Cell fate regulated by nuclear factor-κB- and activator protein-1-dependent signalling in human melanocytes exposed to ultraviolet A and ultraviolet B.

BACKGROUND: Ultraviolet (UV) radiation constitutes an important risk factor for malignant melanoma, but the wavelength responsible for the initiation of this disease is not fully elucidated. Solar UV induces multiple signalling pathways that are critical for initiation of apoptotic cell death as a cellular defence against malignant transformation. OBJECTIVES: To evaluate the involvement of the transcription factors nuclear factor (NF)-κB and activator protein (AP)-1 in the signalling pathways induced by UVA or UVB irradiation in human melanocytes. METHODS: Primary cultures of normal human melanocytes were irradiated with UVA or UVB, and the concomitant DNA damage and redox alterations were monitored. The resulting activation of the NF-κB and AP-1 signalling pathways and subsequent apoptosis were studied. RESULTS: UVB irradiation causes DNA damage detected as formation of cyclobutane pyrimidine dimers, while UVA induces increased levels of 8-hydroxydeoxyguanosine and lipid peroxidation. UVA and UVB initiate phosphorylation of c-Jun N-terminal protein kinase and extracellular signal-regulated kinase, and the apoptosis signalling pathways converge into a common mechanism. Downregulation of c-Jun suppresses AP-1-mediated signalling and prevents apoptosis upstream of lysosomal and mitochondrial membrane permeabilization, whereas inhibition of NF-κB by SN50 increases apoptosis. CONCLUSIONS: We conclude that AP-1 induces proapoptotic signalling, whereas NF-κB is a key antiapoptotic/prosurvival factor in both UVA- and UVB-induced cellular damage in human melanocytes, which might in turn impact melanoma development and progression.

Genotype <21CAs/≥21CAs and allele <21CAs of the MANBA gene in melanoma risk and progression in a Swedish population.

Cutaneous melanoma is characterized by poor patient outcome in its later stages. The search for genetic markers is therefore crucial for the identification of populations at risk for melanoma. Highly polymorphic CA repeats in 3' proximity in the MANBA gene were examined by PCR-capillary electrophoresis in 185 Swedish melanoma patients and 441 tumor-free age- and gender-matched individuals. The associations of the polymorphisms with melanoma risk, the pigment phenotypes of the patients and tumor characteristics were analyzed. A significant difference in allelic distribution between melanoma patients and tumor-free individuals was observed. The frequency of the MANBA genotype <21CAs/≥21CAs was significantly higher in melanoma patients than in the controls. When comparing allele distribution in patients and their matched controls, the allele <21CAs was found to be associated with the female gender (39.8 vs. 31.2%, P=0.041, OR=1.46, 95% CI 1.02-2.10), but not with male gender (34.4 vs. 30.9%, P%0.39). Within the melanoma group, there were no differences in the distribution of the MANBA alleles associated with patient gender or age before or after 55 years at diagnosis, nor was there any association between the MANBA genotype and pigment phenotype or tumor sites. The MANBA allele <21CAs was, however, associated with thin melanomas at diagnosis (Breslow thickness ≤1.5 mm and Clark levels I and II). In conclusion, these data suggest that MANBA polymorphisms might be an indicator of tumor growth and progression and, together with other markers, could be used to identify individuals at increased risk of melanoma.

Low risk of melanoma in patients with atopic dermatitis.

BACKGROUND: There is a possible association between atopy and cancer based on the concept of atopic diseases as a hyper-reactive state of the immune system. Melanoma is an immunogenic tumour, and since patients with atopic dermatitis (AD) are subjected to local and systemic immunosuppressives, it would be expected to find an influence of AD on the melanoma risk. There is a positive correlation between the number of naevi and melanoma risk, and children and adults with AD have fewer naevi than controls although many patients receive ultraviolet treatment. OBJECTIVE: This study aims to investigate the melanoma risk in a retrospective cohort of AD patients compared with the population. STUDY DESIGN: 6280 AD patients born 1935-1979 visited five Dermatology clinics during 1986-2004. Mean follow-up time was 36.7 years (SD 6.9) corresponding to 230 742 person-years at risk. The cohort file was linked to the National Cancer register. RESULTS: Six AD patients with melanoma were identified, and the Poisson regression analysis adjusted for age group, sex and year resulted in an incidence rate ratio of 0.49 (95% confidence interval: 0.27-1.35, P = 0.08) for the AD group compared with the total population in the region. CONCLUSION: A low risk to develop melanoma was found in AD patients. However, the results must be interpreted with caution since the small number of expected cases of melanoma makes the risk estimate sensitive to chance effects. We hypothesize that formation of naevi and progression to melanoma is counteracted by the inflammatory process in the skin of AD patients.

