involucrin and Cell-Transformation--Neoplastic

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

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

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

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

In oral cancers, cytoplasmic BAG-1 overexpression is a marker of poor prognosis. BAG-1 regulates cellular growth, differentiation and survival through interactions with diverse proteins, including the vitamin D receptor (VDR), a key regulator of keratinocyte growth and differentiation. BAG-1 is expressed ubiquitously in human cells as three major isoforms of 50 kDa (BAG-1L), 46 kDa (BAG-1M) and 36 kDa (BAG-1S) from a single mRNA. In oral keratinocytes BAG-1L, but not BAG-1M and BAG-1S, enhanced VDR transactivation in response to 1alpha,25-dihydroxyvitamin D3. BAG-1L was nucleoplasmic and nucleolar, whereas BAG-1S and BAG-1M were cytoplasmic and nucleoplasmic in localisation. Having identified the nucleolar localisation sequence in BAG-1L, we showed that mutation of this sequence did not prevent BAG-1L from potentiating VDR activity. BAG-1L also potentiated transactivation of known vitamin-D-responsive gene promoters, osteocalcin and 24-hydroxylase, and enhanced VDR-dependent transcription and protein expression of the keratinocyte differentiation marker, involucrin. These results demonstrate endogenous gene regulation by BAG-1L by potentiating nuclear hormone receptor function and suggest a role for BAG-1L in 24-hydroxylase regulation of vitamin D metabolism and the cellular response of oral keratinocytes to 1alpha,25-dihydroxyvitamin D3. By contrast to the cytoplasmic BAG-1 isoforms, BAG-1L may act to suppress tumorigenesis.

Topics: Amino Acid Sequence; Cell Differentiation; Cell Line; Cell Nucleus; Cell Transformation, Neoplastic; Cytoplasm; DNA-Binding Proteins; Gene Expression Regulation; Humans; Keratinocytes; Molecular Sequence Data; Osteocalcin; Promoter Regions, Genetic; Protein Isoforms; Protein Precursors; Receptors, Calcitriol; Transcription Factors; Transcriptional Activation; Vitamin D

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

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

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

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

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

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

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

Biopsy specimens from 12 patients who used snuff and had leukoplakia in the mucosa of the oral cavity were studied and compared with specimens from their own nonleukoplakic oral mucosa, as well as with biopsy specimens from corresponding areas of the oral cavity in 12 nonsmoking, nontobacco-using control subjects. The biopsy specimens were processed using standard immunohistochemical rabbit antibody to human involucrin and mouse antibody to human transglutaminase type I as the primary antibodies. A computer-driven light absorbance image analysis system was used to determine the optical density of each of the marker-stained specimens. Optical density measurements were compared using a one-way analysis of variance. The expression of involucrin was significantly higher in the epithelium of the nonsmoking, nontobacco-using control subjects (0.2937 +/- 0.0725 optical density) in comparison with the normal-appearing mucosa (0.2283 +/- 0.0488 optical density) and the leukoplakic mucosa of the snuff-using patients (0.2007 +/- 0.0669 optical density) (p < 0.05). The expression of transglutaminase type I was also significantly higher in the epithelium of the nonsmoking, nontobacco-using controls (0.2308 +/- 0.1381 optical density) than in the patients with leukoplakic mucosa (0.1310 +/- 0.0472 optical density) (p < 0.05). However, there was no difference when compared with the normal-appearing mucosa of the patients in the snuff-using group (0.1686 +/- 0.0323 optical density). This study has shown that involucrin and transglutaminase type I are expressed differently in leukoplakic oral mucosa of snuff users and in normal oral mucosa and that this difference can be measured objectively.

Topics: Adult; Biomarkers; Cell Transformation, Neoplastic; Humans; Keratinocytes; Leukoplakia, Oral; Male; Middle Aged; Mouth Mucosa; Plants, Toxic; Protein Precursors; Tobacco, Smokeless; Transglutaminases

A novel procedure involving the sequential use of two different antisense constructs has been used to inhibit the synthesis of involucrin in a hybrid cell line formed by the fusion of a human cervical carcinoma cell with a normal human keratinocyte (ESH100P6). In this cell line, and other similar hybrids, malignancy, as measured by progressive growth in vivo, is suppressed; and it has been shown that the keratinocyte imposes its own programme of terminal differentiation on the non-malignant hybrid cell. In particular, involucrin, a precursor of one of the major components of the cornified envelope of mature keratinocytes, continues to be produced. When, however, malignant segregants arise in the hybrid cell population, the terminal differentiation programme of the keratinocyte is not expressed and involucrin ceases to be made. It seemed possible that if the synthesis of involucrin, a critical marker of keratinocyte terminal differentiation, could be completely inhibited, this differentiation programme might be disrupted, and the malignant phenotype might then reappear in the non-malignant hybrids. This question was investigated further in the present paper. Total, and specific, inhibition of involucrin synthesis was indeed achieved by a sequential two-step antisense procedure, which might provide a systematic general method for the complete inactivation of other selected target genes.

