Piotr Laidler

Elasticity of normal and cancerous human bladder cells studied by scanning force microscopy.

Abstract Scanning force microscopy was used for the determination of the elastic properties of living cells in their culture conditions. The studies were carried out on human epithelial cells. Two similar lines of normal cells (Hu609 and HCV29) and three cancerous ones (Hu456, T24, BC3726) were measured using the scanning force microscope in order to collect the force versus indentation curves. The BC3726 line originates from the HCV29 cell line which was transformed by the v-ras oncogene. To evaluate their elastic properties, Youngs modulus values were determined. The present study has shown that normal cells have a Youngs modulus of about one order of magnitude higher than cancerous ones. Such a change might be attributed to a difference in the organisation of cell cytoskeletons and requires further studies.

Cancer cell detection in tissue sections using AFM.

Currently, cancer diagnosis relies mostly on morphological examination of exfoliated, aspirated cells or surgically removed tissue. As long as standard diagnosis is concerned, this classical approach seems to be satisfactory. In the recent years, cancer progression has been shown to be accompanied by alterations in mechanical properties of cells. This offers the detection of otherwise unnoticed cancer cell disregarded by histological analysis due to insignificant manifestations. One of techniques, sensitive to changes in mechanical properties, is the atomic force microscopy, which detects cancer cells through their elastic properties. Such measurements were applied to tissue sections collected from patients suffering from various cancers. Despite of heterogeneity and complexity of cancer cell sections, the use of the Youngs modulus as an indicator of cell elasticity allow for detection of cancer cells in tissue slices.

The effect of chitosan on stiffness and glycolytic activity of human bladder cells.

The cells cytoskeleton together with the cell membrane and numerous accessory proteins determines the mechanical properties of cell. Any factors influencing cell organization and structure can cause alterations in mechanical properties of cell (its ability for deformation and adhesion). The determination of the local elastic properties of cells in their culture conditions has opened the possibility for the measurement of the influence of different factors on the mechanical properties of the living cells. The effect of the chitosan on the stiffness of the non-malignant transitional epithelial cells of ureter (HCV 29) and the transitional cell cancer of urine bladder (T24) was determined using scanning force microscopy. The investigations were performed in the culture medium (RPMI 1640) containing 10% fetal calf serum in the presence of the microcrystalline chitosan of the three different deacetylation degrees. In parallel, the effect of chitosan on production of lactate and ATP level was determined. The results showed the strong correlation between the decrease of the energy production and the increase in Youngs modulus values obtained for the cancer cells treated with chitosan.

Comparison of the lectin-binding pattern in different human melanoma cell lines.

Glycosylation is generally altered in tumour cells in comparison with their normal counterparts. These alterations are thought to be important because they contribute to the abnormal behaviour of cancer cells. Therefore, we have comparatively analysed the glycoproteins in cell extracts from human melanoma (primary site – WM35; metastatic sites – WM239, WM9 and A375) cell lines using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and lectin staining. The glycoprotein pattern of the WM35 line differed from that of the other cell lines in having less proteins that reacted with Sambucus nigra, Maackia amurensis and Phaseolus vulgaris agglutinins. A glycoprotein of about 70 kDa had a significantly increased reaction with Sambucus nigra agglutinin in all the cell lines from metastatic sites. In the WM9, WM239 and A375 cell lines, additional bands (160–100 kDa) were stained with Phaseolus vulgaris agglutinin, suggesting that cells from metastatic sites contain more glycoproteins with β1–6 branches. On the other hand, only minor changes in the reaction with Galanthus nivalis agglutinin, a mannose-specific lectin, were detected. Among the proteins showing different lectin staining, one, with an apparent molecular weight of 133 kDa, was recognized by antibodies as N-cadherin. The present results suggest that in human melanoma the expression of branched and sialylated complex type N-oligosaccharides consistently increased in cells from metastatic sites, and support the view that carbohydrates are associated with the acquisition of the metastatic potential of tumour cells.

