Detection of PIGO-deficient cells using proaerolysin: a valuable tool to investigate mechanisms of mutagenesis in the DT40 cell system

Abstract

While isogenic DT40 cell lines deficient in DNA repair pathways are a great tool to understand the DNA damage response to genotoxic agents by a comparison of cell toxicity in mutants and parental DT40 cells, no convenient mutation assay for mutagens currently exists for this reverse-genetic system. Here we establish a proaerolysin (PA) selection-based mutation assay in DT40 cells to identify glycosylphosphatidylinositol (GPI)-anchor deficient cells. Using PA, we detected an increase in the number of PA-resistant DT40 cells exposed to MMS for 24 hours followed by a 5-day period of phenotype expression. GPI anchor synthesis is catalyzed by a series of phosphatidylinositol glycan complementation groups (PIGs). The PIG-O gene is on the sex chromosome (Chromosome Z) in chicken cells and is critical for GPI anchor synthesis at the intermediate step. Among all the mutations detected in the sequence levels observed in DT40 cells exposed to MMS at 100 µM, we identified that ∼55% of the mutations are located at A:T sites with a high frequency of A to T transversion mutations. In contrast, we observed no transition mutations out of 18 mutations. This novel assay for DT40 cells provides a valuable tool to investigate the mode of action of mutations caused by reactive agents using a series of isogenic mutant DT40 cells.

Figure 1. Biosynthesis of the glycosylphosphatidyl inositol (GPI)-anchored protein.

Synthesis of GPI-anchored proteins involves multiple reaction steps. Briefly, the first step of GPI anchor biosynthesis is catalyzed by a multi-subunit GPI-N-acetylglucosaminyltransferase comprised of at least 6 different proteins (PIG-A, PIG-C, PIG-H, PIG-P, PIG-Q, PIG-Y). In addition, DPM2 appears to regulate this first step, followed by de-N-acetylation by the PIG-L. PIG-W then attaches an acyl chain to form glucosamine-(acyl)PI. In the next step, three mannose (Man) residues are added sequentially to glucosamine-(acyl)PI, generating Man-Man-Man-glucosamine-(acyl)PI by PIG-M/PIG-X complex, PIG-V, and PIG-B. After the Man-1 and Man-2 conjugation, PIG-N adds ethanolamine phosphates (EtNP) to the Man-1. In the final step of GPI anchor synthesis, PIG-O/PIG-F and PIG-G/PIG-F complexes attach EtNP to the Man-3 and Man-2, respectively, to generate the mature GPI anchor protein.

Figure 2. Cell survival after PA exposure.

(A) In a low cell density experiment using a 24-well plate (2.5×10³ cells/250 µL/well), DT40 cells were exposed to PA (0.0221–0.125 nM). After a three-day cultivation, cell viability was determined by XTT. Each point represents the mean and S.D. (bars) from three independent experiments. (B) In a high cell density experiment using a 96-well plate (4×10⁴ cells/50 µl/well), DT40 cells were exposed to PA (0.5–1.2 nM). After a seven-day incubation, colony formation was scored visually using an inverted microscope. Each point represents the mean and S.D. (bars) from three independent experiments.

Figure 3. Characterization of PA-resistant (PA^r) DT40 cells and validation of PA selection-based GPI anchor-deficient cell detection assay.

(A) In a low cell density experiment using a 24-well plate (2.5×10³ cells/250 µL/well), the intact DT40 cells and six different clones of DT40 cells that survived from the first PA treatment at 1.2 nM were exposed to PA (0.0221–1.2 nM). After a three-day cultivation, cell viability was determined by XTT. Each point represents the mean and S.D. (bars) from three independent experiments for DT40 cells and single experiment for six different clones of DT40 cells resistant to PA. (B) Using one of the PA^r clones used for Figure 3A, different numbers of PA^r cells (0 to 80 cells/plate) were seeded onto 96-well plates containing intact DT40 cells (40×10³ cells/well) to validate the accuracy of the PA selection step of the assay. The cells were exposed to PA at 1.2 nM. After a seven-day incubation, colony formation was scored visually using an inverted microscope. Plating efficiency was also determined using PA^r cells.

Figure 4. Frequency of GPI anchor-deficient DT40 cells exposed to MMS.

(A) The effect of 24-hour MMS exposure at 0, 1, 3, 10, 30, or 100 µM on cell growth of DT40 cells was evaluated over three days after MMS exposure. The cell growth assay was performed during/after MMS treatment. Each point represents the mean and S.D. (bars) from three independent experiments. (B) The length of the phenotype expression period in the PA^r DT40 cells was optimized after 100 µM MMS treatment for 24 hours. The frequency of PA^r DT40 cells was determined before and after MMS treatment. Each point represents the mean and S.D. (bars) from at least three independent experiments. (C) Frequency of PA^r DT40 cells was determined after exposure to MMS at different concentrations. DT40 cells were exposed to MMS at 0, 1, 3, 10, 30, 40, 60, and 100 µM for 24 hours. The cells were further cultured for five days in fresh medium without MMS. The frequency of PA^r DT40 cells was determined for each group. Each point represents the mean and S.D. (bars) from at least three independent experiments. P<0.05. Discontinued line shows the mean and S.D. of mutational frequency in the control samples (0.5±0.8 mutants/10⁶ cells).