Topics: Animals; Antisense Elements (Genetics); Blotting, Southern; Blotting, Western; Cell Differentiation; Cell Transformation, Neoplastic; Cloning, Molecular; DNA; Humans; Hybrid Cells; Immunoenzyme Techniques; Keratinocytes; Mice; Mice, Nude; Mutagenicity Tests; Neoplasms, Experimental; Protein Precursors; RNA; Transfection

We found that keratinocytes immortalized with human papillomavirus (HPV) type 16 DNA and malignantly converted by H-ras transfection (HPK-1A/ras) exhibit an enhanced ability to synthesize a fibronectin-containing extracellular matrix. Gene expression of fibronectin and thrombospondin was increased in tumorigenic keratinocytes compared to control and immortalized keratinocytes, 6- and 9-fold, respectively. Increased production of soluble and cell surface-associated fibronectin was not specific for HPV 16 transformed keratinocytes. Ad12-SV40-immortalized keratinocytes malignantly converted by H-ras transfection (RHEK-1/ras) also exhibited enhanced expression of fibronectin and thrombospondin, as well as pro-alpha 1 type I collagen. Steady state mRNA levels for autocrine growth-regulatory factors, transforming growth factors alpha and beta 1, were increased in Ad12-SV40 but not HPV 16-transformed human keratinocytes. We then determined whether increased production of fibronectin was associated with aberrant differentiation of transformed keratinocytes. Less than 10% of the HPV 16-transformed cells produced cornified envelopes after suspension-induced differentiation compared to 70% of normal keratinocytes. However, immortalization by HPV 16 DNA was sufficient to confer a differentiation-defective phenotype. Both involucrin mRNA and protein levels were decreased 8-fold in HPV 16-immortalized keratinocytes compared to normal cells and malignant conversion further attenuated involucrin levels. These studies demonstrate that aberrant differentiation is an early event in the transformation of the human keratinocytes and is not the result of enhanced expression of the extracellular matrix proteins. Unlike transformed fibroblastic cell types, up-regulation of fibronectin gene expression and matrix formation is a consistent characteristic of malignantly converted human keratinocytes.

Topics: Cell Differentiation; Cell Division; Cell Line; Cell Line, Transformed; Cell Transformation, Neoplastic; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix Proteins; Genes, ras; Humans; Keratinocytes; Kinetics; Papillomaviridae; Protein Precursors; Transfection

We studied the proliferation and differentiation of human laryngeal papillomas, which are benign tumors induced by human papillomaviruses. Immunofluorescent stains of tissues for a number of differentiation-specific proteins showed abnormal differentiation. Papilloma tissue fragments in vitro showed a slightly decreased fraction of proliferating cells that incorporated tritiated thymidine and a markedly reduced incorporation of tritiated uridine when compared with normal tissue. We propose that papillomavirus infection results in normal basal cell proliferation but abnormal terminal differentiation and that this abnormality significantly contributes to the hyperplasia of the papillomas.

Topics: Cell Division; Cell Transformation, Neoplastic; Filaggrin Proteins; Humans; Immunoblotting; Intermediate Filament Proteins; Keratins; Laryngeal Neoplasms; Neoplasm Proteins; Papilloma; Papillomaviridae; Protein Precursors; Staining and Labeling; Thymidine; Tumor Virus Infections; Uridine

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

The epithelia lining the cyst of five cases of calcifying odontogenic cyst (COC) were evaluated immunohistochemically with the use of monoclonal antibodies (MoAb's) against keratin (PKK1, KL1, K4.62, K8.12) and vimentin, and polyclonal antisera agonist involucrin and filaggrin. Epithelial lining of COC was classified into 1) thin squamous-cell epithelium, 2) ameloblastoma-like, and 3) thin or 4) thick calcifying odontogenic epithelium. Foci consisting of ghost cells or calcified cells were categorized as calcifying epithelial odontogenic tumor (CEOT). Thin squamous-cell epithelium reacted with PKK1, KL1, K4.62, K8.12, and anti-vimentin MoAb's, thus demonstrating the co-expression of keratin and vimentin. Ameloblastoma-like cells showed positive staining with PKK1, KL1, and sometimes with anti-vimentin. Thick calcifying odontogenic epithelial lining showed stratification of cell layers, and the most strikingly reactive zone was the upper intermediate layer, which showed the presence of keratin, involucrin, and a small amount of filaggrin. Cells of this layer might be the most differentiated type of cells in COC. Undifferentiated odontogenic cells of COC masses were characterized by co-expression of keratin and vimentin, and by the absence of involucrin and filaggrin. All ghost cells were devoid of any immunostaining except for filaggrin, which was rarely positive, but eosinophilic or basophilic cells surrounding the ghost cells showed intense staining for all keratin proteins except vimentin.

