involucrin and Head-and-Neck-Neoplasms

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

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

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

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

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

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

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

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

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