Ribonucleotide reductase association with mammalian liver mitochondria.

Deoxyribonucleoside triphosphate pools in mammalian mitochondria are highly asymmetric, and this asymmetry probably contributes to the elevated mutation rate for the mitochondrial genome as compared with the nuclear genome. To understand this asymmetry, we must identify pathways for synthesis and accumulation of dNTPs within mitochondria. We have identified ribonucleotide reductase activity specifically associated with mammalian tissue mitochondria. Examination of immunoprecipitated proteins by mass spectrometry revealed R1, the large ribonucleotide reductase subunit, in purified mitochondria. Significant enzymatic and immunological activity was seen in rat liver mitochondrial nucleoids, isolated as described by Wang and Bogenhagen (Wang, Y., and Bogenhagen, D. F. (2006) J. Biol. Chem. 281, 25791-25802). Moreover, incubation of respiring rat liver mitochondria with [(14)C]cytidine diphosphate leads to accumulation of radiolabeled deoxycytidine and thymidine nucleotides within the mitochondria. Comparable results were seen with [(14)C]guanosine diphosphate. Ribonucleotide reduction within the mitochondrion, as well as outside the organelle, needs to be considered as a possibly significant contributor to mitochondrial dNTP pools.

E2F4 and ribonucleotide reductase mediate S-phase arrest in colon cancer cells treated with chlorophyllin.

Chlorophyllin (CHL) is a water-soluble derivative of chlorophyll that exhibits cancer chemopreventive properties, but which also has been studied for its possible cancer therapeutic effects. We report here that human colon cancer cells treated with CHL accumulate in S-phase of the cell cycle, and this is associated with reduced expression levels of p53, p21, and other G(1)/S checkpoint controls. At the same time, E2F1 and E2F4 transcription factors become elevated and exhibit increased DNA binding activity. In CHL-treated colon cancer cells, bromodeoxyuridine pulse-chase experiments provided evidence for the inhibition of DNA synthesis. Ribonucleotide reductase (RR), a pivotal enzyme for DNA synthesis and repair, was reduced at the mRNA and protein level after CHL treatment, and the enzymatic activity was inhibited in a concentration-dependent manner both in vitro and in vivo. Immunoblotting revealed that expression levels of RR subunits R1, R2, and p53R2 were reduced by CHL treatment in HCT116 (p53(+/+)) and HCT116 (p53(-/-)) cells, supporting a p53-independent mechanism. Prior studies have shown that reduced levels of RR small subunits can increase the sensitivity of colon cancer cells to clinically used DNA-damaging agents and RR inhibitors. We conclude that by inhibiting R1, R2, and p53R2, CHL has the potential to be effective in the clinical setting, when used alone or in combination with currently available cancer therapeutic agents.