BACKGROUND:There is a close correlation between tumour progression and hyaluronan production, either by tumour cells or by stromal cells that are stimulated by tumour-derived factors. Inhibition of tumour stimulation of fibroblast hyaluronan may suppress tumour growth and invasion.OBJECTIVES:To examine the effect of the hyaluronan synthesis inhibitor 4-methylumbelliferone (4-MU) on the growth of and hyaluronan synthesis by fibroblasts and C8161 and MV3 melanoma cell lines, invasion, and inhibition of tumour cell-derived factor activation of fibroblasts.METHODS:Effects of 4-MU on growth and hyaluronan synthesis by fibroblasts and melanoma cells were examined in monolayer culture and fibroblast-contracted collagen lattices, and their effects on the growth and invasion of tumour cells into collagen lattices were also studied.RESULTS:4-MU caused a dose-dependent growth inhibition of fibroblast and melanoma cells with maximum inhibition at 0·5 mmol L(-1) 4-MU. At this dose, 4-MU inhibited (3) H-glucosamine incorporation into fibroblast glycosaminoglycans by 52%, and hyaluronan synthesis by 64%. The relative inhibition was more pronounced when fibroblasts were stimulated with C8161 melanoma cell-conditioned medium. 4-MU reduced the level of hyaluronan in fibroblast-contracted collagen lattices, and inhibited both the growth on and invasion into the lattices by melanoma cells. This growth inhibition appears to be predominantly independent of inhibition of hyaluronan synthesis. The effect on growth inhibition was reversible, and 4-MU had no effect on apoptosis.CONCLUSIONS:4-MU is a potent inhibitor of hyaluronan synthesis, induction of stromal hyaluronan accumulation by tumour cells, and fibroblast and melanoma cell proliferation, and results suggest that 4-MU may have potential as a tumour cell anti-invasive and antiproliferative agent.

Background: C35 is a 12 kDa membrane-anchored protein endogenously over-expressed in many invasive breast cancers. C35 ( C17orf37 ) is located on the HER2 amplicon, between HER2 and GRB7 . The function of over-expressed C35 in invasive breast cancer is unknown. Methods: Tissue microarrays containing 122 primary human breast cancer specimens were used to examine the association of C35 with HER2 expression. Cell lines over-expressing C35 were generated and tested for evidence of cell transformation in vitro . Results: In primary breast cancers high levels of C35 mRNA expression were associated with HER2 gene amplification. High levels of C35 protein expression were associated with hallmarks of transformation, such as, colony growth in soft agar, invasion into collagen matrix and formation of large acinar structures in three-dimensional (3D) cell cultures. The transformed phenotype was also associated with characteristics of epithelial to mesenchymal transition, such as adoption of spindle cell morphology and down-regulation of epithelial markers, such as E-cadherin and keratin-8. Furthermore, C35-induced transformation in 3D cell cultures was dependent on Syk kinase, a downstream mediator of signalling from the immunoreceptor tyrosine-based activation motif, which is present in C35. Conclusion: C35 functions as an oncogene in breast cancer cell lines. Drug targeting of C35 or Syk kinase might be helpful in treating a subset of patients with HER2 -amplified breast cancers.

Nephrogenic systemic fibrosis (NSF) is a fibrotic disease generating intense interest due to its recent discovery, and unknown cause. It appears confined to patients with renal disease and presents as grossly thickened, indurated, tight skin that is woody to palpation. Histologically, the dermis contains thickened collagen bundles, numerous plump fibroblast-like cells, and elevated hyaluronan expression. Recent data suggest a link between the use of gadolinium chelate as an MRI contrast agent and the onset of the disease. Fibroblasts from the lesions of six NSF patients, all of whom were exposed to gadodiamide, were compared with control fibroblasts for hyaluronan and collagen synthesis. Serum from NSF patients was assessed for fibroblast hyaluronan-stimulating activity, collagen synthesis, and gadodiamide for its effect on fibroblast proliferation and matrix synthesis. NSF fibroblasts synthesized excess levels of hyaluronan and collagen compared with control fibroblasts, with up to 2.8-fold and 3.3-fold increases, respectively. NSF patient serum stimulated control fibroblast hyaluronan synthesis by up to 7-fold, and collagen synthesis by up to 2.4-fold. 1 mM gadodiamide added to culture medium stimulated fibroblast growth in a dose-dependent manner, decreasing their doubling time from 28 h to 22 h, and increasing the maximum cell density. Even a short exposure to gadodiamide stimulated cell growth, suggesting that the cells were activated by the gadodiamide. The growth of fibroblasts within contracted collagen lattices was also significantly stimulated by gadodiamide, while fibroblasts exposed to gadodiamide synthesized increased levels of hyaluronan. Control fibroblasts exposed to gadodiamide, and NSF fibroblasts exhibited an extensive pericellular coat of hyaluronan, and expressed alpha-smooth muscle actin. Gadolinium chloride did not affect fibroblast growth. This report demonstrates that NSF fibroblasts synthesize excess levels of hyaluronan and collagen, and that gadodiamide stimulates control fibroblast growth, matrix synthesis, and differentiation into myofibroblasts, suggesting a possible role for gadodiamide in the pathophysiology of NSF.

