Abstract
Aim: To explore kinetic changes in the gene expression profile during radiation-induced mitotic catastrophes. Materials and Methods: Gene expression changes were measured in HPV-infected HeLa Hep2 tumor cells following exposure to 5 Gy of ionizing radiation (60Co). Signaling pathways were explored and correlated to the biological responses linked to mitotic catastrophe. Results: Following irradiation a transient G2-arrest was induced. Anaphase bridge formation and centrosome hyperamplification was observed. These phenotypical changes correlated well with the observed gene expression changes. Genes with altered expression were found to be involved in mitotic processes as well as G2- and spindle assembly checkpoints. Also centrosome-associated genes displayed an increased expression. Conclusion: This study elucidates specific characteristics in the altered gene expression pattern induced by irradiation, which can be correlated to the events of mitotic catastrophe in HeLa Hep2 cells. Therapeutic strategies modulating these alterations might potentiate future therapy and enhance tumor cell killing.
Mitotic catastrophe is considered to be the major cell death mechanism by which solid tumors respond to clinical radiotherapy (1). Mitotic catastrophe occurs during or as a result of aberrant mitosis (2). Aberrant mitosis produces atypical chromosome segregation and cell division and leads to the formation of giant cells with aberrant nuclear morphology (3, 4), multiple nuclei (5) and/or several micronuclei (6). Two important mechanisms for the induction of mitotic catastrophe have been proposed. Firstly, a mitotic catastrophe has been proposed to occur as a consequence of DNA damage and deficient cell-cycle checkpoints. Checkpoint inactivation is frequently a consequence of mutation/inactivation of p53. Furthermore, the p53 protein is frequently functionally-inactivated by viruses including high risk human papillomaviruses (HPVs). HPVs may contribute to malignant transformation causing cervical and other anogenital cancers as well as a sub-population of head and neck cancers (7). The second proposed mechanism for the induction of mitotic catastrophe is centrosome amplification (8). Centrosomes are a major microtubule organizing center and exert an important function by formation of bipolar spindles (9). Hyperamplification of centrosomes may result in multipolar spindles causing abnormal chromosome segregation, and generate cells with multiple micronuclei or binucleated giant cells (10, 11).
An improved understanding of the signaling pathways involved in radiation-induced mitotic catastrophe may help increase the efficacy of radiation therapy. To address this issue, we investigated the kinetic response to ionizing radiation of HeLa Hep2 cells and evaluated if typical cellular phenotypes specific for mitotic catastrophe correlated with alterations in gene expression.
Materials and Methods
Cell lines. HeLa Hep2 cells (CCL-23, American Type Culture Collection (ATCC), Manassas, Virginia, USA)), were grown in DMEM (VWR International, Radnor, Pennsylvania, USA)) (1% penicillin, streptomycin (VWR), 1% L-glutamine (VWR), and 5% Fetal calf serum (VWR)) at 37°C, 5% CO2.
Irradiation. Cells were exposed to absorbed doses of 5 Gy using a Cobalt-60 (Co60) treatment unit (Alcyon II, Best Theratronics Ltd, Ontario, Canada). Dose rate was approximately 0.45 Gy/min in the middle of the treatment period.
Staining of mitotic cells. Anti-phospho-Ser/Thr-Pro Mitotic Protein Monoclonal 2 (MPM-2) antibody (Upstate Cell Signaling Solutions, Millipore, Watford, UK) was used to quantify mitotic cells as earlier described (3). Cells were fixed in PEM buffer (80 mmol/L piperazine-N,N’-bis(2-ethanesulfonic acid) (PIPES), 1 mmol/L (ethylene glycol tetraacetic acid) (EGTA), 4 mmol/L MgCl2*6H2O, 0.2% saponin, pH 7.0 (all from Sigma Aldrich, Gillingham, Dorset, UK) containing 2% paraformaldehyde (Sigma Aldrich). Incubation of primary MPM-2 antibody was followed by Alexa Fluor 488–conjugated goat anti-mouse antibody (Invitrogen, Paisley, UK). Cells were then suspended in propidium iodide (20 mmol/L Tris solution (pH 7.6), propidium iodide (PI) (50 μg/mL), NP40 (0.1%), and RNase (20 μg/mL) (all from Sigma-Aldrich). Data was collected using FACS (BD Biosciences, San Jose, CA, USA), and results were analyzed using CellQuest software (BD Biosciences). Fluorescence microscopy. All fluorescence stainings were examined by confocal laser scanning microscopy using a Leica SP2 confocal microscope equipped with an argon and HeNe laser.
