Pascal Tomakidi

Association of gp91phox homolog Nox1 with anchorage-independent growth and MAP kinase-activation of transformed human keratinocytes.

Among five members of the NADPH oxidase (Nox) family, Nox1 confers mitogenic properties and is implicated to participate in the process of cell transformation. We have established two phenotypes of carcinogenesis model by ethanol treatment of human gingival keratinocytes immortalized with E6/E7 oncogenes of human papillomavirus type16: immortalized (EPI) nontransformed cells with epithelium-like morphology and more advanced transformed (FIB) cells with spindle fibroblastic-shape morphology. FIB membranes possessed a 63-kDa Nox1 protein at higher levels and exhibited 2.8-fold higher capability for superoxide and hydroxyl radical generation, compared with EPI membranes. Both EPI and FIB cells expressed more abundant Nox1 protein at a proliferating stage than that at a quiescent confluent phase. Immunofluorescence staining with an anti-Nox1 antibody showed that immunoreactive materials were distributed in the whole interior of both types of cells, while they were preferentially localized in the nuclei of FIB cells. Nuclei isolated from EPI and FIB cells contained a 63 kDa-Nox1 protein. Compared with EPI cells, FIB cells expressed elevated levels of Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase proteins. Furthermore, JNK2 was constitutively phosphorylated in FIB cells. Together, our data strongly implicate Nox1 in redox-mediated signaling related to cellular activation of human keratinocytes at a more advanced stage of transformation.

Establishment of oral mucosa phenotype in vitro in correlation to epithelial anchorage.

Abstract Cell-matrix interactions and the ordered deposition of basement membrane (BM) components are of major importance for the maintenance of tissue homeostasis in complex epithelia. This aspect was studied in vitro in a coculture system designed as an oral mucosa model. As crucial epithelial features the kinetics of proliferation, expression of site-specific keratins as well as integrin patterns in correlation to synthesis of BM components were assessed by immunohistochemistry and in situ hybridization. Comparison with non-cornified gingiva as tissue of origin revealed different stages of epithelial development, eventually leading to complete reconstruction within a time frame of 1–3 weeks. First, the initial activated stage up to 1 week was characterized by (a) high keratinocyte proliferation, (b) extended expression of the basal cell-specific keratin K5 and (c) a patchy pattern of the differentiation-specific keratins K4 and K13. Second, after 2 weeks the improvement of histoarchitecture correlated to (a) predominant K5 expression in the basal and (b) extension of K4 and K13 within the suprabasal cell compartment, (c) high expression of integrins α3β1 and α6β4 including their ligand laminin-5 and (d) accumulating deposition of basement membrane components. Third, virtually complete tissue normalization at 3 weeks was indicated by (a) restriction of K5 to the basal cell area, (b) regular suprabasal localization of K4 and K13, (c) polarization of integrins to basal and parabasal cells and (d) linear codistribution of collagen IV, “classical” laminin (-1 or -10) and laminin-5 underneath the basal cells. Thus, these organotypic cocultures represent relevant equivalents for non-keratinized oral mucosa with typical gingival differentiation features and in addition suitable models for preclinical trials such as prospective dental material testing.

Assessment of acute cyto- and genotoxicity of corrosion eluates obtained from orthodontic materials using monolayer cultures of immortalized human gingival keratinocytes

