Changes in Keratin Expression during 7,12-Dimethylbenz[a ...cancerres.aacrjournals.org/content/canres/50/14/4441.full.pdf · The normal hamster cheek pouch epithelium expressed K14

[CANCER RESEARCH 50, 4441-4445, July 15. 1990|

Changes in Keratin Expression during 7,12-Dimethylbenz[a]anthracene-inducedHamster Cheek Pouch Carcinogenesis1

Irma B. Gimenez-Conti,2 Dong M. Shin, Albert B. Bianchi, Dennis R. Roop, Waun Ki Hong, Claudio J. Conti, and

Thomas J. SlagaThe University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas 78957 [1. B. G-C., A. B. B., C. J. C., T. J. S.J, and Departmentof Medical Oncology, Houston, Texas 77030 [D. M. S., W. K. H.J, and Baylor College of Medicine, Department of Cell Biology and Department of Dermatology,Houston, Texas 77030 [D. R.J

ABSTRACT

This study was undertaken to explore the expression of keratins in thehamster cheek pouch carcinogenesis model, using monospecific keratinantibodies and a technique that allows immunoblotting analysis of tissuesembedded in paraffin. Changes in keratin expression were correlatedwith histopathological changes and with the expression of the enzyme 7-glutamyl transpeptidase. The right cheek pouch of 20 male golden Syrianhamsters was treated with 0.5% 7,12-dimethylbenz|a|anthracene for 16weeks. As previously described by other laboratories, this treatmentresulted in hyperplastic and dysplastic lesions and benign and malignanttumors. The keratins assayed in this study were K14 (M, 55,000), Kl(M, 67,000), and K13 (M, 47,000).

The normal hamster cheek pouch epithelium expressed K14 in thebasal layer and K13 in the suprabasal and differentiated layers, whereasKl was not detected by either immunohistochemistry or immunoblotting.Concomitant with 7,12-dimethylbenz(a]anthracene-induced hyperplasia,there were some topographical alterations in the distribution of K14. Inthis case, K14 was no longer restricted to the basal layer but was alsoexpressed in differentiated cells. The same pattern was also observed indysplastic lesions and in squamous cell carcinoma. Furthermore, expression of the K13 differentiation-associated keratin was preserved in thishyperplastic epithelium during all the stages of carcinogenesis, includingeither anaplastic or differentiated areas.

In contrast, after 2 weeks of 7,12-dimethylbenz|a]anthracene treatment, Kl expression started as a weak and patchy pattern in suprabasalcells, becoming stronger and more homogeneous at 8 and 16 weeks oftreatment. However, Kl was almost absent in squamous cell carcinoma,where only small very well differentiated areas were stained. We alsoobserved 7-glutamyl transpeptidase-positive foci in earlier stages ofcarcinogenesis, concomitant with the expression of the Kl keratin. However, it was not possible to find a perfect topographical correspondencebetween the two events.

Alterations in the pattern of keratin expression appear to be a commonfeature during the development of squamous cell carcinoma in differentsystems and could be an excellent tool to study carcinogenesis andchemoprevention.

INTRODUCTION

The golden Syrian hamster cheek pouch carcinogenesismodel is probably the best known animal system that closelycompares with events involved in the development of premalig-nancy and malignancy in human oral cancer. Furthermore, itis one of the most well characterized models for SCC3 (1, 2).

The carcinogenesis studies in the hamster cheek pouch areusually carried out either by using multiple applications ofDMBA (usually 0.5% in mineral oil, 3 times a week) or,occasionally, by using other carcinogens or a two-stage protocol(3-5). The 0.5% dose of DMBA induced a hyperplastic response

Received 11/9/89; revised 3/14/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by NIH-CA 43278, the Mary Gough Estate Re

search Fund, and the Olga Keith Weiss Chair.2To whom requests for reprints should be addressed.3The abbreviations used are: SCC, squamous cell carcinoma; GGT, 7-glutamyl

transpeptidase: DMBA, 7,12-dimethylbenz[a)anthracene.

in the pouch epithelium after only a few applications, followedby the appearance of a variety of dysplastic lesions resemblinghuman premalignant lesions after 6-8 weeks of treatment.

Benign and malignant tumors (papillomas and SCC) started todevelop after 10 weeks of treatment.

Although the mechanisms by which chemical carcinogensinduced cancer in this system are not clearly defined, there havebeen some biochemical and molecular studies described (6-9).Probably the most well characterized event has been the induction of GGT, an enzyme that is not normally expressed in thehamster cheek pouch.

