Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

The effects of potential anti-senescence treatment strategies in human T cells

The effect of a reduced oxygen tension culture system (6% O2) on TCCs has been examined. Specifically, the effects of the altered culture conditions on DNA damage levels, in vitro lifespan and proliferative capacity were assessed in six independently-derived human CD4+ TCCs. DNA damage levels over the entire lifespan were significantly lowered by reducing oxygen tension. However, lifespan (total population doublings (PDs) achieved) and proliferative capacity (PDs/week) was reduced for all clones under reduced oxygen tension (mean: lifespan 74.9 PD, proliferative capacity 2.1 PD) when compared to standard culture (20% O2 conditions; mean: lifespan 56.3 PD, proliferative capacity 1.0 PD, p<0.05).

The effect of a synthetic superoxide dismutase mimetic (10µM EUK-134) on TCCs has also been examined. The effects of 10µM EUK-134 on in vitro lifespan and proliferative capacity were assessed in six independently-derived human CD4+ TCCs. Lifespan and proliferative capacity were were significantly reduced (mean: lifespan 53.3 PD, proliferative capacity 0.6 PD) when compared to standard culture (20% O2 conditions; mean: lifespan 74.2 PD, proliferative capacity 2.0 PD, p<0.05). Preliminary data suggests that DNA damage levels over the entire lifespan were significantly lowered in the presence of EUK-134, analysis will be complete by 31/12/2005.

To further investigate these results, the TCC were examined to determine the fraction of the populations which were actively proliferating (using a colorimetric ELISA BrdU incorporation assay), the fraction of the populations which were actively undergoing apoptosis (using a flow cytometry-based Annexin V assay) and cell cycle distribution analysis of the populations (using classical PI flow cytometry technique). Analysis will be complete and results available by 31/12/05.

Results may suggest that reduced oxygen tensions induced in the culture system had an effect on the overall redox balance of TCCs (this is partially verified by the reduced levels of oxidative DNA damage in these clones). This may have led to a detrimental effect on ROS-mediated signalling pathways in the TCCs and thus contributed to the reduced proliferation of the TCCs.

Western blot analysis was utilised to investigate redox sensitive signalling pathways, purified proteins from six independently-derived human CD4+ TCCs were probed at the beginning and end of their in vitro lifespans for the relative levels of phosphorylated- and dephosphorylated-JNK 1/2 MAPK, phosphorylated- and dephosphorylated-p38 MAPK and phosphorylated- and dephosphorylated-ERK MAPK. Preliminary data suggests that there are age-related alterations in MAPK-mediated signalling in TCCs under the various culture conditions employed in WP10. Analysis will be complete and results available by 31/12/05.

The gene for a hsp 70 stress protein (hsp72a1b) has been cloned and preparations made for stable expression in TCC. HTERT constructs have been generated using GFP marker but so far the infection rate of TCC has been much too low to generate any hTERT transfectants. Use of cytokine cocktails to extend lifespan has focussed on IL 15, found to be of benefit for some TCC when combined with IL 2.

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Nottingham Trent University
Clifton Campus
NG11 8NS Nottingham
United Kingdom
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