Differential role of D cyclins in the regulation of cell cycle by influencing Ki67 expression in HaCaT cells.

D-type cyclins are important regulatory proteins of the G1/S phase of the cell cycle however, their specific functions are only partially understood. We show that silencing of individual D-type cyclins has no effect on the proliferation and morphology of Immortalized non-tumorigenic human epidermal (HaCaT) cells, while double and triple D cyclin silencing results in the failure of the cytokinesis leading to the appearance of large multinucleated cells. Both CDC20 and Ki67 mRNA is downregulated in these cells. Ki67 mRNA silenced cells show similar multinucleated cellular phenotype as double or triple D cyclin silenced cells without affecting D cyclin expression, suggesting that Ki67 is necessary for normal G2/M transition. Our data have revealed that cyclin D1 may have a leading role in G1/S phase regulation and suggest an incomplete functional overlap among D cyclins. Our results indicate that beside their well-known functions during the G0-G1/S phase, D-type cyclins play a pivotal role in the regulation of mitosis via influencing Ki67 expression in a downstream manner probably through E2F1 activation in HaCaT cells.

The adherens junction: a mosaic of cadherin and nectin clusters bundled by actin filaments.

Cadherin and nectin are distinct transmembrane proteins of adherens junctions. Their ectodomains mediate adhesion, whereas their cytosolic regions couple the adhesive contact to the cytoskeleton. Both these proteins are essential for adherens junction formation and maintenance. However, some basic aspects of these proteins, such as their organization in adherence junctions, have remained open. Therefore, using super-resolution microscopy and live imaging, we focused on the subjunctional distribution of these proteins. We showed that cadherin and nectin in the junctions of A431 cells and human keratinocytes are located in separate clusters. The size of each cluster is independent of that of the adjacent clusters and can significantly fluctuate over time. Several nectin and cadherin clusters that constitute an individual adherens junction are united by the same actin-filament bundle. Surprisingly, interactions between each cluster and F-actin are not uniform, as neither vinculin nor LIM-domain actin-binding proteins match the boundaries of cadherin or nectin clusters. Thus, the adherens junction is not a uniform structure but a mosaic of different adhesive units with very diverse modes of interaction with the cytoskeleton. We propose that such a mosaic architecture of adherence junctions is important for the fast regulation of their dynamics.

Increased axonal expression of nectin-1 in multiple sclerosis plaques.

Nectin-1 is a cell adhesion molecule that plays a role in interneuronal synapse formation, in axonal guidance during development and possibly in neuron-glia interactions. To better understand axonal changes in MS, nectin-1 expression was determined by immunohistochemistry in normal adult human cerebral white matter (n = 4) and in six MS plaques (three active and three inactive). The intensity of axonal nectin-1 expression was scored on a scale of 0 to 4+. In normal adult cerebral white matter, axons showed weak nectin-1 expression with a score of 1.25 ± 0.50. Axonal nectin-1 expression was significantly stronger within both active (score = 3.33 ± 0.289, p = 0.001) and inactive (score = 2.16 ± 0.29, p = 0.038) MS plaques than in normal white matter. Axons in white matter adjacent to MS plaques showed nectin-1 expression (score = 1.5 ± 0.50) that was not statistically different from normal controls (p = 0.542). These findings raise the possibility that increased expression of nectin-1 in MS lesions plays a role in the pathogenesis of MS through participation in axonal responses to injury and mediation of altered neuron-glia interactions relevant to myelination.

Increased Resistance of Breast, Prostate, and Embryonic Carcinoma Cells against Herpes Simplex Virus in Three-Dimensional Cultures.

