Cytochrome P450 expression and related metabolism in human buccal mucosa

Constituents in food and fluids, tobacco chemicals and many drugs are candidates for oral absorption and oxidative metabolism. On this basis, the expression of cytochrome P450 isozymes (CYPs) and the conversion of CYP substrates were analysed in reference to buccal mucosa. A RT-PCR based analysis of human buccal tissue from 13 individuals demonstrated consistent expression of mRNA for the CYPs 1A1, 1A2, 2C, 2E1, 3A4/7 and 3A5. CYP 2D6 was expressed in six out of the 13 specimens, whereas all samples were negative for 2A6 and 2B6. Serum-free monolayer cultures of the Siman virus 40 large T-antigen-immortalized SVpgC2a and the carcinoma SqCC/Y1 buccal keratinocyte lines expressed the same CYPs as tissue except 3A4/7 and 3A5 (SVpgC2a), and 2C, 2D6 and 3A4/7 (SqCC/Y1). Dealkylation of ethoxyresorufin and methoxyresorufin in both normal and transformed cells indicated functional 1A1 and 1A2, respectively. SVpgC2a showed similar activity as normal keratinocytes for both substrates, whereas SqCC/Y1 showed about 2-fold lower 7-ethoxyresorufin O-deethylation and 7-methoxyresorufin O-demethylation activities. SVpgC2a showed detectable and many-fold higher activity than the other cell types towards chlorzoxazone, a substrate for 2E1. Absent or minute catalytic activity of 2C9, 2D6 and 3A4 in the various cell types was indicated by lack of detectable diclofenac, dextromethorphan and testosterone metabolism (<0.2-0.5 pmol/min/mg). Metabolic activation of the tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and the mycotoxin aflatoxin B1 (AFB1) to covalently bound adducts was indicated by autoradiographic analysis of both monolayer and organotypic cultures of SVpgC2a. In contrast, SqCC/Y1 showed lower or absent metabolic activity for these substrates. Finally, measurements of various non-reactive AFB1 metabolites indicated rates of formation <0.1 pmol/min/mg in both normal and transformed cells. The results indicate presence of several CYPs of which some may contribute to significant xenobiotic metabolism in human buccal epithelium. Notably, metabolic activation of AFB1 was not previously implicated for oral mucosa. Further, the results show that CYP-dependent metabolism can be preserved or even activated in immortalized keratinocytes. Metabolic activity in SVpgC2a under both monolayer and organotypic culture conditions suggests that this cell line may be useful to pharmaco-toxicological and carcinogenesis studies.     

Gene expression of differentiation-specific keratins in oral epithelial dysplasia and squamous cell carcinoma

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 35S-labeled 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.     

Relationship between the expression of differentiation-specific keratins 1 and 10 and cell proliferation in epidermal tumors.

In normal epidermis, the expression of keratins 1 and 10 is associated with the loss of proliferative capacity and the onset of terminal differentiation. Keratins 1 (K1) and 10 (K10) are commonly expressed in the differentiating layer of benign tumors, but are lost during progression from the benign to the malignant state in skin carcinogenesis. Active gene constructs of mouse K1 and K10 were introduced into papilloma and carcinoma cell lines derived from keratinocytes to analyze the consequences of the expression of these keratins on the organization of the endogenous cytoskeletal network and on the mitotic activity of the recipient cells. Exogenous K1 integrated into the preexisting keratin K5/K14 network of both SLC-1 carcinoma and 308 papilloma cells. The formation of a recombinant cytoskeleton was more restricted for K10 than for K1 and appeared to be related to a requirement for cessation of cell division before K10 could integrate. The integration of exogenous K1 filaments into the endogenous keratin network was compatible with sustained proliferation of SLC-1 carcinoma cells in vitro. However, the exogenous gene was not expressed in tumor grafts in vivo. In contrast, stable K1 or K10 transfectants could not be selected in 308 cells, suggesting that benign tumor cells expressing suprabasal keratins cannot sustain proliferation.     

Epidermal morphogenesis and keratin expression in c-Ha-ras-transfected tumorigenic clones of the human HaCaT cell line.

