involucrin and Carcinoma--Squamous-Cell

Clinical and experimental experience indicate that differentiation and malignancy are inversely correlated. However, more recent experimental studies using mouse and human keratinocyte systems have demonstrated that complete or even substantial loss in overall epithelial differentiation is not a prerequisite for malignant growth of cancer cells. Major defects in differentiation are also not a prerequisite for premalignant stages, in particular for cell immortalization, which is considered an early and essential step in the transformation process. Moreover, progressive dedifferentiation, often associated with advanced tumor stages, is also found in immortalized cell lines which are, however, nontumorigenic. On the other hand, malignant cell lines may have maintained a high degree of their normal differentiation program and sensitivity to differentiation modulators. However, to date no transformed keratinocyte cell lines with completely normal differentiation have been observed. Since epidermal keratinization is a very complex process involving many different parameters and is fully expressed only under in vivo conditions, an exact and quantitative comparison of such ill-defined phenomena (differentiation and malignancy) is still problematic. Obviously, both phenomena are under separate control and not causally linked. Nevertheless, a better understanding of factors and mechanisms regulating differentiation and of their disturbance in carcinogenesis would offer new possibilities to design novel tumor therapeutic strategies in the field of differentiation therapy.

Topics: Biomarkers; Carcinoma, Squamous Cell; Cell Differentiation; Cell Survival; Cell Transformation, Neoplastic; Disease Progression; Epidermal Cells; Epithelial Cells; Humans; Keratins; Neoplasm Proteins; Neoplasms; Protein Precursors; Skin Neoplasms; Tumor Cells, Cultured

The objective was to discover whether the oxygen-regulated protein, metallothionein, is expressed in the hypoxic cells of squamous cell carcinomas. Twenty patients with squamous cell carcinoma of the uterine cervix or head and neck were infused with a solution of the hypoxia marker, pimonidazole hydrochloride, at a dose of 0.5 g/m2. The following day, biopsies were collected, formalin fixed, paraffin embedded, and sectioned at 4 microm. Sections from each biopsy were immunostained for pimonidazole binding, metallothioneins I and II, involucrin, and proliferating cell nuclear antigen. A total of 84 biopsies were analyzed. Sixty-four of 84 biopsy sections contained hypoxia. Of the hypoxia-containing sections, 43 of 64 or 67% showed no microregional overlap between hypoxia and metallothionein; 7 of 64 showed overlap; and 14 of 64 showed a combination of overlap and no overlap. On a tumor-by-tumor basis, 5 of 7 head and neck and 7 of 13 cervix tumors showed no overlap between metallothionein and hypoxia at the microregional level. Ranges for the percentage of the area of hypoxia in head and neck (<0.9 to 17%) and cervix (<0.1 to 14%) tumors were similar. In the hypoxia-containing sections, immunostaining for involucrin, a molecular marker for differentiation, overlapped with that for hypoxia in 82% of the cases. The majority of hypoxic cells in squamous cell carcinomas do not express metallothionein protein, although metallothionein is induced by hypoxia in human tumor cells in vitro. Hypoxic cells in human tumors tend to be in regions immunostaining for involucrin, and it seems possible that differentiation of hypoxic cells in squamous cell carcinomas might affect metallothionein I and II expression.

Topics: Biomarkers; Carcinoma, Squamous Cell; Cell Hypoxia; Female; Head and Neck Neoplasms; Humans; Immunohistochemistry; Metallothionein; Neoplasm Staging; Nitroimidazoles; Proliferating Cell Nuclear Antigen; Protein Precursors; Uterine Cervical Neoplasms

This study aimed to evaluate the prognostic significance of expression levels of involucrin (IVL), cytokeratin (CK)-10 and -13 at different intratumor sites (tumor center and invading area) of oral tongue squamous cell carcinoma (OTSCC).. IVL, CK13 and CK10 expression levels were examined in a multicenter cohort of 146 OTSCCs using immunohistochemistry. External mRNA datasets were used for expression analysis and/or to validate survival associations.. External transcriptomic datasets showed downregulation of IVL and KRT13 in oral malignancies including OTSCC as compared to normal controls. The combined loss of IVL and CK13 expression at the invading core but not at the center core was significantly associated with poor differentiation and reduced 5-year overall survival. Multivariate Cox analysis confirmed the loss of CK13 and IVL expression to be an independent prognostic factor. Transcriptomic dataset corroborated immunohistochemistry results.. Combined expression levlels of IVL and CK13 might be useful as prognostic biomarkers in OTSCC.

Topics: Carcinoma, Squamous Cell; Head and Neck Neoplasms; Humans; Keratin-13; Prognosis; Protein Precursors; Squamous Cell Carcinoma of Head and Neck; Tongue Neoplasms

HOXA13 overexpression has been detected in human ESCC tissue and high HOXA13 protein expression is correlated with a shorter median survival time in ESCC patients. Although aberrant expression of HOXA13 in ESCC has thus been established, little is known regarding the functional consequences thereof. The present study aimed to examine to what extent aberrant HOXA13 might drive carcinogenesis in esophageal keratinocytes. To this end, we overexpressed HOXA13 in a non-transformed human esophageal cell line EPC2-hTERT, performed gene expression profiling to identify key processes and functions, and performed functional experiments. We found that HOXA13 expression confers oncogenic hallmarks to esophageal keratinocytes. It provides proliferation advantage to keratinocytes, reduces sensitivity to chemical agents, regulates MHC class I expression and differentiation status and promotes cellular migration. Our data indicate a crucial role of HOXA13 at early stages of esophageal carcinogenesis.

Topics: Carcinogenesis; Carcinoma, Squamous Cell; Cell Adhesion; Cell Differentiation; Cell Line, Transformed; Cell Movement; Esophageal Neoplasms; Esophagus; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histocompatibility Antigens Class I; Homeodomain Proteins; Humans; Keratin-19; Keratinocytes; Neoplasm Proteins; Phosphoproteins; Protein Precursors; Signal Transduction; Spheroids, Cellular; Survival Analysis

The F1F0-ATP synthase, an enzyme complex, is mainly located on the mitochondrial inner membrane or sometimes cytomembrane to generate or hydrolyze ATP, play a role in cell proliferation. This study focused on the role of F1F0-ATP synthase in keratinocyte differentiation, and its relationship with intracellular and extracellular ATP (InATP and ExATP). The F1F0-ATP synthase β subunit (ATP5B) expression in various skin tissues and confluence-dependent HaCaT differentiation models was detected. ATP5B expression increased with keratinocyte and HaCaT cell differentiation in normal skin, some epidermis hyper-proliferative diseases, squamous cell carcinoma, and the HaCaT cell differentiation model. The impact of InATP and ExATP content on HaCaT differentiation was reflected by the expression of the differentiation marker involucrin. Inhibition of F1F0-ATP synthase blocked HaCaT cell differentiation, which was associated with a decrease of InATP content, but not with changes of ExATP. Our results revealed that F1F0-ATP synthase expression is associated with the process of keratinocyte differentiation which may possibly be related to InATP synthesis.

Topics: Adenosine Triphosphate; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line, Transformed; Dermatitis; Gene Expression Regulation; Humans; Keratinocytes; Keratoacanthoma; Keratosis, Seborrheic; Mitochondria; Mitochondrial Membranes; Mitochondrial Proton-Translocating ATPases; Protein Precursors; Prurigo; Psoriasis; Skin; Skin Neoplasms; Warts

To assess the immunocytochemical and immunohistochemical correlation of adhesion (E-cadherin) and cell differentiation (involucrin) molecules in oral leukoplakia and oral squamous cell carcinoma. Cytological samples and biopsies were obtained from male and female patients aged over 30 years with oral leukoplakia (n = 30) and oral squamous cell carcinoma (n = 22). Cell scrapings and the biopsy were performed at the site of the lesion and histological slides were prepared for the immunocytochemical analysis of exfoliated oral mucosal cells and for the immunohistochemical analysis of biopsy tissues using E-cadherin and involucrin. Spearman's correlation and kappa coefficients were used to assess the correlation and level of agreement between the techniques. Immunostaining for E-cadherin and involucrin by both techniques was similar in the superficial layers of the histological sections compared with cell scrapings. However, there was no statistical correlation and agreement regarding the immunocytochemical and immunohistochemical expression of E-cadherin and involucrin in oral leukoplakia (R = 0.01, p = 0.958) (Kappa = 0.017, p = 0.92) or in oral squamous cell carcinoma (R = 0.26, p = 0.206) (Kappa = 0.36, p = 0.07). The immunoexpression of E-cadherin and involucrin in tissues is consistent with the expression patterns observed in exfoliated oral mucosal cells, despite the lack of a statistically significant correlation. There is an association of the histopathological characteristics of leukoplakia with the expression E-cadherin and of the microscopic aspects of oral squamous cell carcinoma with immunohistochemical expression of involucrin.

Topics: Adult; Aged; Antigens, CD; Biomarkers, Tumor; Biopsy; Cadherins; Carcinoma, Squamous Cell; Female; Humans; Immunohistochemistry; Leukoplakia, Oral; Male; Middle Aged; Mouth Neoplasms; Protein Precursors; Reference Values; Statistics, Nonparametric

Esophageal squamous cell carcinoma (ESCC) develops within the squamous epithelial layer and invades the submucosa to the subadventitia that has adipose tissue (AT). AT seems critical to ESCC progression, but the underlying mechanism is unknown. We aimed to address the association between ESCC and AT in vitro. ESCC cells were cultured on rat or human subcutaneous AT-embedded or -non-embedded collagen gel. AT promoted the growth of ESCC cells and inhibited their apoptosis. AT promoted the expression of the squamous differentiation marker involucrin in ESCC cells. AT accelerated the expression of invasion-related factors in poorly differentiated ESCC cells only. AT promoted the expression of phosphorylated-insulin-like growth factor-1 receptor in ESCC cells, whereas it inhibited that of the human epidermal growth factor receptor 2. Insulin-like growth factor-1, but not leptin, adiponectin, or resistin, promoted and inhibited the growth and apoptosis of ESCC cells, respectively. In turn, ESCC cells decreased the production of these adipokines in AT and the number of preadipocytes and mesenchymal stem cell-like cells, which developed from AT. These results suggest that i) AT may influence the progression of ESCC with increased growth or invasion and decreased apoptosis through insulin-like growth factor-1/insulin-like growth factor-1 receptor signaling, ii) AT may affect human epidermal growth factor receptor 2-targeted therapy; and iii) the cancer cells may affect adipokine production in AT.

Topics: Adiponectin; Adipose Tissue; Animals; Apoptosis; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Adhesion Molecules; Cell Line, Tumor; Cell Transformation, Neoplastic; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Filamins; Humans; Hypertrophy; Insulin-Like Growth Factor I; Kalinin; Lipid Metabolism; Matrix Metalloproteinase 14; Matrix Metalloproteinase 9; Microscopy, Electron; Protein Precursors; Rats, Wistar; Receptor, ErbB-2; Resistin; Stromal Cells; Tumor Cells, Cultured

Hematopoietic pre-B-cell leukemia transcription factor-interacting protein (HPIP) is a corepressor of pre-B-cell leukemia homeobox (PBX) 1 and is known to play a role in hematopoiesis. Recently, HPIP was demonstrated to promote breast cancer cell proliferation and hepatocellular carcinoma growth. Moreover, it has been revealed that homeobox and PBX proteins, the expression of which is regulated by HPIP, play key roles in cancer of various organs, including oral squamous cell carcinoma (OSCC). Nevertheless, there has not been any study regarding the role of HPIP in OSCC. This study investigated the expression of HPIP in normal oral mucosa, epithelial precursor lesion (OEPL), and OSCC, and the functional roles of HPIP in OSCC cells and normal keratinocytes.. Immunohistochemical analysis of HPIP, Ki-67, and involucrin was performed in OSCC specimens, and the change in involucrin expression following RNA interference treatment against HPIP was examined by quantitative RT-PCR and Western blot analysis in SCC9 and NHEK cells undergoing extracellular calcium-induced differentiation. Matrigel transwell and cell proliferation assays for both cell lines transfected with HPIP siRNA were also conducted.. HPIP expression increased in OEPL and OSCC specimens. In vitro analysis revealed that HPIP suppressed differentiation and proliferation of SCC9 cells and transwell migration of NHEK cells, while HPIP promoted invasion of SCC9 and proliferation of NHEK cells. However, HPIP has no significant effect on NHEK cell differentiation.. HPIP may play a critical role in oral carcinogenesis and is thus a potential target for anticancer therapy, with particular emphasis on its involvement in differentiation and migration/metastasis.

Topics: Adult; Aged; Calcium; Carcinogenesis; Carcinoma in Situ; Carcinoma, Squamous Cell; Cell Culture Techniques; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Co-Repressor Proteins; Female; Gene Silencing; Humans; Keratinocytes; Ki-67 Antigen; Male; Middle Aged; Mouth Mucosa; Mouth Neoplasms; Precancerous Conditions; Protein Precursors; RNA, Small Interfering; Transcription Factors

Skin squamous cell carcinoma (SCC) is a subtype of very aggressive skin cancers. To investigate if epithelial-mesenchymal transition (EMT), a process for epitheloid cells losing their polarity and cohesiveness and transform into spindle-shaped cells, occurs in skin SCC. By using immunofluorescence, we defined the immunolocalization of vimentin, Keratin 17, β-catenin, E-cadherin, Ki-67 and involucrin, in SCC samples. Our results show reduced activity of involucrin and E-cadherin, and increased expression of Ki-67, β-catenin, Keratin 17 and vimentin in SCC. These data propose that EMT really occurs in poorly differentiated SCC and keratin 17 and involucrin may be another two biomarkers for EMT.

Topics: Aged; beta Catenin; Biomarkers, Tumor; Cadherins; Carcinoma, Squamous Cell; Case-Control Studies; Epithelial-Mesenchymal Transition; Female; Humans; Keratin-17; Ki-67 Antigen; Male; Middle Aged; Protein Precursors; Skin Neoplasms; Vimentin

Zfp191 represses differentiation and keeps various cells in the stem/progenitor stage. Here, we report that a Zfp191 homolog protein, ZNF396, is expressed in basal cell carcinoma (BCC) and possibly represses the expression of a Notch system effector molecule, Hes1 (hairy and enhancer of split-1), and prevents BCC cells from undergoing Notch-mediated squamous cell differentiation. ZNF396 immunoreactivity was found in the nucleus of 35 of 38 cutaneous BCC and 4 of 74 squamous cell carcinoma tissue specimens. In non-tumorous epidermal tissues, ZNF396 immunoreactivity was restricted in basal cells. siRNA-mediated silencing of ZNF396 induced the expression of Notch2, Hes1, and involucrin in cultured BCC cells. Finally, we found that siRNA-mediated silencing of ZNF396 gene inhibited the proliferation of TE354.T basal cell carcinoma cells. ZNF396 might repress Notch-Hes1 signaling axis and prevent tumor cells from undergoing squamous differentiation in BCC.

Topics: Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Down-Regulation; HEK293 Cells; Homeodomain Proteins; Humans; Protein Precursors; Receptor, Notch2; RNA Interference; RNA, Small Interfering; Skin Neoplasms; Transcription Factor HES-1; Transcription Factors

We investigated protein expression and in situ activity of transglutaminases (TGs) in normal skin and various epidermal neoplasms. In normal skin, TG1 protein expression and TG activity were found at keratinocyte cell membranes in upper epidermis and granular layer, respectively. In seborrhoeic keratosis, TG1 protein was expressed evenly throughout tumors, while TG activity increased in gradient fashion from lower tumor area to cornified layer. In squamous cell carcinoma, TG1 protein was expressed at inner side of cell membranes, whereas TG activity was found in cytoplasm predominantly at horn pearls. In basal cell carcinoma, weak TG activity was found in cytoplasm of all tumor cells without the presence of TG1 protein. Immunoblotting and in situ activity assays using specific substrate peptides confirmed that TG2, but not TG1, contributed to the TG activity. These results suggested that different expression and activation of TGs may contribute to characteristics of the skin tumors.

Topics: Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line, Tumor; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Keratinocytes; Keratosis, Seborrheic; Protein Precursors; Skin; Skin Neoplasms; Transglutaminases

Transcription factor c-Jun plays a key role in controlling epithelium cell proliferation, apoptosis and differentiation. However, molecular mechanism and biological functions of c-Jun in squamous differentiation and the progression of esophageal squamous cell carcinoma (ESCC) remain elusive. In this study, we found that c-Jun bound directly to the promoter region, and activated the transcription of differentiation-associated genes including cystatin A, involucrin and SPRR3 in vivo. Ectopic expression of c-Jun enhanced SPRR3 transactivation in KYSE450 cells. Conversely, TAM67, a dominant negative mutant of c-Jun, inhibited SPRR3 transactivation. c-Jun increased expression of SPPR3 mainly via a PKC/JNK pathway in response to TPA in KYSE450 cells. Furthermore, c-Jun was remarkably reduced in esophageal cancer. Interestingly, cystatin A, involucrin and SPRR3 were significantly downregulated as well, and associated with differentiation grade. Expression of c-Jun was correlated with the expression of these genes in normal epithelium and ESCC. Importantly, the expression of these genes was remarkably decreased during the malignant transformation from normal epithelium to low-grade intraepithelial neoplasia (LGIN) or high-grade intraepithelial neoplasia (HGIN). The expression of cystatin A and involucrin was significantly reduced from LGIN to HGIN. These results suggest c-Jun was involved in the regulation of differentiation-associated genes in ESCC. These genes might serve as the potential markers in distinguishing normal epithelium from esophageal squamous intraepithelial neoplasia.

