HIV-positive patients taking antiretroviral drugs, including protease inhibitors, have shown a significant increase in the development of oral complications; these complications are a major health issue for affected patients. The effect of these drugs on oral epithelium growth and differentiation is presently unknown. In this study, we explore for the first time the effect of the HIV protease inhibitor amprenavir on gingival epithelium growth and differentiation.
Methods
Organotypic (raft) cultures of gingival keratinocytes were established and the raft cultures treated with a range of amprenavir concentrations. Haematoxylin and eosin staining was performed to examine the effect of amprenavir on gingival epithelium growth and stratification. The raft cultures were also immunohistochemically analysed to determine the effect of amprenavir on the expression of key differentiation and proliferation markers, including cytokeratins, proliferating cell nuclear antigen (PCNA) and cyclin A.
Results
Amprenavir severely inhibited the growth of gingival epithelium when the drug was present throughout the growth period of the tissue. When the drug was added at day 8, amprenavir treatment altered the proliferation and differentiation of gingival keratinocytes. Expression of the cytokeratins 5, 14, 6 and 10, PCNA and cyclin A was increased; their expression pattern was also altered over time in treated rafts. Biochemically, the tissue exhibited characteristics of increased proliferation in the suprabasal layers of amprenavir-treated tissue.
Conclusions
Our results suggest that amprenavir treatment deregulates the cell cycle/proliferation and differentiation pathways, resulting in abnormal epithelial repair and proliferation. Our system could be developed as a potential model for studying the effects of highly active antiretroviral therapy in vitro.
MoniaciD., GrecoD., FlecchiaG., RaitieriR., SiniccoA.Epidemiology, clinical features and prognostic value of HIV-1 related oral lesions.J Oral Pathol Med1990; 19: 477–481.
3.
BravoI.M., CorrentiM., EscalonaL.Prevalence of oral lesions in HIV patients related to CD4 cell count and viral load in Venezuelan population.Med Oral Patol Oral Cir Bucal2006; 11: E33–E39.
4.
GortmakerS.L., HughesM., CerviaJ.Effects of combination therapy including protease inhibitors on mortality among children and adolescents infected with HIV-1.N Engl J Med2001; 345: 1522–1528.
5.
GranadosJ.M.S., AmadorJ.T.R., MiguelS.F.Impact of highly active antiretroviral therapy on the morbidity and mortality in Spanish human immunodeficiency virus infected children.Pediatr Infect Dis J2003; 22: 863–867.
6.
GreenspanD., CancholaA.J., MacPhailA., CheikhB., GreenspanJ.S.Effect of highly active antiretroviral therapy on frequency of oral warts.Lancet2001; 357: 1411–1412.
7.
GreenspanJ.S., GreenspanD.The epidemiology of the oral lesions of HIV infection in the developed world.Oral Dis2002; 8: 34–39.
8.
HodgsonT.A., GreenspanD., GreenspanJ.S.Oral leasions of HIV disease and HAART in industrialized countries.Adv Dent Res2006; 19: 57–62.
9.
ScullyC., DiosP.D.Orofacial effects of antiretroviral therapies.Oral Dis2001; 7: 205–210.
10.
DiosP.D., ScullyC.Adverse effects of antiretroviral therapy: focus on orofacial effects.Expert Opin Drug Saf2002; 1: 307–317.
11.
FagotJ.P., MockenhauptM., Bouwes-BavinckJ.N., NaldiL., ViboudC., RoujeauJ.C.Nevirapine and the risk of Stevans– Johnson syndrome or toxic epidermal necrolysis.AIDS2001; 15: 1843–1848.
12.
JordanR.A.[Implications of antiretroviral therapy in oral medicine – a review of literature].Schweiz Monatsschr Zahnmed2007; 117: 1210–1216. German.
13.
FungH.B., KirschenbaumH.L., HameedR.Amprenavir: a new human immunodeficiency virus type 1 protease inhibitor.Clin Ther2000; 22: 549–572.
14.
GoodgameJ.C., PottageJ.C.Jr., JablonowskiH.Amprenavir in combination with lamivudine and zidovudine versus lamivudine and zidovudine alone in HIV-1-infected antiretroviral-naive adults.Antivir Ther2000; 5: 215–225.