Significance of glutathione S-transferases M1, T1 and P1 polymorphisms in Swedish melanoma patients.

Polymorphisms of GSTM1, GSTT1 and GSTP1 were examined in melanoma patients and tumor-free individuals. Relationships between the polymorphisms and tumor characteristics and pigment phenotypes of the patients were analyzed. There was no significant difference in GSTM1 null and GSTT1 null genotypes nor GSTP1 GG genotype between melanoma patients and controls. In melanoma patients, these polymorphisms were not correlated with early or later onset of melanomas or gender of the patients. Frequency of GSTM1 null genotype was higher in patients with melanoma >2.5 mm than in those with tumors <1.0 mm, and higher frequency was found in nodular melanoma than in the other tumor types. GSTP1 GG genotype was more often found in the patients with brown and mixed eye color or brown and black hair than those with blue and green eyes or blond hair. It is unlikely that polymorphisms of GSTM1, GSTT1 and GSTP1 are general risk factors for melanoma in the Swedish population. GSTM1 null genotype was correlated with Breslow thickness and tumor type, which might serve as an additional biomarker for a rapid tumor progression. GSTP1 GG increases risk for melanoma in the subgroup of individuals with dark eyes or hair.

Ultraviolet (UV)A- and UVB-induced redox alterations and activation of nuclear factor-kappaB in human melanocytes-protective effects of alpha-tocopherol.

BACKGROUND: Despite compelling evidence that ultraviolet (UV) irradiation causes melanoma the knowledge concerning reaction pathways and signalling transduction in melanocytes is still limited. OBJECTIVES: To evaluate the protective capacity of alpha-tocopherol and beta-carotene during UVA and UVB irradiation of human melanocytes in vitro. METHODS: Primary cultures of normal human melanocytes were irradiated by different wavelengths within the UV spectrum (UVA 6 J cm(-2), UVB 60 mJ cm(-2)). Redox alterations and apoptosis were studied and the protective potential of alpha-tocopherol and beta-carotene was evaluated. RESULTS: UVA and UVB irradiation decreased the intracellular concentration of reduced glutathione and activated the transcription factor nuclear factor (NF)-kappaB, detected as the increased level of the p65 subunit and translocation to the nucleus. This coincided with a rise in the level of gamma-glutamyl-cysteine-synthetase, the rate-limiting enzyme of the glutathione synthesis. UVA and UVB caused apoptotic cell death as detected by nuclear fragmentation and caspase activation 24 h postirradiation. Pretreatment with alpha-tocopherol prevented UVA- and UVB-induced glutathione loss, NF-kappaB translocation and diminished apoptosis, but beta-carotene did not show a similar protective capacity. Further, exposure to alpha-tocopherol by itself reduced cell proliferation rate. CONCLUSIONS: UVA and UVB irradiation affected the intracellular redox state and increased the frequency of apoptosis in human melanocytes in vitro. alpha-Tocopherol might be a useful substance in protecting melanocytes from UV-induced damage.

Frequency and distribution pattern of melanocytic naevi in Estonian children and the influence of atopic dermatitis.