In previous studies we found that uveal melanoma cells grown in extracellular matrix (ECM)-containing three-dimensional (3D) cultures have increased resistance against herpes simplex virus type 1 (HSV-1)-mediated destruction relative to cells cultured without ECM. Using additional tumor cell types including MB-231 human breast cancer cells, PC-3 human prostate cancer cells, and P19 mouse embryonal carcinoma cells, we show here that tumor cell lines other than melanoma are also more resistant to HSV-1-mediated destruction in 3D cultures than cells grown in 2D. We also demonstrate here that one mechanism responsible for the increased resistance of tumor cells to HSV-1 infection in 3D cultures is an ECM-mediated inhibition of virus replication following virus entry into cells. These findings confirm and extend previous observations related to the role of the ECM in tumor resistance against HSV-1 and may lead to improved strategies of oncolytic virotherapy.

Stem cell marker CD271 is expressed by vasculogenic mimicry-forming uveal melanoma cells in three-dimensional cultures.

PURPOSE: Cancer stem cells have increased resistance against a variety of anti-tumor treatment modalities. Vasculogenic mimicry (VM) patterns are present in numerous malignant tumor types, represent the formation of perfusion pathways by tumor cells, and their presence in tumors is associated with adverse outcome. Earlier we have shown that VM-forming tumor cells in three-dimensional (3D) uveal melanoma cultures have increased resistance against cytotoxic agents and oncolytic herpes simplex virus-mediated destruction. The purpose of the current study was to explore the possibility that this increased resistance of VM-forming tumor cells is due to a cancer stem cell phenotype. METHODS: The expression of cancer stem cell marker cluster of differentiation 271 (CD271) was determined in traditional two-dimensional (2D) and 3D cultures of C918 uveal melanoma cells by fluorescent immunocytochemistry. RESULTS: We found that the VM-forming tumor cell subpopulation in 3D cultures expressed CD271. In contrast, cells grown in 2D cultures and tumor cell subpopulations not participating in VM formation in 3D cultures were negative for CD271. CONCLUSIONS: These findings suggest that VM-forming uveal melanoma cells acquire a cancer stem cell-like phenotype that may play a role in the increased therapy resistance of these cells.

Decreased oligodendrocyte nuclear diameter in Alzheimer's disease and Lewy body dementia.

To better understand the pathogenesis of dementia, it is important to understand histopathologic changes in neurodegenerative diseases because they might highlight key aspects of the degenerative process. In this study, the nuclear diameter of neurons and oligodendrocytes in selected temporal lobe areas were determined in autopsy tissue sections from patients with Alzheimer's disease (AD), Lewy body dementia (LBD) and controls. Our morphometric studies targeted neurons in the CA4 region of the pyramidal cell layer of the hippocampus, neurons in the granular layer of the dentate gyrus and oligodendrocytes in parahippocampal white matter. Mean neuronal nuclear diameters were not different among the studied groups. However, our studies revealed a statistically significant reduction of mean oligodendrocyte nuclear diameter in AD and LBD relative to controls. The reduction of the mean nucleus diameter of oligodendrocytes in LBD was independent of the presence of associated AD pathology in LBD. These findings for the first time identify decreased oligodendrocyte nucleus diameter as a morphologic feature of AD and LBD and may lead to a better understanding of the role of oligodendrocytes in AD and LBD pathogenesis.

[Effect of decontaminating solutions on titanium surface: an in vitro study of human epithelial cell culture]. / Dekontamináló anyagok hatása a titánfelszín biointegrációs tulajdonságaira: in vitro humán epithel sejtkultlúra vizsgálatok.

INTRODUCTION: The effects of three different decontaminating solutions in clinical use for peri-implantitis therapy on the chemical structure and surface roughness of commercially pure (CP) Ti were investigated. A further aim was to survey the response of the biological environment to these changes, by examining the attachment and proliferation of human epithelial cells after treatment of the Ti surfaces with these solutions. MATERIALS AND METHODS: CP (grade 4) machined titanium discs (CAMLOG Biotechnologies AG, Switzerland) were treated with 3% H2O2 (5 min), saturated citric acid (pH = 1; 1 min) or chlorhexidine gel (CHX, 5 min). The surface properties were followed through the use of X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The epithelial cell attachment and proliferation was examined by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and bicinchoninic acid (BCA) protein-content assays. RESULTS: XPS showed an intact TiO2 layer on each sample and CHX was adsorbed by the surface, as C-O and/or C=O bond formation was revealed. AFM results gave no significant changes in the roughness after treating the surfaces with the cleaning solutions. While MTT and BCA assays did not show significant differences in epithelial cell attachments, the cell proliferation was significantly increased after H2O2 treatment as compared to CHX (not shown by BCA assays). CONCLUSIONS: The applied decontaminating agents do not damage the Ti surface. H2O2 can be used effectively in decontaminating the implants affected by peri-implantitis, as the human epithelial cell growth was improved, in contrast with CHX.

