Expression of keratins in normal, immortalized and malignant oral epithelia in organotypic culture

Keratins have been extensively studied in tissues and cultured keratinocytes but limited information is available on epithelia reconstructed in vitro. The aim of this study was to examine keratin expression in organotypic epithelia with normal (NOK), immortalized (SVpgC2a) and malignant (SqCC/Y1) human buccal cells. Organotypic epithelia were derived from 10 days of culture at the air-liquid interface of collagen gels containing human oral fibroblasts using a standardized serum-free medium. Sections were stained immunohistochemically with selected mono-specific antibodies to a range of keratins. Organotypic epithelia showed sharp differences in keratin expression and distribution. K4/K13, K1/K10, K6/K16 were variably expressed in NOK and SqCC/Y1 but were not detected in SVpgC2a. K5 was expressed in all organotypic epithelia but K14 was absent in SVpgC2a. K7 and K8 showed variable expression while K18 was expressed uniformly in all epithelia. K19 was expressed consistently in NOK and K20 was distributed heterogeneously in SVpgC2a. Overall, organotypic cultures of normal keratinocytes express many of the same keratins as buccal mucosa. Further, the loss of keratins in SVpgC2a and their retention in SqCC/Y1 have several features in common with the respective keratin profile of oral epithelial dysplasia and well-differentiated oral squamous cell carcinoma. Although qualitative and quantitative differences exist compared to keratin expression in vivo, these cell lines in organotypic culture may serve in studies of the multi-step progression of oral cancer.     

Overexpression of retinoic acid receptor beta in head and neck squamous cell carcinoma cells increases their sensitivity to retinoid-induced suppression of squamous differentiation by retinoids.

Nuclear retinoic acid receptor beta(RARbeta) expression is suppressed in many head and neck squamous cell carcinomas (HNSCCs), and an inverse relationship was found between squamous differentiation and RARbeta expression in such cells. To investigate the role of RARbeta in HNSCC growth and differentiation, we transfected a retroviral RARbeta2 expression vector (LNSbeta) into HNSCC SqCC/Y1 cells, which do not express endogenous RARbeta but do express RARalpha, RARgamma, and retinoid X receptors. Transfected clones expressing RARbeta2 mRNA and protein exhibited enhanced sensitivity to the suppressive effects of all-trans-retinoic acid (ATRA) on squamous differentiation compared with cells transfected with the LNSX vector only; transglutaminase type I level was suppressed after a 3-day treatment with 10(-10) M ATRA in four of five LNSbeta clones, whereas it was not suppressed in LNSX cells even by 10(-6) M ATRA. Similarly, cytokeratin 1 mRNA level was more suppressed in ATRA-treated LNSbeta clones than it was in LNSX cells. This effect was independent of transrepression of activator protein-1. None of the LNSbeta-transfected clones showed an increased growth inhibition by ATRA, 9-cis-retinoic acid, or the synthetic retinoid 6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-naphthale necarboxylic acid. These findings suggest that, in SqCC/Y1 cells, RARbeta mediates suppression of squamous differentiation by ATRA without enhancing its growth-inhibitory effects.     

Immunohistochemical expression of SKALP/elafin in squamous cell carcinoma of human lung

The immunohistochemical expression of skin-derived anti-leukoproteinase (SKALP)/elafin in lung squamous cell carcinoma (SqCC) and in uninvolved bronchial epithelium was studied. The results were compared with the immunohistochemical staining of proliferating cell nuclear antigen (PCNA) and the TDT-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay. SKALP/elafin was expressed in SqCC with high incidence (82.6%). By contrast, the expression was not observed in uninvolved bronchial epithelium. SKALP/elafin expression was located in the cell layers just underneath the cornified envelope of each cell nest, and DNA fragmentation was observed in the same cell layers. PCNA was expressed in the basal layer of each cornified envelope, and both expressions were clearly separated. Immunoreactive score of SKALP/elafin expression was significantly correlated with the differentiation and it tended to increase in accordance with the degree of differentiation. These results suggested that SKALP/elafin was possibly involved in the cell differentiation program, finally leading to keratinization in SqCC of human lung. SKALP/elafin may be a beneficial molecular target for detection or even the development of a new therapeutic method against cancer.     

