Measurement in vivo of proliferation rates of slow turnover cells by 2H2O labeling of the deoxyribose moiety of DNA

摘要

We describe here a method for measuring DNA replication and, thus, cell proliferation in slow turnover cells that is suitable for use in humans. The technique is based on the incorporation of ²H2O into the deoxyribose (dR) moiety of purine deoxyribonucleotides in dividing cells. For initial validation, rodents were administered 4% ²H2O in drinking water. The proliferation rate of mammary epithelial cells in mice was 2.9% per day and increased 5-fold during pregnancy. Administration of estradiol pellets (0–200 μg) to ovariectomized rats increased mammary epithelial cell proliferation, according to a dose–response relationship up to the 100 μg dose. Similarly, proliferation of colon epithelial cells was stimulated in a dose–response manner by dietary cholic acid in rats. Bromodeoxyuridine labeling correlated with the ²H2O results. Proliferation of slow turnover cells was then measured. Vascular smooth muscle cells isolated from mouse aorta divided with a half-life in the range of 270–400 days and die-away values after ²H2O wash-out confirmed these slow turnover rates. The proliferation rate of an adipocyte-enriched fraction from mouse adipose tissue depots was 1–1.5% new cells per day, whereas obese ad libitum-fed ob/ob mice exhibited markedly higher fractional and absolute proliferation rates. In humans, stable long-term ²H2O enrichments in body water were achieved by daily ²H2O intake, without toxicities. Labeled dR from fully turned-over blood cells (monocytes or granulocytes) exhibited a consistent amplification factor relative to body ²H2O enrichment (≈3.5-fold). The fraction of newly divided naive-phenotype T cells after 9 weeks of labeling with ²H2O was 0.056 (CD4⁺) and 0.043 (CD8⁺) (replacement rate <0.1% per day). In summary, ²H2O labeling of dR in DNA allows safe, convenient, reproducible, and inexpensive measurement of cell proliferation in humans and experimental animals and is well suited for slow turnover cells.