BACKGROUND: There is a strong correlation between naevus number and prospective melanoma risk. Melanoma is one of the most rapidly increasing cancers in Estonia and primary prevention programmes for melanoma that target risk behaviour in the sun have so far not been launched. METHODS: The naevus profile was examined in 549/700 9-year-old Estonian children (282 boys and 267 girls) and the presence of active atopic dermatitis (AD) was registered. RESULTS: There was a wide range of naevi (4-121) and a median total body count of 26. There was no difference in naevus count between boys and girls. No dysplastic naevi were found. Thirty-nine of 549 children (7%) had at least one lesion clinically diagnosed as a congenital naevus. Boys had more naevi on the face (median 4) and trunk (median 12) than girls (median 3 and 9, respectively, P < 0.001). Girls had more naevi on the legs compared with boys (median 4 and 3, respectively, P < 0.01). Fifty-four out of 549 (9.8%) had naevi on the palms and 18/549 (3.3%) on the soles. Children with fair skin, freckles and light hair and eye colours had significantly more naevi than those with darker colours. Thirty-one of 549 (6%) children had AD diagnosed on the examination day and they had a lower total naevus count (median 20) compared with children with no AD (median 27, n = 518, P < 0.05). CONCLUSIONS: The naevus situation in Estonian children today might constitute a starting point for evaluating the efficiency of coming preventive measures as a change of naevus number in children might serve as an early marker for a change in melanoma incidence.

Photodynamic therapy of actinic keratosis at varying fluence rates: assessment of photobleaching, pain and primary clinical outcome.

BACKGROUND: Although photodynamic therapy (PDT) is becoming an important treatment method for skin lesions such as actinic keratosis (AK) and superficial basal cell carcinoma, there are still discussions about which fluence rate and light dose are preferable. Recent studies in rodents have shown that a low fluence rate is preferable due to depletion of oxygen at high fluence rates. However, these results have not yet been verified in humans. OBJECTIVES: The objective was to investigate the impact of fluence rate and spectral range on primary treatment outcome and bleaching rate in AK using aminolaevulinic acid PDT. In addition, the pain experienced by the patients has been monitored during treatment. PATIENTS/METHODS: Thirty-seven patients (mean age 71 years) with AK located on the head, neck and upper chest were treated with PDT, randomly allocated to four groups: two groups with narrow filter (580-650 nm) and fluence rates of 30 or 45 mW cm(-2), and two groups with broad filter (580-690 nm) and fluence rates of 50 or 75 mW cm(-2). The total cumulative light dose was 100 J cm(-2) in all treatments. Photobleaching was monitored by fluorescence imaging, and pain experienced by the patients was registered by using a visual analogue scale graded from 0 (no pain) to 10 (unbearable pain). The primary treatment outcome was evaluated at a follow-up visit after 7 weeks. RESULTS: Our data showed a significant correlation between fluence rate and initial treatment outcome, where lower fluence rate resulted in favourable treatment response. Moreover, the photobleaching dose (1/e) was found to be related to fluence rate, ranging from 4.5 +/- 1.0 J cm(-2) at 30 mW cm(-2), to 7.3 +/- 0.7 J cm(-2) at 75 mW cm(-2), indicating higher oxygen levels in tissue at lower fluence rates. After a cumulative light dose of 40 J cm(-2) no further photobleaching took place, implying that higher doses are excessive. No significant difference in pain experienced by the patients during PDT was observed in varying the fluence rate from 30 to 75 mW cm(-2). However, the pain was found to be most intense up to a cumulative light dose of 20 J cm(-2). CONCLUSIONS: Our results imply that the photobleaching rate and primary treatment outcome are dependent on fluence rate, and that a low fluence rate (30 mW cm(-2)) seems preferable when performing PDT of AK using noncoherent light sources.

All-trans retinoic acid (atRA) differentially induces apoptosis in matched primary and metastatic melanoma cells -- a speculation on damage effect of atRA via mitochondrial dysfunction and cell cycle redistribution.

