j clin mol immunocytochemical expression of growth factors ...odontogenesis, particularly in...

J Clin Pathol:Mol Pathol 1997;50:21-27

Immunocytochemical expression of growth factorsby odontogenic jaw cysts

Tiejun Li, Roger M Browne, John B Matthews

AbstractAim-To determine the immunocyto-chemical pattern of expression of trans-forming growth factor (TGF) a, epidermalgrowth factor (EGF), and TGFP in thethree most common types of odontogenicjaw cystMethods-Growth factor expression wasdetected in paraffin wax sections of odon-togenic cysts (27 odontogenic keratocysts,10 dentigerous cysts, and 10 radicularcysts) using a streptavidin-biotin peroxi-dase technique with monoclonal antibod-ies directed against TGFa (clone 213-4.4)and TGFP (clone TB21) and a polyclonalantibody directed against EGF (Z-12).Results-The epithelial linings of all cystsshowed reactivity for TGFa which wasmainly localised to basal and suprabasallayers. Odontogenic keratocyst liningsexpressed higher levels of TGFa thanthose of dentigerous and radicular cysts,with 89% (24127) of odontogenic kerato-cysts exhibiting a strong positive reactioncompared with 50% (five of 10) of denti-gerous and radicular cysts, respectively.EGF reactivity was similar in all cystgroups, weaker than that for TGFa andpredominantly suprabasal. TGFa andEGF were also detected in endothelialcells, fibroblasts and inflammatory cellswithin the cyst walls. The most intenseTGFP staining in odontogenic cysts wasextracellular within the fibrous tissuecapsules, irrespective of cyst type.Conclusions-These results, together withprevious studies ofEGF receptor, indicatedifferential expression of TGFa, EGF andtheir common receptor between the dif-ferent types of odontogenic cyst, suggest-ing that these growth factors (viaautocrine or paracrine, or both, path-ways) may be involved in their pathogen-esis.(JClin Pathol:Mol Pathol 1997;50:21-27)

Keywords: EGF; TGFa; odontogenic cysts; TGFI.

Unit of Oral Pathology,School of Dentistry,University ofBirmingham, StChad's Queensway,Birmingham B4 6NN

Correspondence to:Dr J B Matthews.

Accepted for publication28 October 1996

Transforming growth factor alpha (TGFa) andepidermal growth factor (EGF) are structurallyrelated mitogens that regulate cell growth anddifferentiation.' 2 Both polypeptides are ligandsbound by the external domain of the 170 kDepidermal growth factor receptor (EGF-R)localised to the cell surface membranes ofmany cell types.'4 Ligand-receptor bindingactivates a tyrosine specific protein kinasewhich is part of the intracellular domain of the

EGF-R and leads to intracellular changes pre-paring the cell for DNA synthesis and celldivision.2 3 5 6EGF was first isolated from mouse sub-

mandibular glands and found to accelerateincisor tooth eruption and eyelid opening inthe developing animal.' It is a potent mitogenwhich stimulates the growth and differentiationof a variety of mammalian cells, including epi-thelial cells.' 7 TGFa was originally isolatedfrom retrovirally transformed 3T3 fibroblastsas a factor that stimulated anchorage inde-pendent growth.2 Like EGF, high level expres-sion ofTGFa is mitogenic in cell lines possess-ing EGF receptors.8 Both ligands have beenidentified in various human fetal and adulttissues"'* and high levels of TGFa, and some-times EGF, have been detected in a wide vari-ety of neoplastic tissues including odontogenictumours.'2 13