Targeting the Cancer Initiating Cell: The Ultimate Target for Cancer Therapy

An area of therapeutic interest in cancer biology and treatment is targeting the cancer stem cell, more appropriately referred to as the cancer initiating cell (CIC). CICs comprise a subset of hierarchically organized, rare cancer cells with the ability to initiate cancer in xenografts in genetically modified murine models. CICs are thought to be responsible for tumor onset, self-renewal/maintenance, mutation accumulation and metastasis. CICs may lay dormant after various cancer therapies which eliminate the more rapidly proliferating bulk cancer (BC) mass. However, CICs may remerge after therapy is discontinued as they may represent cells which were either intrinsically resistant to the original therapeutic approach or they have acquired mutations which confer resistance to the primary therapy. In experimental mouse tumor transplant models, CICs have the ability to transfer the tumor to immunocompromised mice very efficiently while the BCs are not able to do so as effectively. Often CICs display increased expression of proteins involved in drug resistance and hence they are intrinsically resistant to many chemotherapeutic approaches. Furthermore, the CICs may be in a suspended state of proliferation and not sensitive to common chemotherapeutic and radiological approaches often employed to eliminate the rapidly proliferating BCs. Promising therapeutic approaches include the targeting of certain signal transduction pathways (e.g., RAC, WNT, PI3K, PML) with small molecule inhibitors or targeting specific cell-surface molecules (e.g., CD44), with effective cytotoxic antibodies. The existence of CICs could explain the high frequency of relapse and resistance to many currently used cancer therapies. New approaches should be developed to effectively target the CIC which could vastly improve cancer therapies and outcomes. This review will discuss recent concepts of targeting CICs in certain leukemia models.

Crystal structure of a covalent intermediate of endogenous human arylsulfatase A.

The structures of human arylsulfatase A crystals soaked in solutions containing 4-methylumbelliferyl phosphate and O-phospho-DL-tyrosine have been determined at 2.7- and 3.2-A resolution, respectively. The formylglycine in position 69, a residue crucial for catalytic activity, was unambiguously identified in both structures as forming a covalent bond to the phosphate moiety. A hydroxyl group is present at the Cbeta of residue 69 and the formation of one out of two possible stereomeric forms is strongly favoured. The structures confirm the importance of the gem-diol intermediate in the arylsulfatases catalytic mechanism. The presence of an apparently stable covalent bond is consistent with the weak phosphatase activity observed for human arylsulfatase A. The structures of the complexes suggest that phosphate ions and phosphate esters inhibit arylsulfatase in non-covalent and covalent modes, respectively. The metal ion present in the active site of arylsulfatase A isolated from human placenta is Ca(2+) and not Mg(2+) as was found in the structure of the recombinant enzyme.

The mechanism of contribution of integrin linked kinase (ILK) to epithelial-mesenchymal transition (EMT)

Integrin linked kinase (ILK) is ubiquitously expressed serine/threonine protein kinase, a binding partner of β1 and β3 integrin subunit as a cytoplasmic effector of integrin receptors that functionally links them to the actin cytoskeleton.We postulate that ILK is important enzyme involved in epithelial-mesenchymal transition (EMT) a critical event in the process of cancer progression. Commonly used EMT molecular markers include among others increased expression of N-cadherin and vimentin, nuclear localization of β-catenin, and the decrease of E-cadherin synthesis. In this study we were able to show that N-cadherin expression in melanoma cells is dependent on ILK signaling and the translocation of β-catenin to the nucleus. Silencing of ILK expression by siRNA significantly inhibited the stabilization and subsequent nuclear translocation of β-catenin and the expression of N-cadherin, a crucial molecule in the EMT, which facilitates association with fibroblast and endothelial cells during invasion of various cancers. The results allow to cautiously speculate on the important role of ILK in the cross-talk between integrins and cadherins accompanying EMT in melanoma.