Topics: Aged; Carcinoma in Situ; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line; Cornified Envelope Proline-Rich Proteins; Cystatin A; Disease Progression; Epithelium; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Gene Expression Regulation, Neoplastic; Genes, Dominant; Genes, jun; Humans; Male; Middle Aged; Mutation; Oligonucleotide Array Sequence Analysis; Protein Kinase C; Protein Precursors; Proto-Oncogene Proteins c-jun; Transcriptional Activation

Atopic dermatitis can result from loss of structural proteins in the outermost epidermal layers, leading to a defective epidermal barrier. To test whether this influences tumour formation, we chemically induced tumours in EPI-/- mice, which lack three barrier proteins-Envoplakin, Periplakin, and Involucrin. EPI-/- mice were highly resistant to developing benign tumours when treated with 7,12-dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). The DMBA response was normal, but EPI-/- skin exhibited an exaggerated atopic response to TPA, characterised by abnormal epidermal differentiation, a complex immune infiltrate and elevated serum thymic stromal lymphopoietin (TSLP). The exacerbated TPA response could be normalised by blocking TSLP or the immunoreceptor NKG2D but not CD4+ T cells. We conclude that atopy is protective against skin cancer in our experimental model and that the mechanism involves keratinocytes communicating with cells of the immune system via signalling elements that normally protect against environmental assaults.DOI: http://dx.doi.org/10.7554/eLife.01888.001.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinoma, Squamous Cell; Cell Communication; Cell Differentiation; Cell Transformation, Neoplastic; Cytokines; Dermatitis, Atopic; Disease Models, Animal; Epidermis; Keratinocytes; Membrane Proteins; Mice, 129 Strain; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mice, Nude; NK Cell Lectin-Like Receptor Subfamily K; Papilloma; Permeability; Plakins; Protein Precursors; Signal Transduction; Skin Neoplasms; Tetradecanoylphorbol Acetate; Thymic Stromal Lymphopoietin; Time Factors

To assess apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) expression in oral squamous cell carcinoma (OSCC) and analyze its clinical and pathological significance.. ASC expression was studied using immunohistochemistry in 119 OSCCs patients. The relationships between ASC expression and clinical and pathological parameters were statistically analyzed. In addition, the relationships between ASC expression and cell differentiation [IVL (involcrin) expression] and apoptosis (TUNEL [TdT-mediated dUTP nick end labeling] positive cell number) were investigated.. ASC expression showed significant correlations with parameters including clinical tumor stage, mode of invasion, and histological differentiation, and had a significant impact on survival of OSCC. The distribution of ASC correlated well with that of IVL. ASC expression was significantly correlated with the TUNEL-positive cell number.. Lower ASC expression correlates with clinical and pathological malignancy and, consequently, poor prognosis of OSCC. ASC has a close association with cell differentiation and apoptosis.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; Biomarkers, Tumor; Carcinoma, Squamous Cell; CARD Signaling Adaptor Proteins; Cell Differentiation; Cell Line, Tumor; Cytoskeletal Proteins; Female; Fluorescent Antibody Technique; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Mouth Neoplasms; Protein Precursors; Real-Time Polymerase Chain Reaction; Retrospective Studies

Aberrant keratinocyte differentiation is a key mechanism in the initiation of cancer. Because activities regulating differentiation exhibit altered or reduced capacity in esophageal cancer cells, it is vital to pinpoint those genes that control epidermal proliferation and terminal differentiation to better understand esophageal carcinogenesis. S100A14 is a member of the S100 calcium-binding protein family and has been suggested to be involved in cell proliferation, apoptosis, and invasion. The present study used immunohistochemistry analysis of S100A14 in clinical specimens of esophageal squamous cell carcinoma (ESCC) to show that decreased S100A14 is strongly correlated with poor differentiation. Furthermore, both mRNA and protein expression of S100A14 was drastically increased upon 12-O-tetra-decanoylphorbol-13-acetate (TPA) and calcium-induced esophageal cancer cell differentiation. Overexpression of S100A14 resulted in a G1-phase cell cycle arrest and promoted calcium-inhibited cell growth. Conversely, decreasing S100A14 expression significantly promoted G1-S transition and prevented the morphologic changes associated with calcium-induced cell differentiation. Molecular investigation demonstrated that S100A14 altered the calcium-induced expression of late markers of differentiation, with the most prominent effect on involucrin (IVL) and filaggrin (FLG). Finally, it was determined that S100A14 is transcriptionally regulated by JunB and that S100A14 and JunB status significantly correlated in ESCC tissue. In summary, these data demonstrate that S100A14 is transcriptionally regulated by JunB and involved in ESCC cell differentiation.. This study further differentiates the molecular mechanism controlling the development and progression of esophageal cancer.

Topics: Calcium; Calcium-Binding Proteins; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Filaggrin Proteins; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Intermediate Filament Proteins; Neoplasm Grading; Neoplasm Staging; Protein Precursors; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transcription Factors

Aberrant contact-inhibited proliferation and differentiation induction couple with tumor severity, albeit with an imprecise association with prognosis. Assessment of contact inhibition and differentiation-promoting culture in this study of normal and immortalized oral keratinocytes (NOK and SVpgC2a, respectively) demonstrated elevated cloning ability and saturation density in the immortalized versus normal state, including consistent absence of differentiated morphological features. Transcriptomic analysis implicated 48 gene ontology categories, 8 molecular networks, and 10 key regulator genes in confluency-induced differentiation of NOK, all of which remained nonregulated in SVpgC2a. The SVpgC2a versus NOK transcriptome enriched 52 gene ontology categories altogether, 18 molecular networks, and 39 key regulator genes, several of which were associated with epithelial-mesenchymal transition. Assessment of the previously described gene sets relative to training data sets of head and neck squamous cell carcinoma samples, one including data on tumor differentiation and patient outcome and one present in the Human Gene Expression Map, identified four genes with association to poor survival (COX7A1, MFAP5, MPDU1, and POLD1). This gene set predicted poor outcome in an independent data set of 71 head and neck squamous cell carcinomas. The present study defines, for the first time to our knowledge, the broad gene spectrum that couples to induction, and loss, of oral keratinocyte differentiation. Bioinformatics assessments of the results relative to clinical data generated novel differentiation-related tumor biomarkers relevant to patient outcome.

Topics: Apoptosis; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Communication; Cell Differentiation; Cell Transformation, Neoplastic; Contractile Proteins; DNA Polymerase III; Electron Transport Complex IV; Extracellular Matrix Proteins; Gene Expression Profiling; Genes, Neoplasm; Genomics; Head and Neck Neoplasms; Humans; Kaplan-Meier Estimate; Keratinocytes; Microarray Analysis; Prognosis; Protein Precursors; RNA Splicing Factors; Tumor Cells, Cultured

Type VII collagen (ColVII) is the main component of anchoring fibrils, attachment structures within the lamina densa of the basement membrane that are responsible for attachment of the epidermis to the dermis in skin. Mutations in the human ColVII gene, COL7A1, cause the severe inherited blistering disorder recessive dystrophic epidermolysis bullosa (RDEB) affecting skin and mucosae, associated with a greatly increased risk of skin cancer. In this study, we examined the effect of loss of ColVII on squamous cell carcinoma (SCC) tumourigenesis using RNAi in a 3D organotypic skin model. Our findings suggest that loss of ColVII promotes SCC migration and invasion as well as regulating cell differentiation with evidence for concomitant promotion of epithelial-mesenchymal transition (EMT). Immunostaining of RDEB skin and a tissue array of sporadic cutaneous SCCs confirmed that loss of ColVII correlates with decreased involucrin expression in vivo. Gene-expression-array data and immunostaining demonstrated that loss of ColVII increases expression of the chemokine ligand-receptor CXCL10-CXCR3 and downstream-associated PLC signalling, which might contribute to the increased metastatic potential of SCCs with reduced or absent ColVII expression. Together, these findings may explain the aggressive behaviour of SCCs in RDEB patients and may also be relevant to non-RDEB skin cancer, as well as other tumours from organs where ColVII is expressed.

Topics: Basement Membrane; Biomarkers; Cadherins; Carcinoma, Squamous Cell; Cell Differentiation; Cell Movement; Collagen Type VII; Humans; Matrix Metalloproteinase 2; Middle Aged; Neoplasm Invasiveness; Protein Precursors; RNA, Small Interfering; Skin Neoplasms; Tissue Culture Techniques

Squamous cell carcinomas (SCC) represent a substantial clinical problem because of increases, frequent recurrences and successive de novo tumors, especially in organ transplant recipients. To improve upon the current surgical and other non-selective therapies, a validated organotypic in vitro model of primary human SCC needs to be developed. Such a model will have obvious advantages over current cell line and animal based approaches, and may render the latter partly obsolete. In a first approach, an explant technique of primary SCC biopsies onto dermal constructs was used to emulate tumor expansion in an in vitro model. Histological analysis revealed the formation of nests of squamous cells, mimicking an invasive morphological feature of primary SCC. Immunohistochemical analysis comprised an array of markers characteristic of keratinocyte (hyper) proliferation (K6, K16, K17 and Ki67), differentiation (K1, K10 and involucrin), basement membrane (collagen types IV and VII, integrins alpha(6) and beta(4) and laminin 332) and SCC (K4, K13 and Axl). The generated human SCC models displayed disturbed differentiation and keratins associated with hyperproliferation, but a low frequency of Ki67 positive cells. Basement membrane composition of the in vitro SCC model resembled that of normal skin. These results show for the first time that in vitro modelling of three-dimensional growth of primary cutaneous human SCC is feasible. This model may provide a platform to develop refined preventive and curative treatments and thereby gain understanding of SCC pathogenesis.

Topics: Adult; Aged; Axl Receptor Tyrosine Kinase; Basement Membrane; Biopsy; Carcinoma, Squamous Cell; Cell Adhesion Molecules; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Female; Fibroblasts; Humans; Integrins; Kalinin; Keratinocytes; Keratins, Type I; Keratins, Type II; Ki-67 Antigen; Male; Middle Aged; Non-Fibrillar Collagens; Oncogene Proteins; Protein Precursors; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Skin; Skin Neoplasms; Tissue Culture Techniques

Basaloid squamous cell carcinoma (BSCC) is a histologically distinct variant of squamous cell carcinoma. It occurs in various sites of the head and neck region and is believed to carry a dismal prognosis. The palate is a very rare site of BSCC development and only three cases have been reported in the international literature. In this report, we present a case of basaloid squamous cell carcinoma of the soft palate. The therapeutic strategy and histological findings are described in detail, including immunohistochemistry with the use of involucrin, an agent used for the first time for BSCC diagnosis. In addition, a brief review of the literature is presented.

Topics: Aged; Biomarkers, Tumor; Biopsy; Carcinoma, Basosquamous; Carcinoma, Squamous Cell; Coloring Agents; Diagnosis, Differential; Humans; Male; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Neoplasm Staging; Palatal Neoplasms; Palate, Soft; Protein Precursors; Radiotherapy, Adjuvant; Reoperation; Tomography, X-Ray Computed

Areca (betel) is an important etiological factor linked to the high prevalence of oral carcinoma and other oral diseases in South Asians. Involucrin is a key component of the cornified envelop and a differentiation marker of keratinocyte. In this study, we found that 5 microg/ml non-toxic areca nut extract (ANE) treatment resulted in the 0.5-fold down-regulation of involucrin and disruption in involucrin distribution in normal human oral keratinocyte (NHOK). Progressive down-regulation of involucrin during oral carcinogenesis was noted. Activation of AKT by 1.7-fold and up-regulation of COX-2 by 2-fold were elicited following ANE treatment in NHOK. Treatment with PI3K/AKT blockers reverted the down-regulation of involucrin. ANE also down-regulated involucrin by 0.6-fold and disturbed both cornified envelope and cell aggregation in calcium-induced differentiated NHOK. However, such phenomena seemed to be independent from the ANE-associated COX-2 activation. The ANE-associated down-regulation of involucrin through AKT pathway could underlie the areca-associated epithelial pathogenesis.

Topics: Adult; Aged; Areca; Blotting, Western; Carcinoma, Squamous Cell; Cells, Cultured; Cyclooxygenase 2; Down-Regulation; Female; Fluorescent Antibody Technique; Humans; Keratinocytes; Male; Middle Aged; Mouth Mucosa; Mouth Neoplasms; Phosphatidylinositol 3-Kinases; Plant Extracts; Precancerous Conditions; Protein Precursors; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Epidemiological evidence suggests that cigarette smoke induces squamous metaplasia in human tracheobronchial epithelium that can progress to lung squamous carcinoma. But it is not well understood how tracheobronchial epithelial cells transduce the signals that mediate cigarette smoke-induced squamous differentiation or squamous metaplasia. In the present study, we found that in vitro cigarette smoke components notably inhibited glycogen synthase kinase 3 (GSK3) and induced the expression of involucrin, a marker of squamous differentiation. The inactivation of GSK3 by two highly selective inhibitors, lithium and SB216763, also significantly enhanced involucrin expression in cultured porcine tracheobronchial epithelial cells (PTBECs). Moreover, we demonstrated that cigarette smoke components significantly promoted activator protein-1 (AP-1) binding activities to the upstream regulatory region of involucrin gene, and similar results were observed by further studies through using GSK3 inhibitors to imitate the effects of cigarette smoke components. Taken together, we conclude that GSK3 is involved in involucrin expression induced by cigarette smoke in PTBEC probably via negatively regulating AP-1 activity, implying a possible mechanism responsible for squamous differentiation induced by cigarette smoke.

Topics: Animals; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Enzyme Inhibitors; Gene Expression; Glycogen Synthase Kinase 3; Indoles; Lithium; Lung Neoplasms; Maleimides; Metaplasia; Precancerous Conditions; Protein Precursors; Respiratory Mucosa; Signal Transduction; Smoke; Swine; Transcription Factor AP-1

p27kip1 belongs to the KIP/CIP family of cyclin-dependent kinase inhibitors and is considered to be a tumor suppressor. Involucrin has been known as a marker of differentiation of squamous cell carcinoma (SCC). The aim of this study was to evaluate the clinicopathologic significance of the expression of p27 and involucrin in esophageal SCC.. Immunohistochemical expression of p27 and involucrin was examined in 70 specimens of esophageal SCC. The correlation of the expression of these proteins and clinicopathologic features was evaluated.. Cellular differentiation in esophageal SCC was significantly correlated with the expression of p27 and involucrin (p = 0.010 and p = 0.002, respectively). Among well, moderately and poorly differentiated SCCs, 45.8 +/- 21.6, 20.0 +/- 15.0 and 10.6 +/- 9.1% of carcinoma cells expressed involucrin, respectively (p < 0.0001 for well vs. poorly, p < 0.0001 for well vs. moderately, and p = 0.042 for moderately vs. poorly). There existed a more powerful statistical difference regarding the histological grade between SCCs with the expression of both p27 and involucrin and tumors with other expression patterns (p = 0.0001).. Expression of both p27 and involucrin can be a powerful biological marker of cellular differentiation of esophageal SCC.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Differentiation; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Differentiation; Cyclin-Dependent Kinase Inhibitor p27; Disease Progression; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; Male; Middle Aged; Protein Precursors; Survival Rate

A suitable model analyzing the behavior of well-differentiated squamous cell carcinoma has not yet been established. We tried to establish such a system using a reconstructed oral mucosa, in which T3M-1 squamous cell carcinoma cells were cultured on 3T3 fibroblast-containing collagen gel. Fibroblasts promoted the stratification and keratinization of T3M-1 cells. During growth, the Ki-67 index of T3M-1 cells with fibroblasts was higher than that of T3M-1 cells alone. Fibroblasts increased the expression of involucrin, a differentiating marker of keratinocytes, in T3M-1 cells. They also promoted the invasion of T3M-1 cells into the gel. When T3M-1 cells alone were cultured in a fibroblast-conditioned (FC) medium, the fibroblast-induced phenomena mentioned above were almost replicated. In addition, epidermal growth factqr (EGF) promoted T3M-1 cells growth, but not the invasion. cDNA microarray analysis showed that FC medium increased the expression of EGF receptor and several other mRNAs of T3M-1 cells. The data suggest that T3M-1 cells, under cancer-stromal fibroblast interaction, undergo invasive growth with their well-differentiated squamous phenotype, and that this interaction may be mediated partly by soluble molecules (e.g., EGF) in an autocrine or paracrine pathway. Our system will probably provide a useful model for analyzing the biological behavior of well-differentiated squamous cell carcinoma.

Topics: 3T3 Cells; Animals; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Coculture Techniques; Collagen; Culture Media, Conditioned; Epidermal Growth Factor; Fibroblasts; Gels; Hepatocyte Growth Factor; Humans; Ki-67 Antigen; Mice; Mouth Neoplasms; Neoplasm Invasiveness; Oligonucleotide Array Sequence Analysis; Protein Precursors; Receptors, Interleukin-1; Stromal Cells

IkappaB kinase (IKK) alpha and beta share the function to phosphorylate IkappaB to activate a transcription factor NF-kappaB. Recent reports, however, revealed differences in the functions of the two kinases. The present study was designed to determine a unique function of IKKalpha on the differentiation of squamous cell carcinoma (SCC). Transfection with IKKalpha gene, but neither IKKbeta nor NF-kappaB gene, inhibited the constitutive expressions as well as extracellular calcium-induced expressions of involucrin and filaggrin, epithelial differentiation markers, in cultured SCC cells. Morphological changes from polygonal to fibroblastic shape were seen in the SCC cells stably expressing green-fluorescent protein (GFP)-fused IKKalpha while intracellular localization of GFP-IKKalpha differed from that of GFP-IKKbeta. Interestingly, phorbol myristate acetate together with IKKalpha gene transfection strongly inhibited the expression of involucrin in SCC cells and induced the phosphorylation of serine residue in IKKalpha, suggesting that protein kinase C is involved in the effect of IKKalpha on the differentiation of SCC cells. In conclusion, high expression of IKKalpha may serve as an intracellular signal to halt the epithelial differentiation of SCC cells.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Transformation, Neoplastic; Filaggrin Proteins; Humans; I-kappa B Kinase; Intermediate Filament Proteins; Mouth Neoplasms; Protein Precursors; Protein Serine-Threonine Kinases

Tissue microarrays allow the simultaneous analysis of many tumours using small-diameter cores sampled from larger blocks of tissue, but may be limited by tumour heterogeneity. This study considers the validation of tissue microarray for the study of four molecules of interest as prognostic factors in head and neck squamous carcinoma, including a consideration of methods for assessing immunocytochemical scoring of microarrays. Tissue microarray blocks were constructed from 100 cases of head and neck squamous carcinoma, taking four cores from different areas of each tumour. Immunocytochemical labelling was performed for cutaneous fatty acid binding protein, involucrin, vascular endothelial growth factor and Ki-67. The extent and intensity of scoring was determined for each core and the degree of agreement determined for results from the assessment of two, three or four cores for each carcinoma. In a subset of 30 representative cases, the labelling in the tissue microarrays was compared with that in whole-tissue sections of the same carcinomas. An adequate sample of carcinoma was achieved in more than 90% of the 400 cores; unsuccessful results were attributed to uneven core alignment or to poor targeting of the tumour tissue in the donor blocks. The degree of agreement in the assessment of extent and intensity of labelling was moderate to good (weighted kappa, range 0.479-0.902) between whole-tissue sections and microarray sections depending on the antigen and the scoring system. Tissue microarray is a reliable tool to demonstrate cellular and molecular alterations in head and neck squamous carcinomas. We recommend using the mean results from four cores for biological studies, with analysis of categorical data based on quartile groups. Concordance with whole-tissue section data is reassuring, but data from microarrays need to be validated against clinical outcomes.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Squamous Cell; Fatty Acid-Binding Proteins; Head and Neck Neoplasms; Histological Techniques; Humans; Immunohistochemistry; Ki-67 Antigen; Middle Aged; Prognosis; Protein Precursors; Sensitivity and Specificity; Vascular Endothelial Growth Factor A

Abnormal expression of Notch1 protein was often found in many kinds of primary tumors, but its correlation with nasopharyngeal carcinoma (NPC) is still unclear. This study was designed to investigate the expression of Notch1 and its downstream proteins P21(WAF1) and Involucrin in NPC, and analyze their correlations with the differentiation of NPC cells.. The expression of Notch1, P21(WAF1), and Involucrin in 101 specimens of NPC and 20 specimens of chronic inflammatory nasopharyngeal mucosa were detected by SP immunohistochemistry.. The positive rates of Notch1, P21(WAF1), and Involucrin were 100%, 90.0%, and 100% in chronic inflammatory nasopharyngeal mucosa, and were 77.2%, 89.1%, and 80.1% in NPC, respectively. The expression of Notch1, P21(WAF1), and Involucrin were significantly suppressed along with descending differentiation of NPC (P=0.000, P=0.026, and P=0.000). The positive rates of Notch1, P21(WAF1), and Involucrin were significantly higher in keratinizing squamous cell carcinoma (KSCC) than in differentiated nonkeratinizing carcinoma (DNKC) and undifferentiated carcinoma (UDC) (P<0.05), and were significantly higher in DNKC than in UDC (P<0.05). The expression of Notch1 in NPC was positively correlated with the expression of P21(WAF1) (r=0.306, P=0.002) and Involucrin (r=0.325, P=0.001). No significant correlation was found between the expression of P21(WAF1) and Involucin.. The expression of Notch1, P21(WAF1), and Involucrin are closely correlated to the differentiation of NPC cells.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Cell Differentiation; Cyclin-Dependent Kinase Inhibitor p21; Epithelium; Female; Humans; Male; Middle Aged; Nasopharyngeal Neoplasms; Nasopharyngitis; Protein Precursors; Receptor, Notch1