Recent studies have suggested that membersof the EGF family (that is, EGF, TGFa, andEGF-R) are involved in regulation and controlof tooth development.'"-18 Administration ofEGF or TGFa to newborn mice acceleratesincisor eruption.' 19 20 Exogenous EGF hasbeen shown to inhibit early mouse tooth mor-phogenesis and cytodifferentiation in vitro, andto decrease tooth size in vivo. 1 2122 Further-more, EGF antisense oligodeoxyribonucle-otides can block murine odontogenesis invitro.'7 Although studies on EGF-R expressionby the tooth germ are contradictory,'4 18 22 onerecent study has demonstrated that EGF-RmRNA and immunoreactive cells are mostlylocated in the epithelial elements of humantooth germ and its expression is subjected totemporospatial variation at different stages oftooth development. 13 As different types ofodontogenic cyst arise from the epithelial rem-nants formed at different stages of tooth devel-opment, they may reflect the cellular andmolecular events occurring during normalodontogenesis, particularly in developmentalcyst types. In recent studies, we have demon-strated differential expression of EGF recep-tors and parathyroid hormone related protein(PTHrP) by, and qualitative and quantitativedifferences in proliferative state between, thedifferent types of odontogenic cyst.2126 Theaim of this study was to determine theimmunocytochemical expression ofTGFa andEGF in different types of odontogenic cyst. AsTGFO, a multifunctional growth factor, hasalso been implicated in tooth development,possibly as a signalling regulator of epithelialdifferentiation,2728 its distribution in differentcyst types was also studied.

21

on April 2, 2020 by guest. P

rotected by copyright.http://m

p.bmj.com

/M

ol Path: first published as 10.1136/m

p.50.1.21 on 1 February 1997. D

ownloaded from

http://mp.bmj.com/

Li, Browne, Matthews

*j t.4.9?Aq

Figure 1 Staining ofan oral squamous cell carwith antibody directed against TGFa preincubaiovernight with buffer (A) and a 10-fold excess ETGFa (B).

MethodsTISSUES

Forty seven odontogenic cysts (27 odckeratocysts, 10 dentigerous cysts,radicular cysts) were retrieved from tlthe Unit of Oral Pathology. Ofodontogenic keratocysts, 10 were

occurring as a solitary lesion inhealthy patients; eight cysts were

lesions within a period of three to 10 yprimary surgery; nine cysts were

patients known to have naevoid bcarcinoma syndrome (NBCCS). Aspecimens were fixed in 10% neutralformalin (18-48 hours), processedand embedded in paraffin wax. Onlycified specimens or specimens that I

decalcified in 10% formic acid for les;hours were chosen to minimise possibsistencies in immunoreactivity as a

such processing variables. Routinely iprocessed specimens of human skinglands and oral squamous cell carcinoused as positive controls.

-.*3 IMMUNOCYTOCHEMISTRYExpression of TGFa, EGF and TGFf wasdetected using a streptavidin-biotin immu-noperoxidase technique (StrAviGen; Bio-Genex, San Ramon, USA) on freshly cut, 5 jmparaffin wax sections that had been flattenedonto polylysine coated glass slides at roomtemperature overnight. Clone 213-4.4 (Onco-gene Science, Uniondale, USA), specific foramino residues 34-50 at the C-terminus ofTGFa, was used at a concentration of 0.5 jg/ml. EGF was detected using a rabbit polyclonalantibody (Z1 2; Santa Cruz Biotechnology)raised against recombinant human EGF andused at a concentration of 2.5 gg/ml. Affinitypurified monoclonal antibody directed againstTGFI (clone TB2 1; mouse IgGjk, Serotec,Oxford, UK), raised against purified TGFI31from human platelets,20 was used at a concen-tration of 0.33 jg/ml. This antibody has beenshown to neutralise the action of TGFf31 onthe growth of various target cells in vitro (minklung epithelial cells; NRK 49F cells) and toreact with recombinant human TGFI31 andpurified human platelet TGFI31 by ELISA. On

rcinoma western blotting, single bands correspondingted to the 12.5 kD monomer and 25 kD dimer5y weight of were observed from crude acid/ethanol extracts

of human platelets after electrophoresis inreducing and non-reducing conditions, respec-tively. At the present time, possible cross-reactivity with other TGFI isotypes and latent

