Previous studies of excised melanocytic skin tumours revealed the involvement of UVR, apoptosis, and a gradual increase in MSI, which correlated with the progression and increased aggressiveness of these tumours.22–24
Based on these studies, we hypothesised that UVR contributes to the molecular pathogenesis of CMM arising on sun-exposed skin by causing MSI, which results in mutations in genes involved in neoplastic transformation and tumour progression. To test our hypothesis, we exposed radial growth phase CMM cell lines to UVA and UVB and examined them for viability, the presence of apoptosis, and MSI using TUNEL and PCR based microsatellite assays.
In agreement with other studies, our data revealed that UVR (in the range capable of causing sunburn in vivo) generally induces cell death and a drastic increase in the apoptotic activity in the irradiated WM cell lines,25–28
suggesting excessive underlying DNA damage beyond the capacity of the repair system.29
This DNA damage may be either in the form of accumulated pyrimidine dimers (induced by UVB) or oxidatively modified bases (induced by UVA). The accumulation of these products as a possible mechanism for the apoptosis seen in WM cell lines concurs with the apoptotic changes induced by UVA and UVB.28,30
Because DNA absorbs little, if any, UVA, the genotoxicity of UVA is mostly the result of the generation of reactive oxygen radicals,6
which have a definitive role in apoptosis.31
In support of this principle, exposure to H2
is a potent inducer of apoptosis in HL-60 cells,32
and oxygen radical scavengers can block DNA nuclear fragmentation and the formation of apoptotic bodies after UVA irradiation.28,33
“Ultraviolet irradiation generally induces cell death and a drastic increase in the apoptotic activity in irradiated Wistar melanoma cell lines, suggesting excessive underlying DNA damage beyond the capacity of the repair system”
In vivo, melanoma cells are traditionally thought to be highly radioresistant. However, several factors may have contributed to the response of the melanoma cells to UVR seen in our study. First, the radioresistance of melanoma cells was noted in an in vivo system, where skin microenvironmental factors probably influence the cells and contribute to this resistance. The in vitro system used here is not affected by changes in the skin microenvironment after UV exposure. Second, ionising rather than non-ionising irradiation (UVR) has been used in the radiotherapy of melanoma.
The MSI-L pattern found in irradiated WM cell lines is in keeping with other studies reporting similar changes in excised melanocytic lesions.8,22
This finding also supports our hypothesis that UVR can induce MSI in these lesions, suggests that MSI can arise early in melanoma pathogenesis, and suggests that UVR may be a driving force in the evolution of at least some CMMs, such as those arising on sun exposed skin. Because MSI may affect the function of the genes in which the microsatellites reside, the presence of MSI in the markers flanking the melanoma susceptibility genes in the 1p and 9p regions warrants further mutational analysis of these genes. Interestingly, UVB produced more MSI than UVA. In this regard, because UVB penetrates less deeply than UVA, the greater effect of UVB on MSI may help explain why most CMMs begin superficially in or near the epidermis.
We suggest three possibilities to support our hypothesis and to explain how UVR was able to induce MSI in the irradiated WM cell lines. First, the presence of MSI after a single exposure to UVR may merely represent a hypermutability state in these cell lines. In this regard, because exposure to UVR in vitro represents a dramatic change in the environment of WM tumour cells, it is conceivable that the cells acquire selective advantages by generating additional mutations, perhaps in the microsatellite repeats, to adjust to the in vitro conditions.34
Second, the presence of MSI after UV exposure may reflect underlying DNA damage by reactive oxygen species. Because UVR inhibits free radical scavenging activity35
and inactivates antioxidant enzymes,5,36
it enhances DNA damage by reactive oxygen radicals. The generation of these radicals is estimated to be responsible for 20 000 hits to DNA/cell/day,37–40
representing a major source of mutations. The role of oxidative stress in melanoma tumorigenesis is supported by three observations, namely: (1) oxygen radical induced DNA damage is involved in some tumours, such as breast cancers41
; (2) CMM cell lines constitutively produce and accumulate hydrogen peroxide at levels comparable to activated polymorphonuclear lymphocytes42
; and (3) exposure of DNA to reactive oxygen species in vitro can induce MSI,43
even in the absence of MMR gene defects, by several mechanisms. One of these mechanisms involves the retardation of DNA polymerase at sites of oxidative base damage, with subsequent strand displacement and misalignment of the growing strands at different positions on the template. Furthermore, strand scission within the microsatellite repeat sequences by reactive oxygen species enhances the possibility of strand misalignment, with the subsequent generation of insertion or deletion loops. The reactive oxygen radicals can also induce single strand breaks in the microsatellite sequences, increasing the formation of slipped strand intermediates.44
The third possibility is that the presence of MSI after UV exposure may reflect underlying DNA replication infidelity. As a part of the response to the UV irradiation of WM cell lines, there is accumulation of damaged DNA photoproducts that stimulate the induction of stress induced secreted proteins in the cell lines.45
Subsequently, these proteins promote activation of the genes encoding extracellular proteins,46
and several yet unidentified proteins. These three protein classes are also involved in inducing replication infidelity, and may favour genetic instability and oncogenic transformation.4,48
In support of this concept, the secretion of these proteins was found to induce genetic instability in mouse T lymphoma cell lines after UVR,4
and some of these proteins are constitutively expressed in several cancer prone syndromes49
and tumour cell lines.50
Take home messages
- Ultraviolet (UV) irradiation induced pronounced apoptosis in radial growth phase melanoma cells, with UVA having a greater effect than UVB
- UV also induced microsatellite instability (MSI) and was more prevalent after UVB than UVA irradiation
- Thus, the ability of erythemogenic UV irradiation to induce both apoptosis and MSI in radial growth phase melanoma cells suggests that it plays a role in the pathogenesis of melanoma
- This instability may reflect a hypermutability state, oxidative stress induced DNA damage, replication infidelity, or a combination of these factors
The absence of LOH after UV irradiation in WM cells is in agreement with previous studies,51
and suggests that the initial genetic events after UVR do not include LOH at an early stage. This observation is in accordance with the fact that LOH is usually associated with exposure to ionising irradiation but not to non-ionising irradiation.52
Of note, further experiments on the same cell lines are suggestive of increased malignant potential. In this regard, UVR was able to induce both point mutations in the mismatch repair genes53,54
and ultrastructural changes in keeping with malignant changes. However, determining whether the cells that survive UVR have in fact an increased tumorigenic potential relative to the parental ones (for example, growth as xenografts in immunosuppressed mice) will require additional experiments. The results of our present study will be useful to compare and contrast with such subsequent investigations.
To the best of our knowledge, our study is the first to suggest the induction of apoptosis and MSI in WM cell lines after exposure to UVR. However, whether MSI reflects a hypermutability state, oxidative stress induced DNA damage, replication infidelity, or a combination of these three must be determined by further investigations.