Initial colonization events and yeast-hyphal transformation by Malassezia furfur were observed using living skin equivalent (LSE) models for growth. Yeast cells were inoculated onto the LSEs which were incubated in CO2-independent media at 37 degreesC for variable lengths of time. Assessment of fungal growth and invasion was by light- and scanning electron microscopy (SEM). Viability counts of M. furfur were determined by a method of washing and serial dilution. Yeast cells had retained their viability and increased in number approximately twofold over a 4-day period of incubation. Yeast-to-hyphal transition was not achieved in this model. Random destruction of the uppermost layers of the stratum corneum was observed in the presence of M. furfur. LSEs therefore appear to be a promising model for mechanisms of growth of cutaneous organisms.

Background Dermal mucin is an amorphous gelatinous substance composed primarily of hyaluronan (HA) and sulphated glycosaminoglycans (GAGs). In primary cutaneous mucinosis, accumulation of mucin is a characteristic feature of lichen myxoedematosus, scleromyxoedema and reticular erythematous mucinosis. Secondary mucinoses are disorders where mucin deposition is an additional finding, and deposition is associated with lupus erythematosus, dermatomyositis, scleroderma and granuloma annulare. The underlying cause of the abnormal mucin deposition is unknown. An increasing number of cases of a fibromucinous scleromyxoedema-like disorder associated with renal dysfunction, recently termed nephrogenic fibrosing dermopathy (NFD), is being reported.Objectives To examine the synthesis of sulphated GAGs and HA by fibroblasts derived from uninvolved and involved skin of a patient with dermatomyositis and two patients with NFD, and the effect of patient serum.Methods GAGs were quantified by a radiometric assay and HA was determined by an enzyme-linked HA-binding protein assay.Results We found that fibroblasts derived from active lesions of NFD synthesize elevated levels of GAGs, and in particular HA, compared with normal controls, while serum from the patient with dermatomyositis and the two patients with NFD stimulates GAG synthesis, including HA synthesis, by both control and patient fibroblasts.Conclusions Fibroblasts from patients with active NFD are either activated to synthesize elevated levels of HA or contain another cell type, possibly derived from circulating fibrocytes. In both disorders, there is additionally a serum-derived factor that stimulates production of sulphated GAGs and HA by fibroblasts.

Hyaluronan, a high molecular weight glycosaminoglycan is associated with cellular proliferation and migration. In a number of different tumour types, there is a close correlation between tumour progression and hyaluronan production, either by the tumour cells or the surrounding stromal cells. We have examined the ability of an aggressive melanoma cell line (C8161) to stimulate the synthesis of fibroblast hyaluronan, and the association of cell-surface CD44 receptors and hyaluronan with invasion. Melanoma cell-conditioned medium (CM) prepared in low glucose medium (1 mg/ml) stimulated the synthesis of fibroblast glycosaminoglycan as measured by [3H] glucosamine incorporation, and the synthesis of hyaluronan as measured using a specific hyaluronan-binding plate assay, while tumour cell-CM prepared in high glucose medium (4.5 mg/ml) inhibited the synthesis of fibroblast glycosaminoglycan. High glucose tumour cell-CM contained large amounts of lactate that appeared to inhibit the tumour-derived factor stimulation of fibroblast glycosaminoglycan synthesis, as removal of the lactate restored the stimulating activity. Melanoma cells seeded on contracted collagen lattices and incubated at the air/liquid interface rapidly formed a multilayered cell mass on the surface, with significant invasion of the gel. Hyaluronan staining was apparent within the collagen gel, and strong staining was seen around the invading tumour cells, but not around those cell layers near the surface. CD44 expression on the tumour cells was confined to those invading cells and corresponded to cellular hyaluronan staining. Hyaluronan staining was also apparent around and between tumour cells invading fibroblast-free collagen lattices. Monolayer cultures of C8161 cells stained strongly for CD44, but few cells stained for hyaluronan, while no detectable hyaluronan was released into the medium. In summary, the C8161 melanoma cells stimulated the synthesis of fibroblast hyaluronan, and in collagen lattices, only the invasive tumour cells expressed CD44 and hyaluronan, either in the presence or absence of fibroblasts.