Several tumorigenic (benign and malignant) clones have been raised from the human epidermal cell line HaCaT after transfection with the c-Ha-ras oncogene (val 12) (P. Boukamp et al., Cancer Res., 50: 2840-2847, 1990). In culture, these HaCaT-ras clones expressed epidermal differentiation markers, such as keratins K1 and 10, at high density or upon depletion of retinoic acid. Accordingly, as HaCaT cells, the clones formed well-differentiated stratified epithelia synthesizing K1 and 10 in surface transplants, while simple and internal epithelial keratins seen in culture were suppressed (as upon retinoic acid depletion in vitro). In transplants of HaCaT cells, in contrast to those of normal keratinocytes, K1 appeared prematurely already in basal cells, while K10 localized rather normally in the suprabasal position. Keratins 1 and 10 were also synthesized in transplants of HaCaT-ras clones (again K1 preceding K10), but both generally shifted toward upper layers. This was particularly evident in thicker transplants of malignant clones. Staining for both keratins persisted "suprabasally" in invasive tissue masses, and this corresponded to their marked expression in solid carcinomas (after s.c. injection), seen by immunofluorescence and two-dimensional gel electrophoresis. Thus, notwithstanding some variations, differentiation potential was not significantly reduced in these clones disregarding levels of ras oncogene expression and malignant properties.     

Frequent expression of the tumor antigen CAK1 in squamous-cell carcinomas.

K1 is a murine monoclonal antibody (MAb) derived from a hybridoma generated by the fusion of splenocytes of BALB/c mice immunized with a human ovarian tumor cell line, OVCAR-3. This antibody reacts strongly with epithelial ovarian tumors and mesotheliomas. The antigen recognized by MAb K1, designated CAK1, has recently been characterized as a 40-kDa protein probably anchored to the cell surface by glycosyl-phosphatidylinositol. Using immunoperoxidase histochemical methods, we examined 37 squamous-cell carcinoma (SqCC) samples from cervix, lung, esophagus and other origins, and 12 normal squamous epithelia of the cervix and esophagus for their reactivity with MAb K1. Of the SqCC specimens, 81% showed K1 reactivity with variable intensity, but none of 12 normal tissue samples of squamous epithelia did so. Two patterns of CAK1 expression in tumor samples were found, i.e., a heterogeneous pattern with strong intensity, and a homogeneous pattern with weak intensity. Three carcinomas in situ of the larynx, vulva and esophagus were moderately positive with K1, suggesting that CAK1 antigen may occur in the early stage of carcinogenesis of SqCC. The expression of CAK1 was also compared with expression of CA125, HER-2/neu, p53 and P-glycoprotein, and MAb K1 was found to react most consistently with SqCC. Since K1 reacts with a majority of cervical and esophageal carcinomas but has no detectable reactivity in normal epithelia of the cervix uteri and esophagus, MAb K1 could be of value as a reagent to help distinguish between normal and neoplastic cells on sections as well as in cytological samples.     

Retinoid status and the control of keratin expression and adhesion during the histogenesis of squamous metaplasia of tracheal epithelium.

We induced vitamin A depletion to define early and late changes during the histogenesis of squamous metaplasia of hamster tracheal epithelium. An early change is the "minimal morphological change" (MMC), in which the mucociliary epithelium is separated from the basement membrane by a continuous layer of basal cells. Immunohistochemistry showed an exclusive localization of the keratins K5 and K14 in basal cells of normal and MMC epithelia. At the MMC stage no staining was observed above the basal layer with antibodies to K5, but upon progression of the lesion to a squamous focus all cells from basal to terminally differentiated were positive for K5 and K14. In contrast, when we used antibodies to the keratins K6 or K13 all cells were negative in the normal epithelium and in the MMC epithelium. Successive layers of suprabasal squamous cells found in squamous metaplasia failed to express normal epidermal differentiation marker keratins K1 and K10 but expressed the proliferation marker keratin K6 and the internal stratified epithelium keratin K13, not normally found in the epidermis or in the trachea. Hamster tracheal epithelial cells could be maintained in culture in serum-free medium for at least 4 weeks in the presence of retinoic acid (RA). In non-RA-containing medium, cells from vitamin A-deficient hamsters showed markedly reduced growth and an increase in the expression of keratins K5, K6, K13, and K14. Since our previous work had implicated retinoids in the control of cell adhesiveness, we were interested to find out whether changes in cell adhesion occur in vitamin A-deficient hamster tracheal epithelial cells, compared to normal cells. Functional assays demonstrated that hamster tracheal epithelial cells, obtained from non-RA-treated tracheas or maintained in culture, displayed reduced attachment to laminin, compared to RA-treated cells. Immunofluorescence studies did not show a decrease either in the alpha 6 integrin subunit, which was localized in the basal aspect of basal cells, or in basement membrane laminin. However, the expression of laminin-binding protein 37 decreased as the epithelium changed from pseudostratified to stratified. Therefore, a coordinated pattern of changes in keratin gene expression, as well as in the expression of laminin-binding protein 37, the precursor to the cell surface laminin receptor 67LR, and in adhesive properties takes place in tracheal epithelium when its phenotype changes from mucociliary to the preneoplastic stage of squamous metaplasia.     