The majority of hypoxic cells in squamous cell carcinomas of the head and neck and cervix express involucrin, a molecular marker for differentiation. This raises the question of whether involucrin is an oxygen-regulated protein and, if so, whether it could serve as an endogenous marker for tumour hypoxia. Consistent with oxygen regulation, involucrin protein was found to increase with increasing hypoxia in confluent cultures of moderately differentiated human SCC9 cells. Cells harvested at the point of confluence and exposed to graded concentrations of oxygen revealed a K(m) of approximately 15 mmHg for involucrin induction. This is similar to K(m)s for HIF-1alpha, CAIX and VEGF. Involucrin induction showed a steep dependence on pO(2) with a transition from minimum to maximum expression occurring over less than an order of magnitude change in pO(2). In contrast to SCC9 cells, involucrin was not induced by hypoxia in poorly differentiated SCC4 cells. It is concluded that involucrin is an oxygen-regulated protein, but that differentiation modulates its transcription status with respect to hypoxia induction.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Differentiation; Cell Hypoxia; Female; Head and Neck Neoplasms; Humans; Oxygen; Protein Precursors; Transcription, Genetic; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Topics: Carcinoma, Squamous Cell; Diagnosis, Differential; Humans; Immunohistochemistry; Keratins; Keratoacanthoma; Protein Precursors; Skin Neoplasms

This study examines differences between cultures of normal human oral epithelial cells and two squamous cell carcinoma cell lines (SCC15 and SCC25) in the expression of structural proteins, adhesion molecules, plasma membrane lipid composition, and intercellular junctions. Based on immunocytochemistry, most normal cell cultures appeared to express more E-cadherin, integrin beta-1, cytokeratin (CK) 14, CK19, and involucrin than SCC cultures. By Western blot analysis, normal cultures expressing high levels of E-cadherin also expressed high levels of involucrin and low levels of CK19. Both SCC cultures demonstrated lower expression of E-cadherin and involucrin, whereas only SCC15 cells showed high levels of CK19. Expression of beta-catenin, an E-cadherin associated protein with potential oncogene function, did not vary among normal and SCC cells. Proportions of saturated fatty acids quantified by thin layer chromatography were higher in the normal cell cultures, than in both SCC cell lines. No morphological differences were evident by transmission electron microscopy (TEM) between normal and SCC cell-cell intercellular junctions. Although no quantitation was attempted, observation suggested that normal cells form more intercellular junctions (TEM observation) and larger intercellular bridges (SEM observation) compared to both SCC cell lines. Of the factors examined, main variations between cultures of normal oral epithelium and the two SCC cell lines examined include the expression of structural and adhesion proteins, lipid composition, and intercellular junctions. The extent of the differences varies according to the stage of terminal differentiation demonstrated by the normal cell cultures.

Topics: Biomarkers, Tumor; Blotting, Western; Cadherins; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line, Tumor; Cells, Cultured; Fatty Acids; Gingiva; Humans; Immunohistochemistry; Integrin beta1; Keratinocytes; Keratins; Microscopy, Electron; Mouth Neoplasms; Protein Precursors

The Yamamoto-Kohama (Y-K) mode of invasion is a useful indicator of the prognosis of squamous cell carcinoma (SCC) of the oral cavity. We have previously reported that the Y-K mode of invasion is related to lymph node metastasis in esophageal SCC. Therefore, to clarify the relationship between the Y-K mode of invasion and survival rate, we histologicaly evaluated specimens obtained by esophagectomy from patients who had not received induction therapy. The results indicated that the overall survival rate of patients with esophageal SCC was significantly lower in the Y-K 4D group. In addition, significantly lower expression of involucrin protein was observed in pStage III cases than in pStage II cases. As a consequence of the poor prognosis of pStage III SCC, it was suggested that involucrin expression was correlated with survival rate. In conclusion, the Y-K mode of invasion and involucrin expression are considered to be informative prognostic indicators in patients with esophageal SCC.

Topics: Carcinoma, Squamous Cell; Esophageal Neoplasms; Humans; Lymph Nodes; Lymphatic Metastasis; Neoplasm Invasiveness; Neoplasm Staging; Prognosis; Protein Precursors; Survival Rate

Squamous differentiation/squamous metaplasia is often associated with endometrial adenocarcinoma and benign lesions, such as endometrial hyperplasia and chronic endometritis. Morules have distinct histological characteristics, and are referred to as squamous metaplasia or squamoid metaplasia.. To focus on the histological characteristics of morules and clarify the difference between morules and squamous differentiation.. Twenty endometrioid carcinomas with morules or squamous differentiation, five adenosquamous carcinomas, and eight non-carcinomatous endometrial lesions with morules were investigated. Numerous antibodies for epithelial membrane antigen (EMA), involucrin, cytokeratins, neuropeptides, and oncofetal antigens were used for immunohistochemistry. In situ hybridisation and polymerase chain reaction were used to detect human papillomavirus (HPV).. The morules observed were uniform cell clusters, with no squamous differentiation. They were immunonegative for epithelial antigens including involucrin, EMA, and cytokeratins, but were positive for neurone specific enolase. A few morules were immunopositive for acetylcholine esterase, and one case was positive for somatostatin; neither oncofetal nor proliferative cell markers, including blood group A, B, and AB, or other neuropeptides were demonstrated in the morules. HPV DNA was not found in either the morules in the carcinomas or in the benign lesions. However, true squamous differentiation tissue in four endometrioid carcinomas and two adenosquamous carcinomas was HPV positive using in situ hybridisation.. Morules are histologically distinct from squamous metaplasia/squamous differentiation tissue. Morules are thought to be neuroectodermal-like cell clusters, and are not infected with HPV. In contrast, some of the true squamous differentiation tissue was associated with HPV infection.

Topics: Adenocarcinoma; Adult; Aged; beta Catenin; Carcinoma, Squamous Cell; Cytoskeletal Proteins; DNA Mutational Analysis; DNA, Viral; Endometrial Neoplasms; Endometrium; Female; Humans; Immunohistochemistry; In Situ Hybridization; Metaplasia; Middle Aged; Papillomaviridae; Protein Precursors; RNA, Messenger; Trans-Activators; Transglutaminases

Protease-activated receptor-1 (PAR-1) is a G-protein-coupled receptor that contributes to multiple signal transduction pathways. Although the functions of PAR-1 in many normal cells, such as platelets and astrocytes, have been well studied, its roles in cancer progression and metastasis have not been fully elucidated, and studies to date appear contradictory.. To clarify the function of PAR-1 in metastasis of squamous cell carcinoma of the head and neck (SCCHN), we examined PAR-1 expression in clinical specimens by immunohistochemistry and in SCCHN cell lines by immunoblotting. Furthermore, par-1 cDNA-transfected SCCHN cell lines were also used to verify PAR-1-mediated pathway.. The metastatic tumors showed a lower percentage of PAR-1-positive cells (46%) and lower levels of PAR-1 expression (median weight index = 10) than node negative primary tumors (80% and median weight index = 60, respectively). In addition, expression level of PAR-1 positively correlated with levels of keratinocyte differentiation markers keratin-1, -10, and -11. Additional studies using sense and antisense par-1 cDNA-transfected SCCHN cell lines illustrated that the presence of PAR-1 was required for the expression of involucrin, a keratinocyte differentiation marker. PAR-1 expression also contributes to activation of the mitogen-activated protein kinase (MAPK) pathway. Blocking MAPK activation by a mitogen-activated protein/extracellular signal-regulated kinase inhibitor, not by a phosphatidylinositol 3'-kinase inhibitor, reduced level of involucrin, suggesting that regulation of involucrin by PAR-1 is partially through the MAPK signaling pathway.. Our study suggests that PAR-1 signaling induces differentiation markers in SCCHN cells, and its expression is conversely correlated with cervical lymph node metastasis.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Differentiation; Disease Progression; DNA, Complementary; Enzyme Inhibitors; Female; Head and Neck Neoplasms; Humans; Immunoenzyme Techniques; Keratinocytes; Lymphatic Metastasis; Male; Middle Aged; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Precursors; Receptor, PAR-1; Signal Transduction; Transfection

Calcium induces both involucrin and transglutaminase-K in normal keratinocytes (NHK) but not in squamous carcinoma cell lines (SCC). The protein kinase C (PKC) agonist phorbol myristoyl acetate potentiates and the PKC antagonist Ro31-8220 blocks the ability of calcium to stimulate the involucrin promoter in normal human keratinocytes but not in SCC4. We thus examined the ability of calcium to regulate the levels of five PKC isozymes in NHK and two SCC. In the normal keratinocytes, the levels of PKC [alpha], PKC [delta], PKC [eta], and PKC [zeta] increased over the first one to two weeks in a calcium-and time-dependent manner. PKC [epsilon] decreased in a time-and calcium-dependent fashion over the three-week period. All five isozymes showed little change during culture in SCC4 at any calcium concentration. Calcium and time of culture had partial effects on SCC12B2, a carcinoma that shows partial differentiation characteristics. Since PKC [alpha] is the only calcium responsive PKC isozyme in keratinocytes and most likely to be directly involved in calcium induced differentiation, we evaluated the effect of inhibiting its production with antisense oligonucleotides on calcium-regulated markers of differentiation. We found that the PKC [alpha] specific antisense oligonucleotide blocked calcium stimulated involucrin promoter activity as well as PKC [alpha], involucrin, and transglutaminase protein production, whereas the sense oligonucleotide control did not. We conclude that although a number of PKC isozymes are regulated during calcium-induced differentiation, PKC [alpha] plays a necessary role in mediating calcium-induced differentiation. Failure to regulate PKC [alpha] in SCC4 may underlie at least part of the failure of calcium to promote differentiation in these cells.

Topics: Calcium; Calcium Signaling; Carcinoma, Squamous Cell; Cell Differentiation; Cell Transformation, Neoplastic; Cells, Cultured; Humans; Infant, Newborn; Isoenzymes; Keratinocytes; Male; Oligonucleotides, Antisense; Protein Kinase C; Protein Kinase C-alpha; Protein Precursors; Transglutaminases

It has been reported previously in cases of adenosquamous carcinoma of the lung in Okinawa, a subtropical island 2000 km south of mainland Japan, that the squamous cell carcinoma components were positive for human papillomavirus (HPV) by non-isotopic in situ hybridisation (NISH). The adenocarcinoma cells adjacent to the squamous cell carcinoma components were enlarged and also positive for HPV. This is thought to indicate that after adenocarcinoma cells are infected with HPV, they undergo morphological changes, and that "squamous metaplasia" follows. In this present study, the effects of HPV transfection into adenocarcinoma cells were examined. The relation between the region expressing the HPV gene and squamous metaplasia was also studied.. Plasmid pBR322 containing HPV type 16 (HPV-16) was transfected into cultured colonic adenocarcinoma (DLD-1) and lung adenocarcinoma (PC-14) cells using the calcium phosphate method. Neomycin was used as a selection marker. The presence of HPV E1, E2, E4, E5, E6, E7, L1, and L2 mRNAs and also transglutaminase 1, involucrin, cyclin dependent kinases (CDKs), cyclins, caspases, apoptosis inducing factor, DNase gamma, Fas, and Fas ligand mRNAs in HPV transfected cells was investigated by means of reverse transcription polymerase chain reaction (RT-PCR). The G0-G1 cell population was analysed by flow cytometry. Morphological examination under light and electron microscopes was also carried out.. The virus transfected cells showed squamous metaplasia when they were injected into severe combined immunodeficient mice, expressing the high molecular weight keratin (Moll's number 1 keratin) and involucrin molecules immunohistochemically, and involucrin and transglutaminase I mRNAs by RT-PCR. The squamous metaplasia was most conspicuous in the HPV transfected DLD-1 cell when compared with HPV transfected PC-14 cells. Squamous metaplasia was most clearly demonstrated in one HPV transfected DLD-1 cell clone, which expressed not only E2 but also E6-E7 fusion gene mRNA. Viral L1 mRNA expression was absent in HPV transfected cell clones, and was not related to squamous metaplasia. The growth rate of HPV transfected cells was reduced. Transfection of the virus into the cultured adenocarcinoma cells increased the G0-G1 cell population greatly, as assessed by flow cytometer analysis. Furthermore, in the virus transfected cells, apoptosis was also observed by means of the terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labelling method.. HPV transfection into adenocarcinoma cells induced clear squamous metaplasia. One of the HPV transfected cell clones that expressed E2 and E6-E7 fusion gene mRNA showed the squamous metaplasia particularly clearly, and apoptosis was also demonstrated.

Topics: Adenocarcinoma; Animals; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Cell Cycle; Cell Differentiation; DNA, Viral; Humans; Keratins; Lung Neoplasms; Metaplasia; Mice; Mice, SCID; Neoplasm Proteins; Neoplasm Transplantation; Papillomaviridae; Protein Precursors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Viral; Transfection; Tumor Cells, Cultured

We report here that human esophageal keratinocyte stem cells are characterized by the expression of the low-affinity neurotrophin receptor p75(NTR) and differentially expressed cell adhesion molecules, the beta1 and beta4 integrins. The candidate stem cells could be fractionated from keratinocytes as a minor cell subset by means of immunocytochemical cell sorting based on the different levels of expression of these cell surface molecules. Flow cytometric analysis revealed that this minor cell subset retained a relatively slow-cycling phenotype in vitro. These cells expressed low levels of involucrin and cytokeratin 13, indicating that the p75(NTR)-positive cell subset is immature relative to the other predominant subpopulations coexpressing beta1 integrin at higher levels. The p75(NTR)-positive cell subset was crucial for achieving longevity and the greatest output of keratinocytes comprising all distinguishable subpopulations in vitro. This process was associated with self-renewal and self-amplification of the p75(NTR)-positive cell subset. These findings strongly implicate p75(NTR) as a stem cell marker, which will be valuable for prospectively investigating stem cell regulation in association with different biological processes including neoplastic transformation of regenerative epithelia.

Topics: Blotting, Western; Carcinoma, Squamous Cell; Cell Adhesion; Cell Cycle; Cell Division; Cell Membrane; Cell Transformation, Neoplastic; Epithelial Cells; Epithelium; Esophagus; Flow Cytometry; Humans; Immunohistochemistry; Integrin beta1; Integrin beta4; Keratinocytes; Keratins; Neoplasms; Phenotype; Propidium; Protein Precursors; Receptor, Nerve Growth Factor; Receptors, Nerve Growth Factor; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells; Subcellular Fractions; Time Factors

Pimonidazole binding (hypoxia) and involucrin expression (differentiation) overlap extensively in squamous cell carcinomas. This study asks whether involucrin might serve as an endogenous marker for tumor hypoxia. A second question is whether differentiation affects hypoxia-inducible metallothionein (MT) expression in normal human epithelia and squamous cell carcinomas as it does in rodent epithelia.. Thirty-four patients with squamous cell carcinoma of the uterine cervix were infused with pimonidazole hydrochloride solution. The next day, multiple biopsies were formalin-fixed, paraffin-embedded and sectioned at 4 micro m. Qualitative and quantitative analyses for involucrin expression, pimonidazole binding, and human MT-IIa mRNA expression were performed.. No overall correlation between the extent of involucrin expression and pimonidazole binding was observed. The lack of correlation was because of heterogeneous patterns of immunostaining for involucrin generally related to tumor grade. Colocalized immunostaining for involucrin and pimonidazole binding was observed in intermediate grade tumors but not in well-differentiated or poorly differentiated tumors. Human MT-IIa mRNA and MT protein were expressed in basal lamina of normal human epithelia and in the proliferative rims of tumor nests.. Colocalization of immunostaining for involucrin and pimonidazole binding is consistent with oxygen regulation, but the lack of involucrin expression in hypoxic regions of poorly differentiated tumors indicates that its transcriptional status with respect to hypoxia induction is altered by cell differentiation. The localization of MT message and protein in the outer rims of most tumor nests indicates that the transcriptional status of metallothionein is also altered by differentiation.

Topics: Biopsy; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line, Tumor; Female; Humans; Hypoxia; Immunohistochemistry; In Situ Hybridization; Metallothionein; Nitroimidazoles; Oxygen; Prognosis; Protein Precursors; Radiation-Sensitizing Agents; RNA, Messenger; Transcription, Genetic; Uterine Cervical Neoplasms

In the literature, few studies based on clinical and histological evaluation analyze skin structural changes after transplantation to the oral cavity. Ten patients affected by squamous cell carcinoma of the oral cavity who were reconstructed with a free forearm flap were included in the current study to analyze skin alterations. The authors performed a histological and ultrastructural evaluation of skin samples from the free forearm flap before transplantation and 18 months after intraoral reconstruction. They analyzed cytokeratin and involucrin distribution, which represent markers of maturation and differentiation of epithelia. The aim of this study was to demonstrate whether skin "mucosalization" occurs. They found that the skin undergoes some morphological changes induced by the intraoral environment. Cytokeratin and involucrin distribution is mostly unchanged. This aspect is in favor of skin structure preservation. Thus, they found that "mucosalization" of the skin is not evident after 18 months.

