Human melanoma HT168 cell line (kind gift of professor József Tímár, Semmelweis University, Hungary) was established from A2058 cell line according to its metastasis formation in immunosuppressed mice (29), while WM35 obtained from ATCC (ATCC^® CRL-1661™, Manassas, VA, USA) originally was isolated from a primary cutaneous melanoma of radial growth phase (30). Cells were nourished with RPMI-1640 culture medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (PAA, Piscataway, NJ, USA), 4.1 g/l glucose, 2 mmol/l L-glutamine (Gibco, Gaithersburg, MD, USA), penicillin (100 U/ml) and streptomycin (100 μg/ml). NHEM cell line (PromoCell GmbH, Heidelberg, Germany) was cultured in Melanocyte Medium (PromoCell GmbH) according to the instructions of the manufacturer. Cells were incubated at 37°C in the presence of 95% air and 5% CO₂ atmosphere and 80% humidity in 25-cm² flasks (PAA Laboratories) until −70% confluence.

Activity of calcineurin and ERK1/2 was inhibited with the continuous application of 2 μM cyclosporine A (CsA) (Sigma-Aldrich) (dissolved in sterile DMSO) and 5 μM PD098059 (PD) (Sigma-Aldrich) (dissolved in sterile DMSO), respectively, started 2 days before confluence. DMSO was administered as vehicle-control but no alterations were shown in any of the experiments comparing with the untreated controls (data not shown).

Histological samples with melanoma metastases (mesenterial lymph node and lung lesions) as well as control tissues from healthy human bodies were collected from cadavers with the assistance of the Pathology and the Forensic Medicine Departments (n=3). The study was approved by the Ethics Committee of University of Debrecen, under licence number 3244-7/2011. For morphological analysis, cell lines were cultured on rectangular cover glasses (Menzel-Gläser, Menzel GmbH, Braunschweig, Germany). Cadaver tissue samples and cell cultures were fixed in Saint-Marie's fixative (99% ethanol and 1% anhydrous acetic acid) for 24 or 1 h, respectively. Tissues were embedded into paraffin and 7-μm thick sections were cut. After removal of paraffin and rehydration in ethanol, PBS supplemented with 1% bovine serum albumin (BSA, Amresco LLC, Solon, OH, USA) at 37°C for 1 h was applied to block nonspecific antibody binding. HA was detected by using a biotinylated HA-binding complex in 5 μg/ml concentration (bHABC was kindly provided by R. Tammi and M. Tammi, Department of Anatomy, University of Kuopio, Kuopio, Finland) at 4°C overnight. The reaction was visualized with Streptavidin-Alexa 555 (2 μg/ml, Invitrogen Corp., Carlsbad, CA, USA) for fluorescence microscopy. Cultures and tissues were mounted in Vectashield Hard Set mounting medium (Vector Laboratories Ltd., Peterborough, UK) containing DAPI to visualise the nuclei of cells. In cadaver tissue samples, monoclonal MelanA antibody (Novocastra Laboratories Ltd., Newcastle, UK) was used to demonstrate melanin-positive cells labelling with an Alexa 488 conjugated anti-mouse antibody (Invitrogen Corporation) at a dilution 1:1,000.

For HAS2, HAS3, RHAMM and CD44 immunocytochemistry cell tissues were incubated with primary antibodies at a dilution described in Table I, at 4°C overnight. For visualisation of the primary antibodies Alexa Flour 488-conjugated goat anti-rabbit (Invitrogen Corp.) and Alexa Fluor 488-conjugated goat anti-mouse (Invitrogen Corp.) secondary antibodies were used at a dilution of 1:1,000. Samples were mounted in Vectashield Hard Set mounting medium (Vector Laboratories, Ltd.) containing DAPI to visualise the nuclei of cells. Photomicrographs of the samples were taken using an Olympus DP72 camera on a Nikon Eclipse E800 microscope (Nikon Corp., Tokyo, Japan). Images were acquired using cellSense Entry 1.5 software (Olympus, Shinjuku, Tokyo, Japan) using constant camera settings to allow comparison of fluorescent signal intensities.