BACKGROUND:There is a current need for a reliable prognostic marker for melanoma patients, particularly those with stage 2 and stage 3 disease, so that adjuvant therapies can be directed appropriately.OBJECTIVES:To establish whether or not the use of tyrosinase-specific or melanA/MART-1-specific reverse transcriptase-coupled-polymerase chain reaction (RT--PCR) of peripheral blood cells detects preclinical disease progression in patients with malignant melanoma.METHODS:Two hundred and ninety-nine patients with melanoma in clinical stages 1--4 were observed in this study. Samples were obtained sequentially from 153 of these patients at 4-week intervals over a period of up to 2 years and correlated with clinical evidence of disease activity. Tyrosinase and melanA/MART-1 amplicons were analysed by agarose gel electrophoresis and Southern blot hybridization subsequent to a single round of amplification.RESULTS:We demonstrated a statistically significant increase in tyrosinase RT--PCR positivity with advancing stage of melanoma progression. The percentage tyrosinase positivity in 910 samples tested was: stage 1, 135 samples, 34% positive; stage 2, 196 samples, 51% positive; stage 3, 423 samples, 50% positive; and stage 4, 156 samples, 65% positive. The positivity rate for individual patients tested sequentially was higher if only one positive test was required to label a patient positive, at 42%, 65%, 82% and 81% for patients in stages 1--4, respectively. However, we did not find a clear pattern of conversion from negativity to positivity in patients who progressed during the study from stage 2 to stage 3 or stage 3 to stage 4, and found no clear evidence of increased positivity rates in the 6-week period following melanoma-related surgery in patients with stage 3 and 4 disease. The positivity rate for melanA/MART-1 was lower for both patients and samples, and no melanA/MART-1-positive sample was negative for tyrosinase.CONCLUSIONS:We conclude that the presence of circulating tyrosinase-positive cells as detected by this method appears to be a discontinuous rather than a continuous phenomenon, even in patients with stage 4 disease. For this reason the assay cannot be recommended as a method of sequentially monitoring individual patients in a clinical setting.

Background Various tumours exhibit glycosaminoglycan rich, and in particular hyaluronan rich matrices surrounding them that facilitate tumour growth and invasion. In many tumours, this matrix is predominantly synthesized by fibroblasts following stimulation by tumour cell-derived factors.Objectives To determine what effect tumour cell-conditioned medium has upon fibroblast glycosaminoglycan synthesis when cells were cultured as monolayers and within contracted collagen lattices.Methods Serum-free conditioned medium from melanoma cell lines (C8161, MV3, A375 and Hs294T) was examined for its ability to stimulate the incorporation of H-3-glucosamine and (SO4)-S-35 into glycosaminoglycans synthesized by fibroblasts.Results Conditioned medium from all four melanoma cell lines exhibited potent glycosaminoglycan-stimulating activity. In monolayer culture, C8161-conditioned medium stimulated a 4.2-fold increase in fibroblast hyaluronan, and a 9.9-fold increase in sulphated glycosaminoglycan synthesis, while (SO4)-S-35 incorporation was increased only 2.1-fold. In collagen lattice cultures, C8161-conditioned medium stimulated a 4.9-fold increase in hyaluronan synthesis, a 5.4-fold increase in sulphated glycosaminoglycans, and a 1.3-fold increase in (SO4)-S-35 incorporation.Conclusions Melanoma cells produce factors that are potent stimulators of fibroblast glycosaminoglycan synthesis, in both monolayer culture and within contracted collagen lattices. Synthesis of both hyaluronan and sulphated glycosaminoglycans with a reduced degree of polymer sulphation is stimulated. Such changes are likely to promote tumour cell proliferation and migration.