In vitro dedifferentiation of melanocytes from adult epidermis.

In previous work we described a novel culture technique using a cholera toxin and PMA-free medium (Mel-mix) for obtaining pure melanocyte cultures from human adult epidermis. In Mel-mix medium the cultured melanocytes are bipolar, unpigmented and highly proliferative. Further characterization of the cultured melanocytes revealed the disappearance of c-Kit and TRP-1 and induction of nestin expression, indicating that melanocytes dedifferentiated in this in vitro culture. Cholera toxin and PMA were able to induce c-Kit and TRP-1 protein expressions in the cells, reversing dedifferentiation. TRP-1 mRNA expression was induced in dedifferentiated melanocytes by UV-B irradiated keratinocyte supernatants, however direct UV-B irradiation of the cells resulted in further decrease of TRP-1 mRNA expression. These dedifferentiated, easily accessible cultured melanocytes provide a good model for studying melanocyte differentiation and possibly transdifferentiation. Because melanocytes in Mel-mix medium can be cultured with human serum as the only supplement, this culture system is also suitable for autologous cell transplantation.

Sortilin is expressed in cultured human keratinocytes and is regulated by cutaneous neuropeptides.

Sortilin, a member of the family of Vps10p domain receptors, has been shown to be able to bind the precursor peptide of nerve growth factor (proNGF). ProNGF interacts with sortilin and the p75(NTR) receptor on the cell surface to form a molecular complex capable of activating an apoptotic cascade. Keratinocytes can secrete proNGF and they have p75(NTR) on their surface. The expression of sortilin in normal human keratinocytes has not yet been clearly shown. In this study, we show that keratinocytes express sortilin mRNA, and the presence of sortilin protein is shown in cultured keratinocytes and in normal human skin. We have also shown that the cutaneous neuropeptides substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, and galanin are able to reduce the expression of sortilin mRNA and sortilin protein in cultured human keratinocytes. In addition, each of the analyzed neuropeptides has the ability to arrest the proNGF-induced apoptosis of human keratinocytes. These results suggest that all the participants in the NGF/proNGF pathway are present in the keratinocytes, and cutaneous neuropeptides can modulate their expressions and actions. The NGF/proNGF balance and its regulation by neuropeptides may have an important role in skin homeostasis.

Effects on titanium implant surfaces of chemical agents used for the treatment of peri-implantitis.

The treatment of peri-implantitis, which causes tissue deterioration surrounding osseointegrated implants, involves surface decontamination and cleaning. However, chemical cleaning agents may alter the structure of implant surfaces. We investigated three such cleaning solutions. Commercially pure (grade 4) machined titanium discs (CAMLOG Biotechnologies AG, Switzerland) were treated with 3% H(2)O(2) (5 min), saturated citric acid (pH = 1) (1 min) or chlorhexidine gel (5 min), and their surface properties were examined by atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). Human epithelial cell attachment (24-h observation) and proliferation (72-h observation) were investigated via dimethylthiazolyl-diphenyltetrazolium bromide (MTT) and bicinchoninic acid (BCA) protein content assays. AFM revealed no significant difference in roughness of the three treated surfaces. XPS confirmed the constant presence of typical surface elements and an intact TiO(2) layer on each surface. The XPS peaks after chlorhexidine gel treatment demonstrated C-O and/or C=O bond formation, due to chlorhexidine digluconate infiltrating the surface. MTT and BCA assays indicated similar epithelial cell attachments in the three groups; epithelial cell proliferation being significantly higher after H(2)O(2) than after chlorhexidine gel treatment (not shown by BCA assays). These agents do not harm the Ti surface. Cleaning with H(2)O(2) slightly enhances human epithelial cell growth, in contrast to chlorhexidine gel.

