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Gene expression of differentiation-specific keratins in oral epithelial dysplasia and squamous cell carcinoma
Oral Oncology 37 (2001) 251–261 www.elsevier.com/locate/oraloncology
Gene expression of differentiation-specific keratins in oral epithelial dysplasia and squamous cell carcinoma B.K. Bloor, S.V. Seddon, P.R. Morgan * Department of Oral Medicine & Pathology, Guy’s, King’s and St. Thomas’ Dental Institute, Guy’s Hospital, London SE1 9RT, UK Received 23 May 2000; accepted 26 July 2000
Abstract The aim of the study was to investigate the differentiation-specific keratins (K4, K13, K1 and K10) in oral epithelial dysplasia and squamous cell carcinoma (SCC). Alterations in keratin gene expression were determined by in situ hybridization using 35Slabeled riboprobes and immunohistochemistry with monoclonal antibodies. In mild dysplasia, both sets of differentiation keratins were expressed in the same group of cells but in moderate lesions, expression of K4 and K13 was reduced in the presence of enhanced K1 and K10 synthesis. In severe dysplasia, neither mRNAs nor proteins were detected. In tumor islands of well and moderately differentiated SCCs, the K4/K13 complex was co-expressed with K1/K10, but in poorly differentiated carcinomas, differentiation keratins were absent. Consequently, mild oral epithelial dysplasia and well differentiated SCC retain an essentially normal pattern of keratin gene expression and hence epithelial differentiation while in severe dysplasia and poorly differentiated SCC keratin gene expression reflects the gross changes in epithelial differentiation and maturation. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Gene expression; Differentiation-specific keratins; K4; K13; K1; K10; In situ hybridization; Immunohistochemistry; Oral epithelial dysplasia; Oral squamous cell carcinoma
1. Introduction The histological features associated with oral epithelial dysplasia and with squamous cell carcinoma (SCC) have been described previously [1-4] and are generally agreed in structural terms. Descriptions are expressed in terms of loss of structural organization and disturbed proliferation (dysplasia) and varying degrees of differentiation (SCC). These pathological changes involve the terminally differentiating cell compartment but it is not clear how they affect the gene expression of differentiation keratins. Knowledge of such gene changes is important for several reasons. First, any disturbance of expression may be out of phase with structural changes. Second, one set of differentiation keratins may substitute for the other, especially if there is a modification of the state of keratinization from normal [5]. Biochemical and immunohistochemical studies have indicated that SCCs express a wider range of keratins * Corresponding author. Tel.: +44-20-7955-4288; fax +44-207955-4455. E-mail address: [email protected] (P.R. Morgan).
than would be expected from the pattern shown by their normal epithelium [6-18]. Poorly differentiated SCCs are generally associated with a reduction in secondary keratins (K4/K13 in non-keratinizing epithelia and K1/ K10 in keratinizing epithelia) and a reduction or total loss of primary keratins (K5, K14 and K15). In contrast, well differentiated SCCs tend to conserve their secondary keratins that are localized to prickle cells within tumor islands. Although protein expression of the differentiation keratins has been demonstrated in oral mucosa [7,1922], the systematic comparison of protein relative to mRNA has not been investigated in malignant oral epithelia. This analysis will provide information on the molecular basis of human keratin expression and the changes that occur in dysplasia and malignancy. Analysis of keratin gene expression involved the use of in situ hybridization (ISH) to determine mRNA levels using specific riboprobes constructed and labeled with 35 S-dUTP for K4, K13, K1 and K10. Protein levels were detected with a standard immunohistochemical approach using monoclonal antibodies to the same keratins.
1368-8375/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S1368-8375(00)00094-4
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2. Materials and methods 2.1. Tissue samples Frozen tissue samples were selected from the Department of Oral Medicine and Pathology, GKT Dental Institute, on the basis of clinical and histopathological analysis. Biopsies of oral epithelial dysplasia were chosen to include mild (n=9), moderate (n=7) and severe (n=7) lesions, avoiding cases with superimposed candidiasis. With the exception of one case from each category, all biopsies originated from sites which are normally non-keratinized. Cases of SCC included well (n=7), moderately (n=9) and poorly differentiated (n= 7) types. One case each of well and poorly differentiated SCCs and two from moderately differentiated tumors originated from keratinized sites. Controls included six specimens of buccal mucosa from patients whose biopsies were recorded normal after histological analysis and two biopsies of normal skin. Each specimen was rapidly frozen onto cork in OCT cryoprotectant, quenched in melting isopentane and stored in liquid nitrogen within 60 min of surgical removal. Serial 5-mm cryostat sections were mounted onto glass microscope slides coated with a 2% solution of 3-aminopropyltriethoxysilane [23]. 2.2. Synthesis and labeling of riboprobes Antisense and sense RNA probes for each of the differentiation keratins were constructed from cDNA clones containing keratin inserts. Fragments of specific DNA for K4 [24] and K13 [25] were selected by PCR amplification and sub-cloned into the pGEM4 transcription vector (Promega, UK). For K1 [26] and K10 [27], cDNA was not sub-cloned, as sequences were mainly non-coding. RNA probes were synthesized for 90 min at 37 C with 200 mCi of 35S-dUTP (Amersham, UK) and the appropriate RNA polymerase. Template DNA was removed at 37 C with a 15-min incubation using five units of DNase I (Roche Diagnostics, UK). Unincorporated 35S-dUTP was eliminated by column chromatography using Nensorb 20 cartridges (Du Pont, USA). Labeled RNA was precipitated with 5% trichloroacetic acid and 20 mM sodium orthophosphate to determine efficiency of labeling.
and 2SSPE. Next, sections were pre-hybridized for 3 h at 45 C in 50% formamide, 2SSPE, 5Denhardt’s solution, 0.05% SDS, 20 mM Tris-HCl (pH 7.4), 100 mM dithiothreitol and 500 mg/ml of sheared salmon sperm DNA. Following this, sections were hybridized for 18 h at 45 C with 4106 cpm/ml of 35S-labeled RNA using the same hybridization buffer as before plus 10% dextran sulfate. After hybridization, unbound probe was removed with 330 min incubations in salt solutions of increasing stringency, including 4SSC (saline sodium citrate), 2SSC and 0.1SSC. Finally, unhybridized target RNA was removed with a 30-min incubation at 37 C with 50 mg/ml of RNase A (Sigma). Next, tissue sections were dehydrated in a series of Ethanol solutions of increasing concentration. Finally, the sensitivity of hybridization and distribution of mRNA transcripts were detected by autoradiography followed by development with K-5 nuclear emulsion (Ilford, UK). 2.4. Immunohistochemistry A standard immunohistochemical technique was applied for the detection of keratin (K) proteins with the following mouse monoclonal antibodies: clones 6B10 (K4) [30], 1C7 (K13) [30], LHK1 (K1) [31] and RKSE60 (K10) [32]. Tissue sections were fixed in acetone for 10 min at 4oC, incubated for 60 min with monoclonal antibody (1:10) followed by 30 min with biotinylated rabbit anti-mouse immunoglobulin (Dako Ltd.; 1:300) and biotin streptavidin-horseradish peroxidase complex (Dako Ltd.; 1:50). Sections were finally developed with 200 mg/ml of 3,30 diaminobenzidine (Sigma) and counterstained with hematoxylin. 2.5. Analysis of keratin gene expression Expression of keratin mRNAs and proteins was recorded using a semi-quantitative 4-point scale as used previously [28,29]. Expression data for normal buccal epithelium and oral epithelial dysplasia are presented in Table 1, results for SCCs being shown in Table 2. These tables represent the range and overall distributions of mRNA and protein in the respective groups. 2.6. Controls for in situ hybridization and immunohistochemistry
2.3. In situ hybridization ISH was carried out according to Su et al. [28,29]. In brief, tissue sections were fixed for 30 min in 4% paraformaldehyde, pre-treated for 5 min in 100 mM glycine followed by 10 min in 100 mM triethanolamine-HCl (pH 8.0) and 0.25% acetic anhydride. Sections were washed for 5 min in 2sodium saline phosphate-EDTA (SSPE) followed by 10 min at 45 C in 50% formamide
Sections of normal buccal mucosa (for detection of K4 and K13) and skin (K1 and K10) were included for comparison with keratin expression in dysplastic and malignant oral epithelia. Negative controls for ISH included 35S-labeled sense RNA probes for K4, K13 and K10, which, having identical sequences to target RNA, should not result in hybridization. For K1, as there is no equivalent sense probe, target RNA was
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B.K. Bloor et al. / Oral Oncology 37 (2001) 251–261 Table 1 Distributions of proteins and mRNAs for K4, K13, K1 and K10 in normal and dysplasticepitheliuma Distribution of differentiation-specific keratins in normal and dysplastic epithelium K4 Protein Normal buccal epithelium (n=6) Parakeratotic mild dysplasia (n=4) Orthokeratotic mild dysplasia (n=5) Parakeratotic moderate dysplasia (n=2) Orthokeratotic moderate dysplasia (n=5) Severe dysplasia (n=7)
Basal Prickle Basal Prickle Basal Prickle Basal Prickle Basal Prickle All cells
OC
+ +++ +OC +++ +OC +OC to +++ +OC to ++ +OC
K13 mRNA 1 3 1OC 3 0 1 to 2 0 1 1OC 1 0
K1
Protein OC
+ +++ +OC +++ +OC +OC to +++ +OC to ++ +OC
K10
mRNA
Protein
mRNA
Protein
mRNA
1 3 1OC 3 0 1 to 2 0 1 1OC 1 0
++* + to +++ +* to +++ ++ + to +++ +OC (28%)
1 2 1OC 1 to 3 1OC 2 to 3 0 2 1 2 to 3 0
+ to ++* + to +++ +* to +++ ++ + to +++ +OC (28%)
1 1 1OC 1 to 3 1OC 2 to 3 0 2 1 2 to 3 0
a to +++, protein expression from negative to high; *, indicates protein expression in columns; 0 to 3, mRNA expression from negative to high; OC, occasional expression from 5 to 30% of cells; percentages in parentheses indicate the proportion of positive cases if not 100% and n indicates the number of cases studied.
