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Nieuwpoort, A. F. van. (2011, March 16). Biochemical and molecular studies of atypical nevi. Retrieved from https://hdl.handle.net/1887/16632
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The central theme of this thesis is that pigment biosynthesis, and especially pheomelanin production, could play an important role in early melanoma progression by inducing ROS, which can result in DNA damage ultimately resulting in mutations driving melanoma progression. In this final chapter the main results of these studies are summarized and discussed.
7.1 Pheomelanin is the predominant pigment in early melanoma development
Investigation of pigment biosynthesis is a relatively new and unexploited field of research. It was only 18 years ago that Thody et al. showed that human melanocytes in the epidermis, irrespective of skin type, contained both pheomelanin and eumelanin [1]. With the available technologies at that time it could not be revealed where exactly melanin production took place [1]. Pigment synthesis was suggested to take place either in melanosomes or in the cytoplasm of the melanocyte. For human hair it was shown that pheomelanin and eumelanin were localized in separate melanosomes, designated as pheomelanosomes and eumelanosomes, within the keratin structures of the hair [2‐4]. The same distinct populations of melanosomes were suggested for melanocytes in the skin [4, 5]. In chapter 2, by applying X‐ray micro analysis (XRMA), we show that human
melanocytes, in contrast to hair, have melanosomes containing a mixture of eu‐
and pheomelanin.
After having identified the location of pigment biosynthesis, we
investigated whether pigment synthesis in cultured melanocytes does reflect the synthesis in vivo (chapters 2 and 3). XRMA showed that pigment synthesis in vitro is indeed comparable to the in vivo situation. Melanogenesis in vitro could be induced by addition of L‐tyrosine to the culture medium.
Especially in melanosomes of melanocytes derived from fair skin individuals (skin type I), adding L‐tyrosine to the medium resulted in an increase of pheomelanin compared to eumelanin. The latter effect was less apparent for melanosomes in melanocytes derived from dark skin type (skin type VI). Based on experiments in chapter 2 we conclude that the preference of pheomelanogenesis over
eumelanogenesis is specifically a characteristic feature of melanocytes in fair skin individuals.
After having investigated pigment synthesis in melanocytes from different skin types, we explored pheomelanin pigment synthesis in melanocytes obtained from the various stages of the Clark melanoma progression model: acquired (benign) nevi, atypical nevi and melanoma (Figure 1 chapter 1 and chapter 3). It was noted that the amount of pheomelanin was significantly elevated in
melanocytes in atypical nevi and melanoma cells compared to normal melanocytes of the same patient. However the amount of pheomelanin in melanoma cells was lower than in atypical melanocytes. On the contrary, melanocytes in benign nevi compared to normal skin melanocytes from the same individual did not show a significant elevation of pheomelanin (chapter 3).
It is not known why melanoma cells have a lower level of pheomelanin than atypical melanocytes but we hypothesize that the high cell division rate, in comparison to the division rate of an atypical melanocyte, could play a role. During the cell division the melanosomes are divided among the two daughter cells, reducing pheomelanin pigment levels in the melanoma cell.
7.2 Pheomelanin, a source of ROS
In chapters 3 and 4 we demonstrated increased levels in basic oxidative stress in atypical melanocytes in comparison to normal melanocytes from the same individual. XRMA analysis on the same cells showed that the FACS observations on increased ROS levels coincided with increased levels of pheomelanin in the atypical melanocytes, suggesting that pheomelanin is the source of increased levels of ROS.
Under conditions of oxidative stress, release of calcium and iron from intracellular stores is known to occur [6, 7]. Our group has shown that melanin precursors can also liberate iron from ferritin, the iron stores in the melanocyte, which can subsequently increase the level of ROS via the Fenton reaction [8, 9].
Next to determining pheomelanin concentrations we also measured the levels of iron and calcium in the melanosomes. In comparison with normal melanocytes, melanoma cells displayed the highest levels followed by atypical melanocytes (figure 4, chapter 3). Iron plays an important role in the energy metabolism and DNA synthesis.
Both processes are elevated in melanoma cells in comparison to normal and atypical melanocytes [10‐12]. The rapid growth as observed in melanoma cells could be an explanation for the increased iron levels.
Free transition metals, such as iron can interact with hydrogen peroxide yielding extremely toxic hydroxyl radicals [13]. These hydroxyl radicals increase the ROS levels in a cell, but also run a risk to damage intracellular structures. It is known that pheomelanin intermediates and their oxidation products leaking from the melanosomes are toxic to cellular organelles such as mitochondria, causing the release of Ca2+, osmotic swelling and malfunctioning of the electron transport chain [14, 15]. Interestingly, in chapters 2 and 3 we saw that melanosomes in melanocytes of fair skin individuals differ in their appearance from melanosomes in melanocytes derived from dark skin individuals.
Melanosomes in fair skin melanocytes were apparently smaller and had a more ellipsoid shape compared to melanosomes obtained from dark skin melanocytes.