Solt and Shklar (10,11) showed that individual GGT-positivecells or doublet cells are detected histochemically as early as 3days after the first DMBA treatment. After 3 weeks of treatment, they were able to detect GGT-positive intraepithelial cell

clones (plaques). GGT activity has also been demonstratedhistochemically in areas of dysplasia, papillomas, and welldifferentiated SCC (12).

Changes in the patterns of keratin expression have also beenobserved in the hamster cheek pouch epithelium during DMBAcarcinogenesis (13). Keratins are a family of several proteins,with molecular weights of 40,000-70,000, that form the intermediate filaments of epithelial cells. The expression of keratinsvaries in the different types of epithelia and is also affected bythe stage of differentiation of a given epithelial cell (14). Reportsfrom several laboratories have shown an alteration in the keratin profile during the carcinogenesis process, including theloss of differentiation-associated keratins and the expression ofaberrant members of the family (15-19).

In the case of the hamster cheek pouch carcinogenesis,changes in the topographic distribution and intensity of im-

munostaining of keratins in different stages of carcinogenesiswere demonstrated (13). However, this study was carried outwith monoclonal antibodies that recognize several subtypes ofkeratins and, therefore, it was not possible to identify the lossor gain of specific keratin species (13, 20).

In the present study, we have explored the expression ofkeratins using immunostaining with monospecific antibodiesand also a technique that allowed immunoblotting analysis oftissues embedded in paraffin. Monospecific antibodies againstmurine keratins were developed by Roop et al. (21), usingsynthetic peptides corresponding to the DNA sequence of the3' nonconserved region of keratin complementary DNA. These

antibodies recognize single members of the keratin family andcan be used to investigated the presence or absence of specifickeratins in histological sections. We have used antibodiesagainst three keratins: K14, which is normally associated withproliferating cells (basal layer); Kl, which is normally expressedin differentiated cells of the epidermis; and K13, which isexpressed in differentiated cells of mucosa. This analysis wascarried out in order to determine the sequence of expression ofkeratins during the hamster cheek pouch carcinogenesis process

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KERATIN EXPRESSION IN HAMSTER CHEEK POUCH CARCINOGENESIS

and to correlate their expression with other markers of prema-lignant progression.

MATERIALS AND METHODS

Twenty-two noninbred male Syrian hamsters, 4-6 weeks old, werepurchased from the NIH. The animals were housed four/plastic disposable cages, with wood chips for bedding. The animals were treatedwith DMBA 3 times/week for 16 weeks. The DMBA was administeredas a 0.5% solution in mineral oil (Sigma Chemical Co., St. Louis, MO),applied to the entire mucosa! surface of the right buccal pouch using aNo. 5 camel hair brush that was dipped once and wiped free of excesssolution at each application. The control animals were treated similarlywith mineral oil alone. At 2, 4, 8, and 16 weeks, groups of animalswere killed with carbon dioxide, the buccal pouches were excised, andrepresentative sections of each buccal pouch and the tumors were fixedin cold acetone for the GGT and keratin studies and in formalin forthe histopathological evaluation.

Gel Electrophoresis, Immunoblotting, and Immunohistochemistry. Inorder to obtain protein samples from the paraffin-embedded tissuesections, we used a recently described technique from our laboratories(22). A total of 0.9 mg of deparaffinized tissue was extracted and loadedinto each well. Electrophoresis was performed in the presence of 0.1%sodium dodecyl sulfate on 7.5% discontinuous polyacrylamide gels,according to the method of Laemmli (23). Gels were transferred ontonitrocellulose paper, as described by Towbin et al. (24). Immunodetec-tion of antigens on nitrocellulose blots was performed using antiseramonospecific for the M, 55,000 (RK14), M, 67,000 (RK1), and M,47,000 (RK13) mouse keratins developed by Roop et al. (21 ). Secondarybiotin-labeled anti-rabbit IgG antibody (Vector Laboratories, Burlin-

game, CA) was applied, and immunoreactive bands were detected usinga biotin-avidin-peroxidase kit (Vector Laboratories, Burlingame, CA).