incubated for 1 h with 50 mg/ml of RNase A prior to hybridization with the K1 antisense probe. Negative controls for immunohistochemistry (IHC) included substitution of primary antibody with an irrelevant antibody of the same class.
3. Results 3.1. Histological appearance of oral epithelial dysplasia and SCC Oral epithelial dysplasias were divided into three categories on the basis of hematoxylin and eosin staining. In mildly dysplastic lesions, stratification was generally well ordered, showing either para- (n=4) or orthokeratinized epithelium (n=5). In some specimens, the epithelial architecture departed little from normal, but in others, bulbous rete processes were present. Nuclear atypia were seldom present, but were sparse and confined mainly to basal cells. The corium was usually associated with a mild lymphoplasmacytic infiltrate. Moderate dysplasias consisted of a para- (n=2) or orthokeratotic (n=5) epithelium, which was often hyperplastic in appearance. Stratification was more disturbed than in mild lesions, prickle cells losing their ordered fashion and basal cells showing loss of polarity. Additional epithelial changes were present in various combinations occupying up to half of the epithelial thickness. These features were not evident in each case, and included bulbous rete processes, hyperchromatic nuclei and pleomorphic cells. Thickening and hyalinization of the basement membrane were sometimes present together with apoptotic basal cell degeneration, thus simulating oral lichen planus. Normal and bizarre
mitoses were limited to basal and lower suprabasal layers. A lymphoplasmacytic infiltrate of varying intensity was present within the superficial corium. Severe dysplasias showed a trend toward parakeratinization irrespective of the site of origin. Rete processes were bulbous, drop-shaped in appearance or showed ‘budding’. Other epithelial changes were present in more than half of the epithelial thickness, with only some of these features being present in each biopsy. These consisted of nuclear hyperchromatism, nuclear and cellular pleomorphism, disordered cell polarity, individual cell keratinization and increased nucleus-tocytoplasm ratio. Bizarre mitoses and apoptoses were observed in basal and suprabasal layers and a lymphoplasmacytic infiltrate of variable intensity was present within the superficial corium. Carcinomas were divided into three categories based on their differentiation pattern. Features of well differentiated SCCs included the presence of well ordered islands and keratin pearls. The corium contained either a focal or diffuse lymphoplasmacytic host response, and in many cases there was epithelial dysplasia in the adjacent epithelium. SCCs were classified as moderately differentiated when tumor islands showed limited maturation, being less well ordered and with individual cell keratinization rather than keratin pearl formation. A light to moderate lymphoplasmacytic infiltrate was often present within the tumor stroma. In poorly differentiated carcinomas, there was no clear distinction between basal and maturation compartments, a high proportion of tumor cells being basaloid in appearance. Keratinization was generally absent in these tumors and there was very limited prickle cell differentiation. Apoptoses and bizarre mitoses were frequent and the host response either minimal or absent.
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1 1 1 1 0 + (43%) + to +++H + to ++OC (56%) 1 to 2 (71%) 1 to 2OC (71%) 1H (33%) 2H (33%) 0 + (43%) +OC to +++H + to ++OC (56%) 1 to 3 (43%) 1 to 2OC (71%) 1H (33%) 2H (33%) 0 + (43%) +OC to +++H + to ++OC (56%) Basal Prickle Basal Prickle All cells Well differentiated SCC (n=7) Moderately differentiated SCC (n=9) Poorly differentiated SCC (n=7)
a to +++, protein expression from negative to high; 0 to 3, mRNA expression from negative to high; OH, mRNA expression from 5 to 30% of cells; H, heterogeneous expression from 40 to 70% of cells; percentages in parentheses indicate the proportion of positive cases if not 100% and n indicates the number of cases studied.
1 to 2H (71%) 1 to 2OC (71%) 1 to 3H (33%) 1 to 3H (33%) 0 + (43%) + to +++H + to ++OC (56%) to 3 to 2OC to 3H (33%) to 3H (33%)
OC
Protein mRNA
H OC
Protein mRNA
H OC
Protein mRNA Protein
H
K1 K13 K4
Distribution of differentiation-specific keratins in tumor islands
Table 2 Distributions of proteins and mRNAs for K4, K13, K1 and K10 in oral squamous cell carcinoma (SCC)a
OC
K10
mRNA
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3.2. Keratin gene expression in normal buccal epithelium Proteins for K4 and K13 were distributed homogeneously in the suprabasal compartment with occasional expression in basal cells (Table 1 & Fig. 1A). 35Slabeled transcripts for K4 and K13 were detected at low level in basal cells but consistently in parabasal and suprabasal cells, the intensity gradually decreasing towards the upper layers (Fig. 1B). Proteins for K1 and K10 were expressed in columns of prickle cells centered over connective tissue papillae (Fig. 1C), their respective transcripts being synthesized in basal, parabasal and deep prickle cells, but at a lower intensity than that expressed for K4 and K13 (Fig. 1D). 3.3. Keratin gene expression in oral epithelial dysplasia In three mild dysplasias with a parakeratinized epithelium including two from buccal and one from alveolar ridge mucosa (Table 1), protein and mRNA expression of K4 and K13 were detected homogeneously in the suprabasal compartment but sparsely in the keratin and basal layers. Expression for K1 and K10 was detected in the prickle cell layer, ranging from a sparse to homogeneous distribution but showing minimal expression in basal cells. In a parakeratotic dysplastic lesion from the floor of mouth in which the keratin layer occupied more than half of the epithelial thickness, proteins for K4 and K13 were expressed heterogeneously in the suprabasal layers (Fig. 1E) although corresponding transcripts were not detected with 35S-labeled probes (Fig. 1F). Proteins for K1 and K10 were expressed at a lower level than for K4 and K13, but respective mRNAs were synthesized at low to moderate intensity (Fig. 1G and H). In orthokeratotic mild dysplasias, two from buccal mucosa and three from the ventral surface of tongue, proteins for K4 and K13 were distributed extensively in the suprabasal compartment and in a few cells of the basal layer. Their respective transcripts were localized in parabasal and prickle cells, the intensity varying from low to moderate levels. Proteins for K1 and K10 were expressed heterogeneously in the prickle cell layer, their corresponding mRNAs being transcribed either at low level or showing a heterogeneous distribution in basal and prickle cells. Expression of these keratins was more varied in moderate than in mild dysplasias (Table 1). In parakeratotic lesions, one each from buccal and alveolar ridge mucosa, proteins for K4 and K13 were distributed sparsely in the suprabasal layer while mRNAs were transcribed at low levels. Proteins and silver grains for K1 and K10 were expressed randomly in the prickle cell layer of buccal mucosa, but were absent from alveolar ridge mucosa. In moderately dysplastic orthokeratotic lesions from the floor of mouth (n=1) and lingual mucosa (n=4), proteins and transcripts for K4 and K13
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Fig. 1. Expression of K4 and K1 in normal (A–D) and mildly dysplastic (E–H) buccal epithelia. (A) In normal epithelium, K4 protein was stained uniformly in the suprabasal compartment and at a lower level of expression in many basal cells. (B) Silver grains for K4 transcript were distributed densely in prickle cells but are at low levels in basal cells. (C) Protein for K1 showed restricted expression in columns of prickle cells over connective tissue papillae whilst transcript was widely distributed in suprabasal cells (D). (E) In a single keratotic lesion showing mild dysplasia, K4 protein was expressed heterogeneously in the prickle cell layer although silver grains for K4 transcript were not detected above background levels (F). In the same lesion, K1 protein showed a less widespread distribution (G) compared with K4 but K1 mRNA was expressed at detectable levels in the suprabasal compartment (H). (Magnification: A, B and D 800, C 200 and E-H 400).
