1 up-regulates type IV collagenase expression in cultured human keratinocytes. J Biol Chem. 1991;26611436- 11441
Fini  MEGirard  MTMatsubara  MBartlett  JD Unique regulation of the matrix metalloproteinase, gelatinase B. Invest Ophthalmol Vis Sci. 1995;36622- 633
Giannelli  GFalk-Marzillier  JSchiraldi  OStetler-Stevenson  WGQuaranta  V Induction of cell migration by matrix metalloprotease-2 cleavage of laminin-5. Science. 1997;277225- 228
Link to Article
Liu  YPSchultz  GSRen  XOTan  DTH MMP2 and MMP9 levels in pterygia and matched superior conjunctiva by gelatin zymography. Invest Ophthalmol Vis Sci. 1998;39S756[abstract]
Baron  STyring  SKFleischmann  WR  et al.  The interferons: mechanisms of action and clinical applications. JAMA. 1991;2661375- 1383
Link to Article
Maskin  SL Regression of limbal epithelial dysplasia with topical interferon. Arch Ophthalmol. 1994;1121145- 1146[letter]
Link to Article
Fung-Rong  HMing-Jet  WSow-Hsong  K Interferon treatment for corneolimbal squamous dysplasia. Am J Ophthalmol. 1998;125118- 119
Link to Article
Vann  RRKarp  CL Perilesional and topical interferon alfa 2-β for conjunctival and corneal neoplasia. Invest Ophthalmol Vis Sci. 1998;39S711[abstract]

Figures

Place holder to copy figure label and caption
Figure 1.

Nuclear p53 immunostaining of altered limbal epithelial cells of pterygia, pingueculae, and limbal tumors with p53 antibodies. A, A specimen of primary medial pterygium of the left eye shows nuclear immunostaining of altered limbal epithelial basal cells (pterygium cells) in the migrating limbus (to the left of the arrowhead pointing to Bowman's layer). Pterygium cells are also migrating onto the corneal basement membrane overlying Bowman's layer. Fibroblasts that have accumulated beneath the migrating limbus do not immunostain for p53 and are making collagenlike material. Fibroblasts immediately next to Bowman's layer appear to be dissolving Bowman's layer (original magnification, ×250). B, In a specimen of recurrent medial pterygium of the left eye, hyperproliferative corneal epithelium overlies Bowman's layer located at the leading edge of the pterygium. An island of fibroblasts (arrowhead) between Bowman's layer and corneal basement membrane does not express p53 and has dissolved Bowman's layer (original magnification, ×325). Directions of basal cell migration: left to right for A and B. C, A specimen of raised pinguecula shows altered limbal cells in limbal epithelium (original magnification, ×425). D, A specimen of conjunctival intraepithelial neoplasia shows dysplastic limbal cells (original magnification, ×250). E, A specimen of carcinoma in situ shows p53-expressing limbal tumor cells with no stromal invasion (original magnification, ×250). F, The p53-expressing limbal tumor cells invade the stroma of a specimen of squamous cell carcinoma (original magnification, ×250).

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Figure 2.

Polymerase chain reaction amplification of genomic DNA (CFTR gene) in specimens of pterygia, pinguecula, and limbal tumors. Lane 1 indicates positive control; lane 2, primary pterygium (A); lane 3, recurrent pterygium (B); lane 4, pinguecula (C); lane 5, conjunctival intraepithelial neoplasia (D); lane 6, carcinoma in situ (E); lane 7, squamous cell carcinoma (F); lane 8, negative control; and lanes M, 123–base pair (bp) DNA ladder. Letters refer to designators in Figure 1.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.

Nested polymerase chain reaction for genomic DNA (CFTR gene) in specimens of pterygia, pinguecula, and limbal tumors. Lane 1 indicates positive control; lane 2, primary pterygium (A); lane 3, pinguecula (C); lane 4, S92-18318; lane 5, S94-18485; lane 6, negative control; and lanes M, 123–base pair (bp) DNA ladder. Letters refer to designators in Figure 1.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.

Polymerase chain reaction amplification for human papillomavirus (HPV) DNA in specimens of pterygia, pinguecula, and limbal tumors. Lane 1 indicates positive control (HPV type 11); lane 2, primary pterygium (A); lane 3, recurrent pterygium (B); lane 4, pinguecula (C); lane 5, conjunctival intraepithelial neoplasia (D); lane 6, carcinoma in situ (E); lane 7, squamous cell carcinoma (F); lane 8, negative control; and lanes M, 123–base pair (bp) DNA ladder. Letters refer to designators in Figure 1.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 5.

