Distinctive Uptake of Neutral Red by Mitotic Cancer Cells

Neutral red stains both normal and cancer mitotic cells, but uptake by living mitotic cancer cells is distinctly higher than in normal cells. This new approach to cancer cell identification is demonstrated in 4 established tumorigenic cancer cell lines: human skin epidermoid carcinoma A431, mouse Cloudman malignant melanoma, human oral epidermoid carcinoma and rat hepatoma. Human Chang liver cells served as normal controls. With epidermal growth factor (EGF) prepulse, neutral red uptake is dramatically enhanced. The possibility of a causal relationship with M-phase specific phosphorylation is discussed.     

Luxol Fast Scarlet C as a Stain for Phase-Contrast Microscopy

Neutral red stains both normal and cancer mitotic cells, but uptake by living mitotic cancer cells is distinctly higher than in normal cells. This new approach to cancer cell identification is demonstrated in 4 established tumorigenic cancer cell lines: human skin epidermoid carcinoma A431, mouse Cloudman malignant melanoma, human oral epidermoid carcinoma and rat hepatoma. Human Chang liver cells served as normal controls. With epidermal growth factor (EGF) prepulse, neutral red uptake is dramatically enhanced. The possibility of a causal relationship with M-phase specific phosphorylation is discussed.     

UV and children's skin

There is indicative epidemiological evidence that exposures of children younger than about 10 years are linked with an increased risk of the development of malignant melanoma as well as non-melanocytic skin cancers later in life. However, an important area of uncertainty relates to lack of knowledge of the sun-sensitivity of children's skin both absolutely and relative to that of adult's skin. For example the thickness of children's skin is very similar to that of adults but due to the nature of the anatomical structure of children's skin, there are indications of children's skin being adversely exposed on the top of the papilla before a significant exposure manifests itself as visible damage to the skin (for example erythema). This might also affect the induction of heavily UV-damaged cells persisting in the basal layer of the epidermis after UV-exposure which are supposed to be keratinocytic epidermal stem cells and may characterize an initiation step of non-melanoncytic skin cancer. For malignant melanoma the number of nevi received in dependence of UV-exposure in childhood is a clear risk factor. Recent data show that the bulge region of hair follicles hosting melanocytic stem cells are located deeper (more protected) in the skin in adults (terminal hair) as compared to pre-pubertal children (vellus hair). This may be an explanation for enhanced risk of malignant melanoma due to UV-exposure in pre-pubertal childhood.     

Polo-like kinase 1 (Plk1) in non-melanoma skin cancers

Polo-like kinase 1 (Plk1) is becoming an increasingly attractive target for cancer management. Plk1 has been shown to be over-expressed in a variety of cancers; however its role in skin cancers is not well-understood. We recently demonstrated that Plk1 is over-expressed in human melanoma and gene-knockdown as well as chemical-inhibition of Plk1 resulted in a significant decrease in melanoma cell viability and growth without affecting the growth of the normal human epidermal melanocytes (NHEMs). Further, the observed anti-proliferative response of Plk1 was found to be accompanied with a significant G2/M cell cycle arrest, mitotic catastrophe and induction of apoptosis in melanoma cells. In this study, we determined the expression profile of Plk1 in non-melanoma skin cancers viz. basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Our data demonstrated that like melanoma, Plk1 is significantly over-expressed in BCC and SCC samples. Further, we also found that compared to normal human epidermal keratinocytes (NHEKs), Plk1 was over-expressed at both the protein and mRNA levels in squamous A253 and A431 cells. In addition, a similar protein expression pattern was found for the downstream targets of Plk1, viz. Cdk1, Cyclin B1 and Cdc25C. We believe that the expression pattern of Plk1 in the various skin cancers, the insusceptibility of normal keratinocytes, to Plk1 inhibition and the easy accessibility for topical applications lends the skin as an attractive tissue for Plk1 based cancer chemoprevention and chemotherapeutic applications.     

