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.
Highlights
Activation of the p53 signaling pathway by ionizing radiation was originally proposed to result either in cell cycle checkpoint activation to facilitate DNA repair and promote survival, or in apoptotic cell death (e.g., [1])
As extensively reviewed recently [2,3,4], a large body of evidence has established that the primary response triggered by moderate doses of ionizing radiation in solid tumor-derived cell lines is a sustained proliferation block, and not apoptosis
We demonstrate that radiosensitivity as measured by multiwell plate colorimetric/fluorimetric assays is markedly skewed towards radioresistance when compared to that measured by growth inhibition (3 days post-irradiation) and colony formation (10 days post-irradiation) assays, and that the response measured by these cell-based assays reflects growth inhibition (SIPS) and not apoptosis or loss of viability
Summary
Activation of the p53 signaling pathway by ionizing radiation was originally proposed to result either in cell cycle checkpoint activation to facilitate DNA repair and promote survival, or in apoptotic cell death (e.g., [1]). Based on this two-armed model, radiosensitivity assessed by multiwell plate colorimetric/fluorimetric assays (e.g., MTT, XTT, CellTiter-Blue) would be expected to reflect cell death. The outcomes of such radiosensitivity (and chemosensitivity) assays are often interpreted to reflect loss of viability and cell death. The proliferation block predominantly reflects stress-induced premature senescence (SIPS) in cells that express wild-type p53 [5,6,7,8,9]
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