For immunohistochemistry, 5-pm sections were deparaffinized and,following a regular immunohistochemistry protocol, one complete setof sections was incubated with RK1 antibody (1:500), another set withthe RK14 antibody (1:500), and a third set with K13 (1:800) antibody.Secondary biotin-labeled anti-rabbit IgG antibody was used, and thereaction was developed with an avidin-biotin-peroxidase complex kit(ABC Vectastaire; Elite-Vector Laboratories). Quantitative evaluationof immunostained sections was performed using an eyepiece disc (Micrometer). Areas expressing normal and abnormal phenotypes weredetermined as a function of the length of epithelial surface. The wholelength of each section was measured and in most cases triplicate sampleswere examined in each slide. One slide/animal (approximately 15.000-nm total epithelial surface length) and 3 to 5 animals/time point wereconsidered for Kl and K14. Results were expressed as a percentage ofpositive (K1) or abnormal pattern (K14) areas per total epithelial length.

GGT Detection. In order to detect GGT on paraffin sections, we useda method described by Ogawa et al. (25). The sections used wereimmediately adjacent to the ones used for the keratin studies.

Histopathology. The histopathological evaluation was performedboth in the hematoxylin- and eosin-stained sections adjacent to theones used for the previously described studies (fixed in acetone) and inthe sections from blocks fixed in formalin (22).

RESULTS

Histopathological Findings. A few days after the initialDMBA treatment, as described by Salley (1), the epithelium ofthe buccal pouch became hyperplastic, with an increment ofnucleated layers and hyperkeratinization. There also was a verystrong inflammatory reaction in the corion.

By 4 weeks of DMBA treatment, the epithelial hyperplasiaincreased and the corion was thicker than the control, withedema, congestion, and polymorphonuclear neutrophil infiltration. At this stage, the presence of ridge edges, which is acharacteristic reaction to irritation of squamous epithelia, wasalso noted. By 8 weeks, there were areas of focalized hyperkar-atosis and hyperplasia with variable degrees of dysplasia, which

are compatible with the histolgica! features of human leuko-plakias.

At week 16 of DMBA treatment, a number of tumors wereobserved by gross examination. Histolgica! analysis of thetumors indicated the presence of different types of lesions. Themost common was a full gradient of tumors from benignpapilloma to invasive SCC. In addition, we have also observedlesions compatible with fibropapillomas. At this time of treatment (16 weeks), 100% of animals presented tumors, and themultiplicity appeared to be 5 to 7 tumors/pouch. The incidenceof multiplicity of tumors seem to be in the range reported byothers (1-5).

These findings seem to be compatible with previous resultsfrom other laboratories (1-5), although a detailed histopathological study of the different premalignant and malignant lesions, as well as their incidence, has not been reported.

GGT Studies. The induction of GGT in DMBA-treated hamster cheek pouch was studied by other laboratories (10, 11, 26).Results of our experiments appear to confirm those previouslyreported, indicating that the GGT-positive reaction is a goodmarker of premalignant lesions. We observed epithelial GGT-positive foci at 4 weeks, which increased in number and sizewith the protracted DMBA treatment. In most SCCs, expression of GGT was also in the form of intratumoral foci (Fig. 1,A-Q.

Keratin Studies. Although the antibodies used are primarilydirected against mouse keratins, we have shown that they cross-react with hamster keratins. We have observed that in hamsterskin the Kl and K14 antibodies presented an immunohisto-chemical distribution identical to that described by Roop et al.( 18) in mouse skin. The corresponding immunoblotting analysisshowed single bands at M, 55,000 for K14 and Mr 67,000 forKl (Fig. 2).

The presence of high molecular weight bands in three samplesas detected by anti-K14 was attributed to incomplete denaturing, since those bands did not appear when the same samplesand additional ones were electrophoresed after denaturing witha higher concentration of -mercaptoethanol and boiling for alonger period of time (data not shown).

Furthermore, in a previous study, the same antibodies weresuccessfully used to detect changes in keratin patterns in organculture of hamster trachea (19).

Both immunohistochemical and immunoblotting analysiswith K13 in the hamster skin showed an absence of reaction(Fig. 2), as was previously observed in other species (18).

The hamster cheek pouch showed a keratin pattern expectedfor oral mucosa epithelium. The K14 proliferation-associatedkeratin was expressed in the basal layer (Fig. \D) and K13,normally associated with terminal differentiation of internalstratified epithelia, was expressed in the differentiating cellcompartments, as described by Nischt et al. (15) in internalepithelium of mouse (Fig. \J). The Kl epidermal differentiation-associated keratin was not expressed in the normal hamstercheek pouch (Fig. 1C).