Stratum corneum is the outermost layer of skin. Its external aspect is in contact with the outside environment and its internal aspect is in contact with the sub-stratum corneum environment. Skin is sterile at birth but soon becomes colonised by a number of microorganisms which make up its flora. Dermatophytes are not considered part of this flora. Dermatophyte fungi have been shown to have keratinolytic, other proteolytic and lipolytic activity (1). Serine proteinases (urokinase and tissue type plasminogen activator) which are involved in extracellular protein cata- bolism have been found in dermatophytes and their relea- se by dermatophytes was suggested to play a major role in the invasion of skin. Sulphitolysis, a process that denatu- res keratin non-enzymatically, has been found during der- matophyte- induced keratinolysis and suggested as a complementary mechanism to keratinolysis. Keratinase has been partially purified from and detected by immuno- electron microscopy in material from tinea pedis caused by Trichophyton rubrum. Using a fluorescent antibody technique keratinase has also been detected in biopsies from the skin of guinea pigs infected experimentally with Trichophyton mentagrophytes. Disintegration of hair thought to be caused by dermatophyte enzyme digestion has been shown in human scalp infection, experimental infection in guinea pigs and in vitro. Thus it seems that dermatophytes have a battery of enzymes able to digest different substrates in their habitat. Dermatophyte fungi invade the stratum corneum. In the skin there are a number of conditions that favor the growth of dermatophytes while others do not. Conditions favorable for growth of dermatophytes include:

Glycosaminoglycans (GAGs), and in particular hyaluronan, are known to play a role in tumour cell migration, invasion and metastasis. Conditioned medium from two human metastatic melanoma cell lines (Hs294T and C8161) shows potent fibroblast GAG-synthesis-stimulating activities which are active in fibroblast cultures derived from different anatomical sites. This ability is not specific to melanoma cells and is observed in several carcinoma cell lines. Initial characterisation studies have demonstrated that the GAG-stimulating activities in the medium conditioned with melanoma cells show a degree of heat and trypsin resistance. Fractionation of the conditioned medium with Amicon ultrafiltration membranes of various molecular weight cut-offs, ranging from 1 to 30 kD, resulted in a total loss of activity. Activity could be regained by recombination of the concentrated fraction with the filtrate, suggesting more than one factor to be involved in GAG stimulation, with a degree of interdependence between the individual fractions. The fraction greater than 30 kD and that less than 1 kD appear to contain the majority of the GAG-stimulating activity.

Fibroblasts grown within contracted collagen lattices synthesize substantially less glycosaminoglycans than fibroblasts grown as monolayers on a plastic substrate, [H-3]glucosamine incorporation into hyaluronate was reduced by 70%, and incorporation into sulphated glycosaminoglycans was reduced by 40%. However, incorporation into heparan sulphate and chondroitin sulphates was reduced by 14 and 49%, respectively, resulting in a substantial change in the proportions of the individual glycosaminoglycans. On the basis of [H-3]glucosamine incorporation, hyaluronate constituted 80% of the total glycosaminoglycans synthesized in monolayer cultures, but only 67% in collagen lattice cultures. Incorporation of (SO4)-S-35 into chondroitin sulphates was reduced by 22%, whereas no change was observed in heparan sulphates following culture within collagen lattices. Exposure of the fibroblast cultures to retinoic acid (10(-6) mol/l) and retinyl propionate (2 x 10(-6) mol/l) resulted in a decrease in the incorporation of [H-3]glucosamine into hyaluronate by up to 41% in monolayer cultures, and 25% in collagen lattice cultures. The retinoids stimulated the incorporation of [H-3]glucosamine into heparan sulphate by up to 72%, and chondroitin sulphates by up to 30%, whereas (SO4)-S-35 incorporation remained essentially unaltered. Only modest changes in the incorporation of both isotopes into fibroblast sulphated glycosaminoglycans were observed following exposure to the retinoids in lattice cultures, Q-Sepharose ion-exchange chromatography at pH 2.0 revealed that there was no change in the degree of polymer sulphation of either chondroitin sulphate or heparan sulphate isolated from collagen lattice cultures compared with monolayer cultures, Retinoic acid (10(-6)mol/l) treatment did, however, reduce the degree of polymer sulphation of heparan sulphates and chondroitin sulphates in both monolayer and lattice cultures. The molecular mass of the sulphated glycosaminoglycans was unaffected by culture conditions, but hyaluronate isolated from collagen lattice cultures had a higher molecular mass than that from monolayer cultures. Retinoic acid treatment had no effect on the molecular mass of the glycosaminoglycans isolated from either culture system.