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Fig. 2. Expression of K4 and K1 in moderate (A–D) and severe (E–H) oral dysplasia. (A) In this moderate dysplasia of lingual epithelium, K4 protein was limited to a few prickle cells and its transcript (B) was not detected. Protein for K1 was expressed uniformly in the suprabasal compartment (C) but not in the keratin layer. Silver grains for K1 (D) were distributed extensively in the deeper prickle cell layers. (E) In this lesion from labial mucosa, K4 protein was absent from the epithelium corresponding to severe dysplasia (right-hand side of field) but was expressed uniformly in the adjacent ‘normal’ epithelium (left-hand side of field). K4 transcript (F) was absent from the dysplastic epithelium but was detected in the normal epithelium. K1 protein (G) was absent from the dysplastic zone but there were a few positive prickle cells in the normal epithelium. Silver grains for K1 (H) were absent from the dysplastic epithelium but were barely detectable in the normal epithelium. (Magnification: A–H 400).
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were either sparsely distributed or absent (Fig. 2A and B). Proteins and mRNAs for K1 and K10 were synthesized, expression varying from sparse to intense in the suprabasal layer (Fig. 2C and D), with silver grains also being expressed at low levels in the basal layer. In severe dysplasias, expression of these keratins was markedly reduced compared with their normal epithelium, with proteins and transcripts being predominantly absent (Fig. 2E–H). In two cases, proteins for K1 and K10 were localized to individual groups of keratinocytes in the suprabasal compartment but in the absence of their corresponding transcripts (Table 2). 3.4. Keratin gene expression in SCC In well differentiated SCCs (Table 2), expression of the differentiation-specific keratins was not homogeneous throughout the tumor, negative tumor islands often being adjacent to those expressing keratins. Proteins for K4 and K13 were expressed randomly as small foci (Fig. 3A and D) or more extensively throughout the tumor islands (Fig. 3C). In five SCCs, silver grains were variably expressed throughout the tumor, but in two lesions, they were absent (Fig. 3B). Proteins for K1 and K10 were expressed intensely in the prickle cell compartment of tumor islands and in some cases, also in the basal compartment (Fig. 3E and G). Silver grains for K1 and K10 were distributed in cells at the periphery and center of tumor islands, although intensity varied from moderate to low (Fig. 3F and H). Keratin expression in moderately differentiated SCCs was also variable and appeared to be independent of the original epithelial phenotype. In five SCCs, proteins for K4 and K13 were distributed randomly in the prickle cell compartment, their corresponding transcripts either being absent or localized to the periphery of tumor islands. As for K4 and K13, proteins for K1 and K10 showed a restricted distribution in prickle cells, transcript varying from weak to moderate expression in cells at the periphery and center of tumor islands. In the remaining four carcinomas, all differentiation keratins were absent. In poorly differentiated SCCs, differentiation-specific keratins were absent although the overlying epithelium showed a near normal profile for the site.
4. Discussion 4.1. Wider distribution of keratin mRNAs than proteins in normal oral epithelia In agreement with previous studies, expression of K4 and K13 proteins was detected in the suprabasal layer of buccal epithelium but K1 and K10 showed a restricted distribution [20,33-35]. Transcripts were
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detected in the basal and suprabasal compartments, implying that these genes are under post-transcriptional control. The widespread localization of K4 mRNA has been reported in esophageal epithelium with non-isotopic ISH [36] and that for K4 and K13 in buccal epithelium with digoxigenin and 35S-labeled probes [35,37]. Consistent expression of K1 and K10 in the basal and lower suprabasal layers has been reported with non-isotopic ISH for buccal epithelium [35] but also in epidermis with both methods [38,39]. Basal expression of K1 and K10 in epidermis has been considered to be associated with delayed onset of differentiation and this could also be the case in buccal epithelium. The current study also emphasizes the importance of the labeling system used to detect mRNA expression. A comparative analysis of 35S- and digoxigenin-labeled probes on cultured cell lines and serial sections of normal oral epithelium, dysplasia and SCC indicated both techniques were comparable in the detection of keratin mRNAs, but only at relatively high levels of expression [40]. Although digoxigenin-labeled probes resulted in better intracellular mRNA localization to individual cells 35S-labeled probes were more sensitive for the detection of mRNAs distributed at low level. Therefore, in the current study, the conventional radioactive approach was chosen over non-isotopic ISH for the detection of keratin mRNAs. 4.2. Variable expression of differentiation keratins in mild and moderate oral epithelial dysplasia is partly dependant on altered epithelial differentiation As disturbed differentiation is a major feature of dysplasia, it was not surprising to find that keratin gene expression was altered in comparison with normal oral epithelia. In mild lesions, derived from non-keratinizing epithelium, both sets of differentiation keratins were expressed in the same group of cells, K4 and K13 being retained from normal epithelium while synthesis of K1 and K10 was enhanced. As mRNAs for K1 and K10 are already present, it is possible that some aspect of the dysplastic change permits increased synthesis of proteins in an epithelium in which these keratins are normally expressed as minor constituents. Seen in this light, the presence of K1 and K10 mRNAs in nonkeratinizing epithelium may reflect an inherent defence mechanism, where these transcripts become readily translatable to proteins when required, possibly providing a protective role. If substantiated, this would be analogous to the synthesis of K6 and K16 in human epidermis. Despite the presence of their mRNAs in normal epidermis, K6 and K16 proteins are synthesized only under conditions of relatively high keratinocyte turnover [41]. It remains to be determined whether such shifts in keratin expression from K4/K13 to K1/K10 are
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Fig. 3. Expression of K4, K13 and K1 in well differentiated oral squamous cell carcinoma (SCC). (A) Protein for K4 was expressed weakly and in a few cells within tumor islands and silver grains (B) for K4 were barely above the background level. K4 protein (C) was distributed heterogeneously in prickle cells of tumor islands, and also in peripheral (basal) cells whereas K13 protein (D) was distributed randomly in the center of tumor islands. Protein for K1 was present heterogeneously in both basal and suprabasal compartments (E) but K1 transcript (F) was expressed mostly in the center of tumor islands. In a different case, K1 protein was distributed heterogeneously in prickle cells and randomly in cells at the periphery of tumor islands (G) but transcript for K1 was located towards the periphery of tumor islands (H) at a relatively low level of synthesis. (Magnification: A–H 400).
