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UVA/B induced redox alterations and apoptosis in human melanocytes
Linköping University, Department of Clinical and Experimental Medicine, Dermatology and Venerology . Linköping University, Faculty of Health Sciences.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Malignant melanoma is one of the most rapidly increasing cancers and accounts for about three-quarter of all skin cancer deaths worldwide. Despite compelling evidence that ultraviolet (UV) irradiation causes melanoma the knowledge how various wavelength spectra affect the balance between proliferation and apoptosis controlling the homeostasis of the melanocyte population is still limited. The aim of this thesis was to elucidate the regulation of UVA/B induced apoptotic signaling in human epidermal melanocytes in vitro in relation to redox alterations and antioxidant photoprotection.

UVA irradiation induced changes in plasma membrane stability, decreased cell proliferation and increased apoptosis. In comparison, melanocyte plasma membrane was markedly resistant to UVB irradiation although apoptosis was triggered. Thus, UVA irradiation should not be overlooked as an etiologic factor in melanoma development. Further, after irradiation with UVA/B we found alterations in redox state manifested by a reduction of intracellular GSH levels, translocation of nuclear factor-κB from the cytosol to the nucleus, an increase of γ-glutamylcysteine synthetase, the rate-limiting enzyme in GSH synthesis, and an increased apoptosis frequency. α-Tocopherol provided photoprotection through several modes of action affecting redox alterations and signaling, stabilizing the plasma membrane, and decreased proliferation and apoptosis rate, while β-carotene did not show the same protective capacity. Altogether, α-tocopherol might be a useful substance in protecting melanocytes from UV induced damage.

We demonstrate UVA/B irradiation to activate the intrinsic pathway of apoptosis in melanocytes where translocation of Bcl-2 family proteins to the mitochondria modulates the apoptosis signal. Interestingly, the anti-apoptotic Bcl-2 family proteins generally thought to be attached to membranes, were localized in the cytosol before UV irradiation and translocated to the mitochondria in the surviving population, which might be a critical event in preventing apoptotic cell death. Lysosomal cathepsins were released to the cytosol acting as pro-apoptotic mediators upstream of activation and translocation of Bax to the mitochondria. When melanocytes were exposed to UVA, p53 participated in apoptosis regulation through interaction with Bcl-2 family proteins, while UVB induced p53-transcriptional activity and apoptosis involving lysosomal membrane permeabilization. Thus, depending on the UV wavelength p53 mediated apoptosis in melanocytes by transcriptional dependent or independent activity. These results emphasize p53 as an important pro-apoptotic component in the regulation of apoptosis.

This thesis gives new insight in the harmful and various effects of different wavelengths within the UV spectrum on human melanocytes in vitro. Improved knowledge of the apoptosis regulatory systems in melanocytes might lead to a better understanding of the formation of pigment nevi and malignant melanoma and, in the future, provide better strategies to prevent and eliminate tumor development and progression.

Place, publisher, year, edition, pages
Institutionen för biomedicin och kirurgi , 2007. , 58 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1004
Keyword [en]
UV, melanocyte, apoptosis, glutathione, p53
National Category
Dermatology and Venereal Diseases
Identifiers
URN: urn:nbn:se:liu:diva-8880ISBN: 978-91-85831-84-5 (print)OAI: oai:DiVA.org:liu-8880DiVA: diva2:23600
Public defence
2007-06-05, Berzeliussalen, Campus US, Ingång 65, Hälsouniversitetet, Linköpings universitet, SE-581 85 Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2007-05-21 Created: 2007-05-21 Last updated: 2017-08-30
List of papers
1. Ultraviolet A and B affect human melanocytes and keratinocytes differently. A study of oxidative alterations and apoptosis
Open this publication in new window or tab >>Ultraviolet A and B affect human melanocytes and keratinocytes differently. A study of oxidative alterations and apoptosis
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2005 (English)In: Experimental Dermatology, ISSN 0906-6705, Vol. 14, no 2, 117-123 p.Article in journal (Refereed) Published
Abstract [en]

Ultraviolet (UV) radiation is an etiologic agent for malignant melanoma and non-melanoma skin cancer, but the spectral range responsible for tumor induction is still to be elucidated. In this study, we compared effects of UVA and UVB irradiation on normal human melanocytes (MCs) and keratinocytes (KCs) in vitro. We demonstrate that UVA irradiation induces immediate loss of reduced glutathione (GSH) in both MCs and KCs. Exposure to UVA also causes reduced plasma membrane stability, in both cell types, as estimated by fluorescein diacetate retention and flow cytometry. Furthermore, we noted reduction in proliferation and higher apoptosis frequency 24 h after UVA irradiation. UVB irradiation of KCs caused instant reduction of reduced GSH and impaired plasma membrane stability. We also found decline in proliferation and increased apoptosis after 24 h. In MCs, on the other hand, UVB had no effect on GSH level or plasma membrane stability, although increased apoptotic cell death and reduced proliferation was detected. In summary, MCs and KCs showed similar response towards UVA, while UVB had more pronounced effects on KCs as compared to MCs. These results might have implications for the induction of malignant melanoma and non-melanoma skin cancer.