PPAR γ regulates MITF and β-catenin expression and promotes a differentiated phenotype in mouse melanoma S91

Melanoma represents one of the most rapidly metastasizing, hence deadly tumors due to its high proliferation rate and invasiveness, characteristics of undifferentiated embryonic tissues. Given the absence of effective therapy for metastatic melanoma, understanding more fully the molecular mechanisms underlying melanocyte differentiation may provide opportunities for novel therapeutic intervention. Here we show that in mouse melanoma S91 cells activation of the peroxisome proliferator activated receptor (PPAR) γ induces events resembling differentiation, such as growth arrest accompanied by apoptosis, spindle morphology and enhanced tyrosinase expression. These events are preceded by an initial transient increase in expression from the Microphthalmia‐associated transcription factor gene, (MITF) promoter, whereas exposure to a PPAR γ ligand‐ ciglitazone that exceeds 8 h, causes a gradual decrease of MITF, until by 48 h MITF expression is substantially reduced. Beta‐catenin, an MITF transcriptional activator, shows a similar pattern of decline during ciglitazone treatment, consistent with previous reports that activated PPAR γ inhibits the Wnt/β‐catenin pathway through induction of β‐catenin proteasomal degradation. We suggest that the PPAR γ‐mediated β‐catenin down‐regulation is likely to be responsible for changes in MITF levels. The data suggest that PPAR γ, besides its well‐established role in mesenchymal cell differentiation towards adipocytes, might regulate differentiation in the melanocytic lineage.

The structure of the oligosaccharides of N-cadherin from human melanoma cell lines.

N-cadherin is calcium-dependent cell adhesion molecule that mediates cell-cell adhesion and also modulates cell migration and tumor invasion. N-cadherin is a heavily glycosylated protein. Many studies have demonstrated that malignant transformation of a number of cell types correlates with changes of cell surface N-linked oligosacharides. We have studied the carbohydrate profile of N-cadherin synthesized in human melanoma cell lines and the effect of this protein and complex N-glycans on in vitro migration of melanoma cells from the primary tumor site—WM35 and from different metastatic sites WM239 (skin), WM9 (lymph node), and A375 (solid tumor). N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterization of its carbohydrate moieties was carried out by SDS-PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and on-blot deglycosylation using PNGase F for glycan release. N-glycans were separated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and their structures identified by the computer matching of the resulting masses with those derived from a sequence database. The assay of in vitro chemotaxic cell migration was performed using QCM™ Cell Invasion Assay (Chemicon).N-cadherin from WM35 (primary tumor site) possessed high-mannose and biantennary complex type glycans with α2–6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines possessed mostly tri- or tetra-antennary complex type glycans. In addition, N-cadherin from WM9 (lymph node metastatic site) and A375 (solid tumor metastatic site) contained heavily α-fucosylated complex type chains with α2,3 linked sialic acid. Blocking of N-cadherin-mediated intercellular interaction by N-cadherin-specific antibodies significantly (of about 40%) inhibited migration of melanoma cells. Inhibition of synthesis of complex type N-glycans by swainsonine (mannosidase II inhibitor) led to 50% decrease of cell migration.The results indicated differences between N-cadherin glycans from primary and metastatic sites and confirmed influence of N-cadherin and complex -type N-glycans on in vitro migration of melanoma cells. Published in 2004.

Characterization of glycosylation and adherent properties of melanoma cell lines

The repertoire of oligosaccharide components of cellular glycoproteins significantly contributes to cell adhesion and communication. In tumor cells, alteration in cellular glycosylation may play a key role in giving rise to invasive and metastatic potential. Over 100 melanoma cell lines deposited in the ESTDAB Melanoma Cell Bank (Tubingen, Germany) were studied for the characteristic glycan composition related to tumor progression. Analysis of: (1) cell adhesion to extracellular matrix proteins—fibronectin, laminin, and collagen; (2) the expression of selected glycosyltransferases—α2,3(Galβ1,3)- and α2,3(Galβ1,4)-sialyltransferases, α1,2- and α1,3-fucosyltransferases, and N-acetylglucosaminyltransferase V; (3) characterization of N-glycans was carried out on uveal (4), primary cutaneous (6), and metastatic (96) melanoma cell lines. Results showed that uveal cells did not adhere to any of the substrates and, in general, possessed less glycans containing α-2,6- and α-2,3-linked sialic acid. The average number of polypeptides bearing β-1,6-branched tri- and tetra antennary glycans(characteristic of the metastatic phenotype)were similar in uveal, primary cutaneous, and metastatic melanoma cell lines. Characterization of N-glycans may open a new perspective in the search for specific glycoproteins that could become targets for the therapeutic modulation of melanoma.