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indeed of functional significance, and whether synthesis of K1 and K10 proteins in non-keratinizing epithelia has a similar function to that of K6 and K16 in epidermis. Co-expression of K4 and K13 with K1 and K10 has been observed in keratoses and lichen planus, implying that increased synthesis of K1 and K10 may be linked with changes in epithelial differentiation [5] in addition to dysplastic change. There are no comparative studies on oral epithelial dysplasia in which differentiation keratins have been investigated at both the levels of transcript and protein. By using similar criteria, changes in keratin protein expression were measured against those in another mucosal disease, cervical intraepithelial neoplasia (CIN). In CIN I, a low-grade lesion analogous to mild oral epithelial dysplasia, expression of K1 and K10 proteins was restricted compared to heterogeneous expression in mild dysplasia, although proteins for K4 and K13 were similarly distributed throughout the epithelium [42,43]. In moderate dysplasia arising from non-keratinizing epithelia, K1/K10 filaments replaced the K4/K13 complex, a finding that accords with the histological changes to para- or orthokeratinization in association with the dysplastic process. This was not the case for one lesion (from alveolar ridge mucosa), as expression of K1 and K10 in the absence of K4 and K13 reflected the normal pattern seen from this type of epithelium. Furthermore, keratin gene expression was independent of the underlying inflammatory response unlike the situation in lichen planus, where epithelial infiltration by lymphocytes resulted in reduced expression of these keratins [5]. A switch from K4/K13 to K1/K10 was not seen in medium-grade CIN II lesions, K4 and K13 being intensely expressed in the basal and suprabasal compartments in the absence of K1 and K10 [42,43]. In the current study and in above IHC studies [42,43] proteins for K4, K13 and K10 were detected on frozen tissue with the same antibodies. Although K4 and K13 showed a similar pattern of expression, the distributions of K1 and K10 proteins were markedly different. It is possible that epithelial architectural differences between oral epithelial dysplasia and cervical neoplasia and an involvement of human papillomavirus (HPV) in the latter may further modulate keratin gene expression. In another study, immortalization of oral keratinocytes by HPV-16 resulted in an alteration in both keratin expression and differentiation [44]. This lends further support to the importance of HPV in affecting keratin gene expression, not only in cervical, but also oral tissue. 4.3. Lack of differentiation keratins as a defining feature of severe oral epithelial dysplasia In severe oral dysplasia, both sets of differentiation keratins were largely absent, although it is interesting to note that all lesions presented with a parakeratinized
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epithelium, a phenotype in which markers of nonkeratinization are retained and those for keratinization are induced [5]. In the current study, few keratinocytes expressed K1 and K10 implying that disrupted epithelial differentiation and stratification are directly responsible for this loss, independent of changes in the epithelial phenotype. Similar limited expression of K1 and K10 was seen in high-grade CIN III lesions in contrast to a heterogeneous distribution of K4 and K13 [42,43]. Although differentiation keratins are lost in severe dysplasia, to compensate for their loss synthesis of other keratins may be induced. Indeed, transcripts and proteins for K7, K8 and K18 have been detected in the basal and suprabasal compartments of severe oral dysplasia, although in normal stratified epithelia these keratins are present only at the transcript level in basal and lower prickle cells [45]. In view of these findings, it may be concluded that keratin gene expression is partially altered from a stratified pattern to a simple one. The comparison of differentiation-specific keratins in different grades of dysplasia has served to emphasize the loss of ordered stratification and differentiation that accompanies the pre-neoplastic change. Loss of K4, K13, K1 and K10 in dysplastic lesions may be regarded in itself, a marker of dysplasia, as normal epithelia express at least two differentiation keratins with the exception of junctional oral epithelium [33,46]. Thus, a lack of these keratins is a more consistent feature of dysplastic epithelium than enhanced expression of K19 [47,48]. 4.4. Co-expression of differentiation keratins in well and moderately differentiated carcinomas In well differentiated SCCs, K4 and K13 showed coexpression with K1 and K10 in the same group of cells, including those at the periphery of tumor islands. As peripheral cells are the counterparts of basal and parabasal cells, it may be concluded that differentiation keratins are transcribed in basal, parabasal and prickle cells similar to expression seen in many normal oral epithelia and that reported for the same proteins in cervical SCC [49]. Well differentiated SCCs derived from non-keratinizing epithelia showed conservation of the K4/K13 complex from normal epithelium but also synthesis of K1 and K10 proteins. The same level of retention was not evident in moderately differentiated SCCs, K4 and K13 being replaced by K1 and K10. Substitution of K4/ K13 with K1/K10 has been detected in similar oral SCCs by SDS polyacrylamide gel electrophoresis [13], immunoblotting [10] and IHC [7,11,12,15,16,50]. Expression of K4, K13 and K10 transcripts has been detected in well differentiated esophageal SCCs [51] but not in moderate lesions. This transition is not in agreement with our findings, and might be accounted for
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variously by the different origins of SCC, differences in grading criteria or in the labeling method, 35S-labeled probes being able to detect low levels of transcript that are often beyond the detection limit of digoxigenin. 4.5. Absence of K4, K13, K1 and K10 in poorly differentiated carcinomas may be linked to loss of epithelial differentiation In poorly differentiated SCCs, differentiation keratins were absent. Impaired epithelial maturation reflected in the absence of ‘true’ prickle cells may influence this lack of keratin expression, a finding that has been reported in SCCs of epidermal and esophageal origin [51,52]. An alternative explanation may be that, due to the loss of stratification, as in severe dysplasia, the pattern of keratin gene expression may be converted to a ‘simple’ program. In support of this, the anomalous expression of simple keratins has been detected in poorly differentiated SCCs by IHC and ISH [6,7,9-11,13,15,42,45].
5. Conclusions The patterns of keratin expression were initially recorded on the basis of protein using both polyclonal and monoclonal antibodies and have therefore not provided the complete picture. Since the development of riboprobes, present studies have helped to extend our knowledge to the mRNA level and enable the systematic comparison of gene expression in both message and protein, and also avoid the complicated problem of ‘‘epitope masking’’ of keratin proteins, which is often encountered with immunohistochemistry. Therefore, this comparative study provides a more complete account of the distribution of keratins. Differentiation keratins showed reduced expression with increasing severity of dysplasia. Increasing grades of SCC were associated with a loss of epithelial differentiation and organization.
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[6] Debus E, Moll R, Franke WW, Weber K, Osborn M. Immunohistochemical distinction of human carcinomas by cytokeratin typing with monoclonal antibodies. Am J Pathol 1984;114:121– 30. [7] Morgan PR, Shirlaw PJ, Johnson NW, Leigh IM, Lane EB. Potential applications of anti-keratin antibodies in oral diagnosis. J Oral Pathol 1987;16:212–22. [8] Vigneswaran N, Peters K-P, Hornstein OP, Haneke E. Comparison of cytokeratin, filaggrin and involucrin profiles in oral leukoplakias and squamous carcinomas. J Oral Pathol Med 1989;18: 377–90. [9] Ermich T, Schulz J, Raabe G, Schumann D. Pattern of oral cytokeratins. III. SDS-electrophoretic analysis and immunoblotting of cytokeratins in leukoplakias and squamous cell carcinomas of the oral mucosa. Biomed Biochim Acta 1989;48:393–401. [10] Vaidya MM, Borges AM, Pradhan SA, Rajpal RM, Bhisey AN. Altered keratin expression in buccal mucosal squamous cell carcinoma. J Oral Pathol Med 1989;18:282–6. [11] Morgan PR, Shirlaw PJ, Lane EB, Leigh IM, Johnson NW. Chapter 9 Cytoplasmic markers of disturbed epithelial differentiation as markers of high risk lesions, with special reference to keratins. In Oral Cancer: Detection of Patients and Lesions at Risk., Johnson N W, editor. Cambridge University Press Cambridge pp. 188–202. [12] Heyden A, Huitfeldt HS, Koppang HS, et al. Cytokeratins as epithelial differentiation markers in premalignant and malignant oral lesions. J Oral Pathol Med 1992;21:7–11. [13] Schulz J, Ermich T, Kasper M, Raabe G, Schumann D. Cytokeratin pattern of clinically intact and pathologically changed oral mucosa. Int J Oral Maxillofac Surg 1992;21:35–9. [14] Moll R. Molecular diversity of cytokeratins: significance for cell and tumor differentiation. Acta Histochem 1992;41:117–27. [15] Ogden GR, Chisholm DM, Adi M, Lane EB. Cytokeratin expression in oral cancer and its relationship to tumor differentiation. J Oral Pathol Med 1993;22:82–6. [16] Ogden GR, Chisolm DM, Hopwood D, Lane EB. Evidence for field change in oral cancer based on cytokeratin expression. Br J Cancer 1993;67:1324–30. [17] Schaafsma HE, van der Velden LA, Manni JJ, et al. Increased expression of cytokeratins 8, 18 and vimentin in the invasion front of mucosal squamous cell carcinoma. J Pathol 1993;170:77– 86. [18] Vaidya MM, Borges AM, Pradhan SA, Bhisey AN. Cytokeratin expression in squamous cell carcinomas of the tongue and alveolar mucosa. Oral Oncol Eur J Cancer 1996;32B:333–6. [19] Clausen H, Moe D, Buschard K, Dabelsteen E. Keratin proteins in human oral mucosa. J Oral Pathol 1986;15:36–42. [20] van Muijen GNP, Ruiter DJ, Franke WW, et al. Cell type heterogeneity of cytokeratin expression in complex epithelia and carcinomas as demonstrated by monoclonal antibodies specific for cytokeratins nos. 4 and 13 Exp Cell Res 1986;162: 97– 113. [21] Morgan PR, Johnson NW, Leigh IM, Lane EB. Structure of gingival epithelium as revealed by monoclonal antibodies to keratins. In: Lehner T, Cimasoni G, editors. Borderland between Caries and Periodontal Disease, Me´decine et Hygie`ne Genevaeary, pp. 47–59. [22] Maeda H, Reibel J, Holmstrup P. Keratin staining pattern in clinically normal and diseased oral mucosa of lichen planus patients. Scand J Dent Res 1994;102:210–5. [23] Maddox DP, Jenkins D. Technical methods 3-aminopropyltriethoxysilane (APES): a new advance in section adhesion. J Clin Pathol 1987;40:1256–60. [24] Leube RE, Bader BL, Bosch FX, et al. Molecular characterization and expression of the stratified-related cytokeratins 4 and 15. J Cell Biol 1988;106:1249–61.