All-trans retinoic acid (atRA) has been suggested to exert its cytotoxicity via apoptosis but the mechanisms behind the damage effects have not been fully understood. In this study, we investigated the cytotoxic effects of atRA in eleven primary and matched metastatic cutaneous melanoma cell lines. All the primary and metastatic melanoma cell lines examined expressed the retinoic acid receptors. The cultured melanoma cells treated with atRA showed dysfunction of mitochondria and altered cell cycle distribution, inhibited cell proliferation and apoptosis. The cytotoxic effects of atRA were dose- and time-dependent. The dysfunction of mitochondria and induction of apoptosis were more pronounced in the primary tumor cells than in the metastatic cell lines from the same patients. The data indicate that the cytotoxic effect of atRA was mediated through dysfunction of mitochondria, alterations in cell cycle and induction of apoptosis. Melanoma in early stage may have better response to atRA adjuvant therapy than the melanoma in late stage, suggesting the early utility of atRA in melanoma chemotherapy.

Expression of oncogenes, tumour suppressor, mismatch repair and apoptosis-related genes in primary and metastatic melanoma cells.

Several genetic alterations have been implicated in the development of malignant melanoma, but the expression of oncogenes, tumour suppressor, mismatch repair and apoptosis-related genes and their interactions in melanoma have not been completely clarified. We simultaneously examined the expression of p73, c-erbB-2, ras, p53, Mdm2, p27, DCC, hMLH-1, hMSH-2, bcl-2, Bax and NF-kappaB, by immunocytochemistry, in both primary and metastatic melanoma cell lines derived from melanoma patients. p73 was expressed in 7/8 cell lines, but stronger expressed in the metastatic cells than in the primary melanoma cells. c-erbB-2 was detected in all 8 cell lines and ras in 2/5 metastases. p53 was found in all the cell lines and Mdm2 in 1/8 of the cell lines. In the same patient, the intensity of p27 expression was decreased from the primary to the metastatic tumours. bcl-2 was expressed in all the cell lines. Bax was absent in 5/8 cell lines. In the same patient, Bax was weakly expressed in the primary tumour but lacking in the metastases. The data demonstrate that overexpression of p73, c-erbB-2, p53 and bcl-2, and loss of Mdm2 and Bax may interact and play important roles in the development and aggressiveness of human melanoma.

Readiness to change sun-protective behaviour.

The incidence of malignant melanoma and non-melanoma skin cancers has increased rapidly in Sweden during the last 20 years. The best-known way to revert this trend is primary prevention. Matching health messages to readiness to change in the population may enhance the effect of community-based prevention. The aims of this study were to investigate readiness to change sun-protective behaviour in two groups (visitors to mobile screening units and beach-goers) and to test a single-item algorithm in assessing the stage of change in sun-protective behaviour. Seven hundred and forty-two visitors to the mobile screening units and 202 individuals on nearby beaches answered a short questionnaire. The assessment of readiness to change was based on stages of change in sun-protective behaviour modified from the Transtheoretical Model of Behaviour Change. As expected, the visitors to the screening units were more often in action/maintenance stages than the beach group for most sun-protective behaviours. In conclusion, the single-item algorithm method appears to be sensitive to assess readiness to change sun-protective behaviour, based on the Transtheoretical Model of Behaviour Change. This method can be incorporated into population surveys and may aid in developing successful skin cancer prevention programmes.

Beta-carotene uptake and bioconversion to retinol differ between human melanocytes and keratinocytes.

beta-Carotene is one of the carotenoids that has been considered to play a role in the natural defense against ultraviolet-induced skin cancer. It is not known whether epidermal cells are able to accumulate beta-carotene and, subsequently, convert it to vitamin A. We used normal cultured human keratinocytes and melanocytes to study the uptake, and possible bioconversion to retinol, of authentic or [14C]beta-carotene. The uptake was much higher in melanocytes than in keratinocytes, corresponding to a fivefold difference in the intracellular fraction after two days of incubation. An increased level of cellular retinol was noted after one day of beta-carotene incubation. The conversion of [14C]beta-carotene to [14C]retinol peaked at 24 hours of incubation in keratinocytes and melanocytes. The results suggest that beta-carotene can function as a local supply of vitamin A in the skin and that melanocytes are especially likely to store beta-carotene.

Teaching non-specialist health care professionals how to identify the atypical mole syndrome phenotype: a multinational study.

