Molecular approach to the nucleo-melanosomal interaction in human melanoma cells

This paper presents evidence that L-tyrosine oxidation products and 5,6-dihydroxyindole, an intermediate of melanin synthesis bind to and modify DNA structure, as tested by extracting cell DNA, using topoisomerase I and denaturation assays. When supercoiled plasmid pCU18 or pBR322 DNAs are treated with 5,6-dihydroxyindole the supercoiled species disappear and are converted to species less mobile in a gel retardation test with respect to relaxed DNA. 5,6-Dihydroxyindole causes an easier acid denaturation of the double helix. The results, that are dose dependent,would point to both intercalation and cross-linking of DNA by 5,6-dihydroxyindole and its oxidation product(s). ³H-L-tyrosine deriving radioactivity, bound to nuclear DNA, is higher at low pH, (5.6) if compared to pH 6.8. The highest radioactivity bound to cell DNA is found during the transition from the amelanotic to the melanotic phenotype in human melanoma cell lines. As a control, the binding of ³H-L-tyrosine radioactivity to human prostate fibroblast DNA was investigated.     

Sister chromatid exchange (SCE) rates in human melanoma cells as an index of mutagenesis

Melanomas are highly clonogenic. Genetic variability and polymorphismof tumour cell populations have been reported. However, no directevidence of imitator activity as a source of genetic polymorphismfor melanoma cells has been described. Some intermediates ofmelanin synthesis are cytotoxic and genotoxic and their mutagenicpower has been described. We show here that the rate of sisterchromatid exchange (SCE) of the line of human melanoma cellsused varies with the concentration of the melanin precursorL-tyrosine, in the culture medium. An increase of melanin synthesisresults in increased SCE rates. The highest values of SCEs arefound in melanotic melanoma cells compared with the amelanoticones. Indeed we present evidence that melanoma cells show higherlevels of SCE when compared with normal human lymphocytes, andto the SCE frequencies derived from the literature on the lymphocytesof familial malignant melanoma, sporadic malignant melanomapatients and the lymphocytes of relatives of familial and sporadicmelanoma patients. ³To whom correspondence should be addressed     

Mutagenicity test for unstable compounds, such as 5,6-dihydroxyindole, using an Escherichia coli HB101/pBR322 transfection system

A mutagenicity test for unstable chemical compounds has been devised. The test makes use of (i) in vitro treatment of plasmid pBR322 with the putative mutagen (ii) subsequent transfection of Escherichia coli HB101; (iii) selection either on tetracycline- or ampicillin-containing Eugon agar (iv) cross-antibiotic replica plating and recovery of single antibiotic resistant colonies (v) restriction analysis of pBR322 isolated from single antibiotic resistant colonies. In this work the test has been used to assess the mutagenicity of 5,6-dihydroxyindole, a cytotoxic intermediate of melanin biosynthesis.     

Changes of lipo-melanosome membrane leakage versus pH, charge and composition.

Liposome models of melanosomes (lipo-melanosomes) were used to investigate how phospholipid composition, charge and medium pH may affect the lipo-melanosome membrane permeability to active oxygen species or melanin synthesis intermediaries. Active oxygen accumulated only at pH 6.4 and was polarographically monitored using superoxide dismutase and/or catalase. Cholesterol appears to increase the O2- accumulation at pH 6.4 while incorporation of positive phospholipids within lipo-melanosomes results in the loss of latency with respect to tyrosinase substrate and intermediates of melanin synthesis.     

Restriction patterns of model DNA treated with 5,6-dihydroxyindole, a potent cytotoxic intermediate of melanin synthesis: effect of u.v. irradiation

The interaction of 5,6-dihydroxyindole, a putative cytotoxicintermediate of melanin synthesis, with model phage DNA hasbeen investigated by using type II restriction endonucleasesand CsCI buoyant density centrifugation. As evidenced by agarosegel electrophoresis and density gradient profiles, the 5,6-dihydroxyindoleor u.v. treated DNAs, restricted or not, are modified. U.v.irradiation enhances 5,6-dihydroxyindole binding to DNA, butno sequence specific binding was observed. The action of L-3,4-dihydroxyphenyl-alanineon the restriction patterns of phage DNA was also investigatedand the effect appeared smaller, by qualitative evaluation,than that produced by 5,6-dihydroxyindole. ²To whom reprint requests should be sent     

Copper deficiency and pigmentation in the rat: morphofunctional aspects.

The effects of a low copper diet on pigmentation, pigment cell and melanosome morphology have been investigated in ACI/T male rats. After a three months treatment the fur and skin pigmentation is reduced as compared to the controls. The melanocytes of the treated rats show the phenotype of active pigment cells while some melanosomes are abnormally differentiated: both lamellar and granular organelles are present in the same pigment cell and mosaic age melanosomes appear. The abnormal melanosome structure expressed by the treated-rat melanocytes is also evident in vitro. After incubation with deoxycholate the melanosomes from the low-copper diet treated rats are much more altered than those from the control rats. The phenotype of the rats starved for copper seems to mimic as regards pigmentation the phenotype of the mouse Mo (mottled) mutation that is an experimental model of the Menkes' kinky hair syndrome. In conclusion copper deficiency seems to affect both the morphology and function of the pigment cells.     

The plant ribosome inactivating protein saporin induces micronucleus formation in peripheral human lymphocytes in vitro

Saporin belongs to the family of plant enzymes known as ribosome inactivating proteins (RIPs) for their property to depurinate the major rRNA, thus leading to inactivation of ribosomes. In this work we analyzed the genotoxic effects of saporin, purified from root cultures of Saponaria officinalis, by evaluating micronucleus formation and by the quantitative determination of cytosolic histone-associated DNA fragments. Saporin induces micronuclei formation in cultured human lymphocytes in a dose dependent manner; treated lymphocytes show a decrease in cell viability and a concomitant increase in the apoptotic response evidenced by the appearance of cytosolic oligonucleosomes. On the other hand saporin treatment failed to induce sister-chromatid exchange (SCE) at any of the doses tested.