B.K. Bloor et al. / Oral Oncology 37 (2001) 251–261 [25] Kuruc N, Leube RE, Moll I, Bader BL, Franke WW. Synthesis of cytokeratin 13, a component characteristic of internal stratified epithelia is not induced in human epidermal tumors. Differentiation 1989;42:111–23. [26] Knapp AC, Franke WW. Spontaneous losses of control of cytokeratin gene expression in transformed, non-epithelial human cells occurring at different levels of regulation. Cell 1989;59:67–79. [27] Rieger M, Franke WW. Identification of an orthologous mammalian cytokeratin gene. High degree of intron sequence conservation during evolution of human cytokeratin 10. J Mol Biol 1988;204:841–56. [28] Su L, Morgan PR, Thomas JA, Lane EB. Expression of keratin 14 and 19 mRNA and protein in normal oral epithelia, hairy leukoplakia, tongue biting and white sponge nevus. J Oral Pathol Med 1993;22:183–9. [29] Su L, Lane EB, Morgan PR. Keratin 14 and 19 expression in normal, dysplastic and malignant oral epithelia — a study using in situ hybridization and immunohistochemistry. J Oral Pathol Med 1996;25:293–301. [30] van Muijen GNP, Ruiter DJ, Ponec M, Huiskens-van der Mey C, Warnaar SO. Monoclonal antibodies with different specificities against cytokeratins. An immunological study of normal tissues and tumors Am J Pathol 1984;114:9–17. [31] Machesney M, Tidman N, Waseem A, Kirby L, Leigh IM. Activated keratinocytes in the epidermis of hypertrophic scars. Am J Pathol 1998;152:1133–41. [32] Smedts F, Ramaekers F, Robben H, et al. Changing patterns of keratin expression during progression of cervical intraepithelial neoplasia. Am J Pathol 1990;136:657–68. [33] Morgan PR, Leigh IM, Purkis PE, et al. Site variation of keratin expression in human oral epithelia — an immunocytochemical study of individual keratins. Epithelia 1987;1:31–43. [34] Sawaf MH, Ouhayoun JP, Forrest N. Cytokeratin profiles in oral epithelia. A review and a new classification J Biol Buccale 1991;19:187–98. [35] Bloor BK, Su L, Shirlaw PJ, Morgan PR. Gene expression of the differentiation-specific keratins (4/13, and 1/10) in normal human buccal mucosa. Lab Invest 1998;78:787–95. [36] Viaene AI, Baert JH. Localization of K4 mRNA in human oesophageal epithelium by non-radioactive in situ hybridization. Histochem J 1994;26:50–8. [37] Su L, Morgan PR. Discrepancies between keratin mRNA and protein expression in mucosal and glandular epithelia by in situ hybridization and immunohistochemistry. Cell Vision J Analyt Morphol 1994;1:146–53.
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[38] Roop DR, Krieg TM, Mehrel T, Cheng CK, Yuspa SH. Transcriptional control of high molecular weight keratin gene expression in multistage mouse skin carcinogenesis. Cancer Res 1988;48:3245–52. [39] Bernerd F, Magnaldo T, Darmon M. Delayed onset of epidermal differentiation in psoriasis. J Invest Dermatol 1992;98:902–10. [40] Bloor BK. Keratin gene expression in oral mucosal diseases: keratoses, lichen planus, dysplasia and squamous cell carcinoma. PhD thesis. University of London 1998. [41] Lane EB, Bartek J, Purkis PE, Leigh IM. Keratin antigens in differentiating skin. Ann NY Acad Sci 1985;455:241–58. [42] Smedts F, Ramaekers F, Troyanovsky S, et al. Basal-cell keratins in cervical reserve cells and a comparison to their expression in cervical intraepithelial neoplasia. Am J Pathol 1992;140:601–12. [43] Smedts F, Ramaekers F, Leube RE, et al. Expression of keratins 1, 6, 15, 16, and 20 in normal cervical epithelium, squamous metaplasia, cervical intraepithelial neoplasia, and cervical carcinoma. Am J Pathol 1993;142:403–12. [44] Sexton CJ, Proby CM, Banks L, et al. Characterization of factors involved in human papillomavirus type 16-mediated immortalization of oral keratinocytes. J Gen Virol 1993;74:755–61. [45] Su L, Morgan PR, Lane EB. Protein and mRNA expression of simple epithelial keratins in normal, dysplastic and malignant oral epithelia. Am J Pathol 1994;145:1–10. [46] Mackenzie IC, Rittman G, Gao Z, Leigh I, Lane EB. Patterns of cytokeratin expression in human gingival epithelia. J Periodont Res 1991;26:468–78. [47] Bartek J, Taylor-Papadimitriou J, Miller N, Millis R. Patterns of expression of keratin 19 as detected with monoclonal antibodies in human breast tissues and tumours. Int J Canc 1985;36:299– 306. [48] Lindberg K, Rheinwald JG. Suprabasal 40 kd keratin (K19) expression as an immunohistologic marker of premalignancy in oral epithelium. Am J Pathol 1989;134:89–98. [49] Smedts F, Ramaekers F, Troyanovsky S, et al. Keratin expression in cervical cancer. Am J Pathol 1992;141:497–511. [50] Ogden GR, McQueen S, Chisolm DM, Lane EB. Keratin prolfiles of normal and malignant oral mucosa using exfoliative cytology. J Clin Pathol 1993;46:352–6. [51] Viaene AI, Baert JH. Expression of cytokeratin mRNAs in squamous cell carcinoma and balloon-cell formation of human oesophageal epithelium. Histochem J 1995;27:69–78. [52] Suo Z, Holm R, Nesland JMSquamous cell carcinomas. An immunohistochemical study of cytokeratins and involucrin in primary and metastatic tumours. Histopathol 1993;23:45–54.
Gene expression of differentiation-specific keratins in oral epithelial dysplasia and squamous cell carcinoma B.K. Bloor, S.V. Seddon, P.R. Morgan * Department of Oral Medicine & Pathology, Guy’s, King’s and St. Thomas’ Dental Institute, Guy’s Hospital, London SE1 9RT, UK Received 23 May 2000; accepted 26 July 2000
Abstract The aim of the study was to investigate the differentiation-specific keratins (K4, K13, K1 and K10) in oral epithelial dysplasia and squamous cell carcinoma (SCC). Alterations in keratin gene expression were determined by in situ hybridization using 35Slabeled riboprobes and immunohistochemistry with monoclonal antibodies. In mild dysplasia, both sets of differentiation keratins were expressed in the same group of cells but in moderate lesions, expression of K4 and K13 was reduced in the presence of enhanced K1 and K10 synthesis. In severe dysplasia, neither mRNAs nor proteins were detected. In tumor islands of well and moderately differentiated SCCs, the K4/K13 complex was co-expressed with K1/K10, but in poorly differentiated carcinomas, differentiation keratins were absent. Consequently, mild oral epithelial dysplasia and well differentiated SCC retain an essentially normal pattern of keratin gene expression and hence epithelial differentiation while in severe dysplasia and poorly differentiated SCC keratin gene expression reflects the gross changes in epithelial differentiation and maturation. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Gene expression; Differentiation-specific keratins; K4; K13; K1; K10; In situ hybridization; Immunohistochemistry; Oral epithelial dysplasia; Oral squamous cell carcinoma
1. Introduction The histological features associated with oral epithelial dysplasia and with squamous cell carcinoma (SCC) have been described previously [1-4] and are generally agreed in structural terms. Descriptions are expressed in terms of loss of structural organization and disturbed proliferation (dysplasia) and varying degrees of differentiation (SCC). These pathological changes involve the terminally differentiating cell compartment but it is not clear how they affect the gene expression of differentiation keratins. Knowledge of such gene changes is important for several reasons. First, any disturbance of expression may be out of phase with structural changes. Second, one set of differentiation keratins may substitute for the other, especially if there is a modification of the state of keratinization from normal [5]. Biochemical and immunohistochemical studies have indicated that SCCs express a wider range of keratins * Corresponding author. Tel.: +44-20-7955-4288; fax +44-207955-4455. E-mail address: [email protected] (P.R. Morgan).
than would be expected from the pattern shown by their normal epithelium [6-18]. Poorly differentiated SCCs are generally associated with a reduction in secondary keratins (K4/K13 in non-keratinizing epithelia and K1/ K10 in keratinizing epithelia) and a reduction or total loss of primary keratins (K5, K14 and K15). In contrast, well differentiated SCCs tend to conserve their secondary keratins that are localized to prickle cells within tumor islands. Although protein expression of the differentiation keratins has been demonstrated in oral mucosa [7,1922], the systematic comparison of protein relative to mRNA has not been investigated in malignant oral epithelia. This analysis will provide information on the molecular basis of human keratin expression and the changes that occur in dysplasia and malignancy. Analysis of keratin gene expression involved the use of in situ hybridization (ISH) to determine mRNA levels using specific riboprobes constructed and labeled with 35 S-dUTP for K4, K13, K1 and K10. Protein levels were detected with a standard immunohistochemical approach using monoclonal antibodies to the same keratins.