Changes in glycosphingolipids accompanying the differentiation of human squamous SQCC/Y1 cells

SqCC/Y1 cells grow as a monolayer in culture and differentiate when maintained in the plateau phase; in the absence of serum these cells differentiate more rapidly. The differentiation is characterized by the stratification of the culture to form a structure consisting of several cellular layers, synthesis of specific keratins, and the attainment of the capacity to form a cornified cell membrane. The stratification process is indicative of the importance of cell-cell interactions during maturation. To study the relationship between membrane glycosphingolipids (GSLs) and the state of differentiation of SqCC/Y1 cells, GSLs were measured in cultures grown in the presence or absence of fetal calf serum. Glycolipids were isolated by diethylaminoethyl-Sephadex and Iatrobeads column chromatographies, and their distributions were determined by high-performance thin-layer chromatography. GM3 was the major ganglioside present in these cells. Other ganglioside components were tentatively identified as GM2, GM1, and GD3. Differences in ganglioside patterns were observed in differentiated cultures; the major changes were accumulation of GD3 and depletion of GM1. The predominant neutral GSLs in SqCC/Y1 cells were identified as Glc beta 1-1Cer, Gal beta 1-4Glc beta 1-1Cer, Gal beta 1-4Gal alpha 1-4Glc beta 1-1Cer, Gal NAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer, and three unknown complex GSLs. Differentiated cultures, however, showed variations in banding patterns, which include an increase in Glc beta 1-1Cer and Gal beta 1-4Glc beta 1-1Cer and a decrease in Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer and Gal NAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta-1Cer. These changes, however, were not observed when the cells were grown in the presence of epidermal growth factor or retinoic acid, factors which inhibit the differentiation process. The findings demonstrate significant changes in glycolipid composition of differentiated SqCC/Y1 cells grown in the absence of serum, suggesting that these lipids may be important to the differentiated state.     

Epidermal growth factor receptor gene expression, protein kinase activity, and terminal differentiation of human malignant epidermal cells

A number of epidermal growth factor (EGF)-resistant clones have been isolated from human epidermoid carcinoma A431 cells (I. King and A. C. Sartorelli, Biochem. Biophys. Res. Commun., 140: 837, 1986). These cells had a higher capacity to enter a pathway of terminal differentiation, as determined by their ability to form cornified envelopes. Thus, after 6 days in culture, 10% of parental A431 cells expressed a differentiated phenotype, while more than 50% of each of the EGF-resistant variants (M1, A5, and A7) formed cornified envelopes. These EGF-resistant clones expressed fewer EGF receptors and had a lower capacity for EGF receptor autophosphorylation than parental A431 cells. Clone M1 had the highest capacity to form cornified cell envelopes and expressed about 10% of the EGF receptor autophosphorylation activity of parental cells. The decrease in the level of EGF receptor autophosphorylation appeared to be due to a decrease in EGF receptor number rather than to a lowering of enzyme activity per se. Southern analysis demonstrated that all of the EGF-resistant variants contained fewer copies of the EGF receptor gene and no apparent gene rearrangement was detected in these variant cells. A corresponding decrease in EGF receptor mRNA was also observed in M1, A5, and A7 cells, with a ratio of 18:1:10:5 for A431, M1, A5, and A7 cells, respectively. In addition, two other malignant epithelial cell lines, SqCC/Y1 and FaDu, contained relatively few copies of EGF receptor genes, had low EGF receptor kinase activity and showed a relatively high capacity to form cornified envelopes. These findings suggest that the level of the EGF receptor was critical to the regulation of the degree of maturation of malignant epidermal cells.     

Inhibitor production by normal rat tracheal epithelial cells influences the frequency of spontaneous and X-ray-induced enhanced growth variants

