Background: In humans, measuring the frequency (f) of cells harboring a mutation is possible for a very small number of sentinel genes and historically has been technically difficult. Measuring the mutation rate (¿)--which represents the probability of new mutations occurring in a gene per cell division-- has been virtually impossible. Nevertheless, f and ¿ may be key parameters in ageing. For example, it is hypothesized: (i) that ¿ increases with age; (ii) that ¿ is elevated in certain premature ageing syndromes; and (iii) that it might be possible to reduce ageing related cancers by decreasing ¿. We have now developed a robust method to measure these parameters using the PIG-A gene, which we hope will greatly facilitate the investigation of these hypotheses. PIG-A is on the X-chromosome- therefore a single inactivating mutation can produce the mutant phenotype. PIG-A mutants lack all GPI-linked membrane proteins, facilitating screening for rare cells with the GPI-null phenotype by flow cytometry. By this approach, we have been able to measure f in granulocytes, and both ¿ and f in B lymphoblastoid cell lines and expanding cultures of ex vivo T lymphocytes from blood samples. Recently, we have shown that it is possible to measure f in human red cells in an additional sentinel gene, XK, which has similar advantages as PIG-A. Specific Aims (a): to determine whether ¿ increases in humans as they age; (b): to determine whether it would be feasible to prevent ageing related cancers with pharmacologic agents that could decrease ¿. For aim (a), volunteer subjects in different age groups will be recruited, and ¿ will be measured directly using PIG-A as a sentinel gene in B lymphoblastoid cell lines and T lymphocyte cultures, in comparison with neonatal cord blood samples. ¿ will also be assessed indirectly, based on analysis of the frequency of granulocytes with PIG-A mutations and red cells with XK mutations as a function of age. For aim (b), using cells from normal older individuals and patients with progeria, the effect on the mutation rate will be determined for three pharmacologic approaches: quenching reactive oxygen species, modulating lamin A using farnesyltransferase inhibitors, and activating SIRT1. Preliminary data: ¿ in cultures of lymphoid cells from normal individuals ranges from 3 to 53 x 10-7 mutations per cell division and is positively correlated with age. ¿ is increased in cells from patients with cancer predisposition syndromes, premature ageing syndromes, and in malignant cell lines. A reduction in the spontaneous mutation rate in human cells has now been demonstrated using dehydroascorbic acid to reduce intracellular reactive oxygen species. Implications: Apart from addressing very important fundamental biological questions regarding ageing and cancer risk, studies under aim (a) are important for planning clinical chemoprevention studies. If ¿ is constant throughout life, then any strategy to reduce mutations may need to start early. If however, as suspected, ¿ starts to increase with age, it may be possible to prevent cancer by reducing ¿ at a later age, once it starts to increase. Studies under aim (b) will be critical for predicting the optimal drug targets and pharmacologic strategy for reducing ¿ clinically.

Narrative: The Department of Veterans' Affairs cares for a large cohort of elderly patients who are prone to cancer because they are elderly. A better understanding of the relationship between ageing and the mutation rate is very much needed to plan studies to prevent cancer in this population. The studies proposed here will help predict at what age intervention to decrease mutations might be required. These studies may also identify drugs that are likely to prevent mutations and cancer.