)ntogenic forms of TGFf has not been tested.and 10 In brief, deparaffinised sections were im-

he files of mersed in 0.3% hydrogen peroxide in bufferthe 27 for 10 minutes to abolish endogenous peroxi-primary, dase activity, washed and then overlayed withotherwise primary antibodies at room temperature forrecurrent two hours. After washing, the sections were

rearsafter further treated with biotinylated multi-linkfrom six (BioGenex, 1 in 75 dilution in buffer contain-

asal cell ing 100% normal human serum; one hour at

U11tissue temperature),

L tffisd with peroxidase-labelled streptavidin (Bio-bouffnere Genex, 1 in 75 dilution; one hour at room tem-routnely perature). Bound peroxidase was visualised byundecal- immersing washed sections in 3,3'-sad been diaminobenzidine (five minutes) and reactions than six products subsequently enhanced by treatment41e incon- with 0.5% copper sulphate (w/v in saline; fiveresult of minutes). Stained sections were lightly coun-fixed and terstained in Meyer's haematoxylin and, salivary mounted in Xam. Phosphate buffered salinemas were (PBS; 0.01 M; pH 7.6) was used for all reagent

dilutions and washes.

Table 1 Immunoreactivity of odontogenic cyst epithelium with antibodies directed against TGFa and EGF

TGFa EGFNumber of

Epithelial lining specimens ++* + - Patternt ++ + - Pattern

Odontogenic 27keratocystSolitary 10 9 1 0 10 0 10 0 0Recurrent 8 7 1 0 1 1 7 0 1Syndrome 9 8 1 0 0 1 8 0 1

Dentigerous cyst 10 5 5 0 2 0 8 2 3tRadicular cyst 10 5 5 0 1 0 8 2 5:

*Number of cases showing degree of reaction: ++, strong; +, weak; -, no staining.tNumber of cases showing predominantly basal and suprabasal staining. All others showed predominantly suprabasal staininig only.tTwo specimens each of dentigerous and radicular cysts were EGF negative.

22



p.bmj.com

/M


p.50.1.21 on 1 February 1997. D

ownloaded from

http://mp.bmj.com/

EGF, TGFa and TGFP in odontogenic cysts

AB

C D~~~ .'

.04K

E F +; .

s ~~~,j XCr~s

'4.

Figure 2 Staining of odontogenic keratocysts (A and D), dentigerous (B) and radicular cysts (C), and epithelial restswithin the connective tissue wall (E) for TGFa. Note the staining pattern of strong basal and granular layer reactivity (D)in some odontogenic keratocyst linings, and strong TGFa reactivity in connective tissue wall associated with the presence ofinflammation (F).

Optimum dilutions, times and temperatureswere determined in preliminary experiments Aon a variety of routinely fixed and processedtissue blocks. Positive staining was abolishedwhen normal mouse IgG, a monoclonalantibody of irrelevant specificity (MRC OX-52, IgG2a anti-rat T cell; Serotec) or PBS wereused as the first layer and when one or bothfsecond layers were omitted. The specificity ofthe reaction was determined by preincubationof antibodies with 10-fold excess by weight oftheir corresponding peptides at 4°C overnight(TGFa, Sigma, Poole, Dorset, UK; EGF,Santa Cruz Biotechnology; TGF,B, Serotec). B

EVALUATION OF TISSUE SECTIONS

Overall, TGFa, EGF and TGFf3 stainingintensity of different cyst linings was scoredsubjectively using a semiquantitative scale:strong (++), weak (+) and negative (-). Inaddition, the distribution of TGFat and EGFstaining within the cyst linings was scored toindicate which epithelial layer(s) exhibited thestrongest reaction: basal and suprabasal; andsuprabasal. Because substantial staining ofTGF,B was also seen in the fibrous tissuecapsules of odontogenic cysts, its staining Figure 3 Staining of an odontogenic keratocyst (A) and aintensity was assessed subjectively for both cyst dentigerous cyst (B) for EGF

23



p.bmj.com

/M


p.50.1.21 on 1 February 1997. D

ownloaded from

http://mp.bmj.com/


0.1TGFa

M EGF

0.08

L)0.06

.00.0

0.02

Simple Rec Syn DC RCOKC OKC OKC

Figure 4 Histogram showing TGFa and EGF absorbancereadings of the epithelial linings of odontogenic cystsmeasured by TV image analysis (mean absorbance (SD)).Rec = recurrent; Syn = syndrome; OKC = odontogenickeratocyst.

linings and fibrous tissue capsules. To assessobjectively the immunoreactivity of TGFu andEGF in odontogenic cyst linings, the absorb-ance ofthe stained cyst linings was determined,at a magnification of x200, using TV imageanalysis (Seescan Prism; Seescan Imaging,Cambridge, UK). This image analysis systemgives true, reproducible absorbance readingsthat are independent of the level ofbackgroundillumination and a linear response over a widerange of absorbance values (0 to +3).3° 31Preliminary titration experiments with EGFand TGFu antibodies showed that the chro-mogen density obtained at working concentra-tions was within this linear response range.Measurements were made in five areas of cystlining in each case. Data were analysed statisti-cally using the Mann-Whitney U-test.