The atypical mole syndrome (AMS) phenotype is the strongest known risk factor for cutaneous melanoma but recognition of the phenotype has been claimed to be problematic and to require specialist assessment. This study determined the ability of previously unskilled doctors and nurses in five countries to recognize the phenotype after brief training. The system used was the AMS scoring system. This incorporates melanocytic naevus counts, clinical atypia of naevi and distribution of naevi. The agreement in scoring between the dermatologist and trained personnel was determined in 986 patients; overall agreement in diagnosis was 94.5% (kappa 0.70, P < 0.0001). The kappa scores in different countries ranged from 0.65 to 0.77 for individual naevus characteristics, indicative of good agreement. Accurate diagnosis of the atypical mole syndrome phenotype is possible by non-specialists. This has implications for collaborative studies of naevi, for screening and for both primary and secondary prevention of melanoma.

Prevalence of pigmented naevi in a Swedish population living close to the Arctic Circle.

The prevalence of common naevi and dysplastic naevi was investigated in a Swedish population with a low incidence of melanoma. A total of 201 subjects aged 30-50 years living in northern Sweden participated. The median number of common naevi per individual was 15, and 11% had dysplastic naevi. Higher numbers of common naevi were found in individuals with dysplastic naevi (median 68) and in those who had spent their childhood in southern Sweden (median 44). The prevalence of common naevi and dysplastic naevi was significantly lower than reported from a previously studied population in southern Sweden, with a melanoma incidence 4 times higher than in the north. The strong variability in naevus phenotype, and in melanoma incidence, between different regions of Sweden seems to be due to different levels of sun exposure rather than to differences in constitutional factors.

Ultraviolet irradiation depletes cellular retinol and alters the metabolism of retinoic acid in cultured human keratinocytes and melanocytes.

Vitamin A is an intrinsic modulator of proliferation and differentiation in human epidermis, and may be destroyed by ultraviolet radiation (UVR) impinging on the skin. To identify the deleterious effects of a perturbed cellular vitamin A status, we investigated the endogenous retinoid concentrations and the metabolism of [3H]retinol and all-trans [3H]retinoic acid in cultured human keratinocytes and melanocytes exposed to UVR, using high performance liquid chromatography. Before UVR the retinoid content was similar in keratinocytes and melanocytes, but the uptake of [3H]retinol was three-fold higher and the uptake of [3H]retinoic acid was 10-fold higher in the melanocytes. In both cell types, UVR (UVA 360 mJ/cm2 plus UVB 140 mJ/cm2) instantaneously reduced the concentration of retinol by about 50% and that of 3,4-didehydroretinol by about 20%. The retinoid concentrations returned to normal within 1-2 days post-irradiation, despite there being no overt increase in the uptake of [3H]retinol or the biosynthesis of 3,4-didehydroretinol. However, in both types of irradiated cells, the accumulation of the biologically most active metabolite, all-trans [3H]retinoic acid, was about 60% higher than in control cells. Furthermore, the metabolism of authentically supplied [3H]retinoic acid was reduced, especially in irradiated keratinocytes, which probably contributed to the restoration of retinoid levels after UV exposure.

Sun-related behaviour and melanoma awareness among Swedish university students.

The relationship between knowledge, attitude and sun-related behaviour among Swedish students was examined in the present study. A total of 296 of 305 questionnaires, distributed among university students (medical school and economy programme) were analysed (157 men, 139 women, mean age 24 years). The percentage of students sunbathing with the intention of getting a tan was 75%. Thirteen per cent reported having experienced at least one painful sunburn every year and 93% stated at least one burn during the last ten years. The majority of the students had used a sun bed, 12% more than ten times during the last year. Subjects with high frequency of sun bed use also scored high on sunbathing and sunburns. Significantly more women (70%) than men (51%) used sunscreen. The overall knowledge of melanoma was high. No difference in knowledge was found between the high- and low-exposure group. Medical students scored higher on knowledge than economy students, but did not differ in exposure score. Our findings reveal an excessive sun exposure among university students. A high level of knowledge of risk does not lead to a sun-protective behaviour. Future preventative campaigns targeting young people must focus on strategies to change attitudes towards tanning as being healthy and attractive.

