Mini ReviewDoes age influence loss of heterozygosity?
Introduction
Age is the greatest carcinogen – as a person enters middle-age they begin a period in which there is an exponential increase in the incidence of many types of cancer with each additional year (DePinho, 2000). The precise mechanism underlying the dramatic age-related rise of cancer remains unclear, but the importance of mutation in cancer presents a likely explanation (Knudson, 2001). It is hypothesized that a small number of genetic events – causing either activation of oncogenes or inactivation of tumor suppressor genes – precede each stage of tumorigenesis (Armitage and Doll, 1954, Armitage, 1957, Knudson, 2001). By middle age, sufficient cellular genomic damage may have accumulated to initiate carcinogenesis.
However, based on the rates of spontaneous mutation observed in human tissue culture cells, the steady accumulation of mutations cannot account for the amount of genetic changes that are present in most tumors (Lengauer et al., 1998). This has led to the hypothesis that one of the early steps in cancer progression is a genetic change causing a switch to a higher than normal rate of mutation, creating a ‘mutator phenotype’ that increases the likelihood of subsequent genetic events (Loeb, 1991; Loeb and Loeb, 2003; Nowak et al., 2002). The relationship between advanced age and the incidence of cancer also raises the question: does aging lead to a higher than normal rate of mutation? If so, is there a specific alteration in genome maintenance responsible for this change? Or is it simply the affect of accumulated mutations at a constant rate over time? To address these questions, we will discuss the affect of aging on loss of heterozygosity, a genetic change that has a significant impact on a cell’s normal function.
In considering events that may lead to oncogenesis, tumor suppressors have had a major impact on the thinking, leading to a focus on identifying mechanisms that result in loss of gene function (Brown, 1997). This is particularly relevant in the context of heterozygosity, where one functional dominant allele is “covering” the phenotype of a defective recessive allele. Loss of normal allele function could occur by any number of means, including point mutation, recombination (that “convert” the normal copy to the mutant version), or gene deletion (see Fig. 1). The importance of this paradigm emerged in the 1980s during studies of tumorigenesis in patients that had a predisposition to retinoblastoma (Cavenee et al., 1983). Patients who were heterozygous at the Rb locus, with one wild-type allele and one non-functional allele in the Rb gene, had a high incidence of tumors that had “lost” the wild-type allele of Rb in somatic cells. As noted above, there are several mechanisms by which the normal allele of Rb could become non-functional. However, the predominant pathways for loss occurred by deletion of the wild-type allele due to loss of an entire chromosome or a portion of it (a hemizygous state), or via a recombination event that replaced the wild-type allele with the non-functional, mutant allele from the homologous chromosome (a homozygous state). Consequently, these genetic changes became known as “loss of heterozygosity” (LOH) events. [Due to limits of methods at the time, point mutations, small deletions, short gene conversions, and epigenetic changes could not be detected. These events are not loss of heterozygosity in the true sense of the word. However, they are frequently classified as LOH events.] Clearly, LOH is not the only mechanism of genomic instability leading to carcinogenesis, however due to its direct link to tumor development and progression, it is important to understand how LOH is affected by aging.
LOH can arise as the result of chromosome mis-segregation, or a cell’s attempt (or inability) to repair DNA damage in the form of a double-strand break (DSB). Cells are equipped with an array of DNA repair pathways to handle such damage (Paques and Haber, 1999). However, in some cases the repair results in changes of the genome, which can manifest as LOH. The relationship between aging and LOH may be considered as two sides of a coin: does LOH (and genomic instability in general) lead to aging, and/or does aging lead to LOH? In this mini-review we will examine whether LOH changes as a function of age, evaluating evidence (from a representative set of data) for changes in the rate of LOH and the means by which it occurs.
Section snippets
Is there an increased rate of LOH with age?
Due to the striking correlation between advanced age and the incidence of cancer, it has long been thought that the rate of DNA damage and subsequent mutations must increase as an organism ages (Loeb, 1991) Although it is easy to speculate about sources of increased endogenous damage, including oxidative stress, telomere dysfunction, an increase in collapsed replication forks, etc. (DePinho, 2000; Finkel and Holbrook, 2000; Lombard et al., 2005), it is difficult to actually determine whether
Does the ability to repair DNA damage diminish with age?
If we accept the premise that there is an age-dependent increase in LOH, then how does this occur? There are two routes by which an increase could arise: more DSB DNA damage and/or a change in the repair of DNA damage. Unfortunately, methods to directly measure DNA damage in the form of DSBs are notoriously insensitive and while surrogate markers (modified or localized repair proteins) are available, they are difficult to accurately quantify (reviewed in Takahashi and Ohnishi, 2005).
Does the method of DNA repair change with age?
Although eukaryotic cells predominantly use NHEJ and HR to resolve DSBs, each of these categories, particularly HR, can be further divided into distinct repair pathways. During HR a template provides the missing DNA sequence information for DSB repair. The cell can use the sister chromatid as the template (sister chromatid exchange), or the homologous chromosome (e.g. gene-conversion or crossing-over). The length of the homologous tract can also vary. For example, in break-induced replication
Conclusions
Although studies of mammalian cells consistently reveal an increased frequency of mutations with advanced age, the experimental data to date do not clearly demonstrate an age-dependent change in the rate of LOH in vivo. However evidence from non-mammalian model organisms has provided some of the first evidence that the rate of LOH does indeed increase with age. Several studies demonstrate an overall functional decline in different types of DNA repair with age. Perhaps the most interesting
Acknowledgements
We thank the members of the Gottschling lab for their comments on the manuscript, and the NIH (R01 AG023779) for support.
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