A cell culture model was used to assay for the induction ofcell populations with enhanced growth capacity in culture inirradiated normal rat tracheal epithelial cells (NTEC). Whencultures were maintained in minimally enriched Ham‘s F-12plus 5% fetal bovine serum, it was noted that increasing theseeding density from 1 to 100 colony forming units per 60 mmdish decreased the frequency of proliferating epithelial foci(PEF) scored 5–6 weeks after seeding, from 2.5 to 0.07%in controls and from 5 to 0.9% in irradiated cultures. Whenlow density (50 colonies per dish) control cultures were fedmedium conditioned by higher density control cultures (300 coloniesper dish) the observed frequency of PEF was 10-fold lower thanthat observed in cultures fed fresh medium or cultures fed mediumconditioned by a carcinogen-induced tracheal cell line. Thesedata suggest that some factor(s) present in conditioned mediumderived from higher density normal cell cultures inhibits theemergence of many PEF. If cultures are maintained in serum-freeenriched Ham’s, many (17–21%) PEF were observedin both control and irradiated cultures. Medium harvested fromhigh density cultures maintained in minimally enriched Ham‘sF-12 (5% FBS) was found to maximally inhibit normal cell thymidineuptake and growth as well as induce cornified envelope formation(terminal differentiation) in proliferating normal cell cultures.Medium harvested from normal cultures maintained in serum-freeenriched Ham’s was not inhibitory to normal cells anddid not induce cornified envelope formation. Subcultured PEF-derivedpopulations and PEF in 4 to 5-week primary cultures also didnot produce inhibitor(s). In addition, subcultured PEF-derivedcells were not induced to form cornified envelopes in the presenceof inhibitory conditioned medium. The data suggest that theelaboration of an inhibitor(s) by normal epithelial cells affectsthe emergence of PEF. Irradiated cells appear to have some growthadvantage over spontaneous PEF in that they are more likelyto survive and form PEF under growth conditions associated withhigher levels of inhibitor(s) in the medium.     

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.     

Modulation of tissue and epidermal transglutaminases in mouse epidermal cells after treatment with 12-O-tetradecanoylphorbol-13-acetate and/or retinoic acid in vivo and in culture

Retinoic acid (RA) induces tissue transglutaminase (TGASE) and inhibits terminal differentiation induced either by calcium ion or by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in primary mouse epidermal cells in culture. The relevance of these effects on cultured cells to the antipromoting action of RA was investigated in female BALB/c and CD-1 mice in vivo. Tissue TGASE was distinguished from epidermal TGASE on the basis of different thermolability at pH 9 or elution from the anion exchanger Mono Q. After topical application of 3 to 5 micrograms (10 to 17 nmol) of RA to the shaved back skin, the specific activity of tissue TGASE increased up to 30-fold primarily in the basal cell fraction of Percoll-separated epidermal cells. Enzyme activity returned to basal levels by 7 days. Treatment with TPA (10 micrograms or 17 nmol/mouse) induced an increase in epidermal TGASE which reached a maximum at 12 h after application, primarily in suprabasal cells. RA applied 1 h before TPA caused no reduction of TPA-induced epidermal TGASE, but the increase in tissue TGASE due to RA was markedly inhibited by TPA. The effects of TPA and RA on TGASE activities in primary epidermal cells in culture were similar to those in vivo except that RA reduced the induction of epidermal TGASE by TPA. In culture the induction of epidermal TGASE by TPA was independent of Ca2+ concentration in the medium above 0.03 mM, but cornified envelope formation was markedly enhanced by Ca2+ above the level required for maintaining a basal cell population (0.03 to 0.05 mM). The TPA-induced formation of cornified envelope in the presence of elevated Ca2+ was completely inhibited by RA if cells were pretreated with RA for 24 h. Our results are consistent with RA causing a reprogramming of epidermal cells that alters their response to differentiation stimuli.     

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.      

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.     

Plasminogen activator mediated degradation of subendothelial extracellular matrix by human squamous carcinoma cell lines