1368-8375/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S1368-8375(00)00094-4
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2. Materials and methods 2.1. Tissue samples Frozen tissue samples were selected from the Department of Oral Medicine and Pathology, GKT Dental Institute, on the basis of clinical and histopathological analysis. Biopsies of oral epithelial dysplasia were chosen to include mild (n=9), moderate (n=7) and severe (n=7) lesions, avoiding cases with superimposed candidiasis. With the exception of one case from each category, all biopsies originated from sites which are normally non-keratinized. Cases of SCC included well (n=7), moderately (n=9) and poorly differentiated (n= 7) types. One case each of well and poorly differentiated SCCs and two from moderately differentiated tumors originated from keratinized sites. Controls included six specimens of buccal mucosa from patients whose biopsies were recorded normal after histological analysis and two biopsies of normal skin. Each specimen was rapidly frozen onto cork in OCT cryoprotectant, quenched in melting isopentane and stored in liquid nitrogen within 60 min of surgical removal. Serial 5-mm cryostat sections were mounted onto glass microscope slides coated with a 2% solution of 3-aminopropyltriethoxysilane [23]. 2.2. Synthesis and labeling of riboprobes Antisense and sense RNA probes for each of the differentiation keratins were constructed from cDNA clones containing keratin inserts. Fragments of specific DNA for K4 [24] and K13 [25] were selected by PCR amplification and sub-cloned into the pGEM4 transcription vector (Promega, UK). For K1 [26] and K10 [27], cDNA was not sub-cloned, as sequences were mainly non-coding. RNA probes were synthesized for 90 min at 37 C with 200 mCi of 35S-dUTP (Amersham, UK) and the appropriate RNA polymerase. Template DNA was removed at 37 C with a 15-min incubation using five units of DNase I (Roche Diagnostics, UK). Unincorporated 35S-dUTP was eliminated by column chromatography using Nensorb 20 cartridges (Du Pont, USA). Labeled RNA was precipitated with 5% trichloroacetic acid and 20 mM sodium orthophosphate to determine efficiency of labeling.
and 2SSPE. Next, sections were pre-hybridized for 3 h at 45 C in 50% formamide, 2SSPE, 5Denhardt’s solution, 0.05% SDS, 20 mM Tris-HCl (pH 7.4), 100 mM dithiothreitol and 500 mg/ml of sheared salmon sperm DNA. Following this, sections were hybridized for 18 h at 45 C with 4106 cpm/ml of 35S-labeled RNA using the same hybridization buffer as before plus 10% dextran sulfate. After hybridization, unbound probe was removed with 330 min incubations in salt solutions of increasing stringency, including 4SSC (saline sodium citrate), 2SSC and 0.1SSC. Finally, unhybridized target RNA was removed with a 30-min incubation at 37 C with 50 mg/ml of RNase A (Sigma). Next, tissue sections were dehydrated in a series of Ethanol solutions of increasing concentration. Finally, the sensitivity of hybridization and distribution of mRNA transcripts were detected by autoradiography followed by development with K-5 nuclear emulsion (Ilford, UK). 2.4. Immunohistochemistry A standard immunohistochemical technique was applied for the detection of keratin (K) proteins with the following mouse monoclonal antibodies: clones 6B10 (K4) [30], 1C7 (K13) [30], LHK1 (K1) [31] and RKSE60 (K10) [32]. Tissue sections were fixed in acetone for 10 min at 4oC, incubated for 60 min with monoclonal antibody (1:10) followed by 30 min with biotinylated rabbit anti-mouse immunoglobulin (Dako Ltd.; 1:300) and biotin streptavidin-horseradish peroxidase complex (Dako Ltd.; 1:50). Sections were finally developed with 200 mg/ml of 3,30 diaminobenzidine (Sigma) and counterstained with hematoxylin. 2.5. Analysis of keratin gene expression Expression of keratin mRNAs and proteins was recorded using a semi-quantitative 4-point scale as used previously [28,29]. Expression data for normal buccal epithelium and oral epithelial dysplasia are presented in Table 1, results for SCCs being shown in Table 2. These tables represent the range and overall distributions of mRNA and protein in the respective groups. 2.6. Controls for in situ hybridization and immunohistochemistry
2.3. In situ hybridization ISH was carried out according to Su et al. [28,29]. In brief, tissue sections were fixed for 30 min in 4% paraformaldehyde, pre-treated for 5 min in 100 mM glycine followed by 10 min in 100 mM triethanolamine-HCl (pH 8.0) and 0.25% acetic anhydride. Sections were washed for 5 min in 2sodium saline phosphate-EDTA (SSPE) followed by 10 min at 45 C in 50% formamide
Sections of normal buccal mucosa (for detection of K4 and K13) and skin (K1 and K10) were included for comparison with keratin expression in dysplastic and malignant oral epithelia. Negative controls for ISH included 35S-labeled sense RNA probes for K4, K13 and K10, which, having identical sequences to target RNA, should not result in hybridization. For K1, as there is no equivalent sense probe, target RNA was
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B.K. Bloor et al. / Oral Oncology 37 (2001) 251–261 Table 1 Distributions of proteins and mRNAs for K4, K13, K1 and K10 in normal and dysplasticepitheliuma Distribution of differentiation-specific keratins in normal and dysplastic epithelium K4 Protein Normal buccal epithelium (n=6) Parakeratotic mild dysplasia (n=4) Orthokeratotic mild dysplasia (n=5) Parakeratotic moderate dysplasia (n=2) Orthokeratotic moderate dysplasia (n=5) Severe dysplasia (n=7)
Basal Prickle Basal Prickle Basal Prickle Basal Prickle Basal Prickle All cells
OC
+ +++ +OC +++ +OC +OC to +++ +OC to ++ +OC
K13 mRNA 1 3 1OC 3 0 1 to 2 0 1 1OC 1 0
K1
Protein OC
+ +++ +OC +++ +OC +OC to +++ +OC to ++ +OC
K10
mRNA
Protein
mRNA
Protein
mRNA
1 3 1OC 3 0 1 to 2 0 1 1OC 1 0
++* + to +++ +* to +++ ++ + to +++ +OC (28%)
1 2 1OC 1 to 3 1OC 2 to 3 0 2 1 2 to 3 0
+ to ++* + to +++ +* to +++ ++ + to +++ +OC (28%)
1 1 1OC 1 to 3 1OC 2 to 3 0 2 1 2 to 3 0
a to +++, protein expression from negative to high; *, indicates protein expression in columns; 0 to 3, mRNA expression from negative to high; OC, occasional expression from 5 to 30% of cells; percentages in parentheses indicate the proportion of positive cases if not 100% and n indicates the number of cases studied.
incubated for 1 h with 50 mg/ml of RNase A prior to hybridization with the K1 antisense probe. Negative controls for immunohistochemistry (IHC) included substitution of primary antibody with an irrelevant antibody of the same class.
3. Results 3.1. Histological appearance of oral epithelial dysplasia and SCC Oral epithelial dysplasias were divided into three categories on the basis of hematoxylin and eosin staining. In mildly dysplastic lesions, stratification was generally well ordered, showing either para- (n=4) or orthokeratinized epithelium (n=5). In some specimens, the epithelial architecture departed little from normal, but in others, bulbous rete processes were present. Nuclear atypia were seldom present, but were sparse and confined mainly to basal cells. The corium was usually associated with a mild lymphoplasmacytic infiltrate. Moderate dysplasias consisted of a para- (n=2) or orthokeratotic (n=5) epithelium, which was often hyperplastic in appearance. Stratification was more disturbed than in mild lesions, prickle cells losing their ordered fashion and basal cells showing loss of polarity. Additional epithelial changes were present in various combinations occupying up to half of the epithelial thickness. These features were not evident in each case, and included bulbous rete processes, hyperchromatic nuclei and pleomorphic cells. Thickening and hyalinization of the basement membrane were sometimes present together with apoptotic basal cell degeneration, thus simulating oral lichen planus. Normal and bizarre
mitoses were limited to basal and lower suprabasal layers. A lymphoplasmacytic infiltrate of varying intensity was present within the superficial corium. Severe dysplasias showed a trend toward parakeratinization irrespective of the site of origin. Rete processes were bulbous, drop-shaped in appearance or showed ‘budding’. Other epithelial changes were present in more than half of the epithelial thickness, with only some of these features being present in each biopsy. These consisted of nuclear hyperchromatism, nuclear and cellular pleomorphism, disordered cell polarity, individual cell keratinization and increased nucleus-tocytoplasm ratio. Bizarre mitoses and apoptoses were observed in basal and suprabasal layers and a lymphoplasmacytic infiltrate of variable intensity was present within the superficial corium. Carcinomas were divided into three categories based on their differentiation pattern. Features of well differentiated SCCs included the presence of well ordered islands and keratin pearls. The corium contained either a focal or diffuse lymphoplasmacytic host response, and in many cases there was epithelial dysplasia in the adjacent epithelium. SCCs were classified as moderately differentiated when tumor islands showed limited maturation, being less well ordered and with individual cell keratinization rather than keratin pearl formation. A light to moderate lymphoplasmacytic infiltrate was often present within the tumor stroma. In poorly differentiated carcinomas, there was no clear distinction between basal and maturation compartments, a high proportion of tumor cells being basaloid in appearance. Keratinization was generally absent in these tumors and there was very limited prickle cell differentiation. Apoptoses and bizarre mitoses were frequent and the host response either minimal or absent.
