Biomedical subjects

J Borovanský

Publications and source records attributed to J Borovanský.

At least 19 recordsLinked to original sources

PubMed2004

Microphthalmia transcription factor: a specific marker for malignant melanoma.

The transcription factor microphthalmia (MITF) is required for the formation of normal melanocytes during embryonic development and for the expression of pigment cell-specific markers, which are the downstream transcriptional targets of MITF. It also seems to be crucial for the survival of malignant melanocytes. The special interest of this review is the possible utility of MITF as a marker of malignant melanoma. Melanocyte-specific isoform of MITF appears to be a unique molecule in the differential diagnosis of melanocytic tumors.

Biomarkers, Tumor↗

PubMed2001

Autofluorescence of melanins induced by ultraviolet radiation and near ultraviolet light. A histochemical and biochemical study.

The induction of autofluorescence of melanins by UV radiation (330-380 nm) and near UV (400-440 nm) light (jointly called UV light) was studied in tissue sections using three commercially available mounting media. Only Immu-Mount (Shandon) was found suitable for this purpose. UV irradiation of melanins in sections mounted in this medium induced strong yellow autofluorescence irrespective of the type of the polymer (eumelanin, neuromelanin, pheomelanin and ochronotic pigment). The phenomenon of autofluorescence induction was also observed with isolated natural and in vitro prepared melanins. It was inhibited by anhydrous conditions, sodium azide and catalase. In parallel experiments, rapid degradation of melanins with an intermediate fluorescent stage was achieved in UV-irradiated sections mounted in media artificially enriched with hydrogen peroxide, or directly in aqueous solutions of H2O2, Na2O2 or HIO4. Oxidations not associated with UV light led to nonfluorogenic breakdown of melanins. These observations indicate that the common mechanism may be an oxidative attack resulting from a concerted action of hydrogen peroxide and UV light leading, through strongly fluorescent intermediates, to a complete bleaching and oxidative breakdown of melanin and melanin-like polymers. Reactive oxygen species (including ozone) are considered to be important reactants in these experiments. Lipopigments differ from melanin-like pigments by their primary autofluorescence, which mostly faded during continuous prolonged irradiation. The only regular exception was melanosis coli pigment, the autofluorescence of which was considerably augmented by UV irradiation. Our results demonstrate a novel type of fluorogen in autofluorescent pigment histochemistry. The implications of the results are discussed especially in the light of the possible presence of melanin-based fluorogens in lipopigments.

Animals↗

PubMed2000

Not only fibroblasts but also melanoma cells express "leucine aminopeptidase" activity.

"Leucine aminopeptidase" (LAP, aminopolypeptidase, EC 3.4.11) activity has been recommended and widely used as a histochemical marker for the identification of contaminating LAP-positive fibroblasts in pigment cell cultures. Using a sensitive biochemical assay with L-leucyl-p-nitroanilide as a substrate we demonstrated that in vitro melanoma cells also exhibit LAP activity. Our comparison of four melanoma cell lines with four fibroblast lines showed that the differences in the enzyme activity were not qualitative but only quantitative. For this reason the specific antibodies, karyological analysis and electron microscopy are recommended as more reliable means in distinguishing fibroblasts from poorly differentiated pigment cells than the LAP-cytochemistry.

Cell Line↗

PubMed1999

Disparate behaviour of two melanosomal enzymes (alpha-mannosidase and gamma-glutamyltransferase).