ResultsSTAINING PATTERN IN CONTROL TISSUES

TGFa and EGF staining was localised to thecytoplasm and mainly detected in epidermis,ducts of salivary glands and epithelial tumourcomponents. In epidermis and oral squamouscell carcinoma, strong reactivity for TGFct was

usually detected in areas showing squamousdifferentiation and in central areas of tumournests. Staining in the basal cell layers was weakor absent (fig IA). TGFu positive staining wasalso found in the cytoplasm of endothelial cells,striated and smooth muscle, macrophages,plasma cells, and fibroblasts. Overall, stainingintensity of TGFa was stronger than that ofEGF. By contrast, the most intense staining forTGFf was found in the connective tissues ofthe positive control specimens and within theepithelium, it was extracellular and usuallyweaker. However, the staining reactions of allthree antibodies were completely abolished bypreincubation with excess corresponding re-

combinant growth factor (fig 1).

TGF(L AND EGF STAINING IN ODONTOGENIC CYSTSTable 1 summarises overall TGFa and EGFstaining intensity and pattern of distributionwithin the epithelial linings of odontogenic

A

B

4J.Aw

rwW; 4 D t;;|.f t*i(... *=< -....-v;-l.~~~~~~~~~~~~.........

*~~~~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~~~~~~~~~~~~.a........w 5

C~~~~~~~~~~~~~~~~~~~~~.

Figure 5 Odontogenic keratocyst (A), dentigerous cyst(B) and radicular cyst (C) stainedfor TGF3.

cysts. The epithelial linings of all cysts showedreactivity for TGFa (fig 2) with those of odon-togenic keratocysts exhibiting a stronger reac-tivity than dentigerous and radicular cysts.Generally, the distribution of TGFa stainingdid not differ between cyst types: mostexhibited predominantly basal and suprabasalreactivity. However, in areas of 10 odontogenickeratocyst linings, strong staining was seen inthe basal and upper cellular layers, with weakerreactivity between the two (fig 2D). There wasno detectable difference between the threesubgroups of odontogenic keratocyst (that is,solitary, recurrent, and NBCCS-associatedcysts) both in terms of epithelial stainingintensity and distribution. TGFa and EGFwere also detected in endothelial cells, fibro-blasts, and inflammatory cells within the cystcapsules. The staining intensity of these cellsfor TGFa seemed to be higher in areas associ-ated with inflammatory foci. In odontogenickeratocysts, there was no apparent alteration inTGFa reactivity exhibited by disordered cystlining associated with areas of inflammation(fig 2F). Epithelial rests also stained stronglyfor TGFa (fig 1 E). In all specimens, EGFreactivity was weaker than that for TGFa, andtwo specimens each of dentigerous cyst andradicular cyst were negative. The distribution

24



p.bmj.com

/M


p.50.1.21 on 1 February 1997. D

ownloaded from

http://mp.bmj.com/

EGI, TGFa and TGF/1 in odontogenic cysts

of EGF was also different from that of TGFa,being predominantly suprabasal (fig 3).Absorbance measurements for TGFa and

EGF expression in different cyst linings, deter-mined by TV image analysis, are summarisedin fig 4, and confirm the subjective assess-ments. The mean values for TGFa weresignificantly higher than those for EGF(p < 0.004) and TGFa absorbance readingsfor all three subgroups of odontogenic kerato-cyst were significantly higher than for dentiger-ous cysts (p < 0.05). Radicular cysts showedgreat variation in TGFa absorbance and wereonly significantly different from the NBCCSodontogenic keratocyst group (p < 0.02). Thismay be partly because of the greater structuralvariability of radicular cyst linings (for exam-ple, intercellular spaces and areas of necrosis)which will affect readings. There were nosignificant differences in EGF absorbancereadings between different cyst types. Withinthe odontogenic keratocyst group, the only sig-nificant difference in TGFa absorbance read-ings was between NBCCS-related lesions andsolitary cysts (p < 0.05).