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1 1 1 1 0 + (43%) + to +++H + to ++OC (56%) 1 to 2 (71%) 1 to 2OC (71%) 1H (33%) 2H (33%) 0 + (43%) +OC to +++H + to ++OC (56%) 1 to 3 (43%) 1 to 2OC (71%) 1H (33%) 2H (33%) 0 + (43%) +OC to +++H + to ++OC (56%) Basal Prickle Basal Prickle All cells Well differentiated SCC (n=7) Moderately differentiated SCC (n=9) Poorly differentiated SCC (n=7)
a to +++, protein expression from negative to high; 0 to 3, mRNA expression from negative to high; OH, mRNA expression from 5 to 30% of cells; H, heterogeneous expression from 40 to 70% of cells; percentages in parentheses indicate the proportion of positive cases if not 100% and n indicates the number of cases studied.
1 to 2H (71%) 1 to 2OC (71%) 1 to 3H (33%) 1 to 3H (33%) 0 + (43%) + to +++H + to ++OC (56%) to 3 to 2OC to 3H (33%) to 3H (33%)
OC
Protein mRNA
H OC
Protein mRNA
H OC
Protein mRNA Protein
H
K1 K13 K4
Distribution of differentiation-specific keratins in tumor islands
Table 2 Distributions of proteins and mRNAs for K4, K13, K1 and K10 in oral squamous cell carcinoma (SCC)a
OC
K10
mRNA
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3.2. Keratin gene expression in normal buccal epithelium Proteins for K4 and K13 were distributed homogeneously in the suprabasal compartment with occasional expression in basal cells (Table 1 & Fig. 1A). 35Slabeled transcripts for K4 and K13 were detected at low level in basal cells but consistently in parabasal and suprabasal cells, the intensity gradually decreasing towards the upper layers (Fig. 1B). Proteins for K1 and K10 were expressed in columns of prickle cells centered over connective tissue papillae (Fig. 1C), their respective transcripts being synthesized in basal, parabasal and deep prickle cells, but at a lower intensity than that expressed for K4 and K13 (Fig. 1D). 3.3. Keratin gene expression in oral epithelial dysplasia In three mild dysplasias with a parakeratinized epithelium including two from buccal and one from alveolar ridge mucosa (Table 1), protein and mRNA expression of K4 and K13 were detected homogeneously in the suprabasal compartment but sparsely in the keratin and basal layers. Expression for K1 and K10 was detected in the prickle cell layer, ranging from a sparse to homogeneous distribution but showing minimal expression in basal cells. In a parakeratotic dysplastic lesion from the floor of mouth in which the keratin layer occupied more than half of the epithelial thickness, proteins for K4 and K13 were expressed heterogeneously in the suprabasal layers (Fig. 1E) although corresponding transcripts were not detected with 35S-labeled probes (Fig. 1F). Proteins for K1 and K10 were expressed at a lower level than for K4 and K13, but respective mRNAs were synthesized at low to moderate intensity (Fig. 1G and H). In orthokeratotic mild dysplasias, two from buccal mucosa and three from the ventral surface of tongue, proteins for K4 and K13 were distributed extensively in the suprabasal compartment and in a few cells of the basal layer. Their respective transcripts were localized in parabasal and prickle cells, the intensity varying from low to moderate levels. Proteins for K1 and K10 were expressed heterogeneously in the prickle cell layer, their corresponding mRNAs being transcribed either at low level or showing a heterogeneous distribution in basal and prickle cells. Expression of these keratins was more varied in moderate than in mild dysplasias (Table 1). In parakeratotic lesions, one each from buccal and alveolar ridge mucosa, proteins for K4 and K13 were distributed sparsely in the suprabasal layer while mRNAs were transcribed at low levels. Proteins and silver grains for K1 and K10 were expressed randomly in the prickle cell layer of buccal mucosa, but were absent from alveolar ridge mucosa. In moderately dysplastic orthokeratotic lesions from the floor of mouth (n=1) and lingual mucosa (n=4), proteins and transcripts for K4 and K13
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Fig. 1. Expression of K4 and K1 in normal (A–D) and mildly dysplastic (E–H) buccal epithelia. (A) In normal epithelium, K4 protein was stained uniformly in the suprabasal compartment and at a lower level of expression in many basal cells. (B) Silver grains for K4 transcript were distributed densely in prickle cells but are at low levels in basal cells. (C) Protein for K1 showed restricted expression in columns of prickle cells over connective tissue papillae whilst transcript was widely distributed in suprabasal cells (D). (E) In a single keratotic lesion showing mild dysplasia, K4 protein was expressed heterogeneously in the prickle cell layer although silver grains for K4 transcript were not detected above background levels (F). In the same lesion, K1 protein showed a less widespread distribution (G) compared with K4 but K1 mRNA was expressed at detectable levels in the suprabasal compartment (H). (Magnification: A, B and D 800, C 200 and E-H 400).
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Fig. 2. Expression of K4 and K1 in moderate (A–D) and severe (E–H) oral dysplasia. (A) In this moderate dysplasia of lingual epithelium, K4 protein was limited to a few prickle cells and its transcript (B) was not detected. Protein for K1 was expressed uniformly in the suprabasal compartment (C) but not in the keratin layer. Silver grains for K1 (D) were distributed extensively in the deeper prickle cell layers. (E) In this lesion from labial mucosa, K4 protein was absent from the epithelium corresponding to severe dysplasia (right-hand side of field) but was expressed uniformly in the adjacent ‘normal’ epithelium (left-hand side of field). K4 transcript (F) was absent from the dysplastic epithelium but was detected in the normal epithelium. K1 protein (G) was absent from the dysplastic zone but there were a few positive prickle cells in the normal epithelium. Silver grains for K1 (H) were absent from the dysplastic epithelium but were barely detectable in the normal epithelium. (Magnification: A–H 400).
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were either sparsely distributed or absent (Fig. 2A and B). Proteins and mRNAs for K1 and K10 were synthesized, expression varying from sparse to intense in the suprabasal layer (Fig. 2C and D), with silver grains also being expressed at low levels in the basal layer. In severe dysplasias, expression of these keratins was markedly reduced compared with their normal epithelium, with proteins and transcripts being predominantly absent (Fig. 2E–H). In two cases, proteins for K1 and K10 were localized to individual groups of keratinocytes in the suprabasal compartment but in the absence of their corresponding transcripts (Table 2). 3.4. Keratin gene expression in SCC In well differentiated SCCs (Table 2), expression of the differentiation-specific keratins was not homogeneous throughout the tumor, negative tumor islands often being adjacent to those expressing keratins. Proteins for K4 and K13 were expressed randomly as small foci (Fig. 3A and D) or more extensively throughout the tumor islands (Fig. 3C). In five SCCs, silver grains were variably expressed throughout the tumor, but in two lesions, they were absent (Fig. 3B). Proteins for K1 and K10 were expressed intensely in the prickle cell compartment of tumor islands and in some cases, also in the basal compartment (Fig. 3E and G). Silver grains for K1 and K10 were distributed in cells at the periphery and center of tumor islands, although intensity varied from moderate to low (Fig. 3F and H). Keratin expression in moderately differentiated SCCs was also variable and appeared to be independent of the original epithelial phenotype. In five SCCs, proteins for K4 and K13 were distributed randomly in the prickle cell compartment, their corresponding transcripts either being absent or localized to the periphery of tumor islands. As for K4 and K13, proteins for K1 and K10 showed a restricted distribution in prickle cells, transcript varying from weak to moderate expression in cells at the periphery and center of tumor islands. In the remaining four carcinomas, all differentiation keratins were absent. In poorly differentiated SCCs, differentiation-specific keratins were absent although the overlying epithelium showed a near normal profile for the site.