Topics: Aged; Carcinoma, Squamous Cell; Female; Humans; Immunohistochemistry; Keratins; Male; Microscopy, Electron, Scanning; Middle Aged; Mouth; Mouth Neoplasms; Protein Precursors; Skin; Skin Transplantation; Surgical Flaps

We report in this study that proliferation inhibition of SCC13 cells by calcipotriol was possibly mediated by its inhibitory effect on autocrine activation of EGF receptor. Based on MTT assay, PCNA staining, DAPI staining, and involucrin immunocytochemical staining, we showed that calcipotriol inhibited cell growth and stimulated differentiation but did not induce apoptosis. Western blot analysis of concanavalin-A-bound fraction demonstrated that calcipotriol specifically dephosphorylated 170- and 66-kDa polypeptides from 8 h posttreatment and complete dephosphorylation was observed at 12 h posttreatment. The 170- and 66-kDa polypeptides were confirmed as EGF receptor and Shc, respectively. Calcipotriol-mediated EGF receptor dephosphorylation required the presence of extracellular calcium. Similar kinetics of the dephosphorylation was also observed in HaCaT cells cultured in medium of high calcium concentration. By BrdU labeling, we also showed calcium dependency of calcipotriol for the inhibition of cell proliferation. Therefore, EGF receptor deactivation by calcipotriol might be a mechanism of action for the inhibition of cell proliferation and the stimulation of differentiation in SCC13 cell and HaCaT cells.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Antineoplastic Agents; Apoptosis; Autocrine Communication; Blotting, Western; Bromodeoxyuridine; Calcitriol; Calcium; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Cell Line; ErbB Receptors; Fluorescent Dyes; Humans; Keratinocytes; Phosphorylation; Proliferating Cell Nuclear Antigen; Protein Precursors; Proteins; Shc Signaling Adaptor Proteins; Signal Transduction; Src Homology 2 Domain-Containing, Transforming Protein 1; Tetrazolium Salts; Thiazoles

Keratinization of the ocular surface epithelium is associated with various disorders impairing vision. We immunohistochemically determined whether the ocular surface epithelia express involucrin, and whether its expression pattern may differ in benign vs. malignant disorders. Expression of cytokeratins was also examined to provide further information relative to the epithelial differentiation.. We evaluated 17 specimens; 6 specimens of the normal ocular surface epithelia, 3 specimens from cases of conjunctival intraepithelial neoplasia (CIN), 6 of conjunctival squamous cell carcinoma (SCC) and 2 of conjunctivae from cases of superior limbic keratoconjunctivitis (SLK).. Corneal epithelium exhibited intracellular immunoreactivity for involucrin. Four of the 6 specimens of bulbar conjunctival epithelium showed involucrin immunoreactivity in the perimembranous region, whereas the fornical conjunctiva was negative. Cornified envelope in SLK specimens was positive for involucrin. The CIN showed its immunoreactivity in the perimembranous region in all levels of the hyperproliferative epithelium without keratinization, i.e., similar to the bulbar conjunctiva. The neoplastic cells of well-differentiated SCC showed involucrin in the perimembranous region, and those of moderately- to poorly-differentiated SCC have involucrin in their cytoplasm. The expression pattern of cytokeratins was unrelated to grade of malignancy in ocular SCC.. The epithelia of normal subjects and of CIN expresses involucrin without keratinization. In contrary, the keratinized SLK epithelium markedly expresses involucrin in the cornified envelope. The subcellular immunolocalization of involucrin in the ocular SCC may help in evaluating the differentiation, i.e., malignancy, of neoplastic cells.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma in Situ; Carcinoma, Squamous Cell; Conjunctiva; Conjunctival Diseases; Conjunctival Neoplasms; Epithelium; Eye Proteins; Female; Filaggrin Proteins; Humans; Immunoenzyme Techniques; Intermediate Filament Proteins; Keratins; Keratoconjunctivitis; Male; Middle Aged; Protein Precursors

When keratinocytes withdraw from the cell cycle, they migrate from the basal to the superficial layers of the epidermis and undergo morphological and biochemical changes during the process of terminal differentiation. These differentiation features of keratinocytes are known to be altered or reduced in esophageal cancer cells. Therefore, we examined the effects of transferring the cyclin-dependent kinase inhibitor p21sdi1 gene into human esophageal cancer cell lines as well as normal keratinocytes using an adenovirus vector system. Ectopic expression of p21sdi1 protein resulted in cell cycle arrest at the G1 phase and produced morphological changes, such as enlarged nuclei and a flattened cellular shape, changes specific to the differentiated phenotype. The human involucrin protein is a specific product of keratinocyte differentiation, which is selectively expressed in the suprabasal epidermal layers. Western blot analysis and immunohistochemical staining demonstrated that involucrin expression was 3- to 5-fold enhanced by the forced expression of p21sdi1 in esophageal cancer cells, whereas only a mild up-regulation up to 1.2-fold occurred in normal keratinocytes. We also found that exogenous introduction of the p2sdi1 gene transcriptionally activated the upstream promoter function of the involucrin gene. These stimulatory effects on involucrin expression were not observed when another cyclin-dependent kinase inhibitor gene, p16(INK4a), was transduced. Moreover, p21sdi1 expression in esophageal cancer cells transduced with p21sdi1 led to a rapid apoptotic cell death after a transient dormant phase, although keratinocytes transduced with p21sdi1 survived longer by terminally withdrawing from the cell cycle. These results may have an important implication for understanding the biology of differentiation-dependent apoptosis in human esophageal squamous cell carcinoma.

Topics: Adenoviruses, Human; Animals; Apoptosis; Carcinoma, Squamous Cell; Cattle; Cell Cycle; Cell Differentiation; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Esophageal Neoplasms; Gene Expression; Gene Transfer Techniques; Genetic Vectors; Humans; Keratinocytes; Protein Precursors; Transcriptional Activation; Tumor Cells, Cultured

The bcl-2 proto-oncogene is a known inhibitor of apoptosis; in normal human stratified squamous epithelium, its expression is restricted to the basal cell layer. To investigate the functional role of bcl-2 protein in the process of differentiation of oral keratinocytes, bcl-2 expression vector was transfected into SCC-25 cells, which normally undergo squamous cell differentiation in vitro while expressing specific differentiation markers, e.g., keratin 10/11 and involucrin. In bcl-2 transfected SCC-25 cells, the expression of these differentiation markers was markedly suppressed. The bcl-2 proto-oncogene may play a critical role in opposing the commitment to terminal differentiation and apoptosis of oral keratinocytes.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Differentiation; DNA Fragmentation; Down-Regulation; Genes, bcl-2; Humans; Immunoenzyme Techniques; Keratinocytes; Keratins; Mouth Neoplasms; Protein Precursors; Proto-Oncogene Mas; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

The chemotherapeutic agent and vitamin A metabolite retinoic acid (RA) has been used to treat many tumor types. The effects of RA are mediated by a family of ligand-dependent transcription factors, the RA receptors and the retinoid X receptors (RXR). Alterations in retinoid receptor expression have been implicated in tumorigenesis. Previous studies have shown lack of RXR-gamma expression in squamous cell carcinoma (SCC) lines. To begin to elucidate the role of RXR-gamma in the malignant transformation of SCCs, we expressed RXR-gamma in SCC lines by stable transfection. SCC lines expressing RXR-gamma produced large numbers of flat cells with abundant cytoplasm, which died and detached from the culture dish. These cells morphologically resembled the differentiated cells of normal stratified squamous epithelium in culture. These cells did not exhibit the characteristic DNA fragmentation pattern of apoptotic cells, nor did they label in a fluorescent apoptosis assay. RNase protection and Western blot analysis revealed induction of RA-responsive involucrin and keratin 10 expression, early markers of terminal differentiation. RXR-gamma expression produced significant reduction in levels of RA-responsive genes including the cyclin-dependent kinase inhibitors p21Cip1/WAF1 and p27Kip1, resulting in increased cdc2 and cdk2 kinase activity and RB phosphorylation. We concluded that RXR-gamma induced terminal differentiation in SCC lines, suggesting a potential tumor suppressor function for this transcription factor.

Topics: Blotting, Western; Carcinoma, Squamous Cell; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cell Death; Cell Differentiation; Cell Size; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Down-Regulation; Enzyme Activation; Genes, Tumor Suppressor; Humans; Keratin-10; Keratins; Microtubule-Associated Proteins; Phosphorylation; Precipitin Tests; Protein Precursors; Protein Serine-Threonine Kinases; Receptors, Retinoic Acid; Retinoblastoma Protein; Retinoid X Receptors; RNA, Messenger; Transcription Factors; Transfection; Tumor Cells, Cultured; Tumor Suppressor Proteins

Carcinoembryonic antigen (CEA), which is a well-known marker for the normal sweat gland apparatus and its neoplasms in the skin, was recently demonstrated in sebaceous neoplasms. The aim of this study was to examine the expression of CEA and related antigens in the other cutaneous keratinous neoplasms and verruca vulgaris. Normal adult skin, squamous cell carcinoma (SCC), senile keratosis, Bowen's disease, basal cell carcinoma (BCC), seborrhoeic keratosis and verruca vulgaris were stained immunohistochemically with a panel of monoclonal and polyclonal antibodies that recognize different epitopes of CEA and related molecules. Localization of the antigens was compared with that of involucrin and proliferating cell nuclear antigen. The strongest expression of CEA-related antigens, other than non-specific cross-reacting antigen (NCA) -50/90, was seen in SCC and verruca vulgaris, while no detectable expression was seen in BCC. Senile keratosis, Bowen's disease and seborrhoeic keratosis showed the predominance of the CEA-related antigens over CEA weakly expressed. Strong expression of both CEA and NCA-50/90 was seen only in SCC. All the expressions were limited to the cells situated in the upper epidermal cell layers of the tumours, at the centre of tumour islands in SCC and along the pseudohorn cysts in seborrhoeic keratosis, where involucrin was coexpressed. We suggest that CEA and related antigens are not only markers for sweat gland differentiation in the skin, as currently accepted, but are also expressed in various cutaneous keratinous neoplasms and verruca vulgaris. The expression may correlate with the terminal differentiation of the tumour cells, the strong coexpression of CEA and NCA-50/90 may correlate with the malignant potential of the tumour types, and the mechanisms that control the expression of CEA and related antigens in the neoplasms may be similar to those operative in verruca vulgaris.

Topics: Adult; Carcinoembryonic Antigen; Carcinoma, Squamous Cell; Humans; Immunoenzyme Techniques; Neoplasm Proteins; Proliferating Cell Nuclear Antigen; Protein Precursors; Skin; Skin Neoplasms; Warts

Nuclear receptors for retinoic acid are important modulators of epidermal cell proliferation and terminal differentiation. Aberrant expression of retinoic acid receptors (RARs) and retinoid X receptors in the epidermis has been associated with altered differentiation capacity and tumor progression. In this study, we describe a human squamous cell carcinoma line, SCC 12F, which displays reduced RARgamma expression and diminished responsiveness to retinoic acid. When compared with normal keratinocytes or other squamous cell carcinoma lines that display normal levels of RARgamma, several measures of cellular response to retinoic acid are altered in SCC 12F cells, including inhibition of cornified envelope formation, reduction of involucrin mRNA expression, and transcriptional regulation of the involucrin gene. Normal patterns of ligand-dependent transcriptional response were restored upon co-transfection of an expression vector containing either RARalpha or RARgamma. Our findings demonstrate that reduced expression of RAR may have direct functional consequences with regard to keratinocyte differentiation and that the defect may be alleviated by reintroduction of functional receptor.

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Gene Expression; Humans; Keratinocytes; Ligands; Protein Precursors; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Transcription, Genetic; Transglutaminases; Tumor Cells, Cultured

In stratifying cultures of human keratinocytes, expression of the proto-oncoprotein c-JUN and the small proline rich 2 (SPRR2) protein, a precursor of the cornified cell envelope, are inversely related. Whereas c-JUN is typically found in basal proliferating cells, SPRR2 is restricted to suprabasal differentiating layers. Malignant keratinocytes (derived from squamous cell carcinoma, SCC) have reduced sprr2 expression, consistent with their low potential to differentiate, and express c-jun at higher levels than normal keratinocytes. A direct relation between c-jun and sprr2 expression was shown in several ways: transient ectopic expression of c-jun inhibits sprr2a promoter activity in normal differentiating cells, whereas in malignant keratinocytes a dominant negative c-jun mutant restored at least partially both the low promoter activity and the expression of endogenous sprr2. These effects are mediated via a 134 bp promoter fragment which does not include the sprr2a AP-1 binding site. Interestingly, in an SCC cell line, constitutively expressing the dominant c-jun mutant, expression of the terminal differentiation marker involucrin is also strongly increased, suggesting that c-JUN is a general modulator of keratinocyte terminal differentiation rather than only affecting the expression of sprr2.

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Cells, Cultured; Cornified Envelope Proline-Rich Proteins; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Genes, jun; Humans; Keratinocytes; Membrane Proteins; Promoter Regions, Genetic; Protein Precursors; Proto-Oncogene Proteins c-jun; Transfection; Tumor Cells, Cultured

Although breast carcinomas are considered to originate from glandular epithelial cells, some exhibit 'squamoid features', comprising stratification with a gradient in the nuclear-cytoplasmic ratio within individual cancer cell nests on microscopy. In parallel with a histological review of squamoid features, we immunohistochemically investigated the expression of involucrin, a marker of terminal squamous differentiation, in 223 breast carcinomas with one to three regional nodal metastases but no distant metastases and analysed their association with other clinicopathological parameters to explore their clinical and biological implications. Squamoid features and involucrin expression, detected in 22% and 27% of cases respectively, correlated with each other and were associated with high-grade atypia, a solid-nest pattern, cancer cell necrosis on histology and negative oestrogen receptor status. The incidence of regional recurrences was higher in patients with involucrin expression, whereas bone metastases were less frequent in groups with squamoid features or with diffuse (> or = 10%) involucrin expression. Both squamoid features and involucrin expression, which were considered to be derived either from differentiation into keratinocytes or from some kind of cellular degeneration caused by high turnover rate, are suggested to influence the biological behaviour of breast cancer cells in vivo, and they may be effective in predicting the most likely recurrence sites.

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Squamous Cell; Cell Differentiation; Female; Humans; Immunohistochemistry; Necrosis; Neoplasm Recurrence, Local; Protein Precursors

In normal skin, proliferation and differentiation are tightly coupled in order to maintain normal architecture in a continually renewing tissue. The temporal and spatial relationships between these two processes in normal, psoriatic, pre-neoplastic and neoplastic skin were investigated by a double immunolabelling technique with Ki67 as a marker of proliferation and involucrin as a marker of terminal differentiation. In normal skin, expression of the two antigens was strictly spatially segregated. In the abnormal, the proportions of cells expressing the antigens were increased with some loss of the spatial segregation, while small numbers of cells showed dual expression suggesting loss of the normal control between proliferation and differentiation. However, the quantitative ratio of proliferation to differentiation in psoriatic and pre-neoplastic skin was similar to the normal; transition to an invasive phenotype, however, was associated with a reversal of this ratio, and this correlated well with the degree of histological differentiation.

Topics: Bowen's Disease; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Humans; Immunohistochemistry; Ki-67 Antigen; Precancerous Conditions; Protein Precursors; Psoriasis; Skin; Skin Neoplasms

We analyzed the effects of three different calcium concentrations on the RNA and functional protein levels of transglutaminase (TGase) and involucrin (INV) over time in culture. We compared the results in normal human keratinocytes with those in a squamous cell carcinoma, SCC4. The highest calcium concentration (1.2 mM) induced the greatest levels of INV and TGase message, INV protein, and rates of CE formation, but not maximal levels of TGase protein. By examining cytosol and membrane fractions of keratinocytes, we found that after synthesis, TGase protein shifts, under the influence of calcium (both 0.1 mM and 1.2 mM), from the cytosol into the membrane in postconfluent cells. However, only 1.2 mM calcium induced significant amounts of TGase activity. These data indicate that elevated calcium (1.2 mM) achieves the expected induction in keratinocyte differentiation by regulation of not only INV and TGase message levels, but also the translation and activation of TGase protein. Our data suggest that this calcium-induced activation of TGase protein occurs while the protein is anchored in the membrane. In contrast, despite ample INV and TGase message levels within SCC4 cells, these RNA levels are not regulated by calcium or translated into protein, suggesting that the transformed phenotype of SCC4 cells results not only in a failure of calcium to regulate gene transcription, but also in a defect within the translation machinery of these differentiation-specific proteins.

Topics: Calcium; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line, Transformed; Cell Membrane; Cytosol; Enzyme Activation; Humans; Keratinocytes; Protein Precursors; Reference Values; RNA, Messenger; Skin Neoplasms; Transglutaminases; Tumor Cells, Cultured

Epidermal growth factor (EGF) is a potent mitogen for keratinocytes. Although the role of the EGF receptor in cell proliferation has been extensively studied, the consequences of EGF receptor activation with respect to cell differentiation remain less well characterized. Our studies demonstrate that stimulation of the EGF receptor substantially suppresses cellular differentiation in squamous cell carcinoma lines that overexpress the EGF receptor, as assessed by an EGF-dependent reduction of cornified envelope formation. Only a modest ligand-dependent decrease in cornified envelope formation was observed in normal keratinocytes. The response is dependent on the concentration of EGF and is evident after 1-2 days of EGF treatment. With extended EGF treatment, the messenger RNA levels for involucrin, a major structural component of the cornified envelope, were unaltered by EGF. In contrast, membrane-associated transglutaminase enzyme activity, which predominantly represents type 1 (keratinocyte) transglutaminase, is markedly inhibited by EGF. The lost of type 1 transglutaminase activity is associated with reduced levels of the messenger RNA and protein. These studies suggest that the functional consequences of EGF receptor activation in squamous cell carcinomas involve not only aberrant growth regulation, but, additionally, reduction of terminal differentiation capacity.

Topics: Blotting, Western; Carcinoma, Squamous Cell; Cell Differentiation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Keratinocytes; Male; Protein Precursors; RNA, Messenger; Transglutaminases; Tumor Cells, Cultured

Squamous carcinoma cells (SCC) fail to differentiate under conditions that are favorable for the growth and differentiation of normal human keratinocytes. Human keratinocytes differentiate from a highly proliferative basal cell to a terminally differentiated cornified cell in culture in the presence of physiological levels of extracellular calcium. 1,25-Dihydroxyvitamin D (1,25[OH]2D3) potentiates this process. Previous studies have shown that the differentiation process in keratinocytes is associated with increased expression of the genes for involucrin and transglutaminase, the products of which participate in cornified envelope formation. The mRNA for involucrin and transglutaminase was not detected in the SCC lines studied (viz. SCC4, 12B2, 12F2, A431, and HACAT) when they were grown in serum free medium. Addition of at least 2% fetal bovine serum for 48 h triggered the expression of these genes, which could then be maintained in the absence of serum. Serum was not required for induction of these genes in keratinocytes. In these cells, 1,25(OH)2D3 stimulated the expression of involucrin and transglutaminase in a concentration-dependent manner, while the SCC lines failed to respond to 1,25(OH)2D3 regardless of whether these cells had been pre-exposed to serum. An important factor that mediates 1,25(OH)2D3-stimulated gene expression is the vitamin D receptor, but vitamin D receptor mRNA levels in all the SCC lines examined were comparable to those in keratinocytes. Furthermore, the vitamin D receptor protein levels in SCC lines as assessed by ligand-binding analysis were comparable to those of keratinocytes. Thus, the mediators of 1,25(OH)2D3 action on gene expression other than the vitamin D receptor may be missing or defective in SCC lines, whereas the mediators of as yet undefined agents in serum may be better expressed in SCC lines than in keratinocytes. Our results indicate that, although SCC lines are capable of expressing the genes for the proteins involved in differentiation, the control of the expression of these genes by 1,25(OH)2D3 is abnormal in SCC despite the presence of a functional vitamin D receptor in concentrations equivalent to those in keratinocytes.