EXPRESSION OF TGF3 IN ODONTOGENIC CYSTSIn contrast to TGFa and EGF expression inodontogenic cysts, the most intense staining ofTGFf was found in the fibrous tissue capsules,irrespective of cyst type, with the majority ofcyst linings exhibiting patchy weak reactivity(fig 5). The epithelial staining appeared to beintercellular and predominantly suprabasal.Consistent strong TGFf staining was detectedin both cellular (fibroblasts, endothelial andinflammatory cells) and intercellular (matrix,intravascular spaces) components of the con-nective tissue walls in all cyst types. In somesections, a stronger intercellular reactivity wasfound in the peripheral part of the tissue withreactivity becoming relatively weaker towardsthe centre.

DiscussionOur results on control tissues are consistentwith published studies using the same antibod-ies on paraffin wax sections.1° 32-3 Further-more, the peptide inhibition studies confirmthe specificity of the staining reactions ob-tained with all three growth factor antibodieson both control and odontogenic cyst speci-mens.The results show that epithelial linings of all

types of odontogenic cyst express TGFa,although the intensity of staining variedbetween cyst types, with odontogenic kerato-cyst linings exhibiting more intense reactivitythan those of dentigerous and radicular cysts.This is consistent with the higher levels ofEGF-R expression," and proliferating cellnuclear antigen (PCNA) and Ki-67 labellingindexes24 25 in odontogenic keratocyst epithe-ium which suggest that these cysts have anintrinsic growth potential not present in theother types of odontogenic cysts. It has beensuggested that coexpression of TGFa andEGF-R in neoplastic cells gives them a growthadvantage over normal cells as antibodiesdirected against TGFa or EGF-R antisense

oligonucleotides have been shown to inhibit theproliferation of several carcinoma cell lines.35.38The strong TGFa reactivity coincident withthe high levels of EGF-R expression inodontogenic keratocyst linings suggest thatTGFa may act as an autocrine growth factor tostimulate cell proliferation or differentiation, orboth, in this cyst type. That such a relation mayexist is also suggested by the significantlyhigher levels of TGFa detected in NBCCS-odontogenic keratocysts compared with soli-tary odontogenic keratocysts as it is known thatsyndrome-related cysts exhibit higher mitoticand Ki-67 labelling indexes.25 39Our previous studies have shown that denti-

gerous cyst linings exhibit a high level ofEGF-R expression and that their stainingintensity is similar to that of the odontogenickeratocyst."3 However, TGFa staining in denti-gerous cyst linings was patchy and weak and itsabsorbance was significantly lower than that ofodontogenic keratocysts (p < 0.05). This dif-ferential expression of TGFt and its receptorEGF-R by dentigerous cyst linings mayindicate that paracrine mechanisms of growthfactor control are a more important feature inthis type of developmental cyst. It is interestingto speculate that this may be directly orindirectly associated with the apparent indo-lence of the epithelial linings of this cyst type,which have significantly lower PCNA andKi-67 labelling indexes.'4

In all cyst types, foci of inflammation in thefibrous capsule were associated with areas ofstrong stromal TGFa reactivity (fibroblasts,inflammatory and endothelial cells) with noapparent difference in the reactivity of theoverlying cyst lining. Interestingly, previousstudies have shown an inverse relation betweenthe presence of inflammation and the stainingintensity of adjacent overlying epithelium forEGF-R." Such differences may reflectepithelial-mesenchymal interactions andgrowth factor/receptor modulation. It is be-lieved that such processes play an importantrole in modulating cell growth and cell interac-tion during normal odontogenesis as differen-tial expression of EGF-R and TGFa has beenshown to relate to the stages of tooth develop-ment in both dental epithelium and dentalmesenchyme." 14 40EGF immunoreactivity in the epithelial

linings of odontogenic cysts was patchy andweaker than that for TGFa, with no detectabledifferences in staining intensity between cysttypes. This difference in reactivity was also afeature ofthe staining results on control tissues.Similar weak or negative EGF reactivity inconjunction with consistent positive TGFastaining has been reported in various normaltissues 4 and tumours,4' including odon-togenic tumours and oral squamous cellcarcinoma.'3 32 Thus, odontogenic cyst liningsare similar to other tissues in that TGFu seemsto be the key factor in the potential autocrineloop for stimulation of EGF-R. However, it isimportant to realise that differences in stainingintensity obtained with two different antibod-ies, for the same or different antigens, do not

