A Novel Measurement of the Rate of Spontaneous Mutations in Human Myeloid Cells.

Abstract

The mutation rate (μ) is likely to be a critical parameter in leukemogenesis, but has not previously been measured in human myeloid cells. Using the PIG-A gene as a sentinel, we have recently measured the mutation rate in B-lymphoblastoid cell lines (BLCLs) and shown that it is elevated in cell lines derived from lymphoid malignancies. Here we have measured μ in normal human CD34+ cells derived from cord blood samples that have been transduced with an AML-ETO fusion gene, growing under the influence of cytokines. PIG-A is an X-linked gene essential for the synthesis of glycosylphosphatidylinositol (GPI). Due to X-inactivation in females and hemizygosity in males, a single mutation can produce the GPI (−) phenotype. From patients with PNH, it is known that a broad spectrum of mutations can inactivate PIG-A. Mutants do not express GPI-linked surface proteins such as CD55 or CD59 and do not bind to the FLAER reagent (which binds GPI directly), but do express transmembrane proteins (e.g. CD45). To measure the rate of new mutations in vitro, we first flow-sorted each culture to collect CD59(+) cells, in order to exclude pre-existing mutants. Then we expanded the collected cells over 3 weeks; using cell counts, we calculated population doublings in culture (d), which ranged from 5.1 to 7.4. Cells were then stained sequentially with biotinylated FLAER, murine anti-CD55 and anti-CD59 antibodies, rabbit anti-mouse PE and streptavidin-PE secondary conjugates, and CD45-FITC. Live cells were identified by forward and side scatter, propidium iodide exclusion, and expression of CD45. The mutant frequency (f) was calculated as the number of GPI (−) cells [which do not bind anti-CD55, CD59 or FLAER] divided by the total number of cells analyzed. An average of 1.2 million cells were analyzed to detect rare mutants. The mutation rate was calculated by the formula μ = f/d. Among 5 cultures derived from 2 different cord blood samples, the mean mutation rate was 12.4 × 10−7 (range 5.1 to 21.4 × 10−7) per cell division. This value is consistent with mathematical models of the mutation rates in human populations as well as our previous experimental determination of μ in BLCLs. We also analyzed 6 cell lines in which p53 had been disrupted by various methods, including shRNA, a dominant-negative p53 mutant, and introduction of HPV E6/E7. Overall, disruption of p53 resulted in a 30% increase in the mutation rate (p=.02). We conclude that spontaneous mutations in human myeloid cultures are rare but measurable and that disruption of p53 results in a modest increase in μ. We predict that the ability to measure this critical parameter in human hematopoietic cells will facilitate the investigation of the role of specific oncogene and tumor suppressor mutations in inducing hypermutability.