Keyword
apoptosis, keratinocyte, melanocyte, oxidative stress, UV irradiation
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14497 (URN)10.1111/j.0906-6705.2005.00238.x (DOI)
Available from: 2008-11-14 Created: 2008-11-14 Last updated: 2017-08-30
2. Ultraviolet (UV) A- and UVB-induced redox alterations and activation of nuclear factor-kappaB in human melanocytes - protective effects of alpha-tocopherol
Open this publication in new window or tab >>Ultraviolet (UV) A- and UVB-induced redox alterations and activation of nuclear factor-kappaB in human melanocytes - protective effects of alpha-tocopherol
2006 (English)In: British Journal of Dermatology, ISSN 0007-0963, Vol. 155, no 2, 292-300 p.Article in journal (Refereed) Published
Abstract [en]

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 α-tocopherol and β-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 α-tocopherol and β-carotene was evaluated.

Results UVA and UVB irradiation decreased the intracellular concentration of reduced glutathione and activated the transcription factor nuclear factor (NF)-κB, detected as the increased level of the p65 subunit and translocation to the nucleus. This coincided with a rise in the level of γ-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 α-tocopherol prevented UVA- and UVB-induced glutathione loss, NF-κB translocation and diminished apoptosis, but β-carotene did not show a similar protective capacity. Further, exposure to α-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. α-Tocopherol might be a useful substance in protecting melanocytes from UV-induced damage.

Keyword
α-tocopherol, β-carotene, melanocyte nuclear factor-κB, reduced glutathione (GSH), ultraviolet A, ultraviolet B
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14498 (URN)10.1111/j.1365-2133.2006.07347.x (DOI)
Available from: 2008-11-14 Created: 2008-11-14 Last updated: 2017-08-30
3. UVA/B induced apoptosis in human melanocytes involves translocation of cathepsins and Bcl-2 family members
Open this publication in new window or tab >>UVA/B induced apoptosis in human melanocytes involves translocation of cathepsins and Bcl-2 family members
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2006 (English)In: Journal of Investigative Dermatology, ISSN 0022-202X, Vol. 126, no 5, 1119-1127 p.Article in journal (Refereed) Published
Abstract [en]

We demonstrate UVA/B to induce apoptosis in human melanocytes through the mitochondrial pathway, displaying cytochrome c release, caspase-3 activation, and fragmentation of nuclei. The outcome of a death signal depends on the balance between positive and negative apoptotic regulators, such as members of the Bcl-2 protein family. Apoptotic melanocytes, containing fragmented nucleus, show translocation of the proapoptotic proteins Bax and Bid from the cytosol to punctate mitochondrial-like structures. Bcl-2, generally thought to be attached only to membranes, was in melanocytes localized in the cytosol as well. In the fraction of surviving melanocytes, that is, cells with morphologically unchanged nucleus, the antiapoptotic proteins Bcl-2 and Bcl-XL were translocated to mitochondria following UVA/B. The lysosomal proteases, cathepsin B and D, which may act as proapoptotic mediators, were released from lysosomes to the cytosol after UVA/B exposure. Proapoptotic action of the cytosolic cathepsins was confirmed by microinjection of cathepsin B, which induced nuclear fragmentation. Bax translocation and apoptosis were markedly reduced in melanocytes after pretreatment with either cysteine or aspartic cathepsin inhibitors. No initial caspase-8 activity was detected, excluding involvement of the death receptor pathway. Altogether, our results emphasize translocation of Bcl-2 family proteins to have central regulatory functions of UV-induced apoptosis in melanocytes and suggest cathepsins to be proapoptotic mediators operating upstream of Bax.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14400 (URN)10.1038/sj.jid.5700124 (DOI)
Available from: 2008-11-13 Created: 2008-11-13 Last updated: 2017-08-30
4. UVA/B mediate divergent p53 apoptosis signaling both dependent and independent of its transcriptional activity in human melanocytes
Open this publication in new window or tab >>UVA/B mediate divergent p53 apoptosis signaling both dependent and independent of its transcriptional activity in human melanocytes
Manuscript (Other academic)
Identifiers
urn:nbn:se:liu:diva-14500 (URN)
Available from: 2007-05-21 Created: 2007-05-21 Last updated: 2010-01-13

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