The fate of radiation induced giant-nucleated cells of human skin fibroblasts
Introduction
Normal mammalian cells usually contain diploid complements of chromosomes that enable conservative genetic integrity during cell replication and recombination. The morphological and functional features of cells remain stable over many generations of cell replication. Conversely, any cellular phenotypic variations, such as the enlargement of the cell nucleus following exposure to ionising radiation will be an indication of genetic instability within the cellular system (Little, 2003).
Radiation-induced genomic instability increases the rate of incidence of genetic alterations in eukaryotic cells. Thus, cells exposed to radiation can survive; however, they give rise to progeny that carry heritable damages that can persist for multiple generations after exposure. This instability might provide a driving force for progression to cancer. One of the biological effects that occurs in response to ionising radiation is the expression of nuclear abnormalities and the related formation of giant-nucleated cells (GCs) (Lyng et al., 1996).
GCs usually contain polyploid complements of chromosomes and maintain the balance between chromosomes, despite changes in total chromosome numbers. However, one or more chromosomes could present in unequal numbers, raising a type of abnormality called aneuploidy, which is one of the leading causes of, and is commonly observed in, cancers (Donnelly et al., 2014, Storchova and Kuffer, 2008).
The enlargement of a cell nucleus results in an increase in the size of other cellular structures. Thereby, the entire cell will be scaled accordingly to maintain the normal ratio of cell size (Webster et al., 2009). The formation of GCs seems to occur during the absence or incompleteness of the mitotic phase. They usually arise by one of, or a combination of, three different mechanisms: (a) mitotic catastrophe (b) cell fusion, or (c) endoreplication process (Storchova and Pellman, 2004).
Most of the cell cycle checkpoints are p53 dependent. Consequently, delayed response of p53 or insufficient DNA repair at the first barrier, the G1-phase, following exposure to ionising radiation results in cells arriving at the next checkpoint, the G2-phase, with accumulated mutations and breaks (Erenpreisa and Cragg, 2001, Erenpreisa et al., 2005). Cells having these complex lesions usually undergo a mitotic catastrophe where they might be partially repaired but are mitotically arrested, with apoptosis and mitotic death features in common (Alexander and Bacq, 1961). However, if the arrested cells remain viable and their growth capacity is increased, they might then enhance the transition from the mitotic to post-mitotic stages (terminal differentiation; fibrosis) (Fournier et al., 2007, Jakob et al., 2000, Lara, 1996, Rodemann, 1993). These terminal differentiation processes can be found in tumours following exposure to ionising radiation (Hanahan and Weinberg, 2011, Tolmach, 1961). Differentiation patterns and fibrotic expression have been also observed in normal human skin fibroblast cell lines following exposure to different qualities of ionising radiation (Fournier et al., 2001). This can be supported by the morphological differentiation studies carried out on the quiescent HH-4 normal human fibroblast cell cultures following exposure to doses between 0 and 7 Gy of γ rays. At various time points of analysis after irradiation, higher rates of mitotic differentiation and morphological changes were detected in the progeny of populations irradiated with 1 Gy compared to those formed at higher doses of γ rays (Rodemann et al., 1991). Similar observations were also found in the collagen synthesis studies that were performed on the asynchronous AG1522 cell cultures after exposure to X-rays or carbon ions (Fournier et al., 1998).
The arrested giant cells are morphologically heterogeneous and usually have a finite lifespan before they become senescent and die. However, some of them are considered viable and mitotically active, based on their morphological and biochemical characteristics. They are also susceptible to potential deficiency of the p53 protein or inactivation of other checkpoint genes, which might lead to chromosomal aberration and an increase in the rate of mutation (Erenpreisa and Cragg, 2010).
The GCs can undergo a prolonged mitotic arrest that significantly increases the length of the cell cycle. The length of the mitotic phase can lead to an increase in the rate of DNA breaks (Bayreuther et al., 1988). Conversely, other authors suggest that the arrest of cells at mitosis for longer periods of time might be a strategy for cell survival, which increases the time available for DNA repair and enables an alternative route to division for cells (Edgar and Orr-Weaver, 2001). However, if these cells fail to remain arrested at mitosis and adapt by undergoing cytokinesis, they are then expected to produce distinctly giant cells (Lanni and Jacks, 1998). A fraction of these cells have been described as living for longer periods with reproducible alterations (Rodemann et al., 1989). This often involves the development of micronucleation and nuclear segmentations and this, in turn, manifests a phenomenon, such as mitotic restitution (Abend et al., 1996, Heddle and Carrano, 1977). The mitotic restitution assists in initiating an endocycle mechanism that helps resistant cells to overcome mitotic catastrophe and reproduce endopolyploidy GCs, as observed in osteosarcoma cell lines (Erenpreisa et al., 2000, Nagl, 1990).