25



p.bmj.com

/M


p.50.1.21 on 1 February 1997. D

ownloaded from

http://mp.bmj.com/


necessarily equate with true quantitative differ-ences in amount of antigen within the section.Many immunocytochemical studies of

TGFI1 have relied on two rabbit polyclonalantibodies (anti-CC and anti-LC) directedagainst unconjugated peptides correspondingto the N-terminal 30 amino acids ofTGFPI1.3 44 The antibodies recognise differentepitopes: anti-CC shows predominantly extra-cellular (mesenchymal) and anti-LC predomi-nantly intracellular (epithelial) reactivity. Theclose correlation of intracellular staining usinganti-LC with in situ localisation of TGFO1mRNA suggests that anti-LC recognisesTGF3 1 at sites of synthesis (latent or precursorform of TGFf1) whereas anti-CC may detectthe active form of TGFOi1 which is bound toextracellular matrix proteins.4" 44 In this study,the pattern of monoclonal antibody TB2 1staining resembles that of anti-CC, mainlyshowing extracellular and mesenchymal reac-tivity on both control and odontogenic cystspecimens. Although the isotype specificity ofTB2 1 is not known, its ability to neutralise theaction of TGF,B1 on the growth of various tar-get cells in vitro suggests that it reacts with theactive form of TGF1.29 Therefore, our resultsmay indicate the ubiquitous presence of theactive form of TGFI3 in the fibrous tissue cap-sules of all odontogenic cyst types. However, itis not possible to know whether this is becauseof locally produced or systemically derivedpeptide or a combination of both. Furtherstudies are required using isotype specificreagents (antibodies and oligonucleotides/riboprobes) to characterise the TGF,B detectedand assess the role of active local synthesis.

In conclusion, our results show the presenceand differential expression of TGFa and EGFin the epithelial linings of odontogenic cysts.Taken together with our previous data onEGF-R expression,23 these results suggest thatmembers of the EGF family (via autocrine orparacrine, or both, pathways) are importantand play a differential role in the initiation andgrowth of odontogenic cysts.

We would like to thank the West Midlands Regional HealthAuthority for financial support and Mrs Christine Wilson forexcellent technical assistance. TJL is partly supported by anOverseas Research Study award.

1 Cohen S. Isolation of a mouse submaxillary gland proteinaccelerating incisor eruption and eye lid opening in thenewborn animal. J Biol Chem 1962;237:1555-62.

2 De Larco JE, Todaro GJ. Growth factors from murinesarcoma virus-transformed cells. Proc Natl Acad Sci USA1978;75:4001-2.

3 Reynolds FH, Todaro GJ, Fryling C, Stephenson JR.Human transforming growth factors induce tyrosine phos-phorylation of EGF-receptors. Nature 1981;292:259-62.

4 Gusterson B, Cowley G, Simth JA, Ozanne B. Cellularlocalisation of human epidermal growth factor receptor.Cell Biol Int 1984;8:649-58.

5 Ushiro H, Cohen S. Identification of phosphotyrosine as aproduct of epidermal growth factor-activated proteinkinase in A-431 cell membranes. J Biol Chem 1980;255:8363-5.

6 Barrandon Y, Green H. Cell migration is essential forsustained growth of keratinocyte colonies; the roles oftransforming growth factor-a and epidermal growth factor.Cell 1987;50:1131-7.

7 Carpenter G. Receptors for epidermal growth factor andother polypeptide mitogens. Ann Rev Biochem 1987;56:881-914.

8 Rosenthal A, Lindquist PB, Bringham TS, Goeddel DV,Derynck R. Expression in rat fibroblasts of a human trans-

forming growth factor alpha cDNA results in transforma-tion. Cell 1986;46:301-9.