Growth and Differentiation Properties of Normal and Transformed Human Keratinocytes in Organotypic Culture

The growth and differentiation of human normal keratinocytes and their transformed counterparts were examined in organotypic cultures in which the keratinocytes were grown at the air-liquid interface on top of contracted collagen gel containing fibroblasts. We developed a modified culture procedure including the use of a mixed medium for keratinocytes and fibroblasts. Normal keratinocytes formed a three-dimensional structure of epithelium that closely resembled the epidermis in vivo , consisting of basal, spinous, granular and cornified layers. Cells synthesizing DNA were located in the lowest basal layer facing the collagen gel. Expressions of proteins involved in epidermal differentiation were examined by immunohistochemical staining and compared with those in skin in vivo . In the organotypic culture, transglutaminase, involucrin and filaggrin were expressed, as in the epidermis in vitro , most prominently in the granular layer. Type IV collagen, a component of basement membrane, was expressed at the interface between the keratinocyte sheet and the contracted collagen gel. Keratinocytes transformed by simian virus 40 or human papilloma virus (HPV) exhibited a highly disorganized pattern of squamous differentiation. In particular, HPV-transformed cells invaded the collagen gel. Organotypic culture is unique in that regulatory mechanisms of growth and differentiation of keratinocytes can be investigated under conditions mimicking those in vivo .     

Delays in malignant tumor development in transgenic mice by forced epidermal keratin 10 expression in mouse skin carcinomas

The keratin cytoskeleton is formed in different epidermal compartments by distinct polypeptides. Basal, proliferative keratinocytes express keratin (K) 5 and K14, whereas, suprabasal, post-mitotic keratinocytes express K1 and K10. Changes in this keratin pattern have been found to occur in hyperproliferative skin disorders and, in particular, throughout mouse epidermal carcinogenesis. Whereas some keratins not found in normal epidermis (K6, K16, K13, and K8) are induced at different stages of tumor development, K1 and K10 expression is lost. To determine whether K1 and K10 loss is just a consequence of the altered differentiation program or an event required for tumor progression, we generated transgenic mice carrying the human keratin 10 gene (hK10) under the control of a bovine keratin 6 gene regulatory region, which is silent in normal skin but is induced and drives transgene expression in hyperproliferative skin keratinocytes and, therefore, in skin tumors. Transgenic animals subjected to a complete carcinogenesis protocol developed tumors that contained various amounts of transgenic hK10. Although no significant difference was found in tumor number or malignancy, tumor onset was significantly delayed in transgenic mice, indicating that the presence of K10 actually impairs tumor development. Mol. Carcinog. 20:3–9, 1997. © 1997 Wiley-Liss, Inc.     

Down-regulation of keratin 14 gene expression after v-Ha-ras transfection of human papillomavirus-immortalized human cervical epithelial cells.

Keratin expression in human cervical squamous cell carcinoma (SCC) lines differed significantly from both normal and human papillomavirus (HPV) immortalized exocervical cells. Keratin 14 (K14) expression, determined by protein synthesis and mRNA levels, was dramatically down-regulated in the cervical SCC lines while keratin 5 (K5) expression was not. K14 expression was similarly down-regulated in an HPV-16 immortalized cervical cell line after tumorigenic transformation with recombinant v-Ha-ras DNA. Cultures derived from nude mouse tumor explants also exhibited an altered keratin profile and the levels of K14 protein synthesis, as well as K14 mRNA, were not detectable. In both cases K5 protein synthesis was not significantly down-regulated. In addition, neoplastic cervical SCC lines exhibited up-regulation of keratins 7, 8, 13, and 19, combined with slight down-regulation of keratins 6 and 16. Epidermal keratinocytes responded in a different manner to exocervical cells. Transfection of human papillomavirus-immortalized epidermal keratinocytes with the BglII N fragment of herpes simplex virus 2 produced a neoplastic cell line, but K5 and K14 expression remained unchanged. Thus, neoplastic transformation of human exocervical cells, both in vivo (spontaneous cervical SCC) and in vitro (HPV-16- and v-Ha-ras-induced cervical SCC), is accompanied by characteristic changes in keratin expression. The specific down-regulation of K14 in these tumorigenic cervical cells, in the absence of significant changes in the expression of K5, implies that the normal coordinate regulation of K5 and K14 gene expression has been uncoupled.     