Staining of centrosomes and DNA. Cells were fixed at −20°C in 95% methanol and 5% acetic acid and incubated with PI solution as described above. Antibodies specific for γ-tubulin were used to visualize centrosomes as earlier described (3). Cells were fixed in PEM buffer containing 2% paraformaldehyde followed by labeling with a monoclonal antibody recognizing γ-tubulin (clone GTU-88, Sigma Aldrich). Alexa Fluor 488 conjugated goat anti-mouse antibody (Invitrogen) was used as secondary antibody. Cells were counterstained with PI as described above.
Extracting total RNA. Cells were harvested at 6, 12, 24, 48, 72 and 96 h following irradiation. RNA was extracted using the RNeasy Mini Kit (QIAGEN, Hamburg, Germany) according to the manufacturer's instructions. Quality was determined using the RNA 6000 Nano assay on the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA integrity value (RIN) for all samples were in the range of 9.5-10.
RNA labeling and gene expression analysis. Total RNA was prepared using the Illumina Total Prep RNA Amplification Kit (Ambion, Austin TX, USA) according to instructions. 250 ng of total RNA was used. Quality was determined using the RNA 6000 Nano assay. Expression profiling was done using HumanRef-8 Bead- Chips (Illumina, San Diego, CA, USA). Raw data were analyzed using GenomeStudio V 3.2.3 (Illumina). Data was normalized using the cubic spline method. Illumina custom error model was used to compute a false discovery rate. p-Values were calculated using the GenomeStudio V 3.2.3 and applying the Illumina custom error model. Cut-off was set to p-values<0.05 and >2-fold change in expression.
Biological functions and pathway analysis. Signaling pathways and processes were explored using MetaCore (Genego Inc., St Joseph, MI, USA). Normalized gene expression data was exported. Filters were set on p<0.05, and fold change 2 (positive and negative).
Results
Induction of G2-arrest. Irradiation induces a transient G2-arrest as seen in Figure 1. The results indicate an initial G2-arrest and a consequent decrease in the fraction of mitotic cells. This arrest is transient and the frequency of mitotic cells returns to control levels approximately 13 h after exposure to irradiation.
Quantification of mitotic cells by FACS, using MPM-2, a monoclonal antibody with specificity for mitosis-specific and cell cycle–regulated phosphoproteins. A temporary decrease in mitotic cells indicates that radiation induce a transient G2-arrest in HeLa Hep2 cells. One representative experiment is presented. C=Control.
Induction of anaphase bridges. Pronounced formation of anaphase bridges were seen approximately 15 h following irradiation. Checkpoint adaptation and entry into mitosis with unrepaired DNA damages is probably the cause of the increased frequency of anaphase bridges observed especially during the first mitosis following irradiation (Figure 2).
Centrosome hyperamplification. Irradiation induces centrosome hyperamplification in HeLa Hep2 cells (Figure 3). We have earlier shown that this centrosome amplification is dose-dependent and is most pronounced in 2 to 3 days after exposure to irradiation (3, 10).
Transcriptional profile of irradiated HeLa Hep2 cells. We investigated the temporal changes in gene expression following irradiation. The number of genes that were significantly altered at different time-points evaluated are presented in Table I. Significantly altered signaling pathways were explored using MetaCore. At 6 and 12 h, only a few genes was significantly altered. From 24 h to 96 h, several genes displayed altered expression. A major part of these genes were found to be involved in cell-cycle progression and regulation, mitotic processes and the G2- and spindle assembly checkpoints. At 96 h pathways involving interferon and other cytokines were significantly changed.
We chose to explore genes that were continuously altered at 24, 48, 72, and 96 h in order to reduce the complexity of interpretation. These time-points were chosen as most cellular events associated with mitotic catastrophe occur during this time. The 10 most significantly changed pathways obtained by MetaCore are displayed in Table II.
Propidium iodide staining of DNA in control and irradiated HeLa Hep2 cells. Irradiated cells displayed an increased frequency of anaphase bridges.
Centrosomes visualized with a γ-tubulin antibody (green) and DNA stained with propidium iodide (red). Left: a mitotic cell with normal number of centrosomes; Right: an irradiated mitotic cell with hyperamplified number of centrosomes.
Differentially expressed genes at 6, 12, 24, 48, 72 and 96 h following irradiation. p≤0.05 and ≥2 fold change in expression.