Giant cells might remain active and stimulate other surrounding cells to join them by a cell fusion mechanism. This process was observed to occur frequently between multinucleated and binucleated cells when they combine instead of separating and forming polyploidy GCs (Huang et al., 2008). The incomplete cytokinesis and mononucleated cell fusion, leading to formation of multinucleated giant cells, frequently occur in tumour cells, as detected in Hodgkin lymphoma cancer cells (Rengstl et al., 2013). Based on the cell type, however, this incompleteness of the cytokinesis might constitute an intermediate stage prior to the formation of mitotically active GCs. This intermediate stage represents the mitotic restitution or endocycle process, which is required for initiation of endoreplication, the predominant mechanism involved in polyploidy GCs formation (Duesberg and Rasnick, 2000). Endoreplication or endoreduplication is an indicator of terminal differentiation and a potential strategy for cell survival that enables an alternative route for cells to form progeny.
The potential fate of the GCs is varied. Some of them might undergo a mitotic arrest and then be terminated out of the cellular system by apoptosis (programmed cell death). Some might bypass the DNA damage repair process and escape the arrest to produce aneuploidy, which is a contributing cause to carcinogenesis (Nigg, 2002). A persistent fraction of GCs might adapt to the mitotic arrest to propagate after as polyploidy GCs. The polyploidy or aneuploidy GCs can adapt to the presence of chromosomal alterations or rearrangements and proliferate in a normal manner, which provides a survival advantage to cells. In addition to the survival strategy, it has been suggested that polyploidization provides a biodosimetric estimate of oncogenesis within heavily injured cells (Schwarze et al., 1984).
This study aims to investigate the fate of randomly selected live giant-nucleated cells developed post-irradiation in a well-defined normal human-diploid AG1522 fibroblast cell line (Azzam et al., 2002, Cornforth et al., 1989, Raju et al., 1991).
Section snippets
Cell culture
AG1522 normal human-diploid skin fibroblasts were purchased from the NIA Aging Cell Repository (CCR) at the Coriell Institute for Medical Research (Camden, USA). The cells were cultured at 37 °C in a humidified atmosphere with 5% CO2 in Eagle's Minimum Essential Medium (E-MEM) supplemented with 15% foetal bovine serum (FBS) and 2.0 mM L-glutamine plus 0.01% penicillin/streptomycin (Lonza, UK). Cells were plated into T75 flasks (Fisher, UK) at a density of 1.2×105 cells/ml and were grown for 5–7
Doubling time calculations
The doubling time () for the AG1522 cells to divide was measured every 24 h from the first subculture using the equation 1.2. The is critical when tracking giant-nucleated cells using live cell fluorescence microscopy to determine their fate after exposure to either X-ray or proton irradiation. It has been reported that the AG1522 cells can have a value of 26 h when they are highly active and this is in agreement with the value of 25.5 h calculated at 6 days after incubation (Zhang et al.,
Conclusions
The aim of this study was to determine the fate of selected fractions of GCs formed in the progeny of irradiated populations at 7, 14 and 21 days following exposure to 0.2, 1 or 2 Gy of X-ray or proton irradiation. The fate of 79 giant-nucleated cells induced in the progeny of X-ray (40 GCs) and proton (39 GCs) populations were determined from fluorescence microscopy images captured over 5 days, and their status was classified as divided, arrested or lost at the end of the observation period.
The
Acknowledgment
This work was funded by the government of Saudi Arabia (Grant no. 176/T). P.H. Regan acknowledges support from the National Measurement Office and the UK Science and Technology Facilities Council. The authors would like to thank Dr Michael Merchant for his contribution to the development of the cell-recognition MATLAB code and helpful discussions during the writing of the manuscript. The authors also thank the Department of Medical Physics of the Royal Surrey County Hospital (Guildford, UK) for
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