To our knowledge, this is the first prospective study addressing the potential contributions of relative telomere length to skin cancer risk. Relative telomere length measured pre-diagnostically in peripheral blood leukocytes (PBLs) was differentially associated with risk of three types of skin cancer (melanoma, BCC, and SCC) in women. These results were not statistically significant and should be interpreted with caution. Further studies are needed to confirm these possible findings.
Telomere shortening plays conflicting roles in cancer development. On one hand, the progressive loss of telomeric repeats with each cell division limits the total number of times a cell can divide. A DNA damage response is triggered once telomeres reach a critical length causing the cell to undergo senescence or apoptosis. Senescent cells express β-galactosidase, develop a characteristic flat and vacuole-rich cytoplasmic morphology, and undergo a permanent and irreversible cell cycle arrest in G1 phase, but remain metabolically active (Mooi and Peeper, 2006
). This constraint on proliferation prevents the accumulation of oncogenic mutations and subsequent malignant transformation. However, if this checkpoint is bypassed, the cell continues to proliferate resulting in additional telomere erosion (Hackett and Greider, 2002
; Wong and Collins, 2003
). Once a threshold length of 12.8 repeats (77 base pairs) is reached, chromosome ends become fusogenic resulting in chromosome instability (Capper et al., 2007
), which may also potentially lead to malignant transformation.
The opposing associations observed between relative telomere length and the various skin cancers may stem from the proliferative characteristics of the corresponding cell types from which the cancers arise and how they deal with telomere shortening. Presumably cutaneous melanocytes are evolutionarily preserved because they produce melanin, which is responsible for constitutional skin pigmentation and protective tanning response to UV. They are characterized by low levels of proliferation and a limited capacity to undergo apoptosis, perhaps due to a high content of anti-apoptotic proteins, such as BCL2 and Slug (Mooi and Peeper, 2006
). Frequently, activating mutations of the BRAF oncogene occur in melanoctyes, resulting in a transient increase in proliferation and the formation of melanocytic nevi (Gray-Schopfer et al., 2006
). Rather than undergo apoptosis, these melanocytes are more likely to senesce in response to oncogenic stress, which allows them to remain functional while preventing the propagation of the oncogenic mutation (Mooi and Peeper, 2006
). It has been hypothesized that mutated cells with longer telomere lengths experience a delay in senescence as a result of the greater replication potential. Prolonged nevus development leads to the increased formation of nevi, which are strongly associated with an increased risk of melanoma (Bastian, 2003
). Consistent with this hypothesis, we observed a positive association between relative telomere length and risk of melanoma.
In contrast to melanocytes, squamous keratinocytes are well differentiated cells that form the uppermost layer of skin. These cells have a lower apoptotic threshold making the apoptotic pathway the predominant protective mechanism, especially when cells are challenged by genotoxic stress such as UV-induced DNA damage. Sunburn cells are squamous keratinocytes undergoing apoptosis. Programmed cell death is also part of the normal terminal differentiation process that squamous keratinocytes undergo to form the water-resistant, outermost protective layer of the epidermis (Nemes and Steinert, 1999
). This process is presumably independent of telomere length. As a result, it is not surprising that we did not observe an association between relative telomere length and SCC risk.
Basal cells, which give rise to BCC, are less susceptible to apoptosis than squamous keratinocytes (Gilchrest et al., 1999
) and have a lower tendency to senescence than melanocytes. The proliferative demand of basal cells coupled with UV-induced damage of telomeric DNA may increase the likelihood of unstable telomeres and trigger chromosomal rearrangements. Under these circumstances, cells with comparatively shorter telomere lengths may potentially reach genomic instability within a smaller number of cell divisions and therefore be at greater risk for malignant transformation. Such a mechanism may explain the association we observed between shorter relative telomere lengths and an increased risk of BCC in our study population.
