Malignant melanoma arises from the pigment producing melanocytes in epidermis and is the most aggressive type of skin cancer. The incidence of malignant melanoma is increasing faster than any other type of cancer in white population worldwide, with a doubling rate every 10-20 years. So far, the only identified external risk factor for malignant melanoma is UV exposure. Elimination of photodamaged cells by apoptosis (programmed cell death) is essential to prevent tumor formation. Melanocytes are considered relatively resistant to apoptosis, however, the regulation of apoptosis in melanocytes is still unknown.The aim of this thesis was to investigate the apoptotic process following ultraviolet (UV) irradiation in primary cultures of human melanocytes. Focus was on regulation of mitochondrial stability by Bcl-2 family proteins and the possible participation of lysosomal proteases, cathepsins. UV irradiation activated the mitochondrial pathway of apoptosis…
Contents
INTRODUCTION
The skin
Epidermal melanin unit
UV irradiation
Skin cancer
Malignant melanoma
Historical aspect
Incidence
Risk factors
Genetics
Melanocyte homeostasis
Apoptosis
Caenorhabditis elegans
Caspases
Apoptotic pathways
Death receptor pathway
Mitochondrial pathway
Crosstalk between the apoptotic pathways
Bcl-2 family
Bcl-2 family proteins in the induction of cytochrome c release
Mcl-1
Mitochondrial membrane permeabilization
Sequestration of Bax in the cytosol
BH3-domain-only proteins
Lysosomes and lysosomal enzymes
Lysosomal membrane permeabilization
Cathepsin B and D involvement in apoptosis
Lysosomal participation in the apoptotic pathways
Stress response
Heat shock protein
c-jun N-terminal kinase (JNK)
p53
AIMS OF THE THESISAND METHODS
Cell culture (paper I-IV)
UV (paper I-IV) and heat (paper III) exposures
Apoptosis detection (paper I-IV)
Annexin V-FLUOS and propidium iodide staining (paper I)
DAPI staining (paper II-IV)
Immunocytochemistry (paper II-IV)
Western blot analysis (paper I-IV)
Subcellular fractionation experiments (paper III)
Cytosolic extraction (paper II-IV)
Caspase activation (paper II-IV)
siRNA transfection (paper III-IV)
Real time polymerase chain reaction (paper I)
Isolation of mitochondria and lysosomes from rat liver (paper III)
Protein insertion into lysosomal and mitochondrial membranes (paper III)
Immunoprecipitation (paper IV)
Microinjection (paper II)
Statistical analysis (paper I, III, IV)
Ethical consideration
RESULTS
Paper I
Paper II
Paper III
Paper IV
DISCUSSION
Apoptosis resistance in melanocytes
Short wavelength UVB effectively induces apoptosis
Keratinocytes protect melanocytes from apoptosis
Protein translocation regulates apoptosis
Pro-apoptotic signaling by lysosomal cathepsin
Bid activation by cathepsins
JNK acts pro-apoptotic upstream of the lysosome
UV induced regulation of Bim and Mcl-1
Hsp70 effectively prevents UVB induced apoptosis
CONCLUSIONS
SIGNIFICANCE OF THE STUDY AND FUTURE PERSPECTIVES
ACKNOWLEDGEMENTS
REFERENCES
Author: Bivik, Cecilia
Source: Linköping University
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