In addition to tyrosinase and its related proteins melanosomes contain a variety of further enzyme activities. Using spectrophotometric methods alpha-mannosidase and gamma-glutamyl-transferase (GGT) were studied in B 16 melanoma, in isolated melanosomes and in tumour host (mice C57BL6J) sera. When compared to the original melanoma tissue (12.8-26.7 nkat/g TP) isolated melanosomes exhibited much higher alpha-mannosidase activity [227-420 nkat/g of total proteins (TP)]. Strong activation by Zn2+ and no influence of Co2+ ions suggested that the dominant form of alpha-mannosidase of the enzyme present in melanosomes was of the acid (lysosomal) type. The GGT activity of isolated melanosomes (168-244 nkat/g TP) was comparable with that of the whole melanoma tissue (203-375 nkat/g TP) . Treatment of melanosomes with detergents (0.1% Triton X-100, 0.5% deoxycholate) revealed striking extractibility differences between the two enzymes investigated in relation to their localization: alpha-mannosidase remained immobilized in the melanized matrix of melanosomes whereas the membrane bound GGT was easily released. Unlike the alpha-mannosidase the GGT serum levels were increasing in relation to the melanoma growth. The demonstration of acid form of alpha-mannosidase in melanosomes is consistent with their lysosomal ranking; the presence of GGT is in keeping with its expected roles both in protection against oxidative stress and in melanogenesis.

Animals↗

PubMed1999

Attempts to induce melanosome degradation in vivo.

Contradiction between repeatedly reported electron microscopic and histochemical observation of melanosome disintegration in the presence of lysosomal enzymes in vivo and failing attempts to induce such degradation by biochemical means in vitro with the aim to explain chemical mechanism(s) of this process belongs to chronically challenging problems in biochemistry of melanin structures. Attempts have been made to bring about melanosome disintegration in vivo by inoculating melanosomes isolated from dog hair into the tissue of amelanotic Bomirski melanoma and into peritoneal cavities of DBA/2 and C57BL/6J mice, and by repeated injection of melanosomes isolated from Bomirski pigmented melanoma into Syrian hamster foot pads. Both histological and electron microscopic observations demonstrated that melanosomes were phagocytized by macrophages and sporadically by fibroblasts. In peritoneal cavities the injected foreign melanosomes remained mostly extracellularly, were surrounded by foreign body multinuclear cells and formed granulomas. There were no convincing signs of degradation of the hair melanosomes, which behaved like inert foreign bodies. Only some phagocytized Bomirski hamster melanoma melanosomes tended to lose their integrity. Our data suggest that melanosomes devoid of their limiting membranes are not necessarily prone to extensive disintegration in vivo. The earlier reported association of lysosomal enzymes with disintegrating melanosomes does not constitute an evidence for their participation in melanosome degradation. Considering the structure of melanins, redox mechanisms (analogous with the metabolism of polycyclic hydrocarbons (DePierre and Ernster, 1978)) seem to be more probably involved in pigment degradation than hydrolytic reactions.

Animals↗

PubMed1998

Tumour tissue is a source of gamma-glutamyl transpeptidase sialoform in the sera of melanoma-bearing mice.

The total serum activity of gamma-glutamyl transpeptidase (GGT) was shown to increase with the growth of transplantable B16 and S91 melanomas in inbred mice. In an effort to define the source of the GGT shed into the bloodstream the physicochemical characteristics of the partially purified GGT isoforms from liver, serum and B16 melanoma were compared. The molecular weights of the serum and melanoma isoforms were identical (86 kDa) and differed from that of the liver isoform (69 kDa). In polyacrylamide gel electrophoresis the serum and melanoma isoforms had a similar mobility which exceeded that of the liver enzyme. Treatment of the enzyme preparations with neuraminidase removed the differences in the electrophoretic mobility of the three GGT isoforms studied. On ion exchange chromatography on a DEAE-Spheron 300 LC column the melanoma and serum isoforms had an affinity to the sorbent unlike the liver isoform. Our observations suggest that melanoma cells express a sialoform of GGT which is responsible for an increase in the total GGT serum activity. Biochemical and histochemical analyses did not reveal any increase in liver GGT production associated with melanoma development. Detection of the GGT isoform of tumour origin in sera ranks GGT among the specific melanoma markers.

Animals↗

PubMed1997

Melanogenesis in transfected fibroblasts induces lysosomal activation.