Extracellular matrix (ECM) produced by bovine corneal endothelial cells was used to investigate the role of the plasminogen activator/plasmin system in the degradation of ECM by human squamous cell carcinoma (SqCCs) and human foreskin epidermal cells (HFEC). SqCCs caused an 8- to 34-fold greater solubilization of 3H-glucosamine-labeled ECM than HFEC. This action in SqCCs was dependent upon the presence of acid-treated serum, indicating that tumor-associated proteinases were sensitive to the inhibitory action of acid-labile proteinase inhibitors present in the serum. SqCC mediated digestion of radiolabeled ECM was decreased by 14- to 55-fold in plasminogen depleted serum, and the addition of 100 micrograms/mL of purified human plasminogen resulted in up to a 30-fold increase in the degradation of the ECM. Inhibitors of this proteinase system and murine monoclonal antibodies (MAb) specific for human urokinase plasminogen activator (uPA) decreased the SqCC mediated digestion of radiolabeled ECM in a concentration dependent manner. SqCCs exhibited 10- to 30-fold higher extracellular uPA levels than HFEC, as assayed by substrate hydrolysis, zymography, micro-ELISA, western analysis, and northern analysis. These findings reflect the differential ability of these cell types to degrade the ECM. In addition, immuno-cross-reactive plasminogen activator inhibitor type I (PAI type 1) and type II (PAI type 2) were identified in cell-free conditioned medium produced by both tumor cells and normal epidermal cells, using a micro-ELISA assay. Indirect immunofluorescence flow cytometry, employing MAbs directed against uPA, detected the presence and localization of uPA on the SqCC cell surface. These findings were specific for uPA, since cell surface associated tissue plasminogen activator was not detected in these cell types under analogous conditions. In addition, partially purified SqCC plasma membrane preparations exhibited 2- to 10-fold higher uPA-like activity than HFEC, as determined by zymography. The findings support the concept that the plasminogen activator system is important in the breakdown of ECM by SqCCs and suggest that regulatory mechanisms involved in this proteolytic system may be important targets for chemotherapeutic intervention to limit tumor cell invasion and metastasis.     

Expression of podoplanin, CD44, and p63 in squamous cell carcinoma of the lung

Recent molecular biological studies have identified podoplanin as a candidate cancer stem cell (CSC) marker in squamous cell carcinoma (SqCC). The purpose of this study was to examine the expression pattern of podoplanin, and the other stem cell markers CD44 and p63, and their relationship to clinico-pathological features including survival in pulmonary SqCC. We examined histologically the expression of podoplanin, CD44, and p63 in 162 consecutive SqCC by immunostaining. Podoplanin expression was observed in 107 (66%) tumors, CD44 in 145 (89.5%), and p63 in 151 (93.2%), respectively. In 95.3% of the podoplanin-positive tumors, tumor cells showing strong expression were localized in the periphery of the tumor nests. However, this peripheral localization was observed in only 55.9% of the CD44-positive and 43% of p63-positive tumors. In 88.8% of the podoplanin-positive tumors, positive cells were localized more peripherally in the tumor nests than CD44- or p63-positive cells and when CD44 and p63 expressions were compared in these podoplanin-positive tumors, p63-positive layers in the periphery of the tumor nests were broader compared to CD44-positive layers. These findings suggest tumor cells are aligned in the "hierarchical distribution pattern" according to the expression of these three markers. Patients who had podoplanin-positive tumors with the "hierarchical pattern" resulted in significantly better overall survival than those who had podoplanin-negative tumors ( P  =   0.043). These results suggest that podoplanin expression would reflect the most immature status in the differentiation process of SqCC, and SqCC with hierarchical expression would be a well-organized tumor group with lower biological aggressiveness based on the CSC concept. ( Cancer Sci 2009)     

Analysis of the expression protein profiles of lung squamous carcinoma cell using shot-gun proteomics strategy

The aim of this study is to globally screen and identify the expression protein profiles of lung squamous carcinoma cell (SqCC) using shot-gun proteomics strategy and to further analyze function of individual proteins by bioinformatics, which may likely result in the identification of new biomarkers and provide helpful clues for pathogenesis, early diagnosis, and progression of lung SqCC. The specific tumor cells were isolated and collected from the tissues of six patients with lung SqCC by laser capture microdissection (LCM). Total proteins from the LCM cells were extracted, digested with trypsin. The sequence information of resulting peptides was acquired by high-performance liquid chromatography (HPLC) and tandem mass spectrometry (TMS). The global protein profiles of lung SqCC cell were identified with BioworksTM software in IPI human protein database. Cellular component, molecular function, and biological process of the all proteins were analyzed using gene ontology (GO). About 720,000 tumor cells were satisfactorily collected from tissues of six patients with lung SqCC by LCM and the homogeneities of cell population were estimated to be over 95% as determined by microscopic visualization. The high resolution profiles including HPLC, full mass spectrum, and tandem mass spectrum were successfully obtained. Database searching of the resulting bimolecular sequence information identified 1982 proteins in all samples. The bioinformatics of these proteins, including amino acids sequence, fraction of coverage, molecular weight, isoelectric point, etc., were analyzed in detail. Among them, the function of most proteins was recognized by using GO. Five candidate proteins, Prohibitin (PHB), Mitogen-activated protein kinase (MAPK), Heat shock protein27 (HSP27), Annexin A1(ANXA1), and High mobility group protein B1 (HMGB1), might play an important role in SqCC genesis, progression, recurrence, and metastasis according to relative literatures. We have successfully isolated the interesting cells and effectively solved the heterogeneous problem of lung SqCC using LCM. The globally expressional proteins of lung SqCC cell were identified by shot-gun proteomics strategy. The five proteins might be hopefully used as markers of lung SqCC.     