O6-methylguanine-DNA methyltransferase activity in human buccal mucosal tissue and cell cultures. Complex mixtures related to habitual use of tobacco and betel quid inhibit the activity in vitro

Extracts prepared from tissue specimens of normal, non-tumourous human buccal mucosa, and cultured buccal epithelial cells and fibroblasts, exhibited O6-methylguanine-DNA methyltransferase (MGMT) activity by catalysing the repair of the premutagenic O6-methylguanine lesion in isolated DNA with rates of 0.2 to 0.3 pmol/mg protein. An SV40 T antigen-immortalized buccal epithelial cell line termed SVpgC2a and a buccal squamous carcinoma line termed SqCC/Y1, both of which lack normal tumour suppressor gene p53 function, exhibited about 50 and 10% of the MGMT activity of normal cells, respectively. The normal, experimentally transformed and tumourous buccal cell types showed MGMT mRNA levels which correlated with their respective levels of MGMT activity. Exposure of buccal cell cultures to various organic or water- based extracts of products related to the use of tobacco and betel quid, decreased both cell survival (measured by reduction of tetrazolium dye) and MGMT activity (measured subsequently to the exposures in cellular extracts). Organic extracts of bidi smoke condensate and betel leaf showed higher potency than those of tobacco and snuff. An aqueous snuff extract also decreased both parameters, whereas an aqueous areca nut extract was without effect. The well- established sulph-hydryl-reactive agent Hg2+, a corrosion product of dental amalgam, served as a positive control and decreased MGMT activity following treatment of cells within a range of 1-10 microM. Taken together, significant MGMT activities were demonstrated in buccal tissue specimens and in the major buccal mucosal cell types in vitro. Lower than normal MGMT activity in two transformed buccal epithelial cell lines correlated with decreased MGMT mRNA and lack of functional p53. Finally, in vitro experiments suggested the potential inhibition of buccal mucosal MGMT activity by complex mixtures present in the saliva of tobacco and betel nut chewers.      

Changes in keratin expression during 7,12-dimethylbenz[a]anthracene-induced hamster cheek pouch carcinogenesis.

This study was undertaken to explore the expression of keratins in the hamster cheek pouch carcinogenesis model, using monospecific keratin antibodies and a technique that allows immunoblotting analysis of tissues embedded in paraffin. Changes in keratin expression were correlated with histopathological changes and with the expression of the enzyme gamma-glutamyl transpeptidase. The right cheek pouch of 20 male golden Syrian hamsters was treated with 0.5% 7,12-dimethylbenz[a]anthracene for 16 weeks. As previously described by other laboratories, this treatment resulted in hyperplastic and dysplastic lesions and benign and malignant tumors. The keratins assayed in this study were K14 (Mr 55,000), K1 (Mr 67,000), and K13 (Mr 47,000). The normal hamster cheek pouch epithelium expressed K14 in the basal layer and K13 in the suprabasal and differentiated layers, whereas K1 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. In this case, K14 was no longer restricted to the basal layer but was also expressed in differentiated cells. The same pattern was also observed in dysplastic lesions and in squamous cell carcinoma. Furthermore, expression of the K13 differentiation-associated keratin was preserved in this hyperplastic epithelium during all the stages of carcinogenesis, including either anaplastic or differentiated areas. In contrast, after 2 weeks of 7,12-dimethylbenz[a]anthracene treatment, K1 expression started as a weak and patchy pattern in suprabasal cells, becoming stronger and more homogeneous at 8 and 16 weeks of treatment. However, K1 was almost absent in squamous cell carcinoma, where only small very well differentiated areas were stained. We also observed gamma-glutamyl transpeptidase-positive foci in earlier stages of carcinogenesis, concomitant with the expression of the K1 keratin. However, it was not possible to find a perfect topographical correspondence between the two events. Alterations in the pattern of keratin expression appear to be a common feature during the development of squamous cell carcinoma in different systems and could be an excellent tool to study carcinogenesis and chemoprevention.     