Sestrin2 Protein Positively Regulates AKT Enzyme Signaling and Survival in Human Squamous Cell Carcinoma and Melanoma Cells

Skin cancer is the most common cancer in the United States and is mainly caused by environmental UV radiation. Reducing skin cancer incidence is becoming an urgent issue. The stress-inducible protein Sestrin2 (Sesn2) plays an important role in maintaining redox and metabolic homeostasis and their related pathologies. However, the role of Sesn2 in cancer remains unclear. Here we show that UVB radiation induces Sesn2 expression in normal human keratinocytes, mouse skin, normal human melanocytes, and melanoma cells. In addition, Sesn2 promotes AKT activation through a PTEN-dependent mechanism. Sesn2 deletion or knockdown sensitizes squamous cell carcinoma (SCC) cells to 5-fluorouracil-induced apoptosis and melanoma cells to UVB- and vemurafenib-induced apoptosis. In mice Sesn2 knockdown suppresses tumor growth from injected human SCC and melanoma cells. Last, as compared with normal skin, Sesn2 is up-regulated in both human skin SCC and melanoma. Our findings demonstrate that Sesn2 promotes AKT activation and survival in response to UVB stress and chemotherapeutics and suggest that Sesn2 is oncogenic in skin SCC and melanoma.     

Crosstalk in skin: melanocytes,keratinocytes, stem cells,and melanoma

In the vertebrate embryo, melanocytes arise from the neural crest, migrate to and colonize the basal layer within the skin and skin appendages. Post-migratory melanocytes are securely attached to the basement membrane, and their morphology, growth, adhesion, and migration are under control of neighboring keratinocytes. Melanoma is a malignant tumor originated from melanocytes or their progenitor cells. During melanocyte transformation and melanoma progression, melanocytes lose their interactions with keratinocytes, resulting in uncontrolled proliferation and invasion of the malignant cells. Melanoma cells at the advanced stages often lack melanocytic features and resemble multipotent progenitors, which are a potential melanocyte reservoir in human skin. In this mini-review, we will summarize findings on cell-cell interactions that are responsible for normal melanocyte homeostasis, stem cell self-renewal, and differentiation. Our ultimate goal is to define molecules and pathways, which are essential for normal cell-cell interactions but deregulated in melanoma formation and progression.     

LASP1, a Newly Identified Melanocytic Protein with a Possible Role in Melanin Release,but Not in Melanoma Progression

The LIM and SH3 protein 1 (LASP1) is a focal adhesion protein. Its expression is increased in many malignant tumors. However, little is known about the physiological role of the protein. In the present study, we investigated the expression and function of LASP1 in normal skin, melanocytic nevi and malignant melanoma. In normal skin, a distinct LASP1 expression is visible only in the basal epidermal layer while in nevi LASP1 protein is detected in all melanocytes. Melanoma exhibit no increase in LASP1 mRNA compared to normal skin. In melanocytes, the protein is bound to dynamin and mainly localized at late melanosomes along the edges and at the tips of the cell. Knockdown of LASP1 results in increased melanin concentration in the cells. Collectively, we identified LASP1 as a hitherto unknown protein in melanocytes and as novel partner of dynamin in the physiological process of membrane constriction and melanosome vesicle release.     

Significance of eIF4E expression in skin squamous cell carcinoma

Cutaneous squamous cell carcinoma (SCC) is a malignant tumour of keratinising epidermal cells. This type of skin cancer is the second leading cause of death after melanoma, and it is the second most common type of non-melanoma skin cancer after basal cell carcinoma. The cellular and molecular events involved in the progression of skin cancers are largely unknown. Increased protein synthesis is necessary for the transition of cells from quiescence to proliferation. Translational control is critical for the proper regulation of the cell cycle, tissue induction and growth. Eukaryotic initiation factor eIF4E, an important regulator of translation, plays critical roles in neo-plastic transformation and cancer progression. We investigated eIF4E expression in 49 skin samples (six normal tissues, eight Bowen diseases, seven stage I, 10 stage II, 13 stage III and five stage IV SCCs). Results obtained demonstrated that all SCC samples, evaluated by SDS-PAGE, Western blotting and cap-affinity chromatography using m7GTP-sepharose, presented eIF4E expression (13.6+/-1.2), whereas, starting from stage 0 (4.1+/-0.9) to stage I (7.4+/-1.4), stage II (12.1+/-2.4), stage III (18.1+/-3.0) and stage IV (26.2+/-3.8) SCCs, a constant and significant increase of protein over expression (P<0.001) was observed. A high expression of eIF4E is correlated with advanced stages. The results presented in this study demonstrate a possible role of eIF4E in SCC.     