Normal melanosome biogenesis requires the association of structural proteins with tyrosinase. 3T3 Swiss fibroblasts transfected with mouse tyrosinase cDNA (line 13.4, clone c) are a unique system in which melanogenesis takes place in the absence of melanosomal structural proteins. Our study confirmed that transfected fibroblasts displayed tyrosinase activity and some of them produced pigment granules. In the absence of melanosomal structural proteins the granules failed both to show a typical ultrastructure and to undergo the usual melanosome ontogenesis. The differentiating agent--dimethyl sulfoxide--increased phaeomelanin production. Pigment was localized in membrane-bound vesicles which were identified as lysosomes by means of immunogold electron microscopy. Cell line 13.4 had higher levels of lysosomal enzymes (beta-hexosaminidase, alpha-mannosidase) than both parental 3T3 cells and clone pKG4 (fibroblasts transfected with the G418 resistance plasmid). Melanosomal proteins act as scavengers of toxic products of melanogenesis, and our results suggest that in their absence cells may employ an alternative mechanism to sequester injurious products.

3T3 Cells↗

PubMed1997

Cytotoxic interactions of Zn2+ in vitro: melanoma cells are more susceptible than melanocytes.

Previous studies have shown that sensitivity to high extracellular levels of Zn2+ is a general feature of cells in vitro and that a prerequisite of the toxic action of zinc is entry into cells via channels that are shared with iron or calcium. As the biochemical and toxicological behaviour of zinc chelate complexes could be different from that of free Zn2+, the effect of chelating agents on zinc transport into human melanoma cell lines was tested. EDTAcal and tetracycline reduced the toxic action of zinc ions in vitro, whereas phenytoin and diethyldithiocarbamate potentiated its effects. D-penicillamine, an effective chelator of zinc in vivo, also exerted a protective action in vitro. Comparison of sensitivity to Zn2+ in vitro between human melanoma lines and several lines of pigment cells from skin of various origins demonstrated that melanoma cells are killed by zinc ions at concentrations which are only partially toxic for normal pigment cells. This is consistent with the repeatedly observed high uptake of 65Zn by melanoma cells.

Chelating Agents↗

PubMed1997

Experimental chemotherapy of murine melanomas: is there a discrepancy compared to clinical experience?

There has been a discrepancy between promising results of experimental chemotherapy in animal melanoma models and clinical response rates. This inconsistency seems to reflect weak points of the assays used so far to monitor the response of melanoma cells to chemotherapeutic agents. Therefore a less usual approach was chosen in the present study: Tumor cells were cultured in peritoneal cavity (B16 melanoma in inbred C57BL/6J mice and Cloudman S91 melanoma in inbred DBA2 mice) to maintain normal in vivo conditions; the animals were receiving the tested agents in i.p. injections and the prolongation of their life span was considered as the principle parameter of therapeutic efficiency of the compounds tested. Previously described therapeutic potency both of vitamins (C, alpha-tocopherol acid succinate) and some phenols (hydroquinone, 4-hydroxyanisole) was confirmed. Benzoate, spin trap N-butyl-alpha-phenyl-nitrone and ammonium chloride as a lysosomotropic agent failed to increase the survival of melanoma-bearing mice. Free radical scavenger methimazole exerted a therapeutic effect in mice with pigmented B16 melanoma. Only classic cytostatic agents--cisplatin and cyclophosphamide--proved its therapeutic effect in both melanoma models studied. These results are in accord with the known resistance of human melanoma to conventional chemotherapy. Measurement of serum activity of gamma-glutamyltransferase was shown to be useful for monitoring therapeutic effect.

Animals↗

PubMed1997

Detection of metals in tissues, cells and subcellular organelles.