15.
RotundaA., HirschR.J., ScheinfeldN., WeinbergJ.M.Severe cutaneous reactions associated with the use of human immunodeficiency virus medications.Acta Derm Venereol2003; 83: 1–9.
16.
FlintS.R., TappuniA., LeighJ., Schmidt-WesthausenA.M., MacPhailL.Markers of immunodeficiency and mechanisms of HAART therapy on oral lesions.Adv Dent Res2006; 19: 146–151.
17.
HawkinsT.HIV drug resistance and you. The limitations of drug resistance testing. Many factors influence the results.Posit Aware2006; Spec no: 13–16.
18.
PreslandR.B., DaleB.A.Epithelial structural proteins of the skin and oral cavity: function in health and disease.Crit Rev Oral Biol Med2000; 11: 383–408.
19.
SquierC.A., KremerM.J.Biology of oral mucosa and esophagus.J Natl Cancer Inst Monogr2001; 29: 7–15.
20.
FrankeW.W., SchillerD.L., MollR.Diversity of cytokeratins: differentiation specific expression of cytokeratin polypeptides in epithelial cells and tissues.J Mol Biol1981; 153: 933–939.
21.
MollR., FrankeW.W., SchillerD.L., GeigerB., KreplerR.The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells.Cell1982; 31: 11–24.
22.
WatanabeS., OsumiM., OhnishiT., IchikawaE., TakahashiH.Changes in cytokeratin expression in epidermal keratinocytes during wound healing.Histochem Cell Biol1995; 103: 425–433.
23.
PaladiniR.D., TakahashiK., BravoN.S., CoulombeP.A.Onset of re-epithelialization after skin injury correlates with a reorganization of keratin filaments in wound edge keratinocytes: defining a potential role for keratin 16.J Cell Biol1996; 132: 381–397.
24.
BloorB.K., SeddonS.V., MorganP.R.Gene expression of differentiation specific keratins (K4, K13, K1 and K10) in oral non-dysplastic keratoses and lichen planus.J Oral Pathol Med2000; 29: 376–384.
25.
BonanP.R.F., KaminagakuraE., PiresF.R.Cytokeratin expression in initial oral mucositis of head and neck irradiated patients.Oral Surg Oral Med Oral Pathol Oral Radiol Endod2006; 101: 205–211.
26.
MeyersC.Organotypic (raft) epithelial tissue culture system for the differentiation-dependent replication of papillomavirus.Methods Cell Sci1996; 18: 201–210.
27.
MeyersC., FrattiniM.G., HudsonJ.B., LaiminsL.A.Biosynthesis of human papillomavirus from a continuous cell line upon epithelial differentiation.Science1992; 257: 971–973.
28.
TomakidiP., FusenigN.E., KohlA., KomposchG.Histomorphological and biochemical differentiation capacity in organotypic co-cultures of primary gingival cells.J Periodontal Res1997; 32: 388–400.
29.
KlausnerM., AyehunieS., BreyfogleB.A., WertzP.W., BaccaL., KubilusJ.Organotypic human oral tissue models for toxicological studies.Toxicol In Vitro2007; 21: 938–949.
30.
MackenzieI.C., RittmanG., GaoZ., LeighI., LaneE.B.Patterns of cytokeratin expression in human gingival epithelia.J Periodontal Res1991; 26: 468–478.
31.
EichnerR., BonitzP., SunT.T.Classification of epidermal keratins according to their immunoreactivity, isoelectric point, and mode of expression.J Cell Biol1984; 98: 1388–1396.
32.
PurkisP.E., SteelJ.B., MackenzieI.C., NathrathW.B., LeighI.M., LaneE.B.Antibody markers of basal cells in complex epithelia.J Cell Sci1990; 97: 39–50.
33.
WeissR.A., EichnerR., SunT.T.Monoclonal antibody analysis of keratin expression in epidermal diseases: a 48 and 56-kdalton keratin as molecular marker for hyperproliferative keratinocytes.J Cell Biol1984; 98: 1397–1406.
34.
BravoR., FrankR., BlundellP.A., Macdonald-BravoH.Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta.Nature1987; 326: 515–517.