Expression of p53 in premalignant and malignant squamous epithelium.

Using three antibodies (JG8, CM-1 and 1081) directed to the p53 protein, strong positivity was found in 16/47 (34.0%) of mucosal squamous cell carcinomas of the head and neck and in two squamous carcinoma cell lines (LICR-LON- HN5 and HN6Rr). The presence of the mutant p53 was confirmed in the cell lines as substitutions in exon 7 (codon 238, TGT greater than AGT) and exon 5 (codon 152, CCG greater than CTG) respectively. Positive staining was seen only in the undifferentiated cells and progressively lost as the cells keratinized, both in the tumour specimens and in the cell lines. Similar results were seen in areas of dysplasia, well removed from the site of the primary tumour. Staining of epidermal lesions showed positivity in 2/12 (16.6%) cases of Bowen's disease, 0/12 (0.0%) cases of solar keratosis, 0/10 (0.0%) basal cell carcinomas and in 3/20 (15.0%) squamous cell carcinomas. These results are discussed in relation to the multifocal origin of squamous cell carcinomas, the role of p53 mutations in squamous cell carcinomas from different sites and the significance of the 'basal' distribution of p53 as a normal growth regulator. The possible significance of the distribution of p53 in squamous epithelium as it relates to papilloma virus infection is also considered.     

Characterization of the immunophenotype of the tumor budding and its prognostic implications in squamous cell carcinoma of the lung

Tumor budding is morphologically defined as infiltration by small clusters of cancer cells. While the biological properties of budding cells in adenocarcinoma (decreased expression of adhesion molecules and of differentiation markers) have been elucidated, those of the cells in squamous cell carcinoma (SqCC) of the lung still remain to be clarified. We examined the clinicopathological data of 217 patients with SqCC of the lung. Furthermore we evaluated the immunohistochemical properties of the budding cells. Tumor budding was observed in 83 (38.2%) patients. A statistically significant difference was observed in overall 5-year survival rates between the cases showing tumor budding and the cases not showing budding (45.6% vs. 64.0%, p<0.001). As compared with cancer cells forming solid nests, budding cells (BCs) exhibited reduced expression levels of the cellular adhesion molecules (E-cadherin; p=0.004, β-catenin; p=0.002) and increased expression levels of laminin-5γ2 (p=0.001). On the other hand, no significant differences in the staining scores for differentiation markers (p63 and podoplanin) were found between BCs and cancer cells forming nests. Multivariate analysis revealed that tumor budding was a significant independent prognostic factor in patients with SqCC of the lung (p=0.022). Tumor budding is an independent adverse prognostic factor in patients with SqCC of the lung. Although budding cells in SqCC exhibited reduced expression levels of the cellular adhesion molecules, the expression levels of specific differentiation markers were retained, suggesting that the budding mechanism in SqCC may differ, at least in part, from that in adenocarcinoma.     

Relationship of myc protein expression to the phenotype and to the growth potential of HOC-7 ovarian cancer cells.