Whilst a patient is undergoing orthodontic treatment, dental appliances based on non-precious metals or titanium remain in the oral cavity for up to several years. Throughout this period the appliance is in either direct or indirect contact with the oral mucosa.To investigate the possibility of cell damage occurring as a result of appliance corrosion, monolayer cultures of immortalized human gingival keratinocytes were assessed for acute cyto- and genotoxicity using the hexosaminidase assay and the Comet assay respectively. The materials tested included 1. a nickel-free wire, 2. a UK-1 bond, 3. nickel-free as well as nickel-containing brackets with and without color signature and 4. a titanium expansion screw. Each of the test materials was corroded in a solution consisting of equal amounts of lactic acid and sodium chloride (0.1 M) for 1, 3, 7 and 14 days. The cell cultures were then exposed to eluates exhibiting the highest ion concentrations.None of the eluates was found to exhibit acute cytotoxicity, regardless of the type of test system used. Qualitative assessment using neutral red dye for live cells and either trypan blue or propidium iodide to disclose dead cells failed to reveal any significant increase in cell damage when exposed cells were compared to control cultures. Unrestricted cell vitality was confirmed by quantifying viable cells through measurement of hexosaminidase enzyme activity. Furthermore, assessment of genotoxicity revealed no apparent DNA damage to immortalized gingival keratinocytes following exposure to the test eluates.Because the materials tested in this study were corroded using the exacting methods normally applied to precious metals or gold-containing alloys, the lack of either acute cyto- or genotoxic effects following exposure to the test eluates indicates that the materials tested exert no adverse effects on cells similar to those of the target tissue exposed to the materials in situ.ZusammenfassungDie während der kieferorthopädischen Behandlung eingesetzten Apparaturen, die aus Nichtedelmetallegierungen oder Titan bestehen, verbleiben häufig über Jahre in der Mundhöhle. Während des gesamten Zeitraums steht die Apparatur in direktem oder indirektem Kontakt mit der Oralmukosa.Um zu untersuchen, ob eine mögliche Zellschädigung durch Korrosion der Apparatur hervorgerufen wird, wurden Monolayerkulturen von immortalisierten humanen Gingivakeratinozyten mit dem Hexosaminidasetest und dem Comet-Assay (Einzelzellgelelektrophorese) auf akute Zyto- und Gentoxizität hin untersucht. Untersucht wurden 1. ein nickelfreier Draht, 2. ein UK-1-Band, 3. nickelfreie und nickelhaltige Brackets mit und ohne Farbmarkierung und 4. eine Dehnschraube aus Titan. Jedes der Untersuchungsmaterialien wurde für einen, drei, sieben und 14 Tage in einer Lösung, die zu gleichen Teilen aus Milchsäure und Natriumchlorid (0,1 M) bestand, korrodiert. Danach wurden die Zellkulturen mit den Eluaten, die die höchsten lonenkonzentrationen aufwiesen, inkubiert.Unabhängig vom Testsystem zeigte keines der Eluate eine akute Zytotoxizität. Die qualitative Untersuchung mit dem Farbstoff Neutralrot, der lebende Zellen färbt, und Trypanblau oder Propidiumjodid, die tote Zellen anfärben, ergab keinerlei Zunahme der Zellschädigung im Vergleich von exponierten zu nichtexponierten Kontrollkulturen. Die uneingeschränkte Zellvitalität wurde durch die Quantifizierung lebender Zellen mittels Messung der Aktivität des Enzyms Hexosaminidase bestätigt. Darüber hinaus ließ die Untersuchung der Gentoxizität nach Eluatexposition keine sichtbare DNA-Schädigung an immortalisierten Gingivakeratinozyten erkennen.Die in dieser Studie untersuchten Materialien wurden unter Bedingungen korrodiert, die normalerweise auf Edelmetalle oder goldhaltige Legierungen angewendet werden. Das Ausbleiben zyto- oder gentoxischer Effekte nach Eluatexposition zeigt, daß die getesteten Materialien keine schädigenden Effekte auf Zellen ausüben, die große Ähnlichkeit zu denen des Zielgewebes aufweisen, das den Materialien in situ ausgesetzt ist.

Metastatic growth of squamous cell carcinomas is correlated with upregulation and redistribution of hemidesmosomal components

Bullous pemphigoid antigen-1 (BPA1) and α6β4-integrin colocalize at the hemidesmosomes in basal-layer keratinocytes of normal squamous epithelia. The expression of these genes was analyzed during the process of tumor cell invasion and metastasis on frozen sections of head and neck biopsies, and the structural appearance of hemidesmosomes was analyzed by electron microscopy. Despite a diminution of hemidesmosomal structures as revealed by electron microscopy, gene expression of BPA1 and α6β4-integrins was distinctly upregulated with the onset of invasive growth, demonstrated at the mRNA level by in situ hybridization. The upregulated gene expression extended to the entire proliferative zone of invasive tumors, including the tumor cells which have lost contact with the basement membrane and no longer display hemidesmosomes. The polarized localization of the BPA1 and α6β4 proteins to the basal aspect of the peripheral tumor cells was largely retained in invasive but nonmetastatic lesions, but was lost upon progression to metastatic growth of head and neck squamous cell carcinomas (SCC), in which pericellular staining extended into many tumor cell layers. The results of this study confirm that expression of BPA1 and α6β4-integrins is elevated in carcinoma cells but is not directed to intact hemidesmosomes. Importantly, this loss of directed localization is an indicator of the capacity to metastasize.

