Radiotherapy serves as adjunctive treatment to chemotherapy and surgical resection of colorectal cancer. However, the cellular response to irradiation varies depending on the expression of tumor suppressor p53, which plays a significant role in the regulation of cell cycle arrest, apoptosis and telomerase activity in various cancers. The present study aimed to investigate cell cycle arrest, apoptosis and telomerase activity with respect to p53 expression in p53 wild-type (+/+) and deficient (−/−) HCT116 colon cancer cell lines following 5 Gy γ-irradiation. Cell cycle arrest and apoptosis were evaluated using flow cytometry. The telomerase activity was measured using a TRAP (telomerase repeat amplification protocol) assay. Following treatment with irradiation, G1/S cell cycle arrest occurred in the p53+/+ cells, whereas the p53−/− cells accumulated in the G2 phase. No differences were observed in the apoptotic ratios between the two cell lines following irradiation. Decreased telomerase activity was observed in the p53+/+ cells, whereas telomerase activity was increased in the p53−/− cells. The results showed that while telomerase activity and G1 cell cycle arrest were regulated depending on the p53 status, G2 arrest and the apoptotic response were promoted via a p53-independent pathway.
Colorectal cancer is one of the leading causes of cancer-associated mortality in the world. According to the linear model of cancer initiation, proposed by Fearon and Vogelstein, cancer is a disease that arises from multiple serial somatic mutations (
The TP53 gene is known as the guardian of the genome or the cellular gatekeeper. The gene contains 11 exons, which encode 2.8 kb mRNA that is translated into a 53 kDa protein. Following exposure to stress conditions, including hypoxia, oncogene activation, DNA damage, nucleotide defects and viral transformation, p53 is subjected to certain post-translational modifications that regulate the subcellular localization and stability of the protein (
p53+/+ and p53−/− HCT116 colon cancer cell lines were cultured in complete McCoy's 5A medium, consisting of 10% fetal bovine serum, 1% penicillin/streptomycin and 1% L-Glutamine at 37°C, in a humidified incubator containing 5% CO2. Once 80–90% confluency was reached in T75 culture flasks, the cells were treated with 5 Gy γ-irradiation (Co60-Dmax) and collected to evaluate the cell cycle, apoptosis and telomerase activity.
Following irradiation, the trypsinized cells were washed and collected by centrifugation. The cell numbers were counted using a hemocytometer. RNase (Sigma, St. Louis, MO, USA) and propidium iodide (Sigma) were added to the cells and mixed using a vortex. Following a 20-min incubation period in the dark at room temperature, the cells were filtered through a nylon mesh (37 μm) and evaluated using flow cytometry (EPICS XL MCL; Beckman Coulter Inc., Brea, CA, USA). The ratios of the cells in the G0/G1, S and/or G2/M phases and the apoptotic cell numbers were evaluated by McCycle software (Phoenix Flow System, San Diego, CA, USA) using dichotomous variable DNA histograms.
Protein lysates were prepared from a CHAPS lysis buffer and quantified using the Bradford method. In order to detect telomerase activity, the TRAPeze XL Telomerase Detection kit (Chemicon, Temecula, CA, USA) was used.
The statistical