The normal keratins expressed in the buccal pouch epithelium were also expressed throughout all stages of carcinogene-sis, including the premalignant (Fig. l, fand A ) and malignant(Fig. l, Fand L) lesions. However, there were some topographical alterations in the distribution of K14 in DMBA-treatedepithelium. In these hyperplastic epithelia, K14 was no longerrestricted to the basal layer but was also expressed in differentiated cells in approximately 90% of the epithelial length (Fig.IE and Table 1). The same pattern was also observed in dys-plastic lesions and SCCs, in which K14 was expressed in all

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Fig. .Paraffin sections of hamster cheek pouch. GGT activity after 4 (A), 8 (B), and 16 weeks (O fDMBA treatment (counterstained with hematoxylin).Immunoreaction with anti-K14 antiserum in normal cheek pouch (D) and after 8 ()and 16 weeks (F) of DMBA treatment. Immunoreaction with anti-Kl antiserumin normal cheek pouch (G) and after 8 (//) and 16 weeks (/) of DMBA treatment. Immunoreaction with anti-K13 antiserum in normal cheek pouch (J) and after 8(.K)and 16 weeks ()of DMBA treatment.

nucleated cells. Also, in the DMBA-induced hyperplastic epithelium there appeared to be an increase in the expression ofKl 3, as observed by immunoblotting and immunohistochemicalanalysis (Figs. 1 and 2). Interestingly, the expression of thisdifferentiation-associated keratin was preserved in all the SCCs,in which K13 was expressed by most tumoral cells, regardlessof their level of anaplasia or differentiation (Fig. \L).

The Kl keratin, which was not normally present in the pouchepithelium, was present in DMBA-treated epithelium startingat 2 weeks of DMBA treatment. Kl staining gave a patchypattern in suprabasal cells that became stronger and more

homogeneous at 8 weeks of treatment (Table 1). However, theKl expression was almost absent in the SCCs, in which onlysmall very well differentiated areas were occasionally stainedwith the anti-Kl antibody (Fig. l, G, H and L).

The expression of Kl was not directly related to a particularhistolgica! pattern. Sometimes it was absent in very hyper-plastic areas at 4 weeks, but it stained strongly positive in otherareas with moderate hyperplasia. Similarly, a clear correlationbetween Kl expression and dysplastic areas was not found.However, some highly hyperplastic or dysplastic areas presented intralesion focal staining for this keratin.

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55kD-

67kD-

45678

12345678

47kD- K13

Fig. 2. Immunoblot analysis of proteins extracted from paraffin sections ofhamster cheek pouch, using monospecific antisera directed against the K14 (A/,55,000), Kl (M, 67,000), and K M (M, 47,000) murine keratins. Lane I, normalmouse skin; lane 2, normal hamster skin; lane 3, normal hamster cheek pouch;lane 4, hamster cheek pouch with DMBA, 2 weeks; lane 5, hamster cheek pouchwith DMBA, 4 weeks; lane 6, hamster cheek pouch with DMBA, 8 weeks; lane7, hamster cheek pouch DMBA, 16 weeks; lane S. hamster cheek pouch tumor(SCC).

Table 1 Quantitative evaluation of changes in keratin patterns in DMBA-treatedhamster cheek pouch epithelium

Weeks of DMBAtreatment2

48

Control (mineral oil alone)K

14, areas withabnormal distribution(%)89.42

18.32

87.58 23.5294.65 6.61

4.34 3.93Kl,

positiveareas(%)37.42

9.28

78.25 14.5284.16 7.04

0Mean percentage SD of 4 or 5 animals; 30 fields (x 40 magnification)

were counted per animal.

DISCUSSION

Keratins are usually expressed in pairs of a type I keratin (M,40,000-60,000; acidic isoelectric point) and a type II keratin(Mr 48,000-70,000; neutral or basic isoelectric point) (27). Inthe epidermis, the pair K5/K14 is expressed by proliferatingcells (basal layer) and the pair K1/K10 is expressed in differentiating cells (suprabasal layers), whereas in the squamousepithelia covering the internal mucosa (oral cavity, esophagus,etc.) the pair K4/K13 is expressed by the suprabasal cells (15,28-31). In our study, we used one member of each of thesemajor pairs of keratins expressed by squamous epithelia. Although the antibodies we used were raised against polypeptides

from mouse keratin complementary DNA sequences, thereseems to be enough homology between the mouse and thehamster keratin genes, because both the immunohistochemicaland the immunoblotting patterns for the three antibodies appear identical.

The pattern of keratins for the hamster cheek pouch wasconsistent with that of the oral mucosa (15). K14 was restrictedto the basal layer, K13 to the suprabasal layer, and there wasno demonstrable immunoreactivity with Kl, by either immu-nohistochemistry or immunoblotting. However, Kl was expressed by the cheek pouch epithelium in a time-dependentfashion in DMBA-treated hamsters.