Nested polymerase chain reaction for human papillomavirus (HPV) DNA in specimens of pterygia, pingvecula, and limbal tumors. Lane 1 indicates positive control (HPV type 11); lane 2, primary pterygium (A); lane 3, recurrent pterygium (B); lane 4, pinguecula (C); lane 5, conjunctival intraepithelial neoplasia (D); lane 6, carcinoma in situ (E); lane 7, squamous cell carcinoma (F); lane 8, negative control; and lanes M, 123–base pair (bp) DNA ladder. Letters refer to designators in Figure 1.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 6.

Possible pathways for the formation of pterygia and limbal tumors. Rb indicates retinoblastoma protein; TGF-β, transforming growth factor β; question mark, it is not known if these mutations are in the fibroblast cells; and up arrow, increase.

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Tables

References

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Dushku  NReid  TW Immunohistochemical evidence that human pterygia originate from an invasion of vimentin-expressing altered limbal epithelial basal cells. Curr Eye Res. 1994;13473- 481
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Cameron  ME Geographic distribution of pterygia. Trans Ophthalmol Soc Aust. 1962;2267- 81
Taylor  HRWest  SKRosenthal  FSMunoz  BMNewland  HSEmmett  EA Corneal changes associated with chronic UV irradiation. Arch Ophthalmol. 1989;1071481- 1484
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Duke-Elder  S Diseases of the outer eye. System of Ophthalmology. Vol 8.Part 1. St Louis, Mo CV Mosby1965;573- 582
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Tabbara  KFKersten  RDaouk  NBlodi  FC Metastatic squamous cell carcinoma of the conjunctiva. Ophthalmology. 1988;95318- 321
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Clear  ASChirambo  MCHutt  MSR Solar keratosis, pterygium, and squamous cell carcinoma of the conjunctiva in Malawi. Br J Ophthalmol. 1979;63102- 109
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Kress  SSutter  CStrickland  PTMukhtar  HSchweizer  JSchwartz  M Carcinogen-specific mutational pattern in the p53 gene in ultraviolet B radiation-induced squamous cell carcinomas of mouse skin. Cancer Res. 1992;526400- 6403
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Dushku  NReid  TW p53 expression in altered limbal basal cells of pingueculae, pterygia, and limbal tumors. Curr Eye Res. 1997;161179- 1192
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Kinzler  KWVogelstein  B Life (and death) in a malignant tumour. Nature. 1996;37919- 20
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Weinberg  RA Oncogenes, antioncogenes, and the molecular bases of multistep carcinogenesis. Cancer Res. 1996;493713- 3721
Werness  BALevine  AJHowley  PM Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science. 1990;24876- 79
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Scheffner  MWerness  BAHuibregste  JMLevine  AJHowley  PM The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell. 1990;631129- 1136
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Tommasino  MCrawford  L Human papillomavirus E6 and E7: proteins which deregulate the cell cycle. Bioessays. 1995;17509- 518
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Kessis  TDSlebos  RJNelson  WG  et al.  Human papillomavirus 16 E6 expression disrupts the p53-mediated cellular response to DNA damage. Proc Natl Acad Sci U S A. 1993;903988- 3992
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Foster  SADemers  GWEtscheid  BGGalloway  DA The ability of human papillomavirus E6 proteins to target p53 for degradation in vivo correlates with their ability to abrogate actinomycin D-induced growth arrest. J Virol. 1994;685698- 5705
Barten  MOstwald  CMilde-Langosch  KMuller  PWukasch  YLoning  T HPV DNA and p53 alterations in oropharyngeal carcinomas. Virchows Arch. 1995;427153- 157
Link to Article
Lam  KYChan  ACChan  KWLeung  MLSrivastava  G Expression of p53 and its relationship with human papillomavirus in penile carcinomas. Eur J Surg Oncol. 1995;21613- 616
Link to Article