Topics: Animals; Calcitriol; Calcium; Carcinoma, Squamous Cell; Cattle; Cell Differentiation; Culture Media; Gene Expression Regulation, Neoplastic; Humans; Keratinocytes; Protein Precursors; Receptors, Calcitriol; RNA, Messenger; RNA, Neoplasm; Transglutaminases; Tumor Cells, Cultured

Non-small cell lung cancer (NSCLC) is fatal in approximately 90% of all cases due to the failure of systemic therapy, secondary to resistance to chemotherapy. In such malignancies new therapeutic paradigms are needed. One such approach takes advantage of normal physiologic growth regulatory mechanisms, such as terminal cellular differentiation or apoptosis. Suramin, as an antineoplastic drug, has shown efficacy in the treatment of prostate cancer and is capable of promoting differentiation in several human cancer cell lines. Little is known about the differentiating effects of suramin in lung cancer. In the present investigation we evaluated the ability of suramin to induce cross-linked envelope (CLE) formation, as a common marker for squamous differentiation and apoptosis, in three representative human non-small cell lung cancer cell lines: NCI-H226 (squamous), NCI-H358 (bronchoalveolar [adenocarcinoma]), and NCI-H596 (adenosquamous). Among agents that we have tested, suramin demonstrated the unique ability to induce spontaneous CLE formation in the two cell lines with squamous features, NCI-H226 and NCI-H596. Suramin induced CLE formation was accompanied by DNA fragmentation, a marker for apoptosis, in NCI-H596 and NCI-H358, but not in NCI-H226. Stimulation of CLE formation by suramin correlated with the rapid induction of both type II transglutaminase (TG) activity and involucrin expression. These parameters were protein synthesis independent, suggesting posttranslational mechanisms of suramin activity. Induction of differentiation/apoptosis markers by suramin did not correlate with its effect on growth. Modulation of signal transduction is a likely candidate mechanism for suramin activity in lung cancer. The relationship between growth, squamous differentiation, and apoptosis is considered.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Calcimycin; Carcinoma, Adenosquamous; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; DNA Fragmentation; Enzyme Inhibitors; Humans; Ionophores; Lung Neoplasms; Neoplasm Proteins; Protein Kinase C; Protein Precursors; Putrescine; Suramin; Transglutaminases; Tumor Cells, Cultured

We examined full thickness specimens of oesophageal squamous dysplasia from both cancer-free and cancer patients using immunohistochemical labelling for cytokeratin subtypes 10/13 and 14 and for involucrin, binding studies for various lectins, and PAS/D staining before and after diastase treatment. We studied specimens from patients with oesophageal carcinoma (52 normal epithelia, and 49 with mild, 38 with moderate, and 32 with severe dysplasia), and 32 specimens from cancer-free patients (five normal epithelia and 16 with mild and 11 with moderate dysplasia). Abnormal cytokeratin expression patterns in atypical cells, i.e. both cytokeratin 10/13 and cytokeratin 14 immunoreactivity in the same cells was detected in 41 of 99 specimens with dysplasias in cancer patients. Helix aspersa, Erythrina cristagalli and Robinia pseudoacacia binding was consistently negative in atypical cells in squamous dysplasia. The non-atypical layer of squamous dysplasia, which was morphologically indistinguishable from the corresponding layer of normal oesophageal squamous epithelium, showed abnormal involucrin expression in 39/ 101 specimens, Helix aspersa binding in 74/106, diastase sensitive PAS staining in 52/110, Erythrina cristaglli binding in 28/107, and Robinia pseudoacacia binding in 16/100. There were no significant differences in the expression of these markers in dysplasia between cancer patients and cancer-free individuals with the exception of increased Robinia pseudoacacia binding in the non-atypical layer in cancer-free patients. The results indicate that abnormal patterns of cytokeratin expression and lectin binding occur not only in atypical cells but also in non-atypical cells in oesophageal squamous dysplasia.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Lectins; Male; Middle Aged; Precancerous Conditions; Protein Binding; Protein Precursors

MIB-1 is an antibody which attaches to the Ki67 antigen expressed by proliferating cells. MIB-1 immunoreactivity may be used to quantify the proliferative component of a tumour. Involucrin is a protein expressed by mature keratinocytes and may be used as a marker of differentiation. The present paper studies the expression of these two markers in a group of patients with squamous carcinoma of the larynx. Tumour cell kinetics were studied in 49 patients with squamous cell carcinoma of the larynx using antibodies to "Ki67' and involucrin. The median potential follow-up for the group was 8.1 years with a minimum follow-up of 5 years. The median MIB-1 index was 32%. The median involucrin index was 56%. Fifteen patients had no or only slight involucrin staining whereas 34 stained intensely for this protein. Involucrin expression was found to be associated with histological grade with those patients expressing involucrin tending to have well differentiated tumours and those not expressing this parameter tending to have poorly differentiated tumours (P = 0.045). There were no other associations between host and tumour factors and the various biological parameters. Survival analysis demonstrated that patients with an involucrin count above the median value had a better 5-year survival than those below the median (89% and 56% respectively) (P < 0.05). In addition, patients with no (or poor) involucrin expression had an increased risk of developing a recurrence at the primary site (P < 0.05). Involucrin appears to be a promising marker of tumour differentiation and survival in squamous carcinoma of the larynx.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Division; Female; Follow-Up Studies; Humans; Ki-67 Antigen; Laryngeal Neoplasms; Larynx; Male; Middle Aged; Prognosis; Protein Precursors; Staining and Labeling; Survival Analysis; Time Factors

The expression of involucrin, a cytoplasmic protein synthesized during squamous maturation, was assessed by immunocytochemistry in different grades of cervical lesions. In normal/benign cervical epithelium and low-grade squamous intraepithelial lesions [SILS or cervical intraepithelial neoplasia (CIN)-1] involucrin showed intense and homogenous cytoplasmic expression in the spinal layers of 75 and 57% of samples, respectively. The basal cell layers showed no expression of involucrin. In high-grade SILs (CIN-2/3) 40% of the samples showed diffuse and focal cytoplasmic expression of involucrin in the differentiated basaloid cells. In the squamous cell carcinomas (SCCs) analyzed, well-differentiated tumors showed intense focal expression in 61% of the cases, moderately differentiated SCCs showed intense expression in 33% of the cases, while poorly differentiated SCCs (PDSCC) showed only a mild focal expression in 7% of cases. With increasing severity of the lesions, patchy expression of involucrin with a mixture of reactive and nonreactive cells predominated. Patterns of immunocytochemical staining for involucrin in cervical lesions of different grades, from low-grade to high-grade SILs, and invasive carcinoma may be of critical importance, if loss of involucrin expression is used as a criterion for neoplastic transformation in cervical epithelium. Our findings suggest that involucrin may be a sensitive marker in identifying the differentiation status of the lesion while the absence of involucrin in PDSCC may be helpful in differential diagnosis.

Topics: Adult; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Differentiation; Female; Humans; Immunohistochemistry; Middle Aged; Protein Precursors; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

The detection of cytokeratins in neoplastic tissues by immunohistochemical methods has numerous diagnostic and investigative applications, because cytokeratins are usually conserved in tumour cells during malignant transformation. Recently, however, it has been reported that progression to malignancy is associated with commencement of expression of low-molecular-weight cytokeratins. In the present study, 42 specimens from 35 cases of squamous cell carcinoma (SCC) of the skin were analysed by immunohistochemical techniques, using polyclonal anti-involucrin antibody and a panel of monoclonal antikeratin antibodies, in order to investigate the nature and differentiation of SCCs. The expression of cytokeratins and involucrin in well-differentiated SCCs was similar to that in normal epidermis. In contrast with well-differentiated SCCs, the expression of differentiation-specific cytokeratins and involucrin was diminished in the immature tumour cells in proportion to the malignancy of the SCCs. Some antibodies, however, stained all tumour cells, irrespective of the degree of malignancy. Furthermore, expression of simple epithelial and non-cornifying stratified squamous epithelial cytokeratins was observed in atypical tumour cells of poorly differentiated SCCs. It is of interest that similar expression was noted in many tumour cells in the lymph node metastases and in some tumour cells in the primary cutaneous lesions. Cytokeratin expression similar to that in normal epidermal keratinocytes was conserved in well-differentiated SCCs, but the expression of cytokeratins changed during progression to malignant transformation. The expression of simple epithelial or non-cornifying stratified squamous epithelial cytokeratins in cutaneous SCCs may be a marker for their capability of invasion and metastatic potential.

Topics: Antibody Specificity; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Humans; Immunohistochemistry; Keratins; Lymph Nodes; Lymphatic Metastasis; Protein Precursors; Skin Neoplasms

Histologic grade seems to be of limited prognostic significance in patients with vulvar carcinoma. However, the study of cytokeratin expression is of potential interest because it allows a more precise evaluation of the degree of squamous differentiation. This study was conducted to investigate whether differences in cytokeratin expression exist between normal vulvar epithelium and vulvar carcinoma and whether these differences are prognostically significant.. The expression of several differentiation markers, i.e., cytokeratin (CK) 10, CK 13, and involucrin, was studied in samples of 41 vulvar carcinomas. The expression of CK 8, 10, 13, and 14 was compared with CK expression in normal vulvar epithelium and was correlated with tumor grade and tumor growth pattern. Tumor growth pattern was considered type A if infiltrating tumor cell nests showed a layer of small, basaloid cells bordering the surrounding mesenchymal tissue and was considered type B if this was not the case. Prognosis was based on whether disease recurred or not.. Sixteen patients had disease recurrence. No prognostic significance of tumor grade was found. Tumor growth pattern was prognostically significant: in patients with a type A tumor, recurrence was observed less often than in patients with a type B tumor (P = 0.03). Cytokeratin 14, typical for basal cells of normal vulvar epithelium, was expressed in all tumors, whereas CK 8 was not expressed in any tumor. A relationship between tumor growth pattern and the concordant expression of differentiation markers was observed: in 55% of type A tumors and in none of type B tumors, concordant expression of CK 10, CK 13, and involucrin was found.. The expression of differentiation markers in vulvar carcinoma is related strongly to the tumor growth pattern, and this pattern is prognostically significant.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Differentiation; Female; Humans; Immunoenzyme Techniques; Keratins; Multivariate Analysis; Prognosis; Protein Precursors; Recurrence; Vulvar Neoplasms

The purpose of the present study was to establish culture conditions for human uterine cervical epithelial cells on permeable support and to determine how it affects cervical cell differentiation. Human ectocervical epithelial cells (hECE), HPV-16 immortalized hECE cells (ECE16-1) and Caski cells were grown on collagen-coated filters. Culture conditions, density of cells in culture and expression of epithelial and cervical-cell phenotypic markers were determined and compared in cells grown on filter and on solid support. Compared with the latter, cultures on filter had a higher cell density, hECE cells stratified to 5-12 cell layers compared to 1-3 on solid support, and cells of all three types expressed intercellular tight junctions. The cytokeratin profiles revealed differences between the three cell types as well as differences within the same cell species when grown on filter, compared to solid support. Of particular importance was the finding of a higher expression of K-13 in hECE grown on filter compared to solid support; K-13 is a marker of ectocervical cell differentiation. The cytokeratin profiles of the cultured hECE, ECE16-1 and Caski cells resembled those of ectocervical, squamous metaplastic and endocervical epithelia, respectively. hECE and ECE16-1 expressed involucrin protein, the level of which in both was higher in cells grown on filter compared to solid support. Polarization of the cultures was determined by morphology (stratification of hECE cells, expression of pseudomicrovilli in the apical cell membrane), selective apical vs. basolateral secretion of [35S]methionine- and [35S]cysteine-, [3H]fucose- and [14C]glucosamine-labeled molecules, and positive short-circuit current (Isc) under voltage-clamp conditions. Confluency of the cultures was determined by measuring transepithelial unidirectional fluxes of inert molecules with different molecular weights (MWs) through the paracellular pathway, and by measuring transepithelial conductance. The results indicated transepithelial permeability of 7-22.10(-6) cm.sec-1, which was 5-100 fold smaller compared to blank inserts, with a cut-off MW of 40-70 kDa for hECE and Caski cells. Transepithelial conductance ranged 18.5 to 51.5 mS.cm-2, indicating a leaky but confluent epithelia. Collectively the results indicate the epithelial nature of the cells and their improved differentiation when grown on filter support; hECE is a model for ectocervical epithelium while ECE16-1 and Caski express phenoty

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Cell Line, Transformed; Cell Polarity; Cells, Cultured; Ceramics; Cervix Uteri; Collagen; Culture Techniques; Epithelial Cells; Epithelium; Female; Humans; Hydrocortisone; Keratins; Permeability; Polytetrafluoroethylene; Protein Precursors; Tretinoin; Tumor Cells, Cultured; Uterine Cervical Neoplasms; Vimentin

The identification of pretreatment markers with predictive significance for the presence of lymph node metastases and treatment outcome in low stage cancer of the uterine cervix is clinically important. Because the presence of differentiation-related markers varies in this type of cancer, the authors investigated whether loss of these markers is related to a poor clinical course.. An indirect immunoperoxidase technique was applied to formalin fixed, paraffin embedded tissue sections of 80 patients with International Federation of Gynecology and Obstetrics Stage IB and IIA primary squamous cell cervical carcinomas for detection of expression of cytokeratin 10 and 13, and involucrin. Comparisons were made of the expression of each of these markers among 40 patients with regional node metastases and 40 age-matched patients with no lymph node metastases. Differences in the frequency of expression of these markers also were analyzed in relation to histopathologic characteristics, recurrence, and survival.. Expression of cytokeratin 10, 13, and involucrin was found in 24, 64, and 53%, respectively, of all patients studied. The authors found no differences between patients with positive regional lymph nodes and those with negative lymph nodes. Expression of cytokeratin 13 and involucrin was associated with tumor grade (P = 0.01). No relationship was found between expression of the markers used and recurrence or survival in the entire group. Within the lymph node-positive group, however, the survival rate of patients with tumors with cytokeratin 13 expression was significantly higher than that of patients with tumors lacking cytokeratin 13 expression (P = 0.02).. Expression of cytokeratin 10, 13, or involucrin in the primary tumor is of no predictive value with respect to the presence of regional lymph node metastases in low stage squamous cell cervical cancer. However, cytokeratin 13 expression appears to be of prognostic significance in patients with positive regional lymph nodes.

Topics: Adult; Aged; Antibodies, Monoclonal; Biomarkers; Carcinoma, Squamous Cell; Female; Humans; Immunoenzyme Techniques; Keratins; Lymphatic Metastasis; Middle Aged; Neoplasm Staging; Prognosis; Protein Precursors; Retrospective Studies; Uterine Cervical Neoplasms

The ability of all-trans-retinoic acid (RA) to modulate the growth and squamous differentiation of four head and neck squamous cell carcinoma cell lines (183, 886, 1483, and SqCC/Y1) was examined, and the relationship of their state of squamous differentiation and RA responsiveness to the expression of cytosolic RA-binding proteins (CRABPs), nuclear RA receptors (RARs), and retinoid X receptors (RXRs) was investigated. RA inhibited proliferation of all but the 183 cell line and suppressed squamous differentiation markers K1 keratin, type 1 transglutaminase, and involucrin mRNAs and proteins to varying degrees in 183, 1483, and SqCC/Y1 cells. Traces of CRABP-I mRNA were detected only in the 886 cells, whereas CRABP-II mRNA was detected in the other three cell lines. RA suppressed CRABP-II expression in SqCC/Y1 cells but had no effect on its expression in the other cell lines. All cell lines expressed mRNAs for RAR-alpha, RAR-beta, RAR-gamma, and RXR-alpha. The RAR-beta mRNA level was lowest in the SqCC/Y1 cells, and RXR-beta and RXR-gamma were not detected in any of the cell lines. RA treatment increased the levels of the three RAR mRNAs in most of the cell lines but had no effect on the RXR mRNAs. The CRABP-II mRNA level in SqCC/Y1 cells was lowest in cells grown in serum-free medium and increased when the cells were grown in medium with 5 or 10% serum. In contrast, the RXR-alpha mRNA level was inversely related to serum concentration. The results show that, in head and neck squamous cell carcinoma cells, there are no simple relationships among the expression of CRABPs, RARs, and RXRs and either squamous differentiation or response to RA-induced growth inhibition or suppression of squamous differentiation.

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Head and Neck Neoplasms; Humans; Keratins; Protein Precursors; Receptors, Retinoic Acid; RNA, Messenger; Transglutaminases; Tretinoin; Tumor Cells, Cultured

Expression of cytoskeletal proteins has been shown to be dependent on the differentiation status of the tissue. In the present study, expression of two cytoskeletal proteins normally present in terminally differentiated keratinocytes, namely cytokeratin types 10/11 and involucrin, were studied in different stages of tumour progression in the oral mucosa. Results showed that cytokeratins 10/11 and involucrin strongly correlated with the differentiation status of cells. High expression was observed in non-dysplastic hyperplastic epithelium as compared to normal, dysplastic and neoplastic epithelium. In addition, various grades of dysplasia showed an inverse correlation with expression of these proteins. Statistical analysis of the results also showed a negative correlation between the differentiation of upper spinal cells and the stage of tumour progression. It therefore appears that the proteins studied may be useful as markers for epithelial carcinogenesis.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Differentiation; Cell Transformation, Neoplastic; Humans; Immunoenzyme Techniques; Keratinocytes; Keratins; Leukoplakia, Oral; Mouth Mucosa; Mouth Neoplasms; Protein Precursors

Somatic cell hybrid cell lines derived from the fusion of human squamous carcinoma cells (FaDu-Hyg) with human keratinocytes were used to examine the relationships between cell differentiation and tumorigenicity. Treatment of the parental keratinocytes or the two hybrid cell lines with the combination of calcium and fetal bovine serum increased the expression of the envelope precursor, involucrin, 4- to 8-fold, whereas it remained unchanged in FaDu-Hyg cells. Similarly, calcium- and serum-treated keratinocytes and the two hybrid cell lines displayed a 7- to 13-fold increase of the activity of membrane-associated type I transglutaminase, whereas transglutaminase activity in FaDu-Hyg cells did not change appreciably. FaDu-Hyg cells were tumorigenic in vivo, but tumorigenicity was suppressed in both hybrid cell lines. Analysis of additional tumor cell lines indicated that the expression of transglutaminase I and involucrin are under separate genetic control and that loss of transglutaminase activity can result either from a lack of protein or from a defect in the activation step. Thus, keratinization of squamous epithelial cells appears to be controlled by several different recessive genes, which cosegregate with but are probably only partly identical with the genes that suppress tumor formation in vivo.

Topics: Animals; Calcium; Carcinoma, Squamous Cell; Cattle; Cell Differentiation; Cell Fusion; Cell Line; Culture Media; Gene Expression; Humans; Hybrid Cells; Keratinocytes; Male; Mice; Mice, Nude; Protein Precursors; Transglutaminases; Transplantation, Heterologous; Tumor Cells, Cultured

More than 95% of lung cancers occur in the bronchi, appearing as adenocarcinoma, squamous carcinoma, large-cell and small-cell carcinoma, or mixed types. Generally, the least aggressive form is squamous cell lung cancer, suggesting the possibility that promotion of squamous cell differentiation may have therapeutic potential for non-small-cell lung cancer, a disease having no effective systemic therapy. Interferons are a group of glycoproteins with known antiproliferative effects, including the ability to induce differentiation in certain cases.. These studies were conducted to determine whether interferon beta induces squamous cell differentiation in non-small-cell lung cancer in vitro.. NCI-H596 adenosquamous cells were grown to confluence to maximize their differentiation potential. Growth and parameters for squamous differentiation (cross-linked envelope competence, transglutaminase activity, and relative involucrin expression) were then measured when the cells were exposed to various concentrations of interferon beta.. Interferon beta inhibited growth of the NCI-H596 cell line and stimulated envelope competence, involucrin expression, and type 2 transglutaminase activity. Alterations in transglutaminase activity and involucrin expression preceded induction of envelope competence and growth suppression.. Interferon beta suppresses the growth and stimulates markers of squamous differentiation in NCI-H596. While the mechanism(s) for such effects are unknown, the sequence of effects suggests a causal relationship between differentiation induction and subsequent growth suppression.. Interferon beta may have clinical usefulness in squamous differentiation strategies for the treatment of non-small-cell lung cancer. More must be learned about the mechanisms whereby interferons and other biologic agents induce differentiation, and clinical trials will be needed to determine whether in vitro results are pertinent in vivo.