While replicative senescence has been hypothesized to halt the initial growth of melanocytic nevi (Bastian, 2003
), experimental evidence suggests otherwise. It is believed that activating mutations in BRAF, which are found in 82% of nevi (Pollock et al., 2003
), initiate the development of nevi with the onset of rapid proliferation. The proliferation is transient as it is generally followed by an increase in expression of p16, resulting in growth arrest and a senescent phenotype in a process known as oncogene-induced scenescence. Telomere attrition does not appear to be the main contributor to the initial growth arrest as telomere length in nevi did not differ significantly from surrounding tissue and nevi generally lack expression of p53 and p21, markers of telomere-induced senescence. These cells can remain arrested for decades (Gray-Schopfer et al., 2006
; Michaloglou et al., 2005
; Mooi and Peeper, 2006
It has also been noted, that atypical nevi and early melanomas express p53 and p21 and that p16 is deleted or silenced in a majority of primary melanomas (Gray-Schopfer et al., 2006
). While telomere attrition may not play a role in the initial formation of melanocytic nevi, these observations suggest replicative senescence may act as a second line of defense in melanocytes. If melanocytes eventually escape oncogene-induced senescence and reenter the cell cycle, cells with short telomeres will reach the critical length earlier causing cells to undergo replicative senescence or apoptosis, which may explain the inverse correlation between number of nevi and age in individuals over 30 years of age (Bataille et al., 2007
). Longer telomeres in melanocytes, which already have two oncogenic mutations (activating BRAF and silenced/deleted p16), may result in delayed senescence or apoptosis providing these cells with a greater opportunity to acquire additional mutations, increasing the probability of malignant transformation. The positive association we observed between relative telomere length and melanoma risk would be consistent with such a mechanism.
In this study, we used self-reported information on nevus counts. The validity of self-reported nevus counts was evaluated in previous studies. For example, the majority of studies on nevus counts have shown substantial agreement between nevus self-counts and dermatologist-counts (Buettner and Garbe, 2000
; Little et al., 1995
; Melia et al., 2000
). The Spearman correlation coefficient was 0.91 between nevus counts by patients and physicians, suggesting quite a good correlation between the two measurements (Mikkilineni and Weinstock, 2000
). Some case-control studies involving self-counts of nevi have demonstrated a substantial correlation of those counts with melanoma risk (Lawson et al., 1994
; Weinstock et al., 1989
). We did not have information on the total number of body moles and used the number of moles on the arms as a proxy for the total body mole counts. We previously examined moles on all four limbs versus one limb in relation to melanoma risk and found that the risks calculated from both calculations were comparable (Bain et al., 1988
We used real-time PCR and DNA derived from PBLs to determine relative telomere length. The observed correlation with age and cigarette smoking confirms that real-time PCR provides a biologically meaningful measure of telomere length. Measuring telomeres by real-time PCR is currently the most economical and versatile high-throughput method. It generates a T/S ratio that is proportional to a cell’s average telomere length. Although the values are not an actual kilobase pair length of telomeres as do Southern Blots, the relative T/S ratio has been confirmed to be highly consistent with the Southern Blot assay, which measures telomere length as terminal restriction fragments (Cawthon, 2002
). In fact, the coefficients of variation between replicates are often below 5% whereas densitometry readings from Southern Blots range between 10-15%. Additionally, the Real Time PCR method does not pick up subtelomeric DNA like Southern blots.
Even though relative telomere lengths were not directly measured in melanocytes and keratinocytes, PBLs represent a good proxy for telomere length in other tissues of the same individual, including skin (Friedrich et al., 2000
). The correlation between the PBL telomere length and mole counts observed in our study and the previous study (Bataille et al., 2007
) further suggest the biological relevance of the relative telomere length measure in the PBL.
In summary, our findings suggest the involvement of telomere length in the development of skin cancer. Distinct genetic constitutions across different types of cells and tissues evoke different DNA damage responses against telomere shortening and malignant transformation. Nevertheless, we cannot exclude the possibility that these findings may be due to chance. Further research is needed to confirm these possible associations.