Cells were washed twice in CMF-PBS, harvested with 0.25% trypsin (Sigma-Aldrich) and resuspended in RPMI-1640 medium in a density of 2×10⁵ cells/ml. Lower wells of 48-well Boyden chemotaxis chamber (Neuro Probe Inc., Gaithersburg, MD, USA) were filled with human umbilical cord HA (1,600 kDa) or Streptomyces HA (300–800 kDa) (Sigma-Aldrich) dissolved in CMF-PBS at a concentration of 400 and 800 μg/ml, respectively and covered with a polycarbonate filter (Neuro Probe Inc.) containing pores with a diameter of 3 μm. Cell suspension (50 μl) was inoculated into the wells on the top of the membrane and the chamber was incubated for 3 h at 37°C in a humidified atmosphere (5% CO₂-95% air). Non-migrated cells were removed from the surface of the membrane and after fixation in methanol, migrated cells were stained with 1% toluidine blue (Sigma-Aldrich) dissolved in water. Membranes were air-dried and mounted with Pertex (Sigma-Aldrich). Absolute cell numbers were counted using a light microscope. Six-wells were counted in each experimental group and three independent assays were performed.

Medium containing 1 μCi/ml [³H]-thymidine (185 GBq/mM) [³H]-thymidine (American Radiolabeled Chemicals, Inc. St. Louis, MO, USA) was added to the cells cultured in wells of 24-well plates for 16 h after 2 days of PD treatment. After washing with PBS, proteins were precipitated with ice-cold 5% trichloroacetic acid for 20 min. After washing with PBS again, cells were harvested using 0.25% trypsin for 10 min. Cells were collected with centrifugation at 2,000 rpm, the pellet was resuspended in 10 μl CMF-PBS and placed into wells of special, opaque 96-well plates (Wallac, Perkin-Elmer Life and Analytical Sciences, Shelton, CT, USA). The plates were placed in an exsiccator containing phosphorous pentoxide in order to absorb moisture. Prior to the measurements, 50 μl scintillation solution (MaxiLight; Hidex, Turku, Finland) was added to each well, and radioactivity was counted by a liquid scintillation counter (Chameleon Microplate Reader, Hidex). Measurements were carried out in 6 samples of each experimental group in 3 independent experiments.

Cell cultures were dissolved in TRIzol (Applied Biosystems, Foster City, CA, USA) and after the addition of 20% RNase free chloroform, samples were centrifuged at 4°C at 10,000 x g for 20 min. Samples were incubated in 500 μl of RNase-free isopropanol at −20°C for 1 h. After washing the centrifuged pellet by 70% ethanol the total RNA was resuspended in RNase-free water and stored at −20°C. The assay mixture for reverse transcriptase reaction containing 2 μg RNA was performed by High Capacity RT kit (Applied Biosystems) according to the manufacturer's instructions. Amplifications were performed in a thermal cycler (Labnet MultiGene™ 96-well Gradient Thermal Cycler; Labnet International, Edison, NJ, USA) in a final volume of 25 μl [containing 1 μl forward and reverse primers (0.4 μM), 0.5 μl dNTP (200 μM), and 5 U of Promega GoTaq^® DNA polymerase in 1X reaction buffer] as follows: 95°C, 2 min, followed by 35 cycles (denaturation, 94°C, 1 min; annealing at optimised temperatures as given in Table II for 1 min; extension, 72°C, 90 sec) and then 72°C, 10 min. The sequences of primer pairs, and further details of polymerase chain reactions, are given in Table II. PCR products were analysed by electrophoresis in 1.2% agarose gel containing ethidium bromide. GAPDH was used as internal control. Optical density of signals was measured by ImageJ 1.40 g freeware and results were normalised to the optical density of untreated control cultures.