Topics: Apoptosis; Carcinoma, Adenosquamous; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Humans; Interferon-beta; Lung Neoplasms; Membrane Proteins; Neoplasm Proteins; Protein Precursors; Transglutaminases; Tumor Cells, Cultured

Syndecans comprise a family of integral membrane proteoglycans that presumably participate in cell-matrix interactions and the modulation of growth factor response. Expression of syndecan-1, a cell surface proteoglycan that binds basic fibroblast growth factor (bFGF) and extracellular matrix components, was studied in cultured human keratinocytes from the oral mucosa and in tissue sections derived from various epithelia and carcinomas of the head and neck. For the study, polyclonal antibodies were raised against the core protein of human syndecan-1. The affinity-purified antibody (designated anti-P117) was shown to react specifically with syndecan-1. Abundant expression of syndecan-1 was detected in frozen sections of various stratified epithelia as well as in cultured keratinocytes. Keratinocytes located in the spinous cell layers showed intense immunoreactivity for syndecan-1, while basal cells stained rather weakly, suggesting that the expression of syndecan-1 could be stimulated during the differentiation of keratinocytes. Cultured human keratinocytes were induced to terminally differentiate by increasing the extracellular calcium concentration of the medium. Parallel to the induction of involucrin expression, the mRNA levels of syndecan-1 were found to increase, suggesting that syndecan-1 is indeed induced during keratinocyte differentiation. The molecular mass and glycosaminoglycan composition of syndecan-1 did not change markedly during calcium-induced differentiation. Malignant transformation was associated with marked reduction of syndecan-1 expression, based on the immunoreactivity of anti-P117 in frozen sections from squamous cell carcinomas (SCCs) of the head and neck.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Animals; Blotting, Northern; Breast; Calcium; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Female; Fibroblasts; Head and Neck Neoplasms; Immune Sera; Immunohistochemistry; Keratinocytes; Mammary Glands, Animal; Membrane Glycoproteins; Mice; Molecular Sequence Data; Precipitin Tests; Protein Precursors; Proteoglycans; RNA, Messenger; Syndecan-1; Syndecans; Tumor Cells, Cultured

The expression of cytokeratins (CK) 1, 4, 5/6, 8, 13, 18, 19 and 20 and involucrin in 42 cases of squamous cell carcinomas from various locations was examined. The tumours expressed CK5/6 in 55%, CK8 in 76%, CK13 in 43% and CK19 in 95% of cases. The CK5/6-positive primary tumours were from uterine cervix, head and neck, lung, skin, oesophagus and urinary bladder, and the CK13-positive primary tumours were from uterine cervix, lung and vulva. Metastatic squamous cell carcinomas from head and neck more frequently expressed CK5/6 and 13, 7/7 (100%) and 6/7 (86%) compared with 3/5 (60%) and 0/5 (0%) in the primary squamous cell carcinomas. Few cases were CK1, CK4 and CK18 immunoreactive. CK20 immunoreactivity was not observed. Involucrin was expressed in 71% of tumours, and most of the involucrin-positive cells were located at the central parts of tumour cell clusters except for one case in which the peripheral cells around tumour cell clusters were positive. Thus, expression of the so-called simple epithelial markers CK8 and CK19 occurs in the majority of squamous cell carcinomas. The absence of CK20 immunoreactivity may be helpful in differential diagnosis.

Topics: Carcinoma, Squamous Cell; Female; Humans; Immunohistochemistry; Keratins; Lung Neoplasms; Lymphatic Metastasis; Mouth Neoplasms; Protein Precursors; Skin Neoplasms; Urinary Bladder Neoplasms; Uterine Neoplasms

Using tetradecanoylphorbol 13-acetate (TPA), a known inducer of epithelial cell differentiation, and Northern blot analysis, we investigated in normal versus well-differentiated malignant keratinocytes the modulation of genes implicated in their growth or differentiated function. In normal keratinocytes transient c-fos induction was detected within 30 min after stimulation and was followed by rapid down regulation of the proto-oncogenes c-myc and epidermal growth factor receptor. Within hours after stimulation mRNA levels for three well-characterized differentiated keratinocyte products, involucrin, interleukin-1 beta (IL1-beta), and fibronectin, were induced, as were those for a growth arrest and DNA damage-inducible (GADD 153) gene and a small proline-rich (SPR1) gene, both known to be associated with differentiation but with of as yet unknown functions. Heat shock protein 70 gene was initially down regulated and was induced only after 48 h. The well-differentiated malignant keratinocyte cell line differed in that the c-fos, GADD, SPR1, and IL1-beta genes had several-fold higher induction, but involucrin mRNA was undetectable and fibronectin mRNA was only minimally induced after TPA stimulation. Malignant cells reached terminal differentiation faster than normal keratinocytes as measured by inability to exclude trypan blue dye, and in situ hybridization using a riboprobe for the differentiation-associated SPR1 gene showed that normal keratinocytes constitutively express this transcript while malignant keratinocytes with virtually identical morphology and growth rate do not. These studies greatly expand our understanding of gene activation and down regulation during normal keratinocyte differentiation and imply that a malignant cell line, even when retaining the phenotype of normal cells, differs in its response to outside stimuli, furnishing at best an imperfect model for investigating the molecular mechanisms of cellular differentiation.

Topics: Carcinoma, Squamous Cell; CCAAT-Enhancer-Binding Proteins; Cell Differentiation; Cornified Envelope Proline-Rich Proteins; DNA Damage; ErbB Receptors; Fibronectins; Gene Expression Regulation; Genes, fos; Genes, myc; Heat-Shock Proteins; Humans; Infant, Newborn; Interleukin-1; Keratinocytes; Kinetics; Male; Membrane Proteins; Protein Precursors; Proteins; RNA, Messenger; Skin Neoplasms; Tetradecanoylphorbol Acetate; Time Factors; Transcription Factor CHOP; Transcription Factors; Transcriptional Activation; Tumor Cells, Cultured

Adenosquamous carcinoma and squamous cell carcinoma (SCC) occur rarely in the liver compared with adenocarcinoma, and the histogenesis and biologic behaviors of these tumors remain unknown. The authors addressed these issues in the current article.. A specimen aseptically obtained from the surgically resected cholangiocellular carcinoma (CCC) was cut into pieces and inoculated into the back of a nude mouse, bilaterally. The developed tumors were resected and serially transplanted into nude mice. The morphologic features and growth kinetics of the nude mouse tumors at different passages were compared.. The authors established a new human CCC nude mouse strain, designated nuKMC-2, from a 64-year-old woman. The original tumor of the patient showed the features of moderately differentiated tubular adenocarcinoma with small sheet-like arrangement of polygonal cells. The initial tumor developed in a nude mouse showed morphologic features similar to the original tumor. With the serial transplantation to nude mice, the components of tubular adenocarcinoma diminished, and all of the nuKMC-2 was replaced by SCC. Doubling times of nuKMC-2 at the 5th and 11th passages were 9.9 and 7.4 days, respectively, which suggested that the tumor with squamous components were more aggressive biologically than the adenocarcinoma.. The results suggested that adenosquamous carcinoma might be a transitional form from adenocarcinoma to SCC and that some of the primary hepatic SCC might originate from adenocarcinomas.

Topics: Adenocarcinoma; Adenoma, Bile Duct; Animals; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Bile Duct Neoplasms; Biomarkers, Tumor; Carcinoma, Squamous Cell; Female; Humans; Immunohistochemistry; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Transplantation; Protein Precursors; Serpins

The squamous cell carcinoma line, SqCC/Y1, like natural squamous epithelia, forms a cornified cell envelope during differentiation which can be directly correlated with an increase in particulate transglutaminase activity. When transglutaminase is activated in these cells by calcium ionophore X-537A, annexin I and involucrin become incorporated into the cornified cell envelope and cannot be extracted with solutions containing sodium dodecyl sulfate (SDS) and beta-mercaptoethanol. This effect is specific for annexin I; thus, the amounts of annexins II and IV that were extractable from cells by SDS and beta-mercaptoethanol did not change following treatment with ionophore X-537A. Annexin I could be cross-linked in vitro to itself and to other endogenous proteins by transglutaminase extracted from the particulate fraction of SqCC/Y1 cells. Immunofluorescence studies showed that cross-linked annexin I and involucrin form an envelope-like structure in SqCC/Y1 cells during differentiation that cannot be extracted by EGTA and Triton X-100. The amount of staining of this envelope structure corresponded directly to the particulate transglutaminase activity of these cells. Annexin I monoclonal and polyclonal antibodies were shown to bind to purified cornified cell envelopes from SqCC/Y1. These studies suggest that particulate transglutaminase regulates a function of annexin I during the differentiation of SqCC/Y1 cells by covalently cross-linking this protein into the cornified cell envelope.

Topics: Annexins; Antibodies; Calcium-Binding Proteins; Carcinoma, Squamous Cell; Cell Differentiation; Enzyme Induction; Membrane Proteins; Microscopy, Fluorescence; Protein Precursors; Transglutaminases

Three human lung squamous carcinoma cell lines (NX002, CX140 and CX143) demonstrate features of squamous differentiation including involucrin synthesis and competence to form cornified envelopes. 12-O-Tetradecanoylphorbol 13-acetate inhibits growth of these cell lines and this growth inhibition is associated with enhanced differentiation.

Topics: Antigens, Neoplasm; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Humans; Keratins; Lung Neoplasms; Protein Precursors; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Although EGF receptor expression is generally elevated in human lung squamous carcinoma, the biological significance of this phenomenon and the role of EGF and TGF-alpha in this disease are poorly understood. We have investigated three human lung squamous carcinoma cell lines (NX002, CX140 and CX143) and have shown, using an antibody (EGFR1) directed against the EGF receptor, that the majority of cells in all three lines express the EGF receptor. Using a ligand binding assay, Scatchard analysis indicated high concentrations (1,300-2,700 fmol mg-1 protein) of a single low affinity binding site (Kd = 3-5 nM) within these lines. Addition of EGF or TGF-alpha at concentrations greater than 0.1 nM resulted in growth inhibition of all three lines and this was associated with an accumulation of cells in the G2/M phase of the cell cycle. Growth inhibitory effects were not explained by an enhancement of cellular differentiation as monitored by involucrin expression and the ability to form cornified envelopes. While the presence of EGF could not be detected in medium conditioned by the NX002 cell line, mRNA for TGF-alpha was detected in all three lines suggesting the possibility of an autocrine loop. These results together with reports of growth inhibition by EGF and TGF-alpha in other systems suggest that EGF and similar molecules might have a growth regulatory role in lung cancer cells and modulation of such may have therapeutic potential.

Topics: Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cell Line; Culture Media; Epidermal Growth Factor; ErbB Receptors; Humans; Immunohistochemistry; Lung Neoplasms; Protein Precursors; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha

Retinoids (vitamin A analogues) inhibit the squamous differentiation of normal and malignant epithelial cells. This study investigated the ability of the head-and-neck squamous-cell carcinoma (HNSCC) cell line 1483 to undergo squamous differentiation in the absence and presence of beta-all-trans retinoic acid (RA). The growth of these cells in culture is accompanied by an increase in keratinocyte transglutaminase, involucrin and keratin KI, 3 established markers of squamous cell differentiation. Higher levels of these differentiation markers were detected in cells cultured in delipidized serum (DLS), from which endogenous retinoids have been extracted, than in cells cultured in fetal bovine serum (FBS), which contains retinoids. Treatment with I microM RA decreased the levels of the various differentiation markers in cells cultured in either FBS or DLS as revealed by immunofluorescent labelling of permeabilized cells and by immunoblotting of cell extracts using specific monoclonal or polyclonal antibodies. The cells' ability to cross-link proteins to form envelopes under the plasma membrane was stimulated in the presence of calcium ionophore but inhibited by RA. These results indicate that the malignant 1,483 HNSCC cells recapitulate the main characteristics of normal squamous-cell differentiation in culture and that RA suppresses this differentiation as it does in normal keratinizing epithelial cells.

Topics: Calcium; Carcinoma, Squamous Cell; Cell Differentiation; Fluorescent Antibody Technique; Head and Neck Neoplasms; Humans; Immunoblotting; Ionophores; Keratinocytes; Keratins; Neoplasm Proteins; Protein Precursors; Retinoids; Transglutaminases; Tretinoin; Tumor Cells, Cultured

We have compared expression of involucrin, E-cadherin and P-cadherin in cultures of normal keratinocytes and in five different lines derived from squamous cell carcinomas (SCCs), using Northern analysis and immunofluorescence. In normal keratinocytes there was an inverse correlation between P-cadherin and involucrin expression, whereas E-cadherin was expressed by both basal and terminally differentiating cells. In SCC lines involucrin expression was lower than in normal keratinocytes, and there was variable expression of P- and E-cadherin: E-cadherin mRNA levels tended to be lower in SCC lines than in normal keratinocytes, whereas P-cadherin levels were similar. Our results are consistent with observations of cadherin expression in vivo and suggest that the cultures provide a useful experimental model for investigating the role of cadherins in determining the spatial organization of normal and neoplastic keratinocytes.

Topics: Cadherins; Carcinoma, Squamous Cell; Cells, Cultured; Fluorescent Antibody Technique; Humans; Keratinocytes; Protein Precursors; RNA, Messenger

Epidermolysis bullosa represents a grouping of inherited skin diseases characterized by epidermal fragility and frequently wounded skin. The recessive dystrophic subtype of epidermolysis bullosa (RDEB) is characterized by extensive dermal scarring after healing of repeated epidermal injuries and by an unusually high incidence of squamous cell carcinoma occurring in chronically wounded skin. In contrast, the simplex form of epidermolysis bullosa usually heals without scarring and does not predispose to malignant neoplasms of the skin. The differences in scarring and the neoplastic potential of these two forms of epidermolysis bullosa prompted us to investigate growth activation and differentiation characteristics in epidermal keratinocytes in individuals with these disorders. The expression of filaggrin, involucrin, cytokeratins, and the growth activation marker psi-3 was examined by immunohistochemistry in skin biopsy specimens from four individuals with epidermolysis bullosa simplex and six individuals with RDEB. Previous experiments using this technique have demonstrated that these antibodies are good markers for identifying growth-activated keratinocytes in wounded and hyperplastic epidermis. All biopsy specimens of healed wounds in skin from patients with RDEB showed epidermis that reacted with antibodies to filaggrin, involucrin, specific cytokeratins, and psi-3 in a growth-activated pattern. This growth-activated phenotype was maintained in keratinocytes from previously wounded skin that had been healed for more than 2 years. The RDEB growth-activated phenotype detected by immunohistochemistry was not associated with microscopically detectable epidermal hyperplasia. In contrast, all cases of epidermolysis bullosa simplex examined showed an epidermal phenotype similar to that of keratinocytes in normal skin. Thus, healing with dermal scar formation in RDEB is associated with a persistent growth-activated immunophenotype of epidermal keratinocytes. This chronic growth activation state or failure of cells to differentiate in a normal fashion may be directly linked to the high incidence of squamous cell cancers in individuals with RDEB.

Topics: Adolescent; Adult; Antigens; Carcinoma, Squamous Cell; Epidermis; Epidermolysis Bullosa; Female; Filaggrin Proteins; Humans; Infant; Infant, Newborn; Intermediate Filament Proteins; Keratinocytes; Keratins; Male; Middle Aged; Protein Precursors; Retrospective Studies; Skin Neoplasms

While some cutaneous squamous cell carcinomas (SCC) arise from predisposing conditions such as burn scars, draining sinuses, and chronic, nonhealing wounds, the vast majority of these tumors arise from actinically damaged epidermis. It has been shown previously that keratinocytes within healing wounds show an "activated" immunophenotype when stained with antibodies to psi-3, involucrin, filaggrin, and cytokeratins. A similar pattern has been seen in keratinocytes from patients with recessive dystrophic epidermolysis bullosa (RDEB), in whom the incidence of cutaneous SCC is markedly increased. We tested the hypothesis that actinic keratoses (AK), recognized as precursors in the development of the majority of SCC, would show a similar activated immunophenotype when stained with the antibody panel described above. We examined 10 AK, biopsied from the facies and extremities of ten patients, ages 60 to 80, with antibodies to psi-3, involucrin, filaggrin, and AE1. All lesions examined had an immunostaining pattern indistinguishable from that seen in keratinocytes from patients with RDEB or within healing wounds. There was suprabasilar staining of keratinocytes with antibodies to psi-3 and AE1. Involucrin and filaggrin was expressed by all keratinocytes above the midstratum spinosum. Within the acrosyringia and acrotrichia, the staining pattern was that of the normal epidermis, i.e., AE1 staining of basal keratinocytes, granular layer staining of involucrin and filaggrin, and absence of psi-3 expression. These data suggest that an activated keratinocyte phenotype is a unifying feature in conditions which predispose to development of cutaneous SCC.

Topics: Aged; Aged, 80 and over; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Epidermis; Female; Filaggrin Proteins; Humans; Intermediate Filament Proteins; Keratinocytes; Keratins; Keratosis; Male; Middle Aged; Phenotype; Precancerous Conditions; Protein Precursors; Skin Neoplasms; Sunburn

The well and poorly differentiated oral squamous carcinomas preferentially express proteins, blood group antigens, and contain associated dendritic Langerhans' cells. Keratin pearls in well-differentiated carcinomas simulate the differentiation pathway of the normal oral squamous epithelium, whereas poorly differentiated carcinomas do not and appear more heterogeneous. Terminally keratinized cells correlate with involucrin and expression of blood group antigens in keratin pearls, a feature that differs from the nonkeratinizing normal epithelium in which such carcinomas arise. Dendritic Langerhans' cells are reduced in number in squamous carcinomas.

Topics: ABO Blood-Group System; beta 2-Microglobulin; Carcinoembryonic Antigen; Carcinoma, Squamous Cell; Dendritic Cells; Fibronectins; HLA Antigens; Humans; Keratins; Laminin; Langerhans Cells; Mouth Neoplasms; Protein Precursors; S100 Proteins

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Differentiation; Drug Evaluation; Female; Humans; Immunoenzyme Techniques; Isotretinoin; Keratins; Male; Micronucleus Tests; Middle Aged; Mouth Neoplasms; Protein Precursors; Transglutaminases

Lung carcinoma cell lines were analyzed in culture and in nude mouse xenograft for both morphological appearance and expression of specific proteins that participate in cross-linked envelope formation during normal squamous cell terminal differentiation. Cross-linked envelope formation, induced by artificial influx of millimolar Ca2+ into the cultured cells, was an exclusive trait of squamous, adenosquamous, and mucoepidermoid carcinomas. Small cell lung carcinoma and non-squamous non-small cell lung carcinoma lines, such as adenocarcinoma and large cell carcinoma, were uniformly negative for cross-linked envelope formation. Involucrin, which is incorporated into the cross-linked envelope by the enzyme transglutaminase, was expressed at highest levels in squamous tumors, but several of the non-squamous non-small cell lung carcinoma lines also expressed comparable amounts. On the other hand, transglutaminase activity was consistently higher in squamous as opposed to non-squamous lines, so that in cell culture, a clear contrast between the groups could be observed. A Mr 195,000 protein that is incorporated into cultured human epidermal cell cross-linked envelopes was also observed in some but not all of the squamous lines. Two forms of transglutaminase are expressed in cultured keratinocytes. One of them, tissue transglutaminase, was expressed in the majority of squamous cell lines even though it is not a normal product of squamous differentiation in vivo. Keratinocyte transglutaminase, which is distinct from the tissue form and is normally expressed during terminal differentiation in squamous epithelia. was measurably present in only one of the six squamous cell lines tested. In nude mouse xenografts, keratinocyte transglutaminase, localized immunohistochemically with a biotinylated mouse monoclonal antibody, was again present only in a minority of the squamous lines whereas involucrin was expressed in all. In contrast to involucrin, keratinocyte transglutaminase is not an obligatory component of squamous differentiation in the pulmonary carcinoma cell lines tested. Its expression may be of value in further refining their classification.

