Nico P.M. Smit

INK4a-deficient human diploid fibroblasts are resistant to RAS-induced senescence

The CDKN2A tumour suppressor locus encodes two distinct proteins, p16INK4a and p14ARF, both of which have been implicated in replicative senescence, the state of permanent growth arrest provoked in somatic cells by aberrant proliferative signals or by cumulative population doublings in culture. Here we describe primary fibroblasts from a member of a melanoma‐prone family who is homozygous for an intragenic deletion in CDKN2A. Analyses of the resultant gene products imply that the cells are p16INK4a deficient but express physiologically relevant levels of a frameshift protein that retains the known functions of p14ARF. Although they have a finite lifespan, the cells are resistant to arrest by oncogenic RAS. Indeed, ectopic expression of RAS and telomerase (hTERT) results in outgrowth of anchorage‐independent colonies that have essentially diploid karyotypes and functional p53. We find that in human fibroblasts, ARF is not induced demonstrably by RAS, pointing to significant differences between the proliferative barriers implemented by the CDKN2A locus in different cell types or species.

Melanin content of cultured human melanocytes and UV-induced cytotoxicity.

Cultured melanocytes originating from persons with different skin phototypes were utilized for measurement of endonuclease sensitive sites induced by UVB and the determination of cell survival after UVA or UVB irradiation. During culture, the melanocytes largely maintained their phenotypic characteristics according to their original skin phototype. Total melanin concentrations were 4.9 times higher in the darker skin phototype (IV-VI) melanocytes when compared to the cells from lighter skin phototypes (I-III). Also phaeomelanin contents were higher (2.5 times) in the skin phototype (IV-VI) melanocytes which implies that the cells from light skin types contain less melanin, but a relatively high proportion of phaeomelanin. After UVB irradiation a stronger induction of endonuclease sensitive sites was found for melanocytes with a lower level of total melanin and a high content of pheomelanin. By measuring the clone forming ability in different melanocyte cultures after UVB irradiation, significant better survival was found in case of the cells with the higher melanin content. Despite the large variations in melanin content, no significant difference in survival after UVA irradiation could be demonstrated in this way. Our results suggest a protective effect of melanin for UVB and indicate the importance of the measurements of melanin content and composition when different parameters of UV-induced damage are studied in melanin producing cells.

Variations in melanin formation by cultured melanocytes from different skin types

Abstract In many laboratories, culturing skin melanocytes has become a routine research activity. However, recent investigations have revealed that the quality and quantity of the pigment formed in the cultured cells may differ significantly from those of the original skin pigment cells. To shed more light on this issue, we examined the influence of different culture media on pigment production. We showed that there were notable passage-to-passage variations in the synthesis of melanin. This was particularly true for phaeomelanin. It is therefore advisable to analyse the melanin in the cells before the start of experiments. In spite of the variations, basic differences in the pigmentation pattern between melanocytes isolated from light-skinned and dark-skinned individuals remained preserved in the corresponding cultures as observed by electron microscopy. Also, the total melanin content was higher in a skin typeu2002VI melanocyte culture than in skin type I and II melanocyte cultures. In contrast to total melanin, the phaeomelanin concentration of skin type VI cells was similar to that of the skin type I melanocytes. With higher l -tyrosine concentrations in the medium, as well as increased eumelanin synthesis, phaeomelanogenesis was also stimulated in all cultures tested. This stimulation was particularly prominent in skin type I melanocytes. Our preliminary experiments also showed that a melanocyte culture from atypical naevus cells exhibited a similar preference for phaeomelanogenesis when pigmentation was stimulated.

Tyrosine and cysteine are substrates for blackspot synthesis in potato

Partially purified blackspot pigments from potato tubers (Solanum tuberosum L.) of two commercial cultivars were subjected to a microassay for melanin, which consisted of specific chemical degradation and subsequent HPLC analysis. Permanganate oxidation yielded pyrrole-2,3,5-tricarboxylic acid, whereas hydrolysis in hydriodic acid liberated aminohydroxyphenylalanine isomers. These results indicate that the polymeric pigments, which have previously been found to contain a protein matrix, carry crosslinked 5,6-dihydroxyindole-2-carboxylic acid and benzothiazine units. This leads to the conclusion that free tyrosine and free cysteine are incorporated in the proteinaceous pigments via the polyphenol oxidase catalysed pathway of melanogenesis in the process of blackspot formation. The findings are in accordance with the hypothesis that the process of blackspot formation is a non-regulated cascade of reactions in disintegrated tuber cells, rather than a finely tuned biosynthesis.

A spectrophotometric assay for routine measurement of mammalian target of rapamycin activity in cell lysates

The mammalian target of rapamycin (mTOR) is an important mediator in the PI3K/AKT signaling pathway. mTOR is the target of immunosuppressive drugs, such as rapamycin and everolimus, that are used in transplant patients but also for the treatment of various cancers. We have developed a method for mTOR activity measurement in cell lysates that measures the phosphorylation of p70 S6 kinase by an enzyme linked immunosorbent assay (ELISA) protocol. Using an optimized lysis composition, activity could be measured in the peripheral blood mononuclear cells (PBMCs) isolated from blood. For the PBMCs, intra- and interassay variations of 7 and 10%, respectively, were found using one lot number of the kit. With different lot numbers, the interassay variation increased up to 21%. Activity remained constant for PBMC pool samples on storage for a period of more than 7 months. Activity could also be measured in CD3+ T-cells isolated from blood. In vitro experiments revealed maximum mTOR inhibition of 30% in PBMCs and 44% in T-cells. The in vitro inhibition in PBMCs could also be demonstrated by Western blotting. The mTOR activity measurements may be used to show in vivo inhibition in renal allograft patients during everolimus treatment and to study mTOR activity in various (tumor) cell types.

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 (β-hexosaminidase, α-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.