Specific detection of glycans on a plasma membrane of living cells with atomic force microscopy.

Among the many alterations of cancer cells is the expression of different surface oligosaccharides. In this work, oligosaccharide expression in living cells (cancer and reference ones) was studied with atomic force microscopy by using lectins as probes. The unbinding force obtained for the same lectin type (concanavalin A or Sambucus nigra) suggested slightly dissimilar structures of binding sites of the same ligand type. For the lectin from Phaseolus vulgaris, a much larger unbinding force indicated a distinct structure of the binding site in cancer cells. The unbinding probability confirmed a higher content of both sialic acid and mannose-containing ligands in cancer and reference cells, respectively. These results demonstrate the potential of atomic force microscopy to directly probe the presence of molecules on a living cell surface, together with the quantitative description of their expression.

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--alpha2,3(Gal beta1,3)- and alpha2,3(Gal beta1,4)-sialyltransferases, alpha1,2- and alpha1,3-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 alpha-2,6- and alpha-2,3-linked sialic acid. The average number of polypeptides bearing beta-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.

Comparative analysis of prostatic acid phosphatase and prostate-specific antigen mRNA levels in hyperplastic prostate stimulated with steroid hormones and growth factors.

Prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) are the markers of human prostatic gland. However, it is still not completely understood if and how, steroid hormones and growth factors affect their expression and metabolism in the respect to the major pathologies of the gland. Appropriate studies were carried out on histopathologically diagnosed benign prostatic hyperplasia--BPH (n = 42) using tissue slices and cells derived from them. They were incubated with steroid hormones: 5-alpha-dihydrotestosterone (DHT), estradiol (E) and growth factors: epidermal growth factor (EGF), basic fibroblastic growth factor (bFGF) under culture conditions for up to 24 hours. P-labelled specific oligonucleotide probes were used to analyze total RNA isolated from each sample for the presence of PAP and PSA mRNAs. DHT, E, bFGF, EGF or both DHT + bFGF and DHT + EGF increased PAP and PSA mRNA levels in a time- and dose-dependent manner. The highest and statistically significant increase (P < 0.001) for PAP mRNA was observed when DHT + bFGF were present in the medium while for PSA mRNA if DHT + EGF were added to the medium. Slow but constant decrease of PAP and PSA mRNA levels was observed in the absence of each of these factors in the incubation medium. The results suggest that early expression of PSA and PAP genes and/or their mRNA stability strongly depend on DHT while differ in their response to EGF and bFGF.