Syndecan‐2 regulates melanin synthesis via protein kinase C βII‐mediated tyrosinase activation

Syndecan‐2, a transmembrane heparan sulfate proteoglycan that is highly expressed in melanoma cells, regulates melanoma cell functions (e.g. migration). Since melanoma is a malignant tumor of melanocytes, which largely function to synthesize melanin, we investigated the possible involvement of syndecan‐2 in melanogenesis. Syndecan‐2 expression was increased in human skin melanoma tissues compared with normal skin. In both mouse and human melanoma cells, siRNA‐mediated knockdown of syndecan‐2 was associated with reduced melanin synthesis, whereas overexpression of syndecan‐2 increased melanin synthesis. Similar effects were also detected in human primary epidermal melanocytes. Syndecan‐2 expression did not affect the expression of tyrosinase, a key enzyme in melanin synthesis, but instead enhanced the enzymatic activity of tyrosinase by increasing the membrane and melanosome localization of its regulator, protein kinase CβII. Furthermore, UVB caused increased syndecan‐2 expression, and this up‐regulation of syndecan‐2 was required for UVB‐induced melanin synthesis. Taken together, these data suggest that syndecan‐2 regulates melanin synthesis and could be a potential therapeutic target for treating melanin‐associated diseases.     

Immunofluorescent studies of epidermal protein during UV induced carcinogenesis.

Previous studies with agar diffusion technique demonstrated that antibodies produced in rabbits by injection of urea extractable proteins of rat cornfied cells cross react with proteins extracted from normal epidermis of hairless mice using the same technique. In the present study we investigated by indirect immunofluorescence microscopy the immunoreactivity of epidermal proteins in normal and ultraviolet light (UVB) induced hyperplasia and malignant transformation. Reactivity to the antibody was seen over the entire epidermis of nontreated skin and hypertrophied epidermis which occurred at 6-8 weeks after initiation of UVB irradiation. However, the reactivity diminished when malignant changes took place in the epidermal cells. Almost complete disappearance of the immunoresponse was observed in squamous cell carcinoma produced by further UVB radiation. These results suggest that the reactivity of this urea extractable protein serves as an additional immunologic marker for normal epidermal cells. Alterations in the immunoreactivity parallels UVB induced carcinogenesis.     

Loss of maspin expression contributes to a more invasive potential in malignant melanoma

Deregulation of protease expression and activity is known to play an important role in tumour progression of malignant melanoma. The serpin maspin, a tumour suppressor in breast and prostate cancer was described as an inhibitor of cell migration and inducer of cell adhesion between the basement membrane and extracellular matrix resulting in inhibition of tumour metastasis. In contrast, overexpression of maspin is correlated with poor prognosis in other cancers. However, little is known about expression, regulation and function of maspin in malignant melanoma. In this study, we found loss of maspin expression in malignant melanoma cells compared with normal human epidermal melanocytes, which was analysed by quantitative real-time PCR, Western blot analysis, immunohistochemistry and microarray. For functional studies, melanoma cell clones stably transfected with a maspin expression vector were tested for changes in proliferation, migration and invasion. Although we could not see differences in proliferation and migration, we detected strongly reduced invasive capacity in the melanoma cell clones in which maspin is re-expressed compared with control. Reduced invasive potential was also detected in three different melanoma cell lines transiently transfected with a maspin expression vector. Furthermore, exogenously added maspin alone was sufficient to reduce invasion in MelIm significantly, indicating that maspin directly inhibits invasion on the cell surface. In summary, we believe that maspin is a tumour suppressor in malignant melanoma.     

Evaluation of cytotoxic properties of organometallic ferrocifens on melanocytes, primary and metastatic melanoma cell lines

Malignant melanoma is one of the most severe forms of skin cancer, and chemotherapeutic agents currently in use are poorly effective in curing the disease. Here we describe the properties of two organometallic ferrocenyl derivatives, ferrocifen (Fc-OH-Tam) and ferrociphenol (Fc-diOH) that show a specific antiproliferative effect on melanoma cells. After a short incubation period, Fc-OH-Tam is highly cytotoxic on melanoma cells but less toxic on melanocytes. Fc-diOH is slightly toxic at a high concentration but no discrepancy is observed between malignant and normal cells. After a long incubation time the latter is highly toxic for malignant cells but not for normal cells while the former was very highly toxic for primary malignant cells and significantly less toxic for normal cells. We also found that oxidative stress is not implicated in the mechanism of cytotoxicity, since both derivatives neither induce reactive oxygen species (ROS) level in melanocytes nor in melanoma cells. Finally, investigation on hair follicle growth revealed that the two organometallic derivatives induced an irreversible ejection of the hair shaft, thus predicting a potential hair loss side effect if used as a chemotherapeutic treatment.     

