Int J Mol Sci. 2017 Feb 8;18(2).

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

Razmik Mirzayans*, Bonnie Andrais, April Scott, Ying W. Wang, Piyush Kumar and David Murray

Department of Oncology, University of Alberta, Cross Cancer Institute, Edmonton, Alberta T6G 1Z2, Canada; bonnie.andrais@ahs.ca (B.A.); april.scott@ahs.ca (A.S); ywwang@ualberta.ca (Y.W.W.); pkumar@ualberta.ca (P.K.); david.murray5@ahs.ca (D.M.)

*Correspondence: razmik.mirzayans@ahs.ca; Tel.: +1-780-432-8897.

 

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 3 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 emergence of viable multinucleated giant cells complicates the interpretation of the experimental data obtained by commonly-used multi-well plate colorimetric assays.

KEYWORDS:  96-well plate XTT; colony forming ability; ionizing radiation; multinucleated giant cells; p21WAF1; p53; premature senescence; proliferation; single-cell MTT; viability

PMID: 28208747

 

Supplementary:

In the mid 1990’s, it was proposed that the principal role of the p53 tumor suppressor protein in determining cell fate following genotoxic stress is to either promote survival by activating cell cycle checkpoints and facilitating DNA repair, or to induce apoptotic cell death. However, as discussed recently [1–3], a large body of evidence from studies with solid tumor-derived cell lines demonstrated that the primary response triggered by moderate, clinically relevant doses of cancer therapeutic agents is a sustained proliferation block, and not apoptosis. The proliferation block predominantly reflects stress-induced premature senescence and the creation of polyploid/multinucleated giant cells (MNGCs), depending on the status of p53, its downstream effector p21WAF1 (p21) and other factors.

Numerous studies reported in the past decade have demonstrated that MNGCs created in solid tumors in response to therapeutic agents can contribute to cancer relapse by first entering a state of dormancy and ultimately giving rise to progeny with stem cell-like properties [1]. MNGCs can give rise to tumor-repopulating cells through depolyploidization as well as nuclear budding or bursting similar to simple organisms such as fungi [1–3]. Shockingly, only a single MNGC has been reported to be sufficient to promote metastatic tumors in a mouse model [4].

We recently reported studies demonstrating that human solid tumor-derived cell lines lacking p21 or wild-type p53 activity respond to moderate doses of ionizing radiation (between 2 and 8 Gy), typically used in the colony formation assay, by exhibiting sustained growth arrest but not apoptosis or other modes of cell death, and that this growth-arrested response is attributed to the emergence of MNGCs [5]. In addition, virtually all MNGCs that remain adherent to the culture dish for long times (e.g., 3 weeks) post-irradiation retain membrane integrity and exhibit the ability to metabolize the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) to its water-insoluble formazan derivative (Figure 1); the majority (>60%) of MNGCs exhibit DNA synthesis.

These observations, together with accumulating evidence connecting therapy-induced multinucleation to disease recurrence [1,2], suggest that identifying pharmacological agents capable of inducing apoptosis of MNGCs before they can give rise to tumor-repopulating progeny may have important clinical ramifications. To this end, we demonstrated that treatment of MNGCs with pharmacological activators of apoptosis (e.g., sodium salicylate) triggers their demise [5].

Caution should be exercised to avoid misinterpreting the radiosensitivity data obtained with widely-used long-term (colony formation) and short-term (multi-well plate colorimetric) assays in terms of loss of viability and hence cytotoxicity.

 

 

Figure 1: Representative bright-field microscopy images showing the metabolic activity of p53 knockout HCT116 colon carcinoma cells before [A] and after exposure to ionizing radiation [B-D], as reported in Int. J. Mol. Sci. 18, 360 (2017). 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. As a negative control, cells in some dishes were first treated with methanol to inhibit their metabolic activity, and then incubated with MTT [C]. All images were acquired at the same magnification.

 

 

References:

[1] R. Mirzayans, B. Andrais, P. Kumar, D. Murray. Significance of wild-type p53 signaling in suppressing apoptosis in response to chemical genotoxic agents: Impact on chemotherapy outcome. Int. J. Mol. Sci. 2017, 18, 928. PubMed PMID: 28452953.

[2] R. Mirzayans, B. Andrais, P. Kumar, D. Murray. The growing complexity of cancer cell response to DNA-damaging agents: Caspase 3 mediates cell death or survival? Int. J. Mol. Sci. 2016, 17, 708. PubMed PMID: 27187358.

[3] R. Mirzayans, B. Andrais, A. Scott, Y.W. Wang, Y.W., D. Muray. Ionizing radiation-induced responses in human cells with differing TP53 status. Int. J. Mol. Sci. 2013, 14, 22409–22435. PubMed PMID: 24232458.

[4] Z. Weihua, Q. Lin, A.J. Ramoth, D. Fan, I.J. Fidler. Formation of solid tumors by a single multinucleated cancer cell. Cancer 2011, 117, 4092–4099. PMID: 21365635.

[5] R. Mirzayans, B. Andrais, A. Scott, Y.W. Wang, P. Kumar, D. Murray. Multinucleated giant cancer cells produced in response to ionizing radiation retain viability and replicate their genome. Int. J. Mol. Sci. 2017, 18, 360. PubMed PMID: 28208747.