Modulation of the terminal differentiation of human squamous carcinoma cells in vitro by all-trans-retinoic acid

A malignant human cell line (SqCC/Y1) derived from a squamous carcinoma of the buccal mucosa is described. It formed a stratified cellular structure with ultrastructural characteristics of a fully differentiated stratified squamous epithelium when cultured in equal parts of Dulbecco's modified Eagle medium and Ham's medium F12, supplemented only with insulin, transferrin, and selenium. After 14 days in culture in this defined medium, 30% of the cells became keratinized (insoluble in detergent), and 75% of the cells were capable of being induced to form cornified cell envelopes. Involucrin, the precursor protein of the cornified cell envelope, could be detected by immunofluorescence only in suprabasal cells. Treatment of SqCC/Y1 cultures with 5 X 10(-8) M all-trans-retinoic acid (RA) completely inhibited stratification and markedly increased cell desquamation. In the presence of RA, less than 10% of the cells became keratinized, and only 15-20% of the cells acquired envelope-forming competence. The fraction of colony-forming cells in RA-treated cultures was tenfold higher than in fully mature cultures. Thus RA appears to be an effective inhibitor of terminal differentiation of SqCC/Y1 cells.     

Expression of intermediate filament proteins by normal and transformed mouse epidermal-keratinocytes in culture

The aberrant expression of embryonic simple epithelial keratins K8 and K18 has been previously detected in chemically-induced mouse skin carcinomas and in cultured epidermal keratinocytes containing H-ras oncogenes. In this study, we report that cell lines derived from benign papillomas do not generally synthesize simple epithelial keratins, regardless of the presence of H-ras gene alterations. These results reinforce our previous suggestion that the expression of these keratins during mouse epidermal carcinogenesis is a marker of malignant transformation. The comparison of the two-dimensional electrophoresis intermediate filament protein profiles of transformed keratinocytes in culture, reveal that during in vitro progression to the malignant phenotype increased synthesis of simple epithelial keratins is associated to a general disturbance on the expression of cytoskeletal proteins.     

Use of keratin antibodies in tumor diagnosis.

The tissue specific expression shown by intermediate filament proteins, their ease of extraction and their antigenicity has led to the use intermediate filament antibodies in diagnostic pathology, particularly antibodies to keratin intermediate filaments because of the predominant involvement of epithelia in cancers. We review the principles of differentiation-specific keratin expression and detail 100 of the best-known monoclonal antibodies to keratins which are currently in use: in many circumstances antibodies can now be reliably used as in situ markers of keratin expression. We also suggest a database scheme for better dissemination of information on available monoclonal antibodies to keratins in the future.     

Early expression of type I K13 keratin in the progression of mouse skin papillomas.

The premalignant evolution of chemically induced mouse skin papillomas is characterized by dysplastic changes, aneuploidy, induction of gamma-glutamyl transpeptidase (GGT), and changes in the expression of keratins, especially differentiation-associated K1. This keratin, which is expressed in normal epidermis and early papillomas, is no longer present in more advanced dysplastic and aneuploid papillomas and in fully invasive carcinomas. More recently, it has been shown that K13, a keratin normally present in internal epithelia but not in epidermis, is aberrantly expressed in epidermal tumors. In the present study, the timing of expression of K13 and its correlation with other markers of premalignant evolution were investigated. Papillomas were induced by SENCAR mice by a single initiating dose of 20 nmol of 7,12-dimethylbenz[a]-anthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA) (2 micrograms twice a week). Tumors were randomly harvested at 10, 20 and 35 weeks of promotion. K13 and K1 expression in papillomas was studied using immunoblotting and immunostaining of consecutive sections, as previously described. As expected from previous studies, the distribution of K1 in papillomas collected at 10 weeks of promotion was restricted to differentiated cells and was uniform throughout the section of the papilloma. Conversely, K13 was expressed only as small foci in 10 out of 21 papillomas (48%). Papillomas of 20 weeks were also positive for K1. Staining for K13 was positive in these papillomas with the exception of only one that was essentially negative, presenting only one small positive focus. Some of the papillomas collected at week 35 were negative for K1, but immunostaining with K13 showed uniform staining of suprabasal cells in all the papillomas studied. In all cases, immunohistochemical results were confirmed by immunoblotting with proteins extracted from 7 microns sections from each paraffin block. These results indicate that keratins K1 and K13 are coexpressed in most papillomas from 10 to 35 weeks of promotion. However, analysis of adjacent sections showed that K13 positive areas are topographically located in the K1 negative areas of the papillomas, suggesting a shift in the differentiation program from epidermal to mucosal types of keratinization. Based on these and previous studies from our laboratory, we conclude that K13 is an early marker of papillomas progression, which occurs before gross chromosomal abnormalities are present in the stem line of the tumors, and precedes dysplastic changes and the onset of GGT expression, and is probably concomitant at the individual cell level with loss of K1.