The E5 protein of the human papillomavirus type 16 down-regulates HLA-I surface expression in calnexin-expressing but not in calnexin-deficient cells

The human papillomavirus type 16 E5 protein (HPV16 E5) down-regulates surface expression of HLA-I molecules. The molecular mechanisms underlying this effect are so far unknown. Here we show that HPV16 E5 down-regulates HLA-I surface expression in calnexin-containing but not in calnexin-deficient cells. Immunoprecipitation experiments reveal that calnexin and HPV16E5 can be co-precipitated and that this association depends on the presence of a wild-type first hydrophobic region of E5. When an E5 mutant (M1) in which the first putative transmembrane helix had been disrupted was used for the transfections calnexin-E5 co-precipitation was strongly impaired. In addition, we show that the M1 mutant is only able to marginally down-regulate HLA-I surface expression compared to the wild-type protein. Besides, we demonstrate that E5 forms a ternary complex with calnexin and the heavy chain of HLA-I, which is mediated by the first hydrophobic region of the E5 protein. On the basis of our results we conclude that formation of this complex is responsible for retention of HLA-I molecules in the ER of the cells.

Discriminating expression of differentiation markers evolves in transplants of benign and malignant human skin keratinocytes through stromal interactions

Accumulating evidence indicates a decisive role for the adjacent stroma in tumour growth and dissemination. However, it is not clear how far altered differentiation such as expression of aberrant keratins and vimentin, common in invasive human carcinomas, may reflect intrinsic cell properties or a response to the tumour environment. We have addressed this by transplanting benign and malignant human HaCaT‐ras keratinocytes, seeded on collagen matrix, onto nude mice. Initially, epithelia derived from benign and malignant cells, being separated from host stroma by collagen, were poorly organized and exhibited the same differentiation markers, as identified by immunofluorescence and in situ hybridization. Epidermal basal and suprabasal keratins were expressed persistently even upon contact with newly formed stroma and malignant cell invasion. In contrast, non‐epidermal keratins (K4/K13, K8/18, K19), which were similarly synthesized by benign and malignant cells in culture and in early transplants, were differentially regulated with increasing stromal vicinity. While both proteins and mRNAs were downregulated in benign epithelia, the malignant, invasive tumour cells continuously expressed these non‐epidermal keratins throughout (K19), suprabasally (K4/13) or at invasive sites (K8/18). Furthermore, the mesenchymal protein vimentin was expressed de novo in invasive areas confronting tumour stroma. Thus, atypical tissue markers, similarly synthesized in isolated cells in vitro, are downregulated in benign but maintained and upregulated in malignant epithelia. This is presumably caused by the neighbouring stroma being permanently activated by malignant epithelia. Copyright

Epithelium and fibroblast-like phenotypes derived from HPV16 E6/E7-immortalized human gingival keratinocytes following chronic ethanol treatment.

Epithelial-mesenchymal transition (EMT) may be critical for neoplastic progression and its eventual tumorigenicity of epithelia. In this context, we investigated whether EMT and EMT-associated features occurred after chronic ethanol treatment of human gingival keratinocytes immortalized with the E6/E7 oncogenes of human papillomavirus (HPV) type 16. Following a nine-week treatment of cells with 30 mM ethanol in keratinocyte growth medium, they were cultured in normal DMEM with 10% serum. These cell populations were able to proliferate in this medium gradually exhibiting elongated morphology indicating that these cells underwent EMT. Control cells without ethanol treatment did not survive subcultures in DMEM. Upon long-term subcultures of ethanol-treated cells, two phenotypes were obtained exhibiting epithelium-like and spindle-shape fibroblast-like morphology (respectively, termed as EPI and FIB cells), the latter indicating EMT. In comparison to EPI cells, the phenotypic transition to FIB cells was concomitant with a decrease in the expression of keratins, desmoplakins and a complete loss of K14. Moreover, FIB cell transition strongly correlates with an increase in the expression of vimentin and simple epithelial keratin K18. These alterations in FIB cells were associated with the ability of these cells to exhibit anchorage-independent growth, while EPI cells exhibited anchorage-dependent growth. Concerning the transformation stage, FIB cells represent a progressively more advanced transformed phenotype which may reflect an early step during HPV- and ethanol-dependent multi-step carcinogenesis.