Cutaneous malignant melanoma in Swedish children and teenagers 1973-1992: a clinico-pathological study of 130 cases.

To assess whether there has been a change in histological features and prognostic factors of primary cutaneous malignant melanoma (CMM) in young individuals in Sweden, an unselected, population-based study was undertaken; 177 cases of primary CMM in persons below 20 years of age were reported to the Swedish National Cancer Registry between 1973 and 1992. In 87% of the cases, original tumor tissue was available for histo-pathological review. The original diagnosis was verified in 88% (n = 126) of these cases. All tumors had histological features similar to adult CMM; 17% had an associated precursor lesion. Superficial spreading melanoma (SSM) was the most common sub-type, constituting 20/36 cases in the first decade and 59/90 in the second. Corresponding figures for nodular melanoma (NM) were 11/36 and 23/90. Only 5 melanomas in situ were diagnosed. In girls, the mean thickness of SSM decreased from 1.5 to 0.6 mm (p < 0.001). Overall mortality was 10%, 22% in the group with CMM diagnosed 0-15 years of age and 8% in individuals 15-19 years. Fatal CMM cases diagnosed below 15 years of age (n = 4) were NM > 1.6 mm thick and in subjects 15-19 years (n = 9) 44% of fatal tumors were NM with a mean thickness of 2.2 mm. Breslow index was the single most important prognostic factor. However, when known prognostic factors were adjusted for in a Cox regression analysis, young age remained an independent risk factor, with a relative death rate of 0.21 for individuals aged 15-19 compared with children < 15 years of age.

Increasing incidence of cutaneous malignant melanoma in children and adolescents 12-19 years of age in Sweden 1973-92.

One-hundred-and-seventy-seven cases of cutaneous malignant melanoma were reported to the Swedish National Cancer Registry in subjects below the age of 20 during the period 1973-92. One-hundred-and-fifty-four (87%) were re-examined histologically, and the original diagnosis of primary cutaneous malignant melanoma was verified in 88%. The age-specific mean annual incidence rate increased to 0.5/100,000 in 1983-92 from 0.2/100,000 in 1973-82. Cutaneous malignant melanoma remained extremely rare in children below the age of 12, where only two cases were found. In subjects aged 12-19, the incidence doubled to 93 cases in the second 10-year period compared to 41 in the first. In boys, most of the melanomas occurred on the trunk, and, in girls, on the legs. Sixty-three percent of the melanomas were of the superficial spreading type, which also was the most rapidly increasing type of melanoma. These results emphasize the importance of surveillance and intensified preventive measures in protecting children and adolescents from the harmful effects of excessive exposure to the sun.

Melanocortin 1 receptor variants in an Irish population.

The identification of an association between variants in the human melanocortin 1 receptor (MC1R) gene and red hair and fair skin, as well as the relation between variants of this gene and coat color in animals, suggests that the MC1R is an integral control point in the normal pigmentation phenotype. In order to further define the contribution of MC1R variants to pigmentation in a normal population, we have looked for alterations in this gene in series of individuals from a general Irish population, in whom there is a preponderance of individuals with fair skin type. Seventy-five per cent contained a variant in the MC1R gene, with 30% containing two variants. The Arg151Cys, Arg160Trp, and Asp294His variants were significantly associated with red hair (p = 0.0015, p < 0.001, and p < 0.005, respectively). Importantly, no individuals harboring two of these three variants did not have red hair, although some red-haired individuals only showed one alteration. The same three variants were also over-represented in individuals with light skin type as assessed using a modified Fitzpatrick scale. Despite these associations many subjects with dark hair/darker skin type harbored MC1R variants, but there was no evidence of any particular association of variants with the darker phenotype. The Asp294His variant was similarly associated with red hair in a Dutch population, but was infrequent in red-headed subjects from Sweden. The Asp294His variant was also significantly associated with nonmelanoma skin cancer in a U.K. population. The results show that the Arg151Cys, Arg160Trp, and Asp294His variants are of key significance in determining the pigmentary phenotype and response to ultraviolet radiation, and suggest that in many cases the red-haired component and in some cases fair skin type are inherited as a Mendelian recessive.