Topics: Adolescent; Adult; Cell Transformation, Neoplastic; Epithelium; Female; Filaggrin Proteins; Humans; Immunohistochemistry; Intermediate Filament Proteins; Jaw Diseases; Keratins; Male; Odontogenic Tumors; Protein Precursors

A pentapeptide, pyroGlu-Glu-Asp-Ser-GlyOH, was previously isolated from mouse skin and shown to inhibit reversibly proliferation of murine keratinocytes, both in intact skin and in culture. In the present report we have shown that proliferation of normal human keratinocytes in culture is also inhibited by the pentapeptide, whether the cells are grown under standard conditions or prevented from stratifying in medium containing a low concentration of calcium ions. An SV40-transformed line of human keratinocytes, SVK14, was completely insensitive to the pentapeptide and a line derived from a squamous cell carcinoma of the oral cavity, SCC-9, was less sensitive than the normal cells. The effect of the pentapeptide on terminal differentiation was determined by measuring the proportion of cells expressing involucrin after a single dose or repeated doses of the pentapeptide: no consistent change in the number of terminally differentiating cells was observed. Cell extracts and conditioned medium from all three cell types contained the epidermal pentapeptide, suggesting a role for the peptide in both autocrine (normal keratinocytes) and paracine (SVK14, SCC-9) growth control. A proportion of the pentapeptide isolated from conditioned medium was phosphorylated; since it was as active as the non-phosphorylated form, when assayed on mouse epidermis, the role of phosphorylation remains to be determined.

Topics: Amino Acid Sequence; Cell Differentiation; Cell Transformation, Neoplastic; Cells, Cultured; Demecolcine; DNA Replication; Growth Inhibitors; Humans; Infant, Newborn; Keratinocytes; Kinetics; Male; Mitosis; Mitotic Index; Molecular Sequence Data; Oligopeptides; Protein Precursors; Pyrrolidonecarboxylic Acid; Thymidine

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

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

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

Previous studies have indicated that some Simian-virus-40-transformed human epidermal keratinocytes (SV40-HE) undergo significant changes in their growth and differentiated properties. To better understand the significance of these changes, we have characterized the keratins of SV40-HE cells by one- and two-dimensional immunoblot analysis using the subfamily-specific AE1 and AE3 monoclonal antikeratin antibodies. The results indicate that our SV40-HE cells have lost the Mr 58,000 (No. 5), Mr 56,000 (No. 6), Mr 50,000 (No. 14/15), Mr 48,000 (No. 16), and Mr 46,000 (No. 17) keratins that are expressed by cultured normal human keratinocytes. Instead, these cells express mainly Mr 52,000 (No. 8), Mr 45,000 (No. 18), and Mr 40,000 (No. 19) keratins, a set highly characteristic of simple epithelial cells. Furthermore, our SV40-HE cells have ceased to express involucrin, another marker for keratinocytes, and have a greatly diminished ability to undergo in vitro stratification. These results suggest that epidermal cells can sometimes lose their keratinocyte features as a consequence of viral transformation. This finding may have important implications regarding the mechanisms of epithelial differentiation and tumorigenesis and in the use of keratinocyte markers for tumor diagnosis.

Topics: Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Epithelial Cells; Humans; Keratins; Molecular Weight; Protein Precursors; Simian virus 40; Skin

Involucrin is a recently recognized structural component of mature squamous epithelial cells. We examined involucrin expression using an immunoperoxidase technique in normal skin and in a variety of epidermal hyperplasias and neoplasms to determine whether distinctive staining patterns existed within these lesions. Four patterns of reactivity were observed: diffuse intracellular staining typical of keratinocytes of the upper third of normal epidermis and epidermal hyperplasias and benign neoplasms; staining at cell borders, seen principally in benign epidermal neoplasms; patchy staining characteristic of squamous cell carcinoma in situ; and absence of staining in benign and neoplastic basaloid epithelium. Invasive nests of squamous cell carcinomas were negative for involucrin reactivity, whereas pseudoinvasive tongues of epithelium at the bases of keratoacanthomas were focally positive. These results suggest that immunoperoxidase staining for involucrin may be useful in distinguishing certain benign from malignant epidermal neoplasms as well as in understanding the altered maturation and kinetics of proliferative processes afflicting keratinocytes.

Topics: Cell Differentiation; Cell Transformation, Neoplastic; Epidermal Cells; Epidermis; Histocytochemistry; Humans; Immunoenzyme Techniques; Protein Precursors; Skin Diseases; Skin Neoplasms

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