4. Discussion 4.1. Wider distribution of keratin mRNAs than proteins in normal oral epithelia In agreement with previous studies, expression of K4 and K13 proteins was detected in the suprabasal layer of buccal epithelium but K1 and K10 showed a restricted distribution [20,33-35]. Transcripts were
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detected in the basal and suprabasal compartments, implying that these genes are under post-transcriptional control. The widespread localization of K4 mRNA has been reported in esophageal epithelium with non-isotopic ISH [36] and that for K4 and K13 in buccal epithelium with digoxigenin and 35S-labeled probes [35,37]. Consistent expression of K1 and K10 in the basal and lower suprabasal layers has been reported with non-isotopic ISH for buccal epithelium [35] but also in epidermis with both methods [38,39]. Basal expression of K1 and K10 in epidermis has been considered to be associated with delayed onset of differentiation and this could also be the case in buccal epithelium. The current study also emphasizes the importance of the labeling system used to detect mRNA expression. A comparative analysis of 35S- and digoxigenin-labeled probes on cultured cell lines and serial sections of normal oral epithelium, dysplasia and SCC indicated both techniques were comparable in the detection of keratin mRNAs, but only at relatively high levels of expression [40]. Although digoxigenin-labeled probes resulted in better intracellular mRNA localization to individual cells 35S-labeled probes were more sensitive for the detection of mRNAs distributed at low level. Therefore, in the current study, the conventional radioactive approach was chosen over non-isotopic ISH for the detection of keratin mRNAs. 4.2. Variable expression of differentiation keratins in mild and moderate oral epithelial dysplasia is partly dependant on altered epithelial differentiation As disturbed differentiation is a major feature of dysplasia, it was not surprising to find that keratin gene expression was altered in comparison with normal oral epithelia. In mild lesions, derived from non-keratinizing epithelium, both sets of differentiation keratins were expressed in the same group of cells, K4 and K13 being retained from normal epithelium while synthesis of K1 and K10 was enhanced. As mRNAs for K1 and K10 are already present, it is possible that some aspect of the dysplastic change permits increased synthesis of proteins in an epithelium in which these keratins are normally expressed as minor constituents. Seen in this light, the presence of K1 and K10 mRNAs in nonkeratinizing epithelium may reflect an inherent defence mechanism, where these transcripts become readily translatable to proteins when required, possibly providing a protective role. If substantiated, this would be analogous to the synthesis of K6 and K16 in human epidermis. Despite the presence of their mRNAs in normal epidermis, K6 and K16 proteins are synthesized only under conditions of relatively high keratinocyte turnover [41]. It remains to be determined whether such shifts in keratin expression from K4/K13 to K1/K10 are
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Fig. 3. Expression of K4, K13 and K1 in well differentiated oral squamous cell carcinoma (SCC). (A) Protein for K4 was expressed weakly and in a few cells within tumor islands and silver grains (B) for K4 were barely above the background level. K4 protein (C) was distributed heterogeneously in prickle cells of tumor islands, and also in peripheral (basal) cells whereas K13 protein (D) was distributed randomly in the center of tumor islands. Protein for K1 was present heterogeneously in both basal and suprabasal compartments (E) but K1 transcript (F) was expressed mostly in the center of tumor islands. In a different case, K1 protein was distributed heterogeneously in prickle cells and randomly in cells at the periphery of tumor islands (G) but transcript for K1 was located towards the periphery of tumor islands (H) at a relatively low level of synthesis. (Magnification: A–H 400).
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indeed of functional significance, and whether synthesis of K1 and K10 proteins in non-keratinizing epithelia has a similar function to that of K6 and K16 in epidermis. Co-expression of K4 and K13 with K1 and K10 has been observed in keratoses and lichen planus, implying that increased synthesis of K1 and K10 may be linked with changes in epithelial differentiation [5] in addition to dysplastic change. There are no comparative studies on oral epithelial dysplasia in which differentiation keratins have been investigated at both the levels of transcript and protein. By using similar criteria, changes in keratin protein expression were measured against those in another mucosal disease, cervical intraepithelial neoplasia (CIN). In CIN I, a low-grade lesion analogous to mild oral epithelial dysplasia, expression of K1 and K10 proteins was restricted compared to heterogeneous expression in mild dysplasia, although proteins for K4 and K13 were similarly distributed throughout the epithelium [42,43]. In moderate dysplasia arising from non-keratinizing epithelia, K1/K10 filaments replaced the K4/K13 complex, a finding that accords with the histological changes to para- or orthokeratinization in association with the dysplastic process. This was not the case for one lesion (from alveolar ridge mucosa), as expression of K1 and K10 in the absence of K4 and K13 reflected the normal pattern seen from this type of epithelium. Furthermore, keratin gene expression was independent of the underlying inflammatory response unlike the situation in lichen planus, where epithelial infiltration by lymphocytes resulted in reduced expression of these keratins [5]. A switch from K4/K13 to K1/K10 was not seen in medium-grade CIN II lesions, K4 and K13 being intensely expressed in the basal and suprabasal compartments in the absence of K1 and K10 [42,43]. In the current study and in above IHC studies [42,43] proteins for K4, K13 and K10 were detected on frozen tissue with the same antibodies. Although K4 and K13 showed a similar pattern of expression, the distributions of K1 and K10 proteins were markedly different. It is possible that epithelial architectural differences between oral epithelial dysplasia and cervical neoplasia and an involvement of human papillomavirus (HPV) in the latter may further modulate keratin gene expression. In another study, immortalization of oral keratinocytes by HPV-16 resulted in an alteration in both keratin expression and differentiation [44]. This lends further support to the importance of HPV in affecting keratin gene expression, not only in cervical, but also oral tissue. 4.3. Lack of differentiation keratins as a defining feature of severe oral epithelial dysplasia In severe oral dysplasia, both sets of differentiation keratins were largely absent, although it is interesting to note that all lesions presented with a parakeratinized
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epithelium, a phenotype in which markers of nonkeratinization are retained and those for keratinization are induced [5]. In the current study, few keratinocytes expressed K1 and K10 implying that disrupted epithelial differentiation and stratification are directly responsible for this loss, independent of changes in the epithelial phenotype. Similar limited expression of K1 and K10 was seen in high-grade CIN III lesions in contrast to a heterogeneous distribution of K4 and K13 [42,43]. Although differentiation keratins are lost in severe dysplasia, to compensate for their loss synthesis of other keratins may be induced. Indeed, transcripts and proteins for K7, K8 and K18 have been detected in the basal and suprabasal compartments of severe oral dysplasia, although in normal stratified epithelia these keratins are present only at the transcript level in basal and lower prickle cells [45]. In view of these findings, it may be concluded that keratin gene expression is partially altered from a stratified pattern to a simple one. The comparison of differentiation-specific keratins in different grades of dysplasia has served to emphasize the loss of ordered stratification and differentiation that accompanies the pre-neoplastic change. Loss of K4, K13, K1 and K10 in dysplastic lesions may be regarded in itself, a marker of dysplasia, as normal epithelia express at least two differentiation keratins with the exception of junctional oral epithelium [33,46]. Thus, a lack of these keratins is a more consistent feature of dysplastic epithelium than enhanced expression of K19 [47,48]. 4.4. Co-expression of differentiation keratins in well and moderately differentiated carcinomas In well differentiated SCCs, K4 and K13 showed coexpression with K1 and K10 in the same group of cells, including those at the periphery of tumor islands. As peripheral cells are the counterparts of basal and parabasal cells, it may be concluded that differentiation keratins are transcribed in basal, parabasal and prickle cells similar to expression seen in many normal oral epithelia and that reported for the same proteins in cervical SCC [49]. Well differentiated SCCs derived from non-keratinizing epithelia showed conservation of the K4/K13 complex from normal epithelium but also synthesis of K1 and K10 proteins. The same level of retention was not evident in moderately differentiated SCCs, K4 and K13 being replaced by K1 and K10. Substitution of K4/ K13 with K1/K10 has been detected in similar oral SCCs by SDS polyacrylamide gel electrophoresis [13], immunoblotting [10] and IHC [7,11,12,15,16,50]. Expression of K4, K13 and K10 transcripts has been detected in well differentiated esophageal SCCs [51] but not in moderate lesions. This transition is not in agreement with our findings, and might be accounted for
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variously by the different origins of SCC, differences in grading criteria or in the labeling method, 35S-labeled probes being able to detect low levels of transcript that are often beyond the detection limit of digoxigenin. 4.5. Absence of K4, K13, K1 and K10 in poorly differentiated carcinomas may be linked to loss of epithelial differentiation In poorly differentiated SCCs, differentiation keratins were absent. Impaired epithelial maturation reflected in the absence of ‘true’ prickle cells may influence this lack of keratin expression, a finding that has been reported in SCCs of epidermal and esophageal origin [51,52]. An alternative explanation may be that, due to the loss of stratification, as in severe dysplasia, the pattern of keratin gene expression may be converted to a ‘simple’ program. In support of this, the anomalous expression of simple keratins has been detected in poorly differentiated SCCs by IHC and ISH [6,7,9-11,13,15,42,45].
5. Conclusions The patterns of keratin expression were initially recorded on the basis of protein using both polyclonal and monoclonal antibodies and have therefore not provided the complete picture. Since the development of riboprobes, present studies have helped to extend our knowledge to the mRNA level and enable the systematic comparison of gene expression in both message and protein, and also avoid the complicated problem of ‘‘epitope masking’’ of keratin proteins, which is often encountered with immunohistochemistry. Therefore, this comparative study provides a more complete account of the distribution of keratins. Differentiation keratins showed reduced expression with increasing severity of dysplasia. Increasing grades of SCC were associated with a loss of epithelial differentiation and organization.
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