The herpes simplex virus-induced demise of keratinocytes is associated with a dysregulated pattern of p63 expression.

p63 plays a pivotal role in the development and maintenance of stratified epithelial tissues. In an effort to gain insight into the pathogenic mechanisms of skin infections caused by HSV-1 and HSV-2, we determined the patterns of p63 expression in primary keratinocytes and in the HaCaT cell line. The levels of DeltaNp63alpha and a 50kDa p73 isoform were decreased, Bax-alpha remained unaffected, while the expressions of the Bax-beta, TAp63gamma and a 44.5kDa p73 isoform were highly increased in both HSV-1-infected HaCaT cells and primary keratinocytes. In contrast, in response to HSV-2 infection the levels of DeltaNp63alpha, a 50kDa p73 isoform and a 44.5kDa p73 protein were decreased, Bax-alpha and TAp63gamma remained unaffected, while the expression of Bax-beta was slightly increased. The knockdown of TAp63 expression enhanced the viability of HSV-1-infected cells. Thus, HSV-1 and HSV-2 modulate the patterns of p63 and Bax expression in a serotype-specific manner. The dysregulated pattern of p63 expression observed in HSV-infected keratinocytes may comprise part of a mechanism by which these viruses perturb the functions of keratinocytes and lead to their demise.

Differential expression of D-type cyclins in HaCaT keratinocytes and in psoriasis.

In this study, we show that the G0-G1/S phase of HaCaT keratinocyte cell cycle is characterized by D1-type cyclin expression, whereas during the repeated rapid turnover of highly proliferating cells, the expression of cyclins D2 and D3 dominates. Knocking down cyclin D1 mRNA resulted in no change of cell proliferation and morphology, indicating that D2 and D3 cyclins could substitute for D1 in driving the cell cycle. Increased numbers of cyclin D1-expressing keratinocytes were found in the basal layers of the lesional psoriatic epidermis compared to both normal and non-lesional epidermis without increased expression of cyclin D1 mRNA, suggesting a possible dysfunction in the degradation of cyclin D1 protein. We also detected a significant increase in cyclin D2 and D3 mRNA expressions in psoriatic epidermis compared to normal epidermis with no difference in protein expressions. Blocking alpha5-integrin function by a neutralizing antibody in HaCaT keratinocytes downregulated the expression of cyclin D1 mRNA without affecting the expressions of cyclin D2 and D3 indicating a regulatory role for alpha5-integrin in the expression of cyclin D1. Our data suggest a possible role for D-type cyclins in the excessive basal-cell proliferation and perturbed keratinocyte differentiation in the psoriatic epidermis.

Human adult epidermal melanocytes cultured without chemical mitogens express the EGF receptor and respond to EGF.

We describe a novel chemical mitogen-free in vitro culture technique for obtaining pure melanocyte cultures using normal human adult epidermis as a source. The culture medium consists equal parts of the commercially available Keratinocyte Basal and AIM-V media (both from Gibco), as basal medium, which is supplemented with fetal bovine serum, bovine pituitary extract and recombinant human epidermal growth factor (EGF). Melanocytes harvested from human adult skin proliferate extensively and can be passaged serially up to 10-15 times using this medium. We have verified the identity of the cultured cells by tyrosinase mRNA expression and TRP-1 protein staining. Moreover, we showed that autologous human serum alone, without additional supplements is able to provide sufficient growth support for the cultured cells in the basal medium, making this culture technique suitable for autologous melanocyte transplantation. In this culture system normal human adult melanocytes expressed both EGF receptor (EGFR) mRNA and protein and EGF showed a dose dependent mitogenic effect on the cells. EGF itself had no significant influence on EGFR mRNA expression.