Cell cultures were washed in physiological NaCl solution and then harvested. After centrifugation (2,000 rpm, 10 min), cell pellets were suspended in 100 μl of homogenization RIPA (radioimmunoprecipitation assay)-buffer (150 mM sodium chloride; 1.0% NP40, 0.5% sodium deoxycholate; 50 mM Tris, pH 8.0) containing protease inhibitors [aprotinin (10 μg/ml), 5 mM benzamidine, leupeptin (10 μg/ml), trypsine inhibitor (10 μg/ml), 1 mM PMSF, 5 mM EDTA, 1 mM EGTA, 8 mM Na-Fluoride, 1 mM Na-orthovanadate]. Samples were stored at −70°C. Suspensions were sonicated by pulsing burst for 30 sec at 40 A (Cole-Parmer, Illinois, USA). For western blotting, total cell lysates were used.

Samples for SDS-PAGE were prepared by the addition of Laemmli electrophoresis sample buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, 0.125 M Tris-HCl pH 6.8) to cell lysates to set equal protein concentration of samples, and boiled for 10 min. Approximately 40 μg of protein was separated by 7.5% SDS-PAGE gel for detection of HAS1, HAS2, HAS3, RHAMM, CD44, and actin. Proteins were transferred electrophoretically to nitrocellulose membranes. After blocking with 5% non-fat dry milk in PBS, membranes were washed and exposed to the primary antibodies (Table I) overnight at 4°C. PhosphoSer/Thr detection kit (Millipore, Billerica, MA, USA) was used to detect phosphorylation level of Ser/Thr amino acid side chains. After washing for 3×10 min in PBST, membranes were incubated with HRP conjugated anti-rabbit IgG (Bio-Rad Laboratories, CA, USA) in 1:1,500 or anti-mouse IgG (Bio-Rad Laboratories) in 1:1,500 dilution. Signals were detected by enhanced chemiluminescence (Millipore) according to the instructions of the manufacturer. Signals were manually developed on X-ray film (Agfa-Gevaert Group, Mortsel, Belgium). Optical density of western blot signals was measured by using ImageJ 1.40 g freeware and the results were normalised to the values of untreated control cultures.

Optical density (OD) of RT-PCR and western blot results were normalised to the inner controls, then the ODs of the control samples of every experimental group and CsA or PD treated cultures of each melanoma cell line were used for statistical analysis. Statistical comparisons between control and test samples were performed by using Student's paired t-test where statistical method reported significant differences among the groups (P<0.05). The data are representative of at least three different experiments. Where applicable, data are expressed as mean ± SEM.

Although presence of HA around epidermal keratinocytes (31) and expression of HAS enzymes by these cells have been described long ago (32,33). Involvement of melanocytes in epidermal HA homeostasis is less evident. Therefore, first we investigated the HA secretion of normal melanocytes locating in the stratum basale of human epidermis. We were able to demonstrate only a weak signal for HA in the basal cell layer of the epithelium (Fig. 1A-a.1). Expression of all the three HAS enzymes was also monitored. We failed to demonstrate either HAS1 (data not shown) or HAS2 in the epidermis of human skin tissue samples (Fig. 1B-b.1), but a well visible HAS3 immunopositivity was detected in the stratum basale where melanocytes reside accompanied with undifferentiated keratinocytes (Fig. 1C-c.1). As tumour invasion and motility are highly dependent on the ECM surrounding the malignant cells and HA has been proven to be one of the extracellular macromolecules which can be responsible for the regulation of infiltration of tissues in the vicinity of primary tumours (34), next we investigated HA and HAS content of melanoma samples. Two differently evolved metastases of melanoma were screened (mesenterial lymph node and lung lesions) and compared with normal tissues from healthy human cadavers. HA was modestly detectable in the normal lung and mesenteric lymph node (Fig. 1A-a.2 and a.4) but a strongly enhanced HA signal was observed either in lymph node (Fig. 1.A-a.3) or lung with melanoma metastases (Fig. 1A-a.5). HAS1 was detectable neither in normal skin, nor in malignant lesions (data not shown). The expression of HAS2 and HAS3 was not detectable with immunohistochemistry in normal lymph nodes (Fig. 1B-b.2 and c.2) or the lung (Fig. 1B-b.4 and c.4), but a strong signal of HAS2 immunopositivity was observed in lymph node melanoma metastases (Fig. 1B-b.3). In spite of the strong autofluorescence signal of elastic fibres in the lung, a well visible colocalization of HAS2 and MelanA was detected (Fig. 1B-b.5). Similarly to HAS2, HAS3 was not detected in the normal healthy tissues with this method (Fig. 1C-c.2 and c.4), but a pronounced expression was demonstrated in metastatic lymph nodes and lung metastases (Fig. 1C-c.3 and c.5). Strong HAS3 immunopositivity was seen close to the nuclei of MelanA expressing cells, as well as in the surrounding stromal cell population.