In this investigation we demonstrate expression of myc oncoproteins in HOC-7 ovarian adenocarcinoma cells. The cells were exposed to differentiation inducing agents such as dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), retinoic acid (RA) and transforming growth factor-beta 1 (TGF-beta 1). Myc protein expression in treated cells was then compared with that in control cultures and in monoclonal HOC-7 sublines, which are characterised by distinct phenotypes. Cells exposed to DMSO and DMF became markedly enlarged and flattened and developed cytoplasmic extensions. They looked similar to a subline, which revealed a less malignant and more differentiated cell phenotype. All four inducers prolonged the cell doubling time and reduced the saturation density to levels, normally found in the more differentiated subline. Furthermore, all inducers except RA elevated extracellular fibronectin, which is characteristic for less malignant epithelial cell phenotypes. All four agents inhibited myc oncoprotein expression reversibly (1% DMSO greater than 0.5% DMF greater than 10 microM RA greater than 10 ng ml-1 TGF-beta 1) and in time-dependent manner. Down-regulation of myc protein expression is, therefore, closely related to inducer-dependent growth reduction of HOC-7 cells and to the development of a less malignant cell phenotype.     

Nonmalignant oral keratinocytes from patients with head and neck squamous cell carcinoma show enhanced metabolism of retinoic acid

BACKGROUND AND OBJECTIVE: Retinoids show promise in the treatment of various (pre)malignancies, including head and neck squamous cell carcinoma (HNSCC). It has been shown that metabolic pathways of retinoids are important in their anticancer effect and that these pathways may change during HNSCC carcinogenesis. We have previously reported that HNSCC cells have a 17-fold greater turnover rate of retinoic acid (RA) than normal oral keratinocytes from noncancer controls, and that the formation of polar metabolites such as 4-oxo-RA and 4-hydroxy-RA is only seen in HNSCC cell lines. We aimed to establish whether this altered retinoid metabolism is an intrinsic characteristic of HNSCC patients. METHODS: The normal mucosa of cancer and noncancer patients was the source of keratinocyte cultures. The cells were exposed to RA for various time periods, and the levels of various retinoids were measured in the culture medium and cell pellets with reverse-phase liquid chromatography. RESULTS: Cells from cancer patients were morphologically normal and showed no genetic aberrations (i.e. loss of heterozygosity). The RA turnover rate in normal oral keratinocytes of cancer patients was 15 times higher (p = 0.003) than that in normal oral keratinocytes of noncancer controls, with average turnover rates of 218.6 and 14.8 pmol/mg protein/h, respectively. Specific profiles of RA metabolites were similar. CONCLUSION: The observed higher RA metabolism in noncancer cells of HNSCC patients suggests that individuals with a relatively high RA turnover have an increased risk of developing HNSCC.     

A subpopulation of cultured human keratinocytes which is resistant to the induction of terminal differentiation-related changes by phorbol, 12-myristate, 13-acetate: evidence for an increase in the resistant population following transformation

We have studied the action of phorbol, 12-myrislate, 13-acetate(PMA) on human keratinocytes grown with lethally-irradiated3T3 cells using medium supplemented with hydrocortisone, choleratoxin and epidermal growth factor. Normal keratinocyte culturesshow a heterogeneous response to PMA; 90–93% of the colony-formingcells lose their colony-forming ability and form cornified envelopeswhen treated for 24 h with doses of 100 nM or less, but theremainder are resistant to doses of 1000 nM. The resistant cellsare the precursors of the sensitive ones and heterogeneity isrestored to those cells and their progeny after 8 days culturein the absence of PMA. Cultures of 3 squamous cell carcinomalines, a SV40-transformed human keratinocyte line, and threeclones of these lines were found to contain 3–17 tunesmore PMA-resistant keratinocytes than the normal strains, andthe size of the PMA-resistant fraction in each line was inverselyrelated to the competence of that line to lose colony-formingefficiency when placed in suspension culture (which is the firstdetectable change in an ordered programme of events resemblingterminal differentiation of the keratinocyte). The number ofcells with cornified envelopes in surface cultures of normalhuman keralinocytes increased from 3% in control cultures to70% in those treated for 6 days with 100 nM PMA. The transformedhuman keratinocyte cultures showed a 3–25-fold smallerincrease in cornified envelope formation when treated with 100nM PMA, and the increase in envelope formation by each linewhen exposed to this dose of PMA was related to the competenceof that line to lose cloning efficiency in suspension culture.No relationship was found between the ability of any human keratinocytestrain or line we studied to metabolically inactivate PMA andtheir resulting response to the compound. The results are discussedin relation to the mechanism of action of PMA as a promoterof epidermal carcinogenesis.