Topics: Animals; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line; Cytosol; Humans; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Protein Precursors; Transglutaminases; Transplantation, Heterologous; Tumor Cells, Cultured

Three cell lines of squamous-cell carcinoma and 3 of large-cell carcinoma origin were investigated for the expression of differentiation markers and functional parameters (proliferation, morphology, cornified envelope formation, involucrin staining, transglutaminase activity, adhesiveness and migration) under normal cell culture conditions and after treatment with the tumor promoter phorbol-12-myristate-13-acetate (PMA). Although all original tumors had been described as poorly differentiated by histological grading, we found significant heterogeneity in the expression of differentiation markers in cell culture. A systematic grading of the cell lines became possible only after PMA stimulation. PMA generally increased expression of differentiation markers in cell lines of comparably low grades of differentiation, as indicated by dose-dependent inhibition of proliferation and cloning efficiency, induction of squamous markers, and decreased adhesiveness and cell motility. In contrast, cell lines of apparently higher differentiation by these criteria showed little response to PMA. The results presented show that the assessment of differentiation capacity by comparison of differentiation markers under normal cell culture and PMA-stimulated conditions in established NSCLC cell lines allows for a refined cell culture grading, which might advance the classification and characterization of such cell lines which, otherwise, appear to be very heterogeneous. It may also help to correlate cellular functions with various states of differentiation in vitro.

Topics: Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Adhesion; Cell Line; Cell Transformation, Neoplastic; Chemotaxis; Dose-Response Relationship, Drug; Humans; Immunohistochemistry; L-Lactate Dehydrogenase; Lung Neoplasms; Phorbol Esters; Protein Precursors; Transglutaminases; Tumor Cells, Cultured

Exploiting the sensitivity of neoplastic keratinocytes to physiological effectors, this work analyzes the degree of coordination among differentiation markers in the established human epidermal squamous carcinoma cell line SCC-13 in comparison to normal human epidermal cells. This analysis showed that overall keratin content was modulated substantially and in parallel with particulate transglutaminase activity in response to variation of calcium, retinoic acid, and hydrocortisone concentrations in the medium. The changes in keratin expression were evident primarily in the striking stimulation by hydrocortisone or calcium and the virtual suppression by retinoic acid of species in the 56-58 kd region, which have not previously been reported subject to such physiological modulation. In contrast, involucrin levels were coordinated only to a limited degree with particulate transglutaminase activity and keratin content. The very low involucrin levels observed in low calcium medium were increased 5- to 10-fold in high calcium medium. However, they were also increased 5- to 30-fold in low calcium medium by retinoic acid, a clear example of uncoupling. Activities of the tissue transglutaminase were altered considerably by the various culture conditions but were not obviously coordinated to keratinocyte markers. In normal epidermal cells, the suppressive effect of retinoic acid was much more evident with particulate transglutaminase than involucrin levels. While calcium had a large stimulatory effect on both markers, hydrocortisone had little or no influence. These results emphasize the potential importance of quantitative analysis of differentiation markers for resolving the contribution of physiological elements in coordination of cellular programming.

Topics: Calcium; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line; Epidermal Cells; Humans; Hydrocortisone; Keratins; Protein Precursors; Transglutaminases; Tretinoin

The distribution of several markers of keratinocyte differentiation was studied in normal epidermis, basal cell carcinomas (BCCs), and squamous cell carcinomas (SCCs) using the immunoperoxidase technique on frozen sections of punch biopsy specimens. As markers a panel of chain-specific monoclonal antibodies (MoAbs) directed against cytokeratin (CK) 4, 8, 10, 13, 18 and 19, a polyclonal antiserum against involucrin, as well as a MoAb against the epidermal growth factor (EGF) receptor were used. In 15 out of 19 BCCs tested, expression of CK 8 was seen. Only a few individual cells in a limited number of BCCs showed positive staining for CK 4, 18, or 19. No expression of CK 10 was seen except for some foci of cell keratinization. Involucrin was not found in BCCs except for some squamous horn cysts. In all BCC cells expression of EGF receptor was found. In the suprabasal layers of normal epidermis from SCC patients, positive staining for CK 10 was seen. A few individual cells in a limited number of SCCs showed positive staining for CK 4, 8, or 18. Involucrin was expressed in the center of SCCs and in the upper layers of normal epidermis. Expression of EGF receptor was found in all SCC cells. These results demonstrate differences in cellular origin and differentiation between BCC and SCC.

Topics: Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Epidermal Cells; Epidermis; ErbB Receptors; Humans; Immunoenzyme Techniques; Keratins; Protein Precursors; Skin Neoplasms

Cell line MDA 886Ln was established from a laryngeal lymph node metastasis. When grown as a multicellular tumor spheroid (MTS), it exhibits squamous differentiation. We studied the effects of beta-all-trans retinoic acid (RA) on the growth, differentiation and glycoprotein content of this MTS model for squamous carcinomas of the head and neck. The growth of MTSs was inhibited in a dose-dependent manner by 10(-6) to 10(-10) M RA. Growth inhibition occurred between 3 and 5 days of RA treatment (10(-6)M). Immunohistochemical and electrophoretic analyses revealed that RA suppressed the morphological markers of squamous differentiation (squames), involucrin expression, and keratin expression. Gly-coprotein expression was examined by metabolic labelling using 3H-glucosamine, in situ labelling of polyacrylamide gels with 125I-labelled wheat-germ agglutinin (WGA), localization of fluorescein isothionate-WGA in frozen sections, and determination of sialyltransferase activity. Treatment using 10(-6) M RA altered glycoprotein expression both biochemically and morphologically, and WGA was shown to bind preferentially to sialic acid residues. The sensitivity of this MTS model to RA treatment and its ability to be analyzed through morphological, immunohistochemical and biochemical techniques suggest that it will prove useful in studying the relationships between growth, differentiation and RA-induced alterations in squamous carcinomas.

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Glycoproteins; Head and Neck Neoplasms; Humans; Keratins; Male; Molecular Weight; Neoplasm Proteins; Organoids; Protein Precursors; Tretinoin; Tumor Cells, Cultured

As the distribution pattern of cytokeratin (CK), filaggrin and involucrin has recently been suggested to discriminate between benign and malignant epithelial growths, biopsies of healthy oral mucosa, leukoplakias without and with dysplasia and squamous cell carcinomas were examined immunohistochemically using a panel of 4 monoclonal antibodies (AB) against different cytokeratin polypeptides (34 beta E12, KL1 and Pkk1) and filaggrin as well as a polyclonal AB to involucrin. Major and statistically significant differences were observed in the profiles of CKs (except Pkk1), filaggrin and involucrin between leukoplakias without and with epithelial dysplasia. However, the alteration in the expression of CKs, filaggrin and involucrin proved to be not a constant feature in leukoplakias with dysplasia as a considerable portion (20-25%) of them revealed the profiles of CKs, filaggrin and involucrin similar to those of benign leukoplakias, and vice versa. Immunostaining of these antigens did not define the diagnosis of dysplasia in leukoplakias more precisely than grading in conventional histology can do so far. However, immunohistochemical sensitivity in detecting a broad range of variation in the abnormal maturation patterns of keratinocytes in leukoplakias with dysplasia can be used to divide these lesions into subgroups to elucidate their prognosis in follow-up studies.

Topics: Carcinoma, Squamous Cell; Epithelium; Female; Filaggrin Proteins; Humans; Hyperplasia; Immunoenzyme Techniques; Intermediate Filament Proteins; Keratins; Leukoplakia, Oral; Male; Middle Aged; Mouth Mucosa; Mouth Neoplasms; Phosphoproteins; Protein Precursors

We used immunoperoxidase methods employing antibodies against involucrin and filaggrin, both markers of squamous terminal differentiation, to study squamous metaplastic transformation in the human endocervix. Expression of involucrin and filaggrin was restricted to squamous metaplastic cells whereas columnar epithelial cells were constantly negative. Immature squamous metaplastic epithelium also showed a positive immunostaining. In mature squamous metaplasia a suprabasal homogeneous staining pattern similar to that found in the exocervical epithelium was detected, although with full-thickness filaggrin immunoreactivity in 45% of cases (P less than 0.05). These results support the hypothesis of an epithelial origin of reserve subcolumnar cells, and suggest that precocious squamous differentiation seems to take place in metaplastic cells of the human endocervix.

Topics: Adult; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cervix Uteri; Epithelium; Female; Filaggrin Proteins; Humans; Intermediate Filament Proteins; Protein Precursors; Uterine Cervical Neoplasms

Expression of involucrin was investigated immunohistochemically in 27 cases of urinary bladder carcinoma. Although no keratinization was observed in the transitional cell carcinomas examined all displayed involucrin staining to various degrees. Involucrin expression in foci of G-I transitional cell carcinomas was classified into 3 types: type 1, a mixture of intensely stained and slightly positive cells; type 2, highly positive cells intermingled with negative tumour cells; and type 3, all tumour cells slightly positive. Undifferentiated cell carcinomas demonstrated an irregular distribution of involucrin of varying staining intensity while deposition in squamous cell carcinomas was limited to keratinized areas.

Topics: Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Humans; Immunoenzyme Techniques; Protein Precursors; Urinary Bladder Neoplasms

Forty nasopharyngeal carcinomas (NPC) were studied by immunohistochemistry using an antibody to involucrin and the following three keratin antibodies: (1) an antibody to low molecular weight keratin reactive with nonsquamous epithelium, (2) a high molecular weight keratin antibody reactive with suprabasal squamous epithelium, and (3) a keratin antibody reactive with full thickness stratified epithelium. In its pattern of reactivity, the last antibody overlaps the low and high molecular weight keratin antibodies and is used as a broad spectrum keratin antibody. By World Health Organization (WHO) classification, the cases in this article included eight keratinizing squamous cell carcinomas, eight nonkeratinizing carcinomas, 20 undifferentiated carcinomas, and four adenocarcinomas. The antibody to broad spectrum keratin had an overall sensitivity of 87.5% and was positive in all eight keratinizing squamous cell carcinomas, seven nonkeratinizing carcinomas (87.5%), 18 undifferentiated carcinomas (90%), and two adenocarcinomas (50%). Low molecular weight keratin antibody stained one additional NPC, which was negative when broad spectrum keratin antibody was used. Involucrin and high molecular weight keratin antibodies demonstrated near parallel staining in all histologic classes; there was marked localization to areas of squamous differentiation. While involucrin is a marker for foci of greater squamous differentiation, broad spectrum keratin antibody may aid in the diagnosis of all histologic subtypes of NPC.

Topics: Adenocarcinoma; Adolescent; Adult; Aged; Antibody Specificity; Carcinoma; Carcinoma, Squamous Cell; Female; Humans; Immunoenzyme Techniques; Keratins; Male; Middle Aged; Nasopharyngeal Neoplasms; Protein Precursors

Two cases of clear cell acanthoma are reported. The expression of carcinoembryonic antigen (CEA), involucrin and keratin proteins in the tumors was investigated immunohistochemically. In 1981, Penneys et al. reported that this tumor was not of sweat gland origin because of the absence of CEA. This study confirmed this, further, the pattern of positive reaction of involucrin also indicated that this tumor is not of sweat duct origin.

Topics: Aged; Carcinoembryonic Antigen; Carcinoma, Squamous Cell; Female; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Protein Precursors; Skin Neoplasms

In the human squamous carcinoma cell line SCC-9, the expression of two markers of keratinocyte differentiation, involucrin and transglutaminase, was greatly stimulated when growing cultures reached confluence. However, the two markers differed temporally in their induction, with transglutaminase reaching maximal levels shortly after confluence and involucrin a week later. If replication was arrested with hydroxyurea prior to confluence, transglutaminase induction occurred within several days but involucrin levels were completely suppressed. Such a striking degree of uncoupling also resulted when the cells were treated with polycyclic aromatic hydrocarbons such as benzo[a]pyrene but not with 2,3,7,8-tetrachlorodibenzo-p-dioxin, a potent inducer of aryl hydrocarbon hydroxylase, or with pyrene. Chronic treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed expression of both transglutaminase and involucrin. However, suppression of the latter (evident in greatly reduced mRNA levels) was considerably more potent and powerful. These findings demonstrate uncoupling of keratinocyte differentiation, potentially useful in analysis of its multiple regulatory influences. They also emphasize the utility of sensitive keratinocyte targets for studying the mechanisms by which model carcinogens disturb the orderly progression of events in their differentiation program.

Topics: Benzo(a)pyrene; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line; Enzyme Induction; Epidermal Cells; Epidermis; Humans; Hydroxyurea; Keratins; Kinetics; Protein Precursors; Tetradecanoylphorbol Acetate; Transglutaminases

Topics: Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Diagnosis, Differential; Female; Humans; Immunoenzyme Techniques; Male; Middle Aged; Prognosis; Protein Precursors; Skin Neoplasms; Staining and Labeling

Squamous cell carcinomas of the urinary bladder and the epithelial lesions associated with infection by Schistosoma haematobium were histopathologically and immunohistochemically described for keratin proteins (TK, 41-65 kDa; KL1, 55-57 kDa; PKK1, 40, 45 and 52.5 kDa), involucrin, and epithelial membrane antigen (EMA). Normal urothelial epithelium was positive for all keratins, and showed absent or slight reactions for involucrin and EMA in superficial umbrella cells. The intestinal type of epithelium was composed of columnar cells and small basal cells; TK was positive in the basal cells, KL1 staining was positive in the columnar cells, whereas PKK1 was negative or slight in the columnar cells. Involucrin was confined to columnar cells. Squamous metaplastic epithelium showed a rather regional keratin distribution: TK was distributed in all layers, KL1 decorated upper spinous and granular layers, but PKK1 did not bind, and involucrin staining existed only in upper spinous and granular cells. Keratin expression in squamous cell carcinomas indicated heterogeneity and its stainability was dependent on the degree of keratinization: The G 1 type revealed strong reaction, the G 2 type showed a similar distribution pattern, but the staining intensity was less, and the G3 type showed irregular staining with decreased intensity. Involucrin staining was limited to keratinized cells of carcinoma as was that for EMA.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Female; Histocytochemistry; Humans; Immunochemistry; Keratins; Male; Membrane Proteins; Middle Aged; Mucin-1; Protein Precursors; Schistosomiasis haematobia; Urinary Bladder Neoplasms

Nasal mucosal biopsy specimens encompassing normal and pathological epithelia were obtained from nickel workers, a population with an increased incidence of carcinomas of the respiratory tract. The immunohistochemical detection of keratins was carried out using monoclonal antibodies AE1 and AE3. The antibody AE1 stained only the basal cells of normal mucociliary epithelium. Regular metaplasias showed an increased stain with AE3; all layers of the surface epithelium were stained. The dysplastic and neoplastic lesions exhibited an extraordinary increase in the staining patterns with AE3 and to a lesser extent with AE1. Involucrin, which was absent from the normal pseudostratified epithelium, appeared in all metaplastic-dysplastic lesions and in keratinized areas of carcinomas. The combined detection of keratins and involucrin proved useful in detecting the degree of maturity and differentiation of preneoplastic and neoplastic lesions of the nasal mucosa.

Topics: Antibodies, Monoclonal; Carcinoma, Squamous Cell; Epithelium; Humans; Immunohistochemistry; Keratins; Microscopy, Electron; Nasal Mucosa; Nickel; Nose Neoplasms; Occupational Diseases; Precancerous Conditions; Protein Precursors

Thirty-eight biopsy specimens were examined for involucrin reactivity by an immunoperoxidase technique. The sampling consisted of specimens diagnosed as normal oral mucosa, reactive epithelial hyperplasia, lichen planus (LP), nonspecific lichenoid stomatitis (NLS), lichenoid dysplasia (LD), carcinoma in situ, and squamous cell carcinoma (SCCa) on routine hematoxylin and eosin examination. Findings were consistent with prior observations of involucrin reactivity in skin and cervical-vaginal mucosa. Specifically, conditions characterized by predominance of mature squamous epithelial cells (superficial layers of normal and hyperplastic oral epithelium, NLS, and LP) exhibited strong involucrin reactivity in such areas. In contrast, atypical or dysplastic lichenoid lesions (LD), as well as carcinoma in situ, despite squamoid differentiation, demonstrated irregular distribution of involucrin, suggesting disturbances in terminal differentiation. Invasive components of SCCa revealed markedly diminished involucrin expression. These findings support prior evidence that LP and LD are biologically distinct lesions. Clinically and microscopically, both may be morphologically similar. However, involucrin reactivity should be helpful in distinguishing difficult cases. Accordingly, we suggest that the use of involucrin immunoreactivity may prove to be a valuable adjunct in the separation of similar lichenoid oral conditions.

Topics: Carcinoma, Squamous Cell; Diagnosis, Differential; Humans; Hyperplasia; Immunoenzyme Techniques; Lichen Planus; Mouth Diseases; Mouth Mucosa; Mouth Neoplasms; Protein Precursors; Stomatitis

The expression of involucrin was examined in 23 skin tumours of hair follicle origin, 17 tumours of sweat gland origin and three tumours of unknown origin, using an immunoperoxidase technique. All tumours from the hair follicle showed a positive reaction for involucrin. In particular keratoacanthoma and the squamous eddies in various tumours stained strongly. Trichofolliculoma, trichilemmoma and pilomatrixoma exhibited characteristic staining patterns which resembled those in the normal hair follicle. On the other hand the majority of the tumours of sweat gland origin did not stain, with restricted positive reactions in areas showing lumen formation or squamous metaplasia. In contrast to the lack of staining in syringoma, a positive reaction was observed in desmoplastic trichoepithelioma, which is histologically similar to syringoma. Clear cell acanthoma, the origin of which is still controversial, showed a staining pattern which indicated that its origin may not be in the sweat gland. These results suggest that testing for involucrin in skin appendage tumours may be very useful for understanding the kinetics of maturation as well as in determining the origin of the tumours.

Topics: Carcinoma, Squamous Cell; Cystadenoma; Hair; Hair Diseases; Humans; Immunoenzyme Techniques; Keratoacanthoma; Protein Precursors; Skin Diseases; Skin Neoplasms; Sweat Gland Neoplasms; Sweat Glands

We examined seven invasive squamous cell carcinomas, five squamous cell carcinomas in situ, four keratoacanthomas, two actinic keratoses, and two seborrheic keratoses by indirect immunofluorescence. We used a panel of three antibodies: one directed against filaggrin, one against involucrin, and one against peptidylarginine deiminase. Anti-involucrin stained all the lesions studied, but the pattern within a given category of lesions was variable and consistent differences between the categories were not observed. Similarly, the antibodies against peptidylarginine deiminase and filaggrin were not able to distinguish differences between the various types of tumors. We conclude that in tumors of epidermis, benign or malignant, products of differentiation are expressed independently of histologic atypia or clinical aggressiveness. Therefore, markers of differentiation do not appear to be reliable indexes for distinguishing benign from malignant lesions.