Since the HA secretion and HAS expression were markedly altered in tissue samples with melanoma metastases we investigated the HA homeostasis of normal human melanocyte (NHEM) in a cell culture system. Similarly to the melanocytes locating in the stratum basale of epidermis, cultured melanocytes secreted only a low amount of HA, hardly detectable with affinity cytochemistry (Fig. 2A). As secretion of HA can be regulated by reversible phosphorylation, we investigated the involvement of ERK1/2 in the HA production and we also aimed to identify the involvement of a possible PP, calcineurin in this process. PD098059 was administered as a MAPK and CsA as a calcineurin inhibitor. Inhibition of PP2B activity resulted in an undetectably low HA production of cultured melanocytes (Fig. 2A). In contrast, the inhibition of ERK1/2 elevated the HA secretion of normal human melanocytes (Fig. 2A). These data suggest that the two enzymes exert the opposite effect on HA production of NHEM cells. In terms of the synthesising enzymes, mRNA of HAS1 was not present (data not shown), while the mRNAs of HAS2 and HAS3 were present in the NHEM cells (Fig. 2B), although only weak signals of protein expression were seen with western blot method (Fig. 2C). HAS2 was hardly detectable in NHEM (Fig. 2E) while HAS3 gave evident, strong signals with immunocytochemistry (Fig. 2F). Inhibition of calcineurin did not alter the mRNA expression of HAS3 and HAS2 (Fig. 2B and D), but reduction in the protein expression of the synthases was observed (Fig. 2C–F). Inhibition of MAPK pathway exerted different effect on HAS expression of NHEM cells. It did not alter the expression of HAS2 (Fig. 2B–E), but resulted in a modest elevation of the mRNA expression (Fig. 2B and D) and a pronounced increase of the protein expression of HAS3 (Fig. 2C, D and F). Interestingly, a strong accumulation of HAS3 was visible around the nuclear area in NHEM cells after ERK1/2 inhibition (Fig. 2F).

It is already known that HA regulates the migration, proliferation and other cell biological features of malignant cells (5), so we aimed to detect the presence of this extracellular matrix component and to identify the HAS enzymes responsible for the production of HA in melanoma cell lines established from various stages of cutaneous melanoma.

HA was detected both intracellularly and pericellularly in the examined cell lines (Fig. 3A-a and d). The detected HA is the product of HAS2 and/or HAS3 enzymes because their mRNA (Fig. 4A and C) and protein expressions (Fig. 4B and D) were proven by reverse transcriptase PCR and western blot analyses, respectively. Neither mRNA nor protein expression of HAS1 was detected (data not shown). Similarly to NHEM, both melanoma cell lines expressed the mRNAs of both HAS enzymes (Fig. 4A) but the protein expression of the synthases showed slight differences. HAS2 protein expression was stronger in WM35 cells (Figs. 3B-a, 4B and D), while it remained weaker in HT168 cells (Figs. 3B-d, 4B and D). HAS3 protein expression showed the opposite pattern, it was stronger in HT168 cells and was almost undetectable in WM35 cells (Figs. 3C-d, 4B and D). In both cell types, HAS3 formed filamentous arrangement in the cytoplasm and positive signals associated to the nuclear region were also detected (Fig. 3C-a and d).

