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Genetic damage in defined human T cells ex vivo and in a number of T cell clones in vitro

The effect of age in culture on the DNA excision repair capacity of five independently derived TCC lines has been analysed to date. Two of the TCC were from a 26 year old donor, two from a 45 year old donor and one from an 80 year old donor. The TCC were cultured from the PD at which they were obtained from P1 until they underwent apoptosis in vitro. Samples were taken for DNA repair analysis at the early in vitro lifespan, middle of lifespan and end of lifespan of the TCC. TCC were treated with physiologically relevant DNA damaging agents; hydrogen peroxide, N-methyl-N -nitro-N-nitrosoguanidine or 254nm ultraviolet irradiation. Following treatment, the amount of DNA damage in the clones was determined over a time course using modified comet assays.

The results obtained revealed a decline related to in vitro age in the DNA repair capacity of clones derived from a 26 and a 45 year old donor. This decline may represent at least a partial explanation for the age related increase in DNA damage in these clones when cultured in vitro.

In contrast, there was no evidence for a decline related to in vitro age in repair capacity in the clones derived from an 80 year old SENIEUR donor. An alternative mechanism must underlie the age related increased in DNA damage in these clones when cultured in vitro.

Analysis of the activity of poly (ADP ribose) polymerase (PARP) within samples of TCCs at different stages of their in vitro lifespan and in PBMCs supplied by P6 and P1 has revealed a decline in PARP capacity in TCC derived from normal healthy donors with in vitro ageing. In contrast, SENIEUR donor-derived TCC did not show any evidence of a decline with increasing age in vitro. PBMCs from NONA (see P6) subjects were found to have higher levels of poly(ADP-ribose) than controls, although the difference was not statistically significant. These results provide some evidence in support of the view that poly(ADP-ribosyl)ation capacity appears to be an important contributor towards longevity.

Modified comet assays used to assess levels of DNA mismatches and the ability of TCCs and ex vivo T cell samples to perform mismatch DNA repair did not reveal any clear pattern of change in DNA mismatch frequency with increasing in vitro age. The ability of the TCC to repair supra-physiological levels of induced DNA mismatches (following treatment with acridine mutagen ICR-191) was also investigated. The results revealed a modest age-related decline in the efficiency of the MMR system in TCC derived from a 26 and a 45 year old donor. In contrast, results obtained from TCC derived from an 80 year old SENIEUR donor showed a more efficient MMR capacity with increasing age in vitro.

A novel fluorescent in situ hybridisation (FISH) technique in combination with the comet assay (Comet-FISH) was established and used to assess telomeric DNA integrity and repair in six independently-derived TCCs. Results revealed an age-related decline in telomeric integrity with increasing in vitro age of TCCs derived from a newborn and a 31 year old donor, and no age-related decline in telomeric integrity in TCC derived from a NONA donor. Further analysis of TCC and ex vivo samples will validate this novel technique, which could be a very effective means to analyse telomeric DNA integrity and repair in a host of cell types. Publication is in preparation.

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