Modulation of the epidermal growth factor receptor by the human papillomavirus type 16 E5 protein in raft cultures of human keratinocytes.

It has been shown that the E5 protein of the human papillomavirus type 16 modulates epidermal growth factor receptor downregulation in monolayer cultures of human keratinocytes and mouse fibroblasts. We have now analysed the effect of this protein on the expression, the distribution and the activation of EGF receptors in raft cultures derived from an E5-transfected human keratinocyte cell line. The epithelia generated in these cultures were stratified and exhibited suprabasal expression of cytokeratins 1 and 10, which are known markers of early epidermal differentiation. In situ hybridization with an antisense riboprobe to the human papilloma virus type 16 E5 protein revealed a homogeneous gene expression within the entire epithelium of E5-transfected but not empty vector-transfected control cultures. Treatment of serum-starved rafts with EGF for 48 hours led to a strong decrease of suprabasal EGF receptors in control cultures, but not in rafts of E5-expressing cells. Under these conditions, no activated receptors were observed in control cultures, but activated receptors were still present in E5-raft cultures. Our results indicate that human papilloma virus type 16 E5-mediated modulation of EGF receptor expression occurs in a time- and structure-dependent manner in epithelial equivalents of human keratinocytes.

Connexin 43 expression is downregulated in raft cultures of human keratinocytes expressing the human papillomavirus type 16 E5 protein.

Abstract. A decrease in gap junction-mediated cell-to-cell communication has previously been observed in monolayer cultures of human keratinocytes (HaCaT cells) expressing the human papillomavirus type 16 E5 (HPV16 E5) gene and attributed to the reduced phosphorylation of connexin 43, the most abundant connexin in HaCaT cells. In line with this observation, we have now analyzed the effect of HPV16 E5 on connexin 43 expression in raft cultures produced by transfected HaCaT cells. These keratinocytes transcribe HPV16 E5 under the control of a dexamethasone-inducible promoter. Our results show that treatment with dexamethasone leads to an almost complete disappearance of connexin 43 in rafts expressing the E5 gene but not in control rafts. In our study we discuss the possible effects of this downregulation on cell-cell communication and cellular malignant transformation.

Elevated gene expression of MMP-1, MMP-10, and TIMP-1 reveal changes of molecules involved in turn-over of extracellular matrix in cyclosporine-induced gingival overgrowth.

In humans, pathogenesis in cyclosporine A (CsA)-induced gingival overgrowth (GO) includes the accumulation of extracellular matrix (ECM) constituents, viz., collagen type-1 and type-3 and proteoglycans, in subgingival connective tissue. However, whether this increase is associated with alterations of molecules pivotal for the turn-over of collagens and proteoglycans remains unclear. The present study explores the status of matrix metalloproteinase MMP-1 and MMP-10, which are important for fibrillar collagen and proteoglycan turn-over, and their tissue inhibitor TIMP-1, on their gene expression and protein levels in frozen sections derived from GO and matched normal tissue. In situ hybridization (ISH) revealed elevated levels of MMP-1 gene expression in the connective tissue of GO compared with normal tissue. This elevation also applied to MMP-10 and TIMP-1, the latter exhibiting the strongest gene transcription in the deep connective tissue. These differences detected by ISH were corroborated by quantitative reverse transcription/polymerase chain reaction; relative gene expression analysis indicated a 1.9-fold increase for MMP-1, a 2.3-fold increase for MMP-10, and a 4.8-fold increase for TIMP-1. Detection of the protein by indirect immunofluorescence showed that normal gingival tissue was devoid of all three proteins, although they were detectable in GO tissue, with emphasis on TIMP-1. Analysis of our data indicates elevated levels of MMP-1 and-10, and particularly TIMP-1. With respect to TIMP-1, this elevation may in turn lead to alterations in ECM turn-over by abrogating MMP-1 and MMP-10, thereby contributing to ECM accumulation associated with GO.