Topics: Carcinoma, Squamous Cell; Filaggrin Proteins; Fluorescent Antibody Technique; Humans; Hydrolases; Intermediate Filament Proteins; Keratins; Keratoacanthoma; Precancerous Conditions; Protein Precursors; Protein-Arginine Deiminase Type 4; Protein-Arginine Deiminases; Skin; Skin Neoplasms

Fifteen keratoacanthomas and fifteen squamous cell carcinomas of the skin were examined by immunoperoxidase methods for involucrin and both 45- and 63-kilodalton keratins. Keratoacanthomas showed a relatively homogeneous staining pattern for involucrin; all cells except basal cells stained with mild to moderate intensity. Squamous cell carcinomas disclosed a highly irregular involucrin staining pattern with marked variation in staining intensity from cell to cell. Staining patterns for keratin proteins did not appear to distinguish between keratoacanthomas and squamous cell carcinomas. The 45-kilodalton keratin pattern showed diffuse staining within both keratoacanthomas and squamous cell carcinomas, and the 63-kilodalton keratin pattern consisted of focal staining, mostly of dyskeratotic cells. These results suggest that involucrin may serve as a diagnostic aid in differentiating between squamous cell carcinomas and keratoacanthomas. In addition, other lesions in the differential diagnosis of keratoacanthoma and squamous cell carcinoma were also examined for involucrin.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Epidermis; Histocytochemistry; Humans; Immunoenzyme Techniques; Keratins; Keratoacanthoma; Middle Aged; Neoplasm Proteins; Protein Precursors; Sebaceous Glands; Skin Diseases; Skin Neoplasms; Sweat Glands

Involucrin has been recognized recently as a marker of terminal differentiation of squamous epithelial cells and also as a useful marker for keratinization; its expression in epithelial tumors of oral and pharyngeal mucosa and skin was examined. Involucrin in normal oral mucosa and skin was restricted to the granular and upper spinous layers and was absent in the basal layer. Hyperkeratosis was characterized by strong positive staining for involucrum in spinous and granular cell layers. A similar pattern was noted in seborrheic keratosis and verruca vulgaris. Condyloma acuminatum specimens revealed slight staining, whereas Paget cells were negative. Calcifying epitheliomas of Malherbe were usually unreactive. Papillomas exhibited the regular distribution of involucrin, as found in normal squamous epithelium. Basal cell carcinomas were generally negative, whereas squamous cell carcinomas showed an irregular distribution of involucrin. Immunohistochemical staining for involucrin may be useful for identification of keratinizing cells in epithelial tumor foci, just as is the use of monoclonal antibody to keratin KL1.

Topics: Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Epithelium; Humans; Immunoenzyme Techniques; Keratins; Mouth Diseases; Mouth Mucosa; Mouth Neoplasms; Papilloma; Pharyngeal Neoplasms; Protein Precursors; Skin Diseases; Skin Neoplasms; Staining and Labeling

A431 malignant keratinocytes, although derived from a muco-cutaneous carcinoma of the vulva, fail to achieve terminal epidermal differentiation in culture as shown by their inability to form cornified envelopes. Even after culture in a serum-free medium (MCDB 153) containing no retinoic acid and a high (10(-3) M) calcium concentration (conditions known to facilitate epidermal differentiation), the cells do not become competent as shown by the fact that subsequent treatment with a calcium ionophore is unable to provoke the formation of cornified envelopes. Nevertheless, A431 cells are able to synthesize the envelope precursor involucrin. The block in formation of cornified envelopes is thus not due to a lack in involucrin. The results described here suggest that the absence of cross-linking of this molecule is due to a lowered epidermal membrane-bound transglutaminase activity in A431 cells when compared to normal human keratinocytes. In other respects, EGF, which inhibits the proliferation of A431 cells, enhances involucrin accumulation in these cells, although in normal human keratinocytes it stimulates growth and reduces involucrin synthesis. These results suggest that involucrin synthesis is triggered by the arrest of growth.

Topics: Calcium; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line; Cell Membrane; Cells, Cultured; Culture Media; Epidermal Growth Factor; Epidermis; Female; Humans; Protein Precursors; Transglutaminases; Tretinoin

Involucrin is a precursor of the cross-linked envelope protein of the human stratum corneum. Its appearance in the upper layers of the epidermis reflects normal differentiation of keratinocytes. This study uses an immunoperoxidase technique for localization of involucrin in paraffin sections of normal conjunctiva, conjunctival dysplasia, carcinoma in situ, and invasive carcinoma. Clinicoimmunocytochemical correlations are presented. The results demonstrate that the distribution patterns of involucrin differ in precancerous and cancerous conjunctival lesions: normal limbal conjunctiva shows involucrin only in the three superficial cell layers; the fornix conjunctivae contains no involucrin. All 23 conjunctival dysplasias show an involvement also of deeper layers of the epithelium, sparing the basal layers. Three carcinomas in situ and one invasive squamous cell carcinoma, however, demonstrate an involvement of all layers of the epithelium. The involucrin staining pattern helps in histologic differential diagnosis of epithelial lesions of the conjunctiva.

Topics: Biopsy; Carcinoma; Carcinoma in Situ; Carcinoma, Squamous Cell; Conjunctiva; Conjunctival Neoplasms; Epithelium; Humans; Immunoenzyme Techniques; Neoplasm Invasiveness; Precancerous Conditions; Protein Precursors

Ninety-three cervical conization specimens with condyloma or intraepithelial neoplasia were stained by the peroxidase-antiperoxidase technique for involucrin. Diffuse, homogeneous suprabasal staining was observed in the ectocervical squamous mucosa and mature squamous metaplasia. In immature squamous metaplasia, staining was limited to cells with apparent squamous differentiation. Although diffusely reactive in the upper layers of condyloma and cervical intraepithelial neoplasia (CIN) grade I, the stain was uneven in the former and lacking in the parabasal layers of the latter. The staining intensity, distribution, and pattern were more variable in CIN grade II and grade III. With increasing severity, a patchy pattern with a mixture of reactive and nonreactive cells predominated. Although immunoreactivity with involucrin could not distinguish immature squamous metaplasia from neoplasia, the staining patterns in CIN correlated with extent of disease, degree of squamous differentiation, and cellular disorganization.

Topics: Carcinoma in Situ; Carcinoma, Squamous Cell; Condylomata Acuminata; Female; Humans; Immunoenzyme Techniques; Protein Precursors; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

Human lung tumor cell lines established from the major histological types of lung cancer were examined by immunofluorescent staining techniques for their patterns of intermediate filament (keratin, vimentin, and neurofilament triplet protein) expression. All cell lines examined, both small cell lung carcinoma (SCLC) and non-SCLC (squamous cell carcinoma, adenocarcinoma, large cell carcinoma, and mesothelioma) contained keratin, consistent with their epithelial derivation. These lung carcinoma cell lines also expressed vimentin, the characteristic intermediate filament of mesenchymal cells in vivo. In light of the proposed neuroectodermal origin of SCLC, cell lines were also studied for neurofilament expression. Two of four SCLC tumor cell lines, as well as non-SCLC cell lines, showed no reactivity with antibodies to neurofilament triplet protein. Two of the SCLC cell lines stained weakly with anti-neurofilament antibody. Examination of specific keratin patterns in human lung tumor cell lines by selective immunoprecipitation with keratin antiserum and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that small-sized keratin proteins (Mr 44,000 to 52,000) were present in cell lines derived from SCLC and non-SCLC types of lung cancer. Tumor cell lines exhibiting squamous differentiation by light microscopic criteria (i.e., intracellular keratin, intercellular bridging, "pearl" formation, and/or individual cell keratinization) also displayed a preponderance of intermediate-sized keratins (Mr 57,000 and 59,000) and exhibited another feature of terminal keratinocyte differentiation (cross-linked envelope formation). Mesothelioma cell lines had varying keratin profiles. The presence of keratin proteins in all SCLC cell lines examined argues against a neuroectodermal origin for these tumors and is consistent with the notion that these tumors arise from a common bronchial "stem cell," similar to that from which other types of bronchogenic carcinomas arise.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Neoplastic; Cytoskeleton; Fluorescent Antibody Technique; Humans; Intermediate Filament Proteins; Keratins; Lung Neoplasms; Mesothelioma; Mice; Microfilament Proteins; Molecular Weight; Protein Precursors; Vimentin

Lesions of solar keratoses and squamous cell carcinoma maintained their histological appearance and an increased tritiated thymidine autoradiographic labelling index after being grafted on to nude mice. However, the values for their mean epidermal thickness and individual cell size appeared to decrease slightly during the 24-week period of study. As judged by the immunolocalization of involucrin antibodies, the grafts maintained a human epidermal antigenic profile. However, immunolocalization studies with HLA antibodies showed only a patchy positivity in the original premalignant lesions and were negative after grafting. These results indicate the potential value of the nude mouse as a model for studying the progress of premalignant and malignant skin lesions in an immunologically privileged non-human site and further indicate that solar keratoses can be maintained independently of systemic donor influences.

Topics: Animals; Antibodies; Carcinoma, Squamous Cell; Female; HLA Antigens; Keratosis; Mice; Mice, Nude; Neoplasm Transplantation; Precancerous Conditions; Protein Precursors; Skin Neoplasms; Thymidine

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.

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Cell Line; Culture Media; Humans; Neoplastic Stem Cells; Protein Precursors; Tretinoin

Immunohistochemical staining for involucrin, a cytoplasmic protein synthesized during squamous maturation, was assessed in histologic sections from hysterectomy and cone biopsy specimens from patients with cervical neoplasia. In normal and condylomatous squamous epithelium, diffuse cytoplasmic staining was seen in the suprabasal layers, with no staining of the basal cells. Staining was absent in two cases of cervical intraepithelial neoplasia (CIN), grade III, in which the lesions were composed entirely of undifferentiated cells and markedly decreased in cases involving large numbers of basal cells. In 19 of 23 cases (83 per cent) of CIN, however, focal staining for involucrin was seen in large differentiated cells in the more superficial layers, and in two cases of keratinized CIN diffuse suprabasal staining was observed. Similarly, strong staining for involucrin was present in differentiated areas in one case of microinvasive squamous cell carcinoma and in 93 per cent of cases of infiltrating squamous cell carcinoma. These findings suggest that involucrin is a marker for maturation in cervical squamous epithelial neoplasms. Patterns of immunohistochemical staining for involucrin in keratinized dysplasia and differentiated squamous carcinomas should be taken into consideration if loss of involucrin staining is used as a criterion for neoplastic transformation of cervical epithelium, as has been proposed.

Topics: Carcinoma, Squamous Cell; Cervix Uteri; Female; Histocytochemistry; Humans; Immunoenzyme Techniques; Neoplasm Invasiveness; Protein Precursors; Uterine Cervical Neoplasms

Involucrin is a soluble protein precursor of the cross-linked envelope present in the submembranous zone of human stratum corneum, and subsequently demonstrated in stratified squamous epithelia. The immunoperoxidase technique was used to assess the distribution of involucrin in 107 normal and 318 abnormal tissues. With few exceptions, involucrin was restricted to squamous epithelia, urothelium, some skin appendages and thymic Hassall's corpuscles. In normal squamous epithelium and normal urothelium, staining was most intense in the superficial layers where it was concentrated at the cell periphery and gradually decreased toward the basal layer. This orderly staining pattern was maintained in benign squamous and urothelial lesions and in grade I papillary urothelial carcinomas. Higher grade papillary urothelial carcinomas, infiltrating urothelial and squamous carcinomas, and in situ urothelial and squamous carcinomas demonstrated abnormal staining patterns for involucrin that are described. Foci of squamous differentiation in adenocarcinomas and other epithelial malignancies stained intensely for involucrin. Brenner tumours of the ovary and Walthard rests of the fallopian tube, lesions of uncertain histogenesis but possibly urothelial-related, also stained for involucrin. Results of this study suggest that involucrin is a sensitive and specific marker for squamous and urothelial differentiation, staining patterns for involucrin may be helpful in distinguishing benign from malignant urothelial and squamous lesions, and presence of involucrin may be helpful in determining the histogenesis of selected lesions.

Topics: Carcinoma in Situ; Carcinoma, Squamous Cell; Cell Differentiation; Cell Transformation, Neoplastic; Epithelium; Humans; Immunoenzyme Techniques; Neoplasms; Protein Precursors; Urinary Bladder Neoplasms

A study was undertaken to determine whether immunoperoxidase stains for keratin and involucrin, the latter a protein present in cells of stratified squamous epithelium that have differentiated beyond the basal stage, distinguish any differences in squamous cells present in the adenoacanthoma from those in the adenosquamous carcinoma of the uterine corpus. Forty-eight tumors were studied, of which 33 were adenoacanthomas and 15 adenosquamous carcinomas. The patients with adenoacanthomas were slightly younger (mean 61.5 vs. 64.5 years) and had tumors that were generally better differentiated than the adenosquamous carcinomas. The squamous epithelium in every tumor, regardless of histologic type, stained positively for keratin. There were no obvious differences in staining when tumors were stratified for histologic type, grade, or location within the tumor. The glandular portion of both tumor types stained irregularly, but nonetheless positively, for keratin in 71% of the cases. Involucrin was detected in 57% of adenoacanthomas and 87% of adenosquamous carcinomas. The deeper or more central portion of the squamous morules stained only if the more superficial or peripheral areas were positive. The extent of the involucrin staining was less in the adenosquamous carcinomas than in the adenoacanthomas. The glandular component of the tumors did not stain for involucrin. It is concluded that no qualitative differences in the staining reactions with respect to keratin and involucrin distinguish the adenoacanthomas from the adenoaquamous carcinoma. These findings support the argument that the adenoacanthoma and adenosquamous carcinoma represent a spectrum of squamous differentiation in a single tumor type.

Topics: Adenocarcinoma; Adult; Aged; Carcinoma, Squamous Cell; Cell Differentiation; Diagnosis, Differential; Female; Humans; Immunoenzyme Techniques; Keratins; Middle Aged; Protein Precursors; Uterine Neoplasms

Involucrin is a precursor of cross-linked protein of human stratum corneum, and its appearance in the upper layers of the epidermis is a function of the normal differentiation of the keratinocyte. Cases of basal cell and squamous cell carcinoma were evaluated for the presence of involucrin using immunoperoxidase techniques on paraffin sections. Basal cell carcinomas were negative for involucrin with staining restricted to squamous horn cysts, while squamous cell carcinomas stained strongly, particularly in large keratinized cells. Cases of squamous cell carcinoma in situ (Bowen's disease) revealed increased staining for involucrin with staining of dyskeratotic cells at all levels in the epithelium. Abnormal patterns of staining were also noted in non-neoplastic epidermis adjacent to carcinomas. Immunohistochemical staining for involucrin identifying abnormal or premature keratinization is a sensitive marker for dyskeratosis in squamous epithelia and may have applications in the histopathologic evaluation of skin specimens.

Topics: Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Epidermis; Histocytochemistry; Humans; Immunoenzyme Techniques; Keratins; Protein Precursors; Skin Neoplasms

Involucrin accumulation and ionophore-assisted envelope formation, markers of keratinocyte differentiation, were found to be highly dependent on culture conditions in the malignant epidermal keratinocyte line, SCC-13, derived from a human squamous cell carcinoma. In confluent cultures, approximately one-half of the cells were competent to form envelopes when grown in medium without hydrocortisone or retinyl acetate supplementation. Addition of hydrocortisone to the medium during growth resulted in up to 90% competence, while addition of retinyl acetate instead resulted in as low as 10% competence. Hydrocortisone partially antagonized the effect of retinyl acetate when both agents were added together. Involucrin levels, measured by radioimmunoassay, were modulated essentially in parallel with envelope competence under the various conditions tested. When the cells were grown in medium supplemented with hydrocortisone, the levels shortly after confluence were over 50-fold higher than in sparse cultures. Regardless of hydrocortisone or retinyl acetate addition, less than 1% of the cells were competent in sparse cultures of growing cells, but up to 90% exhibited this property after growth arrest in serum-free medium containing hydrocortisone. High levels of competence were correlated with cessation of cell division but not with loss of colony-forming efficiency; under optimal conditions, two-thirds of the cells were capable of both envelope formation and colony initiation. Normal human epidermal cells showed a 4- to 5-fold increase in envelope competence from sparse to confluent culture but were insensitive to the suppressive effect of retinyl acetate. The results suggest that some potential differentiated character of malignant keratinocytes may be suppressed in vivo by physiological agents such as vitamin A.

Topics: Carcinoma, Squamous Cell; Cell Division; Cell Line; Cell Membrane; Diterpenes; Humans; Hydrocortisone; Keratins; Protein Precursors; Radioimmunoassay; Retinyl Esters; Skin; Time Factors; Vitamin A

Involucrin is a precursor of the cross-linked envelope protein or marginal band present in human stratum corneum. This study uses immunohistochemical techniques for localization of involucrin in histologic sections from 91 lung tumors in order to evaluate the usefulness of involucrin as a tumor marker in lung neoplasms. Although involucrin is absent from bronchial epithelium, it is expressed in cultured tracheal epithelial cell colonies and in bronchial mucosa with squamous metaplasia. Involucrin was present in all 25 cases of squamous and adenosquamous carcinoma. Staining was focal in 12 cases of squamous cell carcinoma and was most marked in the larger neoplastic cells in the center of squamous cell nests. Only two of 20 cases of adenocarcinoma revealed focal staining for involucrin, and these cases may represent adenosquamous variants. Six of 12 cases of large cell undifferentiated carcinoma stained for involucrin, indicating squamous differentiation, and seven cases of malignant mesothelioma were negative. Isolated involucrin-positive cells were present in two of 16 cases of small cell anaplastic carcinoma and one of 11 carcinoid tumors, identifying variants of neuroendocrine tumors with dual differentiation. Patterns of localization of involucrin in paraffin and frozen sections were compared with staining for cytokeratins in parallel sections. Immunohistochemical localization of involucrin comprises a specific marker for squamous differentiation in lung tumors.

Topics: Adenocarcinoma; Carcinoma; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Humans; Lung Neoplasms; Mesothelioma; Protein Precursors; Staining and Labeling

Involucrin, a protein subunit of keratinocyte cross-linked envelopes, is a distinctive marker for suprabasal differentiation in stratified squamous epithelium. Immunoperoxidase staining for involucrin was used to evaluate paraffin sections of tissue obtained by colposcopically directed biopsies of infectious, metaplastic, and dysplastic lesions of the cervix and vagina. Areas of normal squamous epithelium, papillary and flat condyloma acuminatum, and mature and immature squamous metaplasia showed positive staining in 99 per cent of samples lacking significant inflammation and in 60 per cent of those with moderate or severe inflammation. In contrast, only 19 per cent of the squamous cell dysplasias, even those without much inflammation, showed positive staining, and no area with moderate or severe inflammation showed positive staining. These findings indicate that expression of involucrin is modulated by cellular pathologic features and microenvironment. We suggest that immunoperoxidase staining for involucrin may be useful in distinguishing mild dysplasia from immature metaplasia and flat condyloma in some biopsy specimens in which routine histologic examination yields an indeterminate diagnosis.

Topics: Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cervix Uteri; Female; Immunoenzyme Techniques; Methods; Protein Precursors; Staining and Labeling; Uterine Cervical Dysplasia; Vagina