Dna Damage Accumulation In T Cells As A Function Of

A more general approach to DNA damage and repair can also be informative in clonal models and ex vivo studies, several of which have shown that T cells in vivo accumulate DNA damage and mutations (point and chromosomal), for example, in free-living healthy humans aged 35 to 69 years. However, when the same genetic damage endpoints were examined in a group of healthy, older than average (75-80 years) humans, genetic damage levels similar to the levels present in subjects aged 35 to 39 years were found, and significantly less than in subjects aged 65 to 69 years (King et al., 1997). These and many other data support the importance of maintenance of genomic stability, as a determinant factor promoting health and longevity. This hypothesis is supported by the results of work from others in premature aging conditions or in groups of successfully aged humans (centenarians) (Franceschi et al., 1995).

Extensive use has been made of the in vitro human peripheral blood-derived CD4+ T cell clone model of immunosenescence described in this chapter in order to examine DNA damage accumulation under chronic antigenic stress (Hyland et al., 2001). Alkaline comet assays revealed low levels of DNA damage as the clones progressed through their in vitro life span, with a significant increase in DNA damage in the majority of the clones immediately prior to the end of their life spans. The results of modified comet assays for the detection of oxidized purines and pyrimidines revealed an age-related increase in oxidative DNA damage in the TCC as they aged in culture. Figure 66.3 illustrates levels of oxidative purine damage in 11 peripheral blood-derived CD4+ T cell clones as a function of their age in vitro. Similar results have been found for levels of oxidative pyrimidine damage.

It is interesting to speculate what the biological consequences of genetic damage in T lymphocytes might be. There is much evidence that above a threshold level, genetic damage can cause cell cycle arrest in dividing cells, this effect being mediated via cyclin-dependent kinases and their inhibitors, or indeed induce apoptosis. Since T cells are required to undergo numerous rounds of replication following stimulation by a specific antigen prior to mounting an immune response, the impact of an age-related increase in genetic damage in T cells could be to reduce proliferative capacity of the T cells or inhibit capacity altogether, or indeed result in T cell death by apoptosis. It is interesting to note here that in studies with the CD4+ TCC model, approximately three to six days after the oldest weekly samplings of the T cells for analysis, and therefore when DNA damage levels were highest, the T cells die by apoptosis. It is not yet known if the age-related increase in genetic damage within T cells in vivo is sufficient to result in T cell replicative arrest or apoptosis. However, the results of previous studies have suggested that T cells containing mutations in genes coding for normal cellular metabolism may be selected against in vivo. T cells containing such mutations might have a reduced proliferative capacity, lowered response to proliferative stimuli, or may become nonviable. For example, Podlutsky et al. (1996) demonstrated that human T lymphocytes containing mutations in the HPRT gene have reduced proliferation rates in vitro. The work of Dempsey et al. (1983) to quantify the number of lymphocytes containing HPRT gene mutations in carriers of the Lesch-Nyhan mutation, found that only 1 to 9% of lymphocytes carried a mutation. This percentage is much lower than would be predicted (50%) for such carriers, suggesting that HPRT-negative mutant lymphocytes are selected against in vivo.

Protocol for the measurement of DNA damage by the comet assay

A suitable technique to examine levels of DNA damage (DNA single-strand breaks and alkali-labile lesions) in TCC is the alkaline comet assay, originally developed by Singh et al. (1988) and modified by Collins et al. (1993). In the latter, T cells embedded on slides are treated with either formamidopyrimidine glycosylase (FPG), which recognizes oxidatively modified purines, or with endonuclease III (ENDO III), which recognizes oxida-tively modified pyrimidines. These enzymes nick DNA at the sites of oxidatively damaged nucleotides, creating single-strand breaks that can be detected with the alkaline comet assay.

1. Comet assays are performed at 4°C to minimize the repair of existing basal levels of DNA damage present in the T cells.

2. Cells are embedded in a 1% agarose gel on frosted microscope slides (2 x 104 cells/gel), and lysed for at least one hour in a high salt alkaline buffer (2.5 M NaCl, 0.1 M EDTA, 0.01 M Tris, 1%(v/v) Triton X-100, pH 10).


How to Stay Young

How to Stay Young

For centuries, ever since the legendary Ponce de Leon went searching for the elusive Fountain of Youth, people have been looking for ways to slow down the aging process. Medical science has made great strides in keeping people alive longer by preventing and curing disease, and helping people to live healthier lives. Average life expectancy keeps increasing, and most of us can look forward to the chance to live much longer lives than our ancestors.

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