The current state of art in the detection of metals in tissues, cells and their subcellular fractions has been reviewed. Both classic chemical techniques (including titration, spectrophotometric, electrochemical and isotope methods, neutron activation analysis, mass spectrometry, inductively coupled plasma) and cell biology techniques (i.e. conventional histochemical methods, autometallography, autoradiography and microanalytical techniques using beam of electrons or protons, laser, X-ray or ion beam) were discussed. This survey of analytical techniques is, wherever possible, illustrated by examples documenting how our knowledge on metals in pigment cells and their subcellular fractions has been growing.

Animals↗

PubMed1996

Free radical activity of melanins and related substances: biochemical and pathobiochemical aspects.

Human body contains a number of natural pigments-authentic melanins, soluble melanins, neuromelanins, lipofuscins-having a similar origin-oxidation and polymerization of precursor phenols, and sharing many common characteristics among which dominates their free radical activity. Related substances can be formed from exogenous compounds introduced at some occasions to the organism. The functional properties of the pigments seem to be determined by their localization, i.e. cellular or extracellular, and as regards those intracellular ones by their subcellular compartmentalization. All the intracellular pigments mentioned in this study are associated with lysosomal enzymes at some stage of their development. A special attention was paid to a neglected group of soluble melanins. It was proposed that methylation may decide about the fate of precursor molecules whether they will join the soluble melanin pool or will be excreted as melanogens. Participation of the pigments in various biochemical and pathobiochemical processes has been reviewed.

Animals↗

PubMed1996

Approaches to prove the melanoma origin in amelanotic human melanoma cell lines.

The human melanoma B-HM8 cell line was derived from highly pigmented malignant skin melanoma. After 5 weeks of cultivation it entirely lost the pigmentation and has remained amelanotic since. Electron microscopy revealed neither premelanosomes nor melanosomes and the cells did not release detectable amount of dopa-oxidase activity into culture medium. Immunocytochemical studies using the polyclonal anti-S-100 antibody and detection of alpha-mannosidase activity in culture medium proved the melanoma origin of B-HM8 cells. Chromosomal changes in the karyotype of these cells were typical for human melanoma with chromosomes No. 1, 5, 7, 9, and 11 involved most frequently.

DNA, Neoplasm↗

PubMed1995

Ganglioside profiles of experimental melanomas and of their melanosomal fractions.

We compared ganglioside profiles of animal tumours (B16 and Cloudman S91 murine melanomas, Bomirski Syrian hamster melanoma), which are widely used as models of human melanoma, and of their melanosomal fractions. A ganglioside fraction was extracted and purified and the amount of each component ganglioside was assessed by thin-layer chromatography. GM3 was the dominant ganglioside species in the murine melanomas studied. Unlike human melanomas, the GD3 expression in mouse melanomas was low. GD3 and GM3 were major gangliosides in Bomirski hamster melanoma. Alkali-labile O-acetyl-GD3, a melanoma-specific ganglioside, was detected only in Bomirski melanoma. GD2, which in human melanoma is seen as a distinct signal of tumour progression, was not found in the animal melanomas studied. Melanosomes isolated from B16 and Bomirski melanomas contained GM3 and GD3 as their major ganglioside components. These data extend the group of common antigenic determinants shared by melanosomes and cell surface of pigment cells.

Animals↗

PubMed1994

Evaluation of serum sialic acid fractions as markers for malignant melanoma.

Increased total serum sialic acid levels have proved to be a useful non-specific marker of various neoplastic diseases including melanoma. In some malignancies, specific fractions of sialic acid were reported to be more reliable for monitoring disease progression. In this study various fractions of sialic acid were measured in the sera of Bomirski melanoma-bearing Syrian hamsters using the modified thiobarbituric acid method of Skoza and Mohos. Significantly higher serum levels of total sialic acid, 601.0 +/- 49.1 micrograms/ml, were demonstrated in animals with melanoma in comparison with control hamsters, 463.1 +/- 33.0 micrograms/ml (P < 0.01). Free sialic acid concentration was found to be negligible compared with that found in the sera of melanoma patients. Lipid bound sialic acid measurements carried out according to Bhatavdekar et al. were shown to be influenced by interfering substances. Hence, only the estimation of total sialic acid in sera can be recommended for monitoring melanoma progression. Bomirski melanoma proved to be a good model of human melanoma.

