Multinucleated Giant Cancer Cells Produced in Response to Ionizing Radiation Retain Viability and Replicate Their Genome

Razmik Mirzayans,David Murray,Bonnie Andrais,April Scott,Ying Wang,Piyush Kumar

doi:10.3390/ijms18020360

Razmik Mirzayans, David Murray + Show 4 more

Open Access

https://doi.org/10.3390/ijms18020360

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Abstract

Loss of wild-type p53 function is widely accepted to be permissive for the development of multinucleated giant cells. However, whether therapy-induced multinucleation is associated with cancer cell death or survival remains controversial. Herein, we demonstrate that exposure of p53-deficient or p21WAF1 (p21)-deficient solid tumor-derived cell lines to ionizing radiation (between 2 and 8 Gy) results in the development of multinucleated giant cells that remain adherent to the culture dish for long times post-irradiation. Somewhat surprisingly, single-cell observations revealed that virtually all multinucleated giant cells that remain adherent for the duration of the experiments (up to three weeks post-irradiation) retain viability and metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and the majority (>60%) exhibit DNA synthesis. We further report that treatment of multinucleated giant cells with pharmacological activators of apoptosis (e.g., sodium salicylate) triggers their demise. Our observations reinforce the notion that radiation-induced multinucleation may reflect a survival mechanism for p53/p21-deficient cancer cells. With respect to evaluating radiosensitivity, our observations underscore the importance of single-cell experimental approaches (e.g., single-cell MTT) as the creation of viable multinucleated giant cells complicates the interpretation of the experimental data obtained by commonly-used multi-well plate colorimetric assays.

Highlights

Most human cell types that express wild-type p53 respond to moderate doses of ionizing radiation (e.g., 8 Gy) by exhibiting sustained nuclear accumulation of p21WAF1, which down regulates apoptosis and switches on the stress-induced premature senescence (SIPS) program [1,2,3,4]
This does not appear to be likely because using lower cell numbers for selected cell lines (e.g., HCT116p53−/−) did not impact
This does not appear to be likely because using lower cell numbers for selected cell lines (e.g., HCT116p53−/−) did not impact on the degree of radiosensitivity as measured by this assay

Summary

Introduction

Most human cell types that express wild-type p53 respond to moderate doses of ionizing radiation (e.g., 8 Gy) by exhibiting sustained nuclear accumulation of p21WAF1 (hereafter p21), which down regulates apoptosis and switches on the stress-induced premature senescence (SIPS) program [1,2,3,4]. In a series of landmark studies [7,8,9], these authors demonstrated that exposure of human cervical carcinoma (HeLa) cells to ionizing radiation results in the development of multinucleated giant cells (MNGCs) that grow very slowly, if at all, but remain viable and secrete growth stimulating factors. This observation prompted the development of the feeder layer clonogenic assay, in which a “lawn” of heavily-irradiated feeder cells (which encompass MNGCs) is inoculated in a culture dish to promote the growth of test cells given graded doses of radiation [8]. HeLa cells do not undergo SIPS after exposure to ionizing radiation [3]

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