We furthermore observed that in both atypical melanocytes and melanoma cells numerous melanosomes showed altered morphology.
In addition to altered melanosomes in atypical melanocytes, we saw atypia of mitochondria. The latter observation is consistent with the findings of Langer et al. who detected structurally damaged mitochondria in both atypical nevi and melanoma compared to normal skin [16]. Our findings suggest that pheomelanogenesis not only has a profound effect on the structure of the melanosomes itself, but also gives rise to structurally damaged mitochondria and subsequently release of calcium.
7.3 Reduced management of ROS in melanocytes
In general cells can cope with a certain level of ROS induced oxidative stress and have special enzymes and mechanisms to regulate and manage these levels. One such enzyme with a ROS reducing capacity is glutathione [17, 18].
During pheomelanogenesis the amino acid L‐cysteine is incorporated. Apart from playing a role in pheomelanogenesis, L‐cysteine is also an essential component for glutathione. Our experiments show that atypical melanocytes with increased pheomelanogenesis show a significant decrease in the amount of glutathione (chapter 4). Thus, in addition to ROS generating intermediates of pheomelanin also a decrease of ROS reducing mechanisms in the atypical melanocyte could be responsible for the increased ROS levels.
Next to glutathione, other ROS reducing enzymes, are catalase and superoxide dismutase. Maresca et al. found a low activity of catalase in conjunction with pheomelanin synthesis in fair skin melanocytes [19].
Unexpectedly, Sander et al. detected a higher expression and activity of the antioxidant enzymes catalase and superoxide dismutase in melanoma cells in comparison with acquired nevi and normal skin in vivo [20]. Studies performed by Applegate et al. showed that cultured melanoma cells have a higher level of glutathione in comparison with normal skin [21, 22]. It has been suggested that this enhanced antioxidant defense in melanoma cells is a feedback mechanism that protects them from excessive cellular ROS generation [23, 24].
Since ROS levels are elevated in atypical melanocytes, together with lowered levels of glutathione, it appears that atypical melanocytes have not acquired this antioxidant defence yet. However, from current research it is not yet clear how the switch in levels of the antioxidant enzyme levels between atypical melanocytes and melanoma cells can be explained.
7.4 Oxidative stress induced DNA damage
Conger and Fairchild were the first to describe that ROS can diffuse into the nucleus and react with deoxyribonucleic acid (DNA) giving rise to oxidative DNA damage [25]. A method to detect DNA damage, especially strand breaks, is the comet assay. In previous studies, our group showed that cultured atypical melanocytes display higher basic levels of DNA damage in comparison to normal melanocytes [26]. An explanation for this increased level of DNA damage could be the result of pheomelanin precursors reacting with mitochondria resulting in increased calcium levels, which we detected in atypical melanocytes. Calcium is known to play a role in antioxidant defence and Panayiotidis et al. and Barbouti et al. reported that low levels of calcium protect the cell against ROS induced damage, while high levels of calcium induce DNA damage [27, 28]. High levels of calcium induce cleavage of DNA by activation of calcium dependent nucleases [29].
Therefore, the detected elevated levels of calcium can explain the elevated basic levels of DNA damage in atypical melanocytes observed.
However, our group did not investigate in these first comet assay experiments whether the observed DNA damage in the atypical melanocytes was related to oxidative stress. By optimizing the comet assay, we now show that the increased DNA damage observed in atypical melanocytes is caused by oxidation of purines (figure 6, chapter 4).
Oxidative DNA damage could imply increased susceptibility for DNA to become mutated [30, 31]. Despite the fact that an oxidative stress driven tumour progression hypothesis has been suggested in the genesis of several tumour types or diseases such as e.g. Alzheimer [6, 31‐33], evidence for melanoma is still lacking.
The sustained oxidative stress generating oxidised DNA damage feeds the hypothesis that due to this DNA damage, mutations in critical genes may occur that thrive subsequent stages of melanoma progression. However, more research is needed to explicitly prove that genetic alterations initiating cancer or more specific melanoma are the result of oxidative DNA damage. At least the mutation signature in the nevus and melanoma related BRAF and NRAS genes favours this hypothesis.
Approximately 90% of all nevi harbor activating BRAF mutations [34, 35].
The fact that most nevi initially proliferate but then stop growing and never progress into melanomas implicates that the BRAF V600E mutation drives senescence [36]. Several studies anticipate that V600E BRAF bearing melanocytes require additional genetic changes for these senescent melanocytes to re‐enter the cell cycle [35, 36]. Loss of p16INK4a activity or other, yet to be identified hits, has been envisioned to collaborate with these BRAF mutations for melanocytes to resume proliferation [36]. It is unclear whether these additional genetic changes are acquired in a fully senescent melanocyte or a in a melanocyte that is on its way to become senescent.