Akasofu  MOda  Y Immunohistochemical detection of p53 in cervical epithelial lesions with or without infection of human papillomavirus types 16 and 18. Virchows Arch. 1995;425593- 602
Link to Article
He  DZhang  DKLam  KY  et al.  Prevalence of HPV infection in esophageal squamous cell carcinoma in Chinese patients and its relationship to the p53 gene mutation. Int J Cancer. 1997;72959- 964
Link to Article
Ngan  HYTsao  SWLiu  SSStanley  M Abnormal expression and mutation of p53 in cervical cancer: a study at protein, RNA and DNA levels. Genitourin Med. 1997;7354- 58
Markowska  JNowak  MBar  MHarlozinska  A Expression of p53 and coexistence of HPV in premalignant lesions and in cervical cancer. Eur J Gynaecol Oncol. 1995;16368- 372
Dolmetsch  AMAlcocer  CEScull  JJMartins  MCStockl  FABurnier  MN The presence of human papilloma virus in pterygia. Invest Ophthalmol Vis Sci. 1996;37S43[abstract]
McDonnell  JMMcDonnell  PJSun  YY Human papillomavirus DNA in tissues and ocular surface swabs of patients with conjunctival epithelial neoplasia. Invest Ophthalmol Vis Sci. 1992;33184- 189
Alcocer  CBScull  JJMartins  MC  et al.  The presence of human papilloma virus in association with epithelial neoplasia of the conjunctiva. Invest Ophthalmol Vis Sci. 1996;37S625[abstract]
Karcioglu  ZAIssa  TM Human papilloma virus in neoplastic and non-neoplastic conditions of the external eye. Br J Ophthalmol. 1997;81595- 598
Link to Article
Tabrizi  SNMcCurrach  FEDrewe  RHBorg  AJGarland  SMTaylor  HR Human papillomavirus in corneal and conjunctival carcinoma. Aust N Z J Ophthalmol. 1997;25211- 215
Link to Article
Dushku  NAlbert  DMReid  TW The use of PCR to test for human papilloma virus DNA in p53 expressing limbal stem cells of pinguecula, pterygia, and limbal tumors. Invest Ophthalmol Vis Sci. 1998;39S543[abstract]
Vojtesek  BBartek  JMidgley  CALane  DP An immunochemical analysis of the human nuclear phosphoprotein p53. J Immunol Methods. 1992;151237- 244
Link to Article
Man  YGTavassoli  FA A simple epitope retrieval method without the use of microwave oven or enzyme digestion. Appl Immunohistochem. 1996;4139- 141
Taylor  CRShi  SRChaiwun  SYoung  LImam  SACote  RJ Strategies for improving the immunohistochemical staining of various intranuclear prognostic markers in formalin-paraffin sections: androgen receptor, estrogen receptor, progesterone receptor, p53 protein, proliferating cell nuclear antigen, and Ki-67 antigen revealed by antigen retrieval techniques. Hum Pathol. 1994;25263- 270
Link to Article
Lovelace  CZhang  JVanek  PCollier  G Detecting apoptotic cells in situ. Biomed Prod. 1996;2176- 77
Ting  YManos  MM Detection and typing of genital human papillomavirus. PCR Protocols A Guide to Methods and Applications. Orlando, Fla Academic Press Inc1990;356- 367
Spencer  WH Ophthalmic Pathology: An Atlas and Textbook. 3rd ed. Philadelphia, Pa WB Saunders Co1985;
Blum  HF Carcinogenesis by Ultraviolet Light.  Princeton, NJ Princeton University Press1959;
Buschke  WFriedenwald  JSMoses  SG Effects of ultraviolet irradiation on corneal epithelium: mitosis, nuclear fragmentation, post-traumatic cell movements, loss of tissue cohesion. J Cell Compr Physiol. 1945;26147- 164
Link to Article
Coroneo  MTFilipic  M Focal elastotic degeneration in the angle and ciliary body of eyes with pterygia. Invest Ophthalmol Vis Sci. 1997;39S1082[abstract]
Schermer  AGalvin  SSun  TT Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol. 1986;10349- 62
Link to Article
Wei  ZGWu  RLLavker  RMSun  TT In vitro growth and differentiation of rabbit bulbar, fornix, and palpebral conjunctival epithelia: implications on conjunctival epithelial transdifferentiation and stem cells. Invest Ophthalmol Vis Sci. 1993;341814- 1828
Lavker  RMDong  GCheng  SZKudoh  KCostarelis  GSun  TT Relative proliferative rates of limbal and corneal epithelia: implications of corneal epithelial migration, circadian rhythm, and suprabasally located DNA-synthesizing keratinocytes. Invest Ophthalmol Vis Sci. 1991;321864- 1875
