Impact of Premature Senescence on Radiosensitivity Measured by High Throughput Cell-Based Assays

Abstract

In most p53 wild-type human cell types, radiosensitivity evaluated by the colony formation assay predominantly reflects stress-induced premature senescence (SIPS) and not cell death (Int. J. Mol. Sci. 2017, 18, 928). SIPS is a growth-arrested state in which the cells acquire flattened and enlarged morphology, remain viable, secrete growth-promoting factors, and can give rise to tumor-repopulating progeny. The impact of SIPS on radiosensitivity measured by short-term assays remains largely unknown. We report that in four p53 wild-type human solid tumor-derived cell lines (HCT116, SKNSH, MCF7 and A172): (i) the conventional short-term growth inhibition assay (3 days post-irradiation) generates radiosensitivity data comparable to that measured by the laborious and time-consuming colony formation assay; (ii) radiation dose-response curves obtained by multiwell plate colorimetric/fluorimetric assays are markedly skewed towards radioresistance, presumably reflecting the emergence of highly enlarged, growth-arrested and viable cells; and (iii) radiation exposure (e.g., 8 Gy) does not trigger apoptosis or loss of viability over a period of 3 days post-irradiation. Irrespective of the cell-based assay employed, caution should be exercised to avoid misinterpreting radiosensitivity data in terms of loss of viability and, hence, cell death.

Keywords: CellTitre-Blue; MTT; XTT; apoptosis; colony forming ability; ionizing radiation; p53 signaling; premature senescence; proliferation; viability.

Figure 1

Radiosensitivity of the indicated cell lines evaluated by growth inhibition and colony formation assays. Growth inhibition and colony forming ability measurements were performed 3 days and 10 days post-irradiation, respectively. Bars, standard error (SE). CFA, colony forming ability.

Figure 2

Viability of the indicated cell lines before (0 Gy) and 3 days after radiation exposure, evaluated by the trypan blue-exclusion assay. Bars, SE.

Figure 3

Depiction of anticipated outcome of multiwell plate colorimetric (XTT) assays after treatments that result in cytostatic (growth inhibition) effect only (e.g., 5 Gy of ionizing radiation), or cytostatic plus cytotoxic effects (e.g., >40 μM cisplatin). The example is given for a culture with a control population doubling time of 24 h. In this case, for “control” wells (upper left well), seeding 2000 cells per well (day 0) and incubating them with growth medium for 3 days (upper right well) will result in 16,000 cells/well at the time of optical density measurement. For “radiation” wells (middle left well) and “cisplatin” wells (lower left well), the number of cells per well is expected not to increase from day 0 to day 3, due to induction of growth arrest in virtually all cells (irradiation) or induction of growth arrest plus loss of viability (cisplatin treatment). It is important to note that most solid tumor-derived cell lines (e.g., HCT116) have shorter population doubling times than 24 h [22].

Figure 4

Representative bright-field microscopy images depicting the metabolic activity of MCF7 cultures before (control) and at indicated times after 8-Gy irradiation. Metabolic activity was measured by the ability of the cells to convert the yellow MTT to its purple formazan metabolite, appearing as dark granules and crystals. Upper row: images were acquired after incubation of cells with MTT for ~1 h. Middle row: following incubation with MTT, cells were fixed in methanol for 0.5 min to dissolve the MTT metabolite. The resulting purple medium was removed before acquiring images of cells. Lower row: MTT treated and methanol fixed cells were mildly stained with trypan blue (TB) to visualize their morphology. All images were acquired at the same magnification. The border of some cells is marked for clarity.

Figure 5

(A) Representative images of A172 cells used for image analysis. The images of MTT metabolites were acquired as described in Figure 4 legend (upper row). The images were then converted to grayscale and inverted. Blue and red ovals mark some regions of interest (reflecting MTT metabolites) and corresponding background regions used for image analysis, respectively. (B) Densitometric evaluation of MTT metabolic activity for the indicated cultures, expressed as signal intensity for regions of interest (cells) after corresponding background corrections. Mean values for at least 30 cells are presented for each sample. Bars, SE. ROI, region of interest; BG, background.

Figure 6

Radiosensitivity of the indicated cell lines evaluated by the 96-well plate XTT (solid squares) and CellTiter-Blue (open squares) assays. Bars, SE. The ID₅₀ values for colorimetric (this Figure), growth inhibition and colony formation (Figure 1) assays are shown. CTB, CellTiter-Blue; CFA, colony forming ability; ID₅₀, inhibiting dose 50%.

Figure 7

(A) Representative flow cytometry profiles of SKNSH cells stained with Annexin V (FITC) before and 3 days after irradiation. (B) The percentages of Annexin V-positive cells in the indicated cell lines before and 3 days after irradiation. Bars, SE.

Figure 8

(A) Representative flow cytometry profiles of HCT116 cells assessed for Annexin V positivity after incubation in the absence (control) or presence of leptomycin B (2 μM) for 3 days. (B) The percentages of Annexin V-positive cells in HCT116 cultures incubated in the absence or presence of leptomycin B (2 μM) or cisplatin (60 μM) for 3 days. Bars, SE.