Seven out of 10 pathways are related to the cell cycle and especially mitotic processes. The remaining pathways are related to DNA damage regulation of the G2/M checkpoint. A total of 44 genes were found to have an increased gene expression at all time-points (Table III).
The 10 most significantly altered pathways at 24, 48, 72 and 96 h following irradiation. p-Values were calculated using MetaCore.
Several of these genes were associated with centrosome regulation, spindle assembly checkpoint and G2- to M-phase progression (Table IV).
The 44 genes which had a continuously changed expression at 24, 48, 72 and 96 h following irradiation. p≤0.05 and fold change ≥2. p-Values were calculated using the GenomeStudio software.
Discussion
Mitotic catastrophe is considered the most common mode of cell death in solid tumors in which p53 function is impaired (12). In the present study we analyzed the gene expression profile in HeLa Hep2 cells passing through a mitotic catastrophe. HeLa Hep2 cells have wild type p53, but the protein level is reduced by the human papilloma virus protein E6 to give no apparent p53 response.
Genes associated with SAC, centrosomes and G2/M arrest. Fold changes at 24, 48, 72 and 96h following 5 Gy of irradiation are presented.
We observed a transient G2-arrest in irradiated HeLa Hep2 cells (Figure 1) followed by a re-entry into the cell cycle with unrepaired DNA damage, probably as a consequence of an impaired G2/M checkpoint and adaptation (13). Maintenance of the G2-arrest is dependent on p53 and its downstream signaling proteins (14).
Cells with impaired p53 function have a higher expression level of these downstream genes (15), which is in agreement with our results and probably contribute to enhance checkpoint adaptation process. Checkpoint adaptation cause premature mitotic entry of cells with unrepaired DNA damage and induction of a prometaphase arrest (13). In agreement with this observation, one of the most significantly affected signaling pathways induced by irradiation of HeLa Hep2 cells involved the prometaphase (Table II). We observed an increased expression of several genes implicated in the regulation of the G2/M checkpoint including PLK1, CCNB, CDC25B and CDC25C (Table IV). All these mitosis-promoting factors have been described to accumulate during G2-arrest and consequently trigger checkpoint adaptation (13). Impaired checkpoints, checkpoint adaptation and premature entry into mitosis are probably the explanations for increased frequency of anaphase bridges and lagging chromosomal material that we observed especially during the first mitosis following irradiation (Figure 2). Anaphase bridges may be generated when broken chromosomes, induced by irradiation, fuse. We and others have reported that hyperamplification of centrosomes occurs after exposure to radiation (Figure 3) (8, 10, 16). In the present study multiple genes involved in centrosome amplification were up-regulated following irradiation (Table IV). Genes known to be involved in centrosome duplication like CDK2, CCNE, CCNA, AURKA and CDC25 (17-19) were all up-regulated at multiple time-points following radiation. (20). Furthermore, KIF11 and KIFC1 have crucial roles in the formation of bipolar spindles (21, 22). Additionally, PLK1 is an important kinase that controls centrosome maturation. Reduced activity of PLK1 results in functional defects of centrosomes and failure to form bipolar mitotic spindles (23). Genes significantly changed and involved in the spindle assembly checkpoint (SAC) are presented in Table IV. SAC is important for DNA damage induced mitotic catastrophe since this checkpoint prevents cells from entering anaphase until all chromosomes are properly attached to the bipolar mitotic spindles (17, 24). The spindle checkpoint proteins inhibit CDC20 and consequently the onset of the anaphase (24). The results of this study clearly demonstrate a relation between radiation-induced alterations in gene expressions and the subsequent execution of the mitotic catastrophe.
Novel therapeutic strategies currently aim at centrosome and G2/M checkpoint associated regulators with the intention to target and inactivate them, thus promoting mitotic catastrophe and consequently increasing cell death (25). Several inhibitors are currently investigated in pre-clinical and clinical studies including aurora kinases (26, 27), PLK1 (11), survivin (28) and kinesin family members (KIF11, KIFC1) (21, 29). This study indicates that therapeutic strategies combining these inhibitors with irradiation might potentiate therapy and enhance tumor cell kill.
Acknowledgements
This project was financed by the Swedish Cancer Research Council, the Lions Foundation in Umeå, the county of Västerbotten and the University of Umeå.
Footnotes
-
This article is freely accessible online.
- Received April 23, 2014.
- Revision received May 28, 2014.
- Accepted June 3, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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