Animals↗

PubMed1994

Thymidine kinase in malignant melanoma.

Thymidine kinase (EC 2.7.1.21) is an enzyme supporting DNA synthesis under conditions of increased cell proliferation. Although it has proved to be a useful marker for various malignant diseases, it has not been tested in malignant melanoma. Thymidine kinase activity was measured by means of a radioenzymic assay in two classical animal models of melanoma disease--B16 and Cloudman S91 melanoma-bearing mice. Tumour cell proliferation was assessed histochemically by measuring the expression of proliferating cell nuclear antigen (PCNA). Tumour cytosolic specific thymidine kinase activity was found to be higher in less pigmented Cloudman S91 melanoma than in differentiated, ie pigmented B16 melanoma, relative to the proliferative activity of the two tumours. Serum thymidine kinase levels were increased in melanoma-bearing animals of both types compared with healthy mice; this also reflected the efficacy of the therapy: cyclophosphamide-treated B16 melanoma-bearing mice in which the tumour development was slowed down had significantly lower serum enzyme levels in comparison with the non-treated group and the same levels compared with control, healthy mice. Our results suggest that serum thymidine kinase levels might be used as a marker to follow the effect of melanoma therapy.

Animals↗

PubMed1994

Zinc in pigmented cells and structures, interactions and possible roles.

Zinc is a feature trace element of pigment cells and tissues. Organelles, in which melanin is synthesized and stored, i.e. melanosomes, represent a zinc reservoir at the subcellular level. In order to understand function of metals in tissues, cells and their constituents, knowledge is needed on metal interactions with intracellular targets. The possible zinc ligands in pigment cells include melanin, metallothionein, melanotransferrin, B700 and related proteins, ferritin, zinc enzymes and low molecular weight ligands. Areas of a special interest in relation of pigment cells and structures to zinc--such as zinc effect on melanogenesis, zinc excretion and buffering by melanosomes, zinc function in free radical processes as well as zinc role in melanomas--have been reviewed. High level of zinc in pigment cells may indicate a physiological defense against the potential danger of oxidative stress.

Animals↗

PubMed1993

Melanosome--a sophisticated organelle.

Melanosome is a clear out organelle of its own. The experience of the authors in the melanosome investigation has been reviewed and put to context with the literature data. Melanin moiety, a prominent feature of melanosome, was found to vary between 20-60 weight %. Melanosome concentration in pigment tissues was demonstrated to be remarkably high which explains their capacity to perform various functions both cytoprotective and cytotoxic-ascribed to the presence of melanin. Putative relation of melanosomes to other cell organelles was critically analysed. Systems of melanosome maturation were explained. The ultrastructure of melanosomes determined by the quality of their protein matrix was described and basic morphometric data summarized. The importance of the compartmentalization of melanogenesis was underlined and the fragility of melanosomal limiting membrane shown. The melanosome degradation repeatedly observed by electron microscopy has not been confirmed by biochemical means.

Animals↗

PubMed1993

Morphometric parameters of isolated melanosomes.

Basic morphometric parameters of melanosomes isolated from mouse hair, hamster eye and hamster hair are given and tabelized with those published formerly to enlarge the widest published collection of melanosomes isolated from different types of pigmented tissues up to 17 types. The splitting of melanosomes isolated from mixed tissue of hamster eyes into two subpopulations confirmed the presence of double origin of melanosomes in the eye. The close correspondence of subpopulations of hamster eye melanosomes with hamster hair and hamster melanoma (type Bomirski) respectively supports the hypothesis of high structural stability of melanosomes even under pathological conditions.

Animals↗