Intercellular crosstalk in human malignant melanoma

Incidence of malignant melanoma is increasing globally. While the initial stages of tumors can be easily treated by a simple surgery, the therapy of advanced stages is rather limited. Melanoma cells spread rapidly through the body of a patient to form multiple metastases. Consequently, the survival rate is poor. Therefore, emphasis in melanoma research is given on early diagnosis and development of novel and more potent therapeutic options. The malignant melanoma is arising from melanocytes, cells protecting mitotically active keratinocytes against damage caused by UV light irradiation. The melanocytes originate in the neural crest and consequently migrate to the epidermis. The relationship between the melanoma cells, the melanocytes, and neural crest stem cells manifests when the melanoma cells are implanted to an early embryo: they use similar migratory routes as the normal neural crest cells. Moreover, malignant potential of these melanoma cells is overdriven in this experimental model, probably due to microenvironmental reprogramming. This observation demonstrates the crucial role of the microenvironment in melanoma biology. Indeed, malignant tumors in general represent complex ecosystems, where multiple cell types influence the growth of genetically mutated cancer cells. This concept is directly applicable to the malignant melanoma. Our review article focuses on possible strategies to modify the intercellular crosstalk in melanoma that can be employed for therapeutic purposes.     

Dermis‐derived stem cells: a source of epidermal melanocytes and melanoma?

Human multipotent dermal stem cells (DSCs) have been isolated and propagated from the dermal region of neonatal foreskin. DSCs can self-renew, express the neural crest stem cell markers NGFRp75 and nestin, and are capable of differentiating into a wide variety of cell types including mesenchymal and neuronal lineages and melanocytes, indicative of their neural crest origin. When placed in the context of reconstructed skin, DSCs migrate to the basement membrane zone and differentiate into melanocytes. These findings, combined with the identification of NGFRp75-positive cells in the dermis of human foreskin, which are devoid of hair, suggest that DSCs may be a self-renewing source of extrafollicular epidermal melanocytes. In this review, we discuss the properties of DSCs, the pathways required for melanocyte differentiation, and the value of 3D reconstructed skin to assess the behavior and contribution of DSCs in the naturalized environment of human skin. Potentially, DSCs provide a link to malignant melanoma by being a target of UVA-induced transformation.     

Opposite regulation of tenascin-C and tenascin-X in MeLiM swine heritable cutaneous malignant melanoma

Interactions between tumour cells and surrounding extracellular matrix (ECM) influence the growth of tumour cells and their ability to metastasise. It is thus interesting to compare ECM composition in tumours and healthy tissues. Using the recently described MeLiM miniature pig model of heritable cutaneous malignant melanoma, we studied the expression of two ECM glycoproteins, the tenascin-C (TN-C) and tenascin-X (TN-X), in normal skin and melanoma. Using semiquantitative RT-PCR, we observed a 3.6-fold mean increase of TN-C RNAs in melanoma compared to normal skin. Both stromal and tumour cells synthesise TN-C. On the contrary, TN-X RNAs decreased 30-fold on average in melanoma. This opposite regulation of TN-C and TN-X RNAs was confirmed at the protein level by indirect immunofluorescence. Whereas pig normal skin displayed a discrete TN-C signal at the dermo-epidermal junction, around blood vessels and hair bulbs, the swine tumour showed enhanced expression of TN-C in these areas and around stromal and tumour cells. In contrast, normal skin showed a strong TN-X staining at the dermo-epidermal junction and in the dermis, whereas this signal almost completely disappeared in the tumour. The results presented here describe a dramatic alteration of the ECM composition in swine malignant melanoma which might have a large influence on tumourigenesis or invasion and metastasis of melanoma cells.     

