Targeted and Persistent 8-Oxoguanine Base Damage at Telomeres Promotes Telomere Loss and Crisis.

Telomeres are essential for genome stability. Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere shortening. Although telomeres are hypersensitive to ROS-mediated 8-oxoguanine (8-oxoG) formation, the biological effect of this common lesion at telomeres is poorly understood because ROS have pleiotropic effects. Here we developed a chemoptogenetic tool that selectively produces 8-oxoG only at telomeres. Acute telomeric 8-oxoG formation increased telomere fragility in cells lacking OGG1, the enzyme that removes 8-oxoG, but did not compromise cell survival. However, chronic telomeric 8-oxoG induction over time shortens telomeres and impairs cell growth. Accumulation of telomeric 8-oxoG in chronically exposed OGG1-deficient cells triggers replication stress, as evidenced by mitotic DNA synthesis at telomeres, and significantly increases telomere losses. These losses generate chromosome fusions, leading to chromatin bridges and micronucleus formation upon cell division. By confining base damage to the telomeres, we show that telomeric 8-oxoG accumulation directly drives telomere crisis.

The inverse relationship between reduced folate carrier function and pemetrexed activity in a human colon cancer cell line.

Pemetrexed, a new generation antifolate recently approved for the treatment of mesothelioma and non-small cell lung cancer, is an excellent substrate for the reduced folate carrier (RFC). To explore the carrier's effect on pemetrexed activity, RFC was inactivated in HCT-15 colon cancer cells by mutagenesis and PT632 selective pressure. A clone (PT1) was obtained with a glycine to arginine substitution at amino acid 401, resulting in the loss of RFC function. PT1 cells were resistant to PT632 (178-fold), methotrexate (4-fold), and ZD1694 (Tomudex, raltitrexed; 20-fold), but were 3-fold collaterally sensitive to pemetrexed when grown in 25 nmol/L of 5-formyltetrahydrofolate. PT1 cells transfected with wild-type RFC had antifolate sensitivities comparable to that of wild-type HCT-15 cells, indicating that the RFC mutation was the sole basis for resistance. Folate pools were contracted in PT1 cells by 32% or 60%, as measured by radiolabeling intracellular folates or by an enzyme binding assay, respectively. This was reflected in marked (6.5-fold) collateral sensitivity to trimetrexate. The initial uptake of pemetrexed in PT1 cells was markedly reduced ( approximately 85%) but intracellular pemetrexed levels increased to approximately 60% and approximately 70% to that of wild-type cells after 2 hours and 6 days, respectively. There was increased pemetrexed inhibition of glycinamide ribonucleotide transformylase and, to a lesser extent, thymidylate synthase in PT1 cells growing in 5-formyltetrahydrofolate based on nucleoside protection analyses. Hence, loss of RFC function leads to collateral sensitivity to pemetrexed in HCT-15 cells, likely due to cellular folate pool contraction resulting in partial preservation of pemetrexed polyglutamylation and increased target enzyme inhibition. micro