Although not normally present in internal epithelia, the Kl/K10 keratin pair has been previously shown in some hyperker-atinized mucosa (forestomach, dorsal tongue, cervix). Thesekeratins are also normally coexpressed in other internal humanepithelia (14, 15, 30, 32) (anus, vagina, exocervix). In thehamster cheek pouch, Kl expression occurs after DMBA treatment, concomitant with a marked hyperplasia and also anaugmentation of K13. Whether the presence of K l in the pouchepithelium is a simple consequence of hyperplasia or is a specificevent of DMBA carcinogenesis events remain to be elucidated.The fact that human oral and lung premalignant lesions expressed this type of keratin (30) suggests that they may constitute a necessary step in the pathway to the development ofSCC. Although Kl was present in most dysplastic stages, it wasessentially absent in the SCC stage, with the exception of smallwell differentiated areas of the tumors. Conversely, K13 doesnot seem to be affected by malignant transformation and wasstrongly expressed in the SCC stage.

The loss of Kl has been previously reported in several humantumors and has been studied in detail in the mouse skin two-Stage carcinogenesis system (17, 18, 25). Loss of Kl occurs inpremalignant stages of tumor development and seems to followthe development of gross numerical chromosomal abnormalities. The loss of Kl also seems to precede the appearance ofGGT foci, since GGT staining was always present in Kl-negative areas of papillomas (17). Recently, Nischt et al. (15)showed that K13, a keratin not normally expressed in epidermis, was present in all SCCs and some papillomas. The aberrantexpression of this keratin appears to be a very early event inpapilloma development.4 In the mouse skin system, therefore,the loss of a differentiation-associated keratin (Kl) and theaberrant expression of a different differentiation keratin (K13)seem to be characteristic of malignant progression. Interestingly, in this study, the same pattern was found in the SCCs ofthe hamster buccal pouch, in which Kl, which had been gainedin the premalignant stage, was lost in the fully malignant cellsbut K13 was strongly present in all the tumors. Therefore, itappears that, independent of the keratin patterns of the tissueof origin, the SCCs tend to lose Kl and express K13 freely.The reason for this particular behavior is not known; however,it can be speculated that it is probably related to the incompleteand faulty patterns of differentiation observed in the SCCs.

Unlike mouse skin, in which the GGT expression seems tobe a late phenomenon, in the hamster cheek pouch small focican be detected 3 days after DMBA treatment. Soil and Shklar(11) have speculated that the GGT foci may represent premalignant clones. In our study, we also observed GGT foci inearlier stages of carcinogenesis, concomitant with the expression of Kl keratin. However, it was not possible to find aperfect topographical correspondence between the two events.

41. B. Gimenez-Conti, el al. unpublished observations.

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KERATIN EXPRESSION IN HAMSTER CHEEK POUCH CARC1NOGENESIS

At a later stage of carcinogenesis, GGT was usually found indysplastic lesions, some of which had lost Kl keratin.

The expression of K14 was also altered during the carcinogenesis process. These alterations consisted primarily ofchanges in the topographical distribution. During DMBA-in-duced hyperplasia, K14 is no longer restricted to the basal layerbut is also expressed in suprabasal layers. These patterns havebeen previously described in mouse papillomas, in which im-munohistochemistry and in situ hybridization showed that K14expression is not shut off as epidermal cells move to moresuperficial layers of the epithelium. It has been speculated thatthis phenomenon is related to the lack of response to differentiation signals by the genetically altered (initiated) keratinocyte(18).

Alterations in the pattern of keratins appeared to be a common feature in the development of SCC in different systems.These alterations probably reflect abnormal differentiation patterns and are excellent tools to monitor the process of carcinogenesis, as well as for studies of chemoprevention and chemotherapy of premalignant lesions.

ACKNOWLEDGMENTS

The authors wish to thank Judy Chesner for technical assistance,Judy Ing for art work, and Mary Lou Fendley for typing this manuscript.

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1990;50:4441-4445. Cancer Res Irma B. Gimenez-Conti, Dong M. Shin, Albert B. Bianchi, et al. ]anthracene-induced Hamster Cheek Pouch Carcinogenesis

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Changes in Keratin Expression during 7,12-Dimethylbenz[a ...cancerres.aacrjournals.org/content/canres/50/14/4441.full.pdf · The normal hamster cheek pouch epithelium expressed K14