Treatment of melanoma cells with 2 μM CsA notably decreased the HA secretion (Fig. 3A-b and e), while the inhibition of ERK 1/2 pathway with administration of 5 μM of PD098059 increased the HA production of both cell lines (Fig. 3A-c and f). Parallel with the HA production, application of CsA also decreased HAS2 protein expression (Figs. 3B-b and e, 4B and D). Presence of the dominantly expressed HAS3 isoform of aggressive metastatic HT168 cells was significantly elevated by PD098059 treatment but no alteration was detected after the administration of CsA (Figs. 3C-c and f, 4B and D). Expression of HAS3 isoform showed only a modest decrease in WM35 cells after PP2B inhibition (Figs. 3C-b, 4B and D). At the level of ~60 kDa molecular weight we detected increased Ser/Thr phosphorylation at the presence of CsA. Administration of PD098059 exerted an opposite effect it resulted in weaker signal for phosphorylated proteins at the same molecular weight. These observations suggest altered phosphorylation and the likelihood of a consequent change in the enzymatic activity of HAS2 and/or HAS3 under the effect of the applied enzyme inhibitors (Fig. 4E and F).

It has been published that both CD44 and RHAMM have important roles in the signal transduction of melanoma cells (35). In vitro, both of these receptors can play a role in the regulation of the migration of melanoma cell lines. Expression of mRNA and protein of RHAMM and CD44 were demonstrated in both cell lines (Fig. 5A–D). RHAMM gave strong intracellular signals and the observed filamentous organization of the positivity suggests association to cytoskeletal elements. CD44 also showed cytoplasmic positivity but membrane associated signals were also detected (Fig. 5E-a and F-a). Protein expression of RHAMM was higher in HT168 cell line than in WM35, while CD44 exhibited similar expression pattern both in HT168 and WM35 cell lines (Fig. 5B and D). Not every cell of the WM35 cells showed RHAMM positivity with immunocytochemistry (Fig. 5E-a). Administration of 2 μM CsA did not significantly alter the protein expression of RHAMM in HT168 and in WM35 melanoma cells (Fig. 5B, D and E-b and e). In contrast, the application of 5 μM PD098059 intensely elevated the protein expression of RHAMM (Fig. 5B, D and E-c and f). Furthermore, none of the inhibitors had prominent effect on the CD44 protein expression (Fig. 5B, D and F).

Since incorporated HA can be partly responsible for the regulation of proliferation of tumour cells (5) we investigated the effects of inhibitors on cellular division of WM35 and HT168 melanoma cells. We have previously published results that CsA reduces the proliferation of both cell lines (28). In contrast, PD098059 elevated the proliferation rate of HT168 cells (Fig. 6A).

As strong HA signals localized in the pericellular matrix of melanoma cells either in the melanoma cell cultures or in the tissue samples containing melanoma metastases, a supportive effect of HA on cell motility seemed very likely (5). Hence, we investigated the migratory properties and invasiveness of HT168 and WM35 melanoma cell lines. An in vitro migration assay was performed in Boyden chamber in the presence of hyaluronic acid (a higher, 1,600 kDa and a lower, 300–800 kDa molecular weight HA solution) as a chemoattractant. We did not find significant differences between the migrations of these cell lines towards different size HA chemoattractants (Fig. 6B). As a result of the 2 μM CsA or 5 μM PD098059 treatments, the average number of the migrated HT168 cells toward lower molecular weight of HA was elevated but no significant alteration was shown in the presence of 1,600 kDa HA (Fig. 6D). While the administration of CsA markedly diminished the migration of WM35 cells, the presence of PD098059 significantly facilitated the migration toward 300–800 kDa HA (Fig. 6C). In contrast, cell motility in the presence of the 1600 kDa HA was not significantly altered by PD098059 administration (Fig. 6C).