Emerging molecular targets in melanoma invasion and metastasis

Metastatic cutaneous melanoma accounts for the majority of skin cancer deaths due to its aggressiveness and high resistance to current therapies. To efficiently metastasize, invasive melanoma cells need to change their cytoskeletal organization and alter contacts with the extracellular matrix and the surrounding stromal cells. Melanoma cells can use different migratory strategies depending on varying environments to exit the primary tumour mass and invade surrounding and later distant tissues. In this review, we have focused on tumour cell plasticity or the interconvertibility that melanoma cells have as one of the factors that contribute to melanoma metastasis. This has been an area of very intense research in the last 5 yr yielding a vast number of findings. We have therefore reviewed all the possible clinical opportunities that this new knowledge offers to both stratify and treat cutaneous malignant melanoma patients.     

Tryptophan hydroxylase expression in human skin cells

We attempted to further characterize cutaneous serotoninergic and melatoninergic pathways evaluating the key biosynthetic enzyme tryptophan hydroxylase (TPH). There was wide expression of TPH mRNA in whole human skin, cultured melanocytes and melanoma cells, dermal fibroblasts, squamous cell carcinoma cells and keratinocytes. Gene expression was associated with detection of TPH immunoreactive species by Western blotting. Characterization of the TPH immunoreactive species performed with two different antibodies showed expression of the expected protein (55-60 kDa), and of forms with higher and lower molecular weights. This pattern of broad spectrum of TPH expression including presumed degradation products suggests rapid turnover of the enzyme, as previously reported in mastocytoma cells. RP-HPLC of skin extracts showed fluorescent species with the retention time of serotonin and N-acetylserotonin. Immunocytochemistry performed in skin biopsies localized TPH immunoreactivity to normal and malignant melanocytes. We conclude that while the TPH mRNA and protein are widely expressed in cultured normal and pathological epidermal and dermal skin cells, in vivo TPH expression is predominantly restricted to cells of melanocytic origin.     

Melanoma,Growth Factors,and Cutaneous Paraneoplastic Syndromes

Paraneoplastic syndromes are systemic reactions in patients with cancers that are unrelated to tumor size or location. Cutaneous paraneoplastic syndromes include proliferative, metabolic, and inflammatory skin disorders. Both systemic and cutaneous paraneoplastic reactions may occur in patients with malignant melanoma. Cancers, including melanoma, may produce growth factors, which may be responsible for proliferative cutaneous paraneoplastic syndromes. A patient with malignant melanoma we previously reported, who had the sudden onset of acanthosis nigricans, skin tags (acrochordons), and seborrheic keratoses provides a model for proliferative cutaneous paraneoplastic syndromes. High levels of α-TGF were found in the patient's urine prior to melanoma removal. The increased level of α-TGF declined after the melanoma was removed, and a corresponding clinical improvement in his acanthosis nigricans, skin tags, and seborrheic keratoses occurred. In the skin lesions, EGF receptors were abnormally present throughout all epidermal layers prior to melanoma removal, and returned to their normal distribution in the basal layers after surgery. Ectopic growth factor production by malignant melanomas and other epithelial neoplasms may cause rare, but distinctive cutaneous paraneoplastic lesions. The model of melanoma, cutaneous paraneoplastic syndromes, and growth factors may provide understanding of both cutaneous lesions associated with neoplasia, and benign cutaneous lesions.     

Cutaneous gene therapy: the graft takes

Prospects of ex vivo cutaneous gene therapy rely on stable corrective gene transfer in epidermal stem cells followed by engraftment of corrected cells in patients. In the case of cancer prone genodermatoses, such as xeroderma pigmentosum, cells that received the corrective gene must be selected. However, this step is potentially harmful and can increase risks of immune rejection of grafts. These obstacles have recently been overcome thanks to the labeling of genetically modified stem cells using a small epidermal protein naturally absent in stem cells. This approach was shown to be respectful of the fate of epidermal stem cells that retained full growth and differentiation capacities, as well as their potential to regenerate normal human skin when grafted in a mouse model in the long term. These progresses now open realistic avenues towards ex vivo cutaneous gene therapy of cancer prone genodermatoses such as xeroderma pigmentosum. However, major technical improvements are still necessary to preserve skin appendages which would contribute to aesthetic features and comfort of patients.