In order to become immortal cells have to fully overcome senescence by maintaining a minimal telomere length, which can be achieved by activation of hTERT. In conclusion, BRAF V600E is an early event in melanomagenesis, but cannot cause melanoma on its own.
7.5 Gene expression profiling of normal and atypical melanocytes
In order to identify and gain more insight in altered genes or pathways underlying the early transition of a normal melanocyte into an atypical melanocyte we compared genome‐wide gene expression in a large set of short term cultures of normal melanocytes and atypical melanocytes from the same individuals (chapter 5). Surprisingly the gene expression differences between single genes in the two subtypes of melanocytes were extremely small and we therefore had to apply Gene Ontology (GO) analysis, a method to determine effects of biologically related genes that reinforce each other. The most significant differentially expressed GO‐
categories between normal and atypical melanocytes turned out to be vesicle, especially mitochondria, related and included genes encoding mitochondrial ribosomal proteins (MRPs).
The elevated expression of MRPs fit extremely well in the context of our earlier ROS related observations, since MRPs play a role in the maintenance of mitochondrial DNA by detecting DNA damage [37]. MRPs also regulate the energy providing electron transport chain of the cell [37, 38]. Especially lowered
expression of MRPS16, 22, 28 and MRLP37 as found in our study (chapter 5) could affect a proper functioning of the electron transport chain resulting in
accumulated electrons which leak into the cytosol of the cell where they can give rise to ROS [38].
Also the lower expression of genes in atypical melanocytes compared to normal melanocytes involved in the hydrogen ion transporter activity sustain the role of ROS in melanoma progression. The resulting elevated pH in organelles and the cytosol of the atypical melanocyte has consequences for many cellular processes such as pigment synthesis, DNA synthesis and proliferation [39]. With respect to pigment synthesis, elevated pH results in increased pheomelanogenesis resulting in further accumulation of ROS. The newly formed ROS can react with the mitochondrial membrane resulting in structural damaged mitochondria [38]. These findings are therefore in line with our earlier observation on the morphological changes observed in mitochondria and the elevated levels of ROS in atypical melanocytes in comparison to normal melanocytes [8, 40]
There is increasing evidence for the supportive role of ROS in melanoma development published by various groups [41‐50]. The common denominator in all these studies is pigment biosynthesis related ROS production. Meyskens et al.
showed that melanoma cells have higher intracellular levels of ROS in comparison with normal melanocytes but furthermore showed that melanin itself becomes progressively more oxidized and starts to function as a pro‐oxidant [45]. Oxidation of melanin can be further increased by binding of metals, such as iron [41].
This is in line with our observations of higher iron levels in atypical melanocytes and melanoma [8]. Thus, through conversion by the Fenton reaction of these melanin‐metal complexes even more ROS is produced [46].
7.6 Protein profiling of normal and atypical melanocytes
Gene expression profiling could reveal alterations in genes underlying cellular processes resulting in cellular behavioural changes e.g. processes driving tumour progression. Ultimately mRNA is translated into proteins and enzymes which are the real players in cellular functions. In order to verify whether the
chapter 6, 2D‐DIGE in combination with a laser scanner was applied to study the protein expression differences on the same set of normal and atypical melanocytes used in chapter 5.
In line with the biochemical results of chapters 3 and the gene expression results of chapter 5 most of the lower expressed proteins observed in the atypical melanocyte are dedicated to manage the oxidative status of the cell and to store iron in ferritin [8]. The same holds true for the antioxidant enzymes such glutathione‐S‐transferase, ferritin heavy chain and cytochrome b‐c1 complex subunit Rieske for which diminished levels of protein were observed in the atypical melanocytes.
We also found proteins expressed at a much lower levels which were related to the cytoskeleton, which we did not detect with our gene expression study. Changes in filament organisation in general have been reported in response to oxidative stress, heat shock and extracellular calcium, which have been
associated to cancer cell mobility [51, 52]. Interestingly, Mirabelli et al. and Bellomo et al. showed that cytoskeleton filaments not only become oxidised by ROS, but also by quinones [53, 54]. Especially in the atypical melanocyte this oxidation by quinones can be of importance. In chapters 3 and 4 we determined that atypical melanocytes display elevated levels of pheomelanin intermediates (which are quinones) as a result of increased pheomelanin synthesis [8, 40]
Therefore the increased pheomelanin production in the atypical melanocytes could be regarded as a plausible cause underlying several of the observed protein differences. This observation provides additional evidence that pigment
biosynthesis related ROS production could play an important role in early melanoma progression.
7.7 Concluding remarks
The results obtained in this thesis suggest that the most explicit differences between normal and atypical melanocytes are subtle changes in pigment biosynthesis and the functioning of the antioxidant system. Impairment of the antioxidant system and increased levels of pheomelanin result in increased levels of oxidative stress. It is anticipated that these increased levels of oxidative stress contribute to early melanoma development by inducing DNA mutations, but additional studies are required to prove this hypothesis.
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