9 Coffey RJ, Derynck R, Wilcox JN, Bringham TS, GoustinAS, Moses HL, et al. Production and autoinduction oftransforming growth factor-a in human keratinocytes.Nature 1987;328:817-20.

10 Yasui W, Ji Z-Q, Kuniyasu H, Ayhan A, Yokozaki H, Ito H,et al. Expression of transforming growth factor alpha inhuman tissues; immunohistochemical study and Northernblot analysis. VirchowsArchA 1992;421:513-19.

11 Miettinen PJ. Transforming growth factor-a and epidermalgrowth factor expression in human fetal gastrointestinaltract. PediatrRes 1993;33:481-4.

12 Derynck R, Goeddel DV, Ullirich A, Gutterman JU,Williams RD, Bringman TS, et al. Synthesis of messengerRNAs for transforming growth a and 1 and the epidermalgrowth factor receptor by human tumours. Cancer Res1987;47:707-12.

13 Heikinheimo K, Voutilainen R, Happonen R-P, MiettinenPJ. EGF receptor and its ligands, EGF and TGFa, indeveloping and neoplastic human odontogenic tissues. Int3'Dev Biol 1993;37:387-96.

14 Partanen AM, Thesleff I. Localization and quantitation ofI"'-epidermal growth factor binding in mouse embryonictooth and other embryonic tissues at different developmen-tal stages. Dev Biol 1987;120:186-97.

15 Partanen AM, Thesleff I. Growth factors and toothdevelopment. IntJT Dev Biol 1989;33: 165-72.

16 Kronmiller JE, UpholtWB, Kollar EJ. Expression of epider-mal growth factor mRNA in the developing mousemandibular process. Arch Oral Biol 199 1;36:405-10.

17 Kronmillar JE, Upholt WB, Kollar EJ. EGF antisense oligo-deoxynucleotides block murine odontogenesis in vitro. DevBiol 1991;147:485-8.

18 Hu C-C, Sakakura Y, Sasano Y, Shum L, Bringas P, Werb Z,et al. Endogenous epidermal growth factor regulates thetiming and pattern of embryonic mouse, molar tooth mor-phogenesis. Int _J Dev Biol 1992;36:505-16.

19 Tam JP. Physiological effects of transforming growth factorin the newborn mouse. Science 1985;229:673-5.

20 Rhodes JA, Fitzgibbon DH, Macchiarulo PA, Murphy RA.Epidermal growth factor-induced precocious incisor erup-tion is associated with decreased tooth size. Dev Biol 1987;121:247-52.

21 Partanen AM, Ekblom P, Thesleff I. Epidermal growth fac-tor inhibits morphogenesis and cell differentiation incultured mouse embryonic teeth. Dev Biol 1985;111:84-94.

22 Abbott BD, Pratt RM. EGF receptor expression in thedeveloping tooth is altered by exogenous retinoic acid andEGF. Dev Biol 1988;128:300-4.

23 Li T-J, Browne RM, Matthews JB. Expression of epidermalgrowth factor receptors by odontogenic jaw cysts. VirchowsArch A 1993;432:137-44.

24 Li T-J, Browne RM, Matthews JB. Quantification of PCNA+cells within odontogenic jaw cyst epithelium. J Oral PatholMed 1994;23:184-9.

25 Li T-J, Browne RM, Matthews JB. Epithelial cell prolifera-tion in odontogenic keratocysts; A comparative immunocy-tochemical study of Ki67 in simple, recurrent and basal cellnaevus syndrome (BCNS) associated lesions. Jf Oral PatholMed 1995;24:221-6.

26 Li T-J, Browne R M, Matthews JB. Immunocytochemicalexpression of parathyroid hormone related protein(PTHrP) in odontogenic jaw cysts. Br J Oral MaxillofacialSurg (in press).

27 Vaahtokari A, Vainio S, Thesleff I. Associations betweentransforming growth factor 131 RNA expression andepithelial-mesenchymal interactions during tooth morpho-genesis. Development 1991;113:985-94.