35.
PaganoM., PepperkokR., VerdeF., AnsorgeW., DraettaG.Cyclin A is required at two points in the human cell cycle.EMBO J1992; 11: 961–971.
36.
DesdouetsC., Sobczak-ThepotJ., MurphyM., BrechotC.Cyclin A: function and expression during cell proliferation.Prog Cell Cycle Res1995; 1: 115–123.
37.
BernerdF., MagnaldoT., DarmonM.Delayed onset of epidermal differentiation in psoriasis.J Invest Dermatol1992; 98: 902–910.
38.
FuchsE., GreenH.Changes in keratin gene expression during terminal differentiation of the keratinocytes.Cell1980; 19: 1033–1042.
39.
dos SantosJ.N., de SousaS.O.M., NunesF.D., SottoM.N., de AraujoV.C.Altered cytokeratin expression in actinic cheilitis.J Cutan Pathol2003; 30: 237–241.
40.
BloorB.K., TidmanN., LeighI.M.Expression of keratin K2e in cutaqneous and oral lesions: association with keratinocytes activation, proliferation and keratinization.Am J Pathol2003; 162: 963–975.
41.
WetzelsR.H., KujipersH.J., LaneE.B.Basal specific and hyperproliferation-related keratins in human breast cancer.Am J Pathol1991; 138: 751–763.
42.
MorganP.R., SuL.Intermediate filaments in oral neoplasia. 1. Oral cancer and epithelial dysplasia.Eur J Cancer B Oral Oncol1994; 30: 160–166.
43.
UsuiM.L., UnderwoodR.A., MansbridgeJ.N., MuffleyL.A., CarterW.G., OlerudJ.E.Morphological evidence for the role of suprabasal keratinocytes in wound reepithelialization.Wound Repair Regen2005; 13: 468–479.
44.
MansbridgeJ.N., KnappA.M.Changes in keratinocytes maturation during wound healing.J Invest Dermatol1987; 89: 253–263.
45.
StolerA., KopanR., DuvicM., FuchsE.Use of monospecific antisera and cRNA probes to localize the major changes in keratin expression during normal and abnormal epidermal differentiation.J Cell Biol1988; 107: 427–446.
46.
LundqvistK., SchmidtchenA.Cyclin A expression in chronic leg ulcers.Acta Derm Venereol2006; 86: 61–62.
47.
ThomsonP.J., GoodsonM.L., BoothC., CraggN., HamadahO.Cyclin A activity predicts clinical outcome in oral precancer and cancer.Int J Oral Maxillofac Surg2006; 35: 1041–1046.
48.
NelsonW.G., SunT.T.The 50- and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies.J Cell Biol1983; 97: 244–251.
49.
CelisJ.E., CelisA.Cell cycle dependent variations in the distribution of the nuclear protein cyclin proliferating cell nuclear antigen in cultured cells: subdivision of S phase.Proc Natl Acad Sci U S A1985; 82: 3262–3266.
50.
ZuberM., TanE.M., RyojiM.Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.Mol Cell Biol1989; 9: 57–66.
51.
BujiaJ., SudhoffH., HollyA., HildmannH., KastenbauerE.Immunohistochemical detection of proliferating cell nuclear antigen in middle ear cholesteatoma.Eur Arch Otorhinolaryngol1996; 253: 21–24.
52.
GirodS.C., PfeiffertP., RiesJ., PapetH.D.Proliferative activity and loss of function of tumour suppressor genes as biomarkers in diagnosis and prognosis of benign and preneoplastic oral lesions and oral squamous cell carcinoma.Br J Oral Maxillofac Surg1998; 36: 252–260.
53.
KingM.D., ReznikD.A., O'DanielaC.M.Human papillomavirus associated oral warts among human immunodeficiency virus seropositive patients in the area of highly active antiretroviral therapy: an emerging infection.Clin Infect Dis2002; 34: 641–648.
54.
CameronJ.E., MercanteD., O'BrienM.The impact of highly active antiretroviral therapy and immunodeficiency on human papillomavirus infection of the oral cavity of human immunodeficiency virus seropositive adults.Sex Transm Dis2005; 32: 703–709.