Miller  SJLavker  RMSun  TT Keratinocyte stem cells of cornea, skin and hair follicle: common and distinguishing features. Dev Biol. 1993;4217- 240
Link to Article
Jakobiec  SA Corneal tumors. Kaufman  HBarron  BAMcDonald  MBWaltman  SReds.The Cornea. London, England Churchill Livingstone Inc1988;563- 598
Cotran  RSKumar  VRobbins  SL Robbins Pathologic Basis of Disease. 4th ed. Philadelphia, Pa WB Saunders Co1989;832- 834
Gigi-Leitner  OGeiger  BLevy  RCzemobilsky  B Cytokeratin expression in squamous metaplasia of the human uterine cervix. Differentiation. 1986;31191- 205
Link to Article
Reid  TWDushku  N Pterygia and limbal epithelial cells: relationship and molecular mechanisms. Prog Retin Eye Res. 1996;15297- 329
Link to Article
Dushku  NAlbert  DMReid  TW Classification of p53 expression in limbal-cells of pingueculae, pterygia, and premalignant and malignant limbal tumors. Invest Ophthalmol Vis Sci. 1997;38S1082[abstract]
Coroneo  MT Pterygium as an early indicator of ultraviolet insolation: a hypothesis. Br J Ophthalmol. 1993;77734- 739
Link to Article
Dushku  NReid  TW Immunohistochemical evidence that pterygia originate from Rb and TGFβ-expressing, p53-transformed, limbal basal stem cells. Invest Ophthalmol Vis Sci. 1995;36S1027
Kria  LOhira  AAmemiya  T Immunohistochemical localization of basic fibroblast growth factor, platelet derived growth factor, transforming growth factor-β and tumor necrosis factor-α in the pterygium. Acta Histochem. 1996;98195- 201
Link to Article
Ren  XLiu  YPTan  DTHSchultz  GS Elevated expression of TGFβ and EGF system in pterygia tissues and matched superior conjunctiva . Invest Ophthalmol Vis Sci. 1998;395509[abstract]
Pasquale  LRDorman-Pease  MELutty  GAQuigley  HAJampel  HD Immunolocalization of TGF-β1, TGF-β2, and TGF-β3 in the anterior segment of the human eye. Invest Ophthalmol Vis Sci. 1993;3423- 30
Roberts  ABSporn  MBAssoian  RKSmith  JMRoche  NSWakefield  LM Transforming growth factor type β: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci U S A. 1986;83416- 417
Link to Article
Liotta  LAStetler-Stevenson  WSteeg  PS Metastasis suppressor genes. DeVita  VTHellman  SRosenberg  SAeds.Oncology. Philadelphia, Pa JB Lippincott1991;85- 100
Seiffert  PSekundo  W Capillaries in the epithelium of pterygium. Br J Ophthalmol. 1998;8277- 81
Link to Article
Dameron  KMVolpert  OVTainsky  ABouck  N Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1. Science. 1994;2651582- 1584
Link to Article
Kay  EPLee  HKPark  KSLee  SC Indirect mitogenic effect of transforming growth factor-β on cell proliferation of subconjunctival fibroblasts. Invest Ophthalmol Vis Sci. 1998;39481- 486
Van der Zee  EEverts  VBeertsen  W Cytokines modulate routes of collagen breakdown. J Clin Periodontol. 1997;24297- 305
Link to Article
Salo  TLyons  JGRahemtulla  FBirkedal-Hansen  H Transforming growth factor-β1 up-regulates type IV collagenase expression in cultured human keratinocytes. J Biol Chem. 1991;26611436- 11441
Fini  MEGirard  MTMatsubara  MBartlett  JD Unique regulation of the matrix metalloproteinase, gelatinase B. Invest Ophthalmol Vis Sci. 1995;36622- 633
Giannelli  GFalk-Marzillier  JSchiraldi  OStetler-Stevenson  WGQuaranta  V Induction of cell migration by matrix metalloprotease-2 cleavage of laminin-5. Science. 1997;277225- 228
Link to Article
Liu  YPSchultz  GSRen  XOTan  DTH MMP2 and MMP9 levels in pterygia and matched superior conjunctiva by gelatin zymography. Invest Ophthalmol Vis Sci. 1998;39S756[abstract]
Baron  STyring  SKFleischmann  WR  et al.  The interferons: mechanisms of action and clinical applications. JAMA. 1991;2661375- 1383
Link to Article
Maskin  SL Regression of limbal epithelial dysplasia with topical interferon. Arch Ophthalmol. 1994;1121145- 1146[letter]
Link to Article
Fung-Rong  HMing-Jet  WSow-Hsong  K Interferon treatment for corneolimbal squamous dysplasia. Am J Ophthalmol. 1998;125118- 119
Link to Article
Vann  RRKarp  CL Perilesional and topical interferon alfa 2-β for conjunctival and corneal neoplasia. Invest Ophthalmol Vis Sci. 1998;39S711[abstract]

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