28 Heikinheimo K, Happonen R-P, Miettinen PJ, Ritvos 0.Transforming growth factor 132 in epithelial differentiationof developing teeth and odontogenic tumours. J Clin Invest1993;91:1019-27.

29 Shi W-K, Ye Q-W, Bao L-P. Biological characterisation of amonoclonal antibody TB21 against TGF,B 1. Acta Biol ExptSinica 1993;26:141-50.

30 Lawrence GM, Beesley ACH, Matthews JB. The use ofcontinuous monitoring and computer-assisted imageanalysis for the histochemical quantification of hexokinaseactivity. Histochem J 1989;21:557-64.

31 Mason GI, Matthews JB. In situ determination of differentdehydrogenase activity profiles in the linings of odon-togenic keratocysts and radicular cysts. Histochem 71996;28: 187-93.

32 Christensen ME, Therkildsen MH, Poulsen SS, Bretlau P.Immunoreactive transforming growth factor alpha and epi-dermal growth factor in oral squamous cell carcinomas. .7Pathol 1993;169:323-8.

33 Terada T, Ohta T, Nakanuma Y Expression of transforminggrowth factor-alpha and its receptor during human liverdevelopment and maturation. Virchows Arch A 1994;424:669-75.

34 Scott PG, Dodd CM, Tredget EE, Ghahary A, RahemtullaF. Immunohistochemical localization of the proteoglycansdecorin, biglycan and versican and transforming growthfactor-,B in human post-burn hypertrophic and maturescars. Histopathology 1995;26:423-31.

35 Rodeck U, Williams N, Murthy U, Herlyn M. Monoclonalantibody 425 inhibits growth stimulation of carcinoma cellsby exogenous EGF and tumour-derived EGF/TGF-a. _7Cell Biochem 1990;44:69-79.

36 Sizeland AM, Burgess AW. The proliferation of morpho-logic responses of a colon carcinoma cell line (LIM 1215)

26



p.bmj.com

/M


p.50.1.21 on 1 February 1997. D

ownloaded from

http://mp.bmj.com/

EG1, TGFa and TGF/3 in odontogenic cysts

require the production of two autocrine factors. Mol CellBiol 1991;11:4005-14.

37 Derynck R. The physiology of transforming growthfactor-a. Adv Cancer Res 1992;58:27-52.

38 Karnes Jr WE, Walsh JH, Wu SV, Kim RS, Martin MG,Wong HC, et al. Autonomous proliferation of colon cancercells that coexpress transforming growth factor a and itsreceptor. Gastroenterology 1992;102:474-85.

39 Woolgar JA, Rippin JW, Browne RM. A comparativehistological study of odontogenic keratocysts in basal cellneavus syndrome and control patients. Oral Pathol 1987;16:75-80.

40 Cam Y, Neumann MR, Ruch JV. Immunolocalization oftransforming growth factor 1, and epidermal growth factorreceptor epitopes in mouse incisors and molars with ademonstration of in vitro production of transforming activ-ity. Arch Oral Biol 1990;35:813-22.

41 Diaz-Ruiz C, Perez-Tomas R, Cullere X, Domingo J.Immunohistochemical localization of transforming growthfactor-a and epidermal growth factor-receptor in the mes-onephros and metanephros of the chicken. Cell Tissue Res1993;271:3-8.

42 Barton CM, Hall PA, Hughes CM, Gullick WJ, LemoineNR. Transforming growth factor alpha and epidermalgrowth factor in human pancreatic cancer. Pathol1991;163:111-16.

43 Flanders KC, Thompson NL, Cissel DS, Obberghen-Schilling EV, Baker CC, Kas ME, et al. TGFJI1;histochemical localisation with antibodies to differentepitopes. Cell Biol 1989;108:653-9.

44 Thompson NL, Flanders KC, Smith JM, Ellingsworth LR,Roberts AB, Sporn MB. Expression of TGF,B1 in specificcells and tissues of adult and neonatal mice. Cell Biol1989;108:661-9.

27



p.bmj.com

/M


p.50.1.21 on 1 February 1997. D

ownloaded from

http://mp.bmj.com/

j clin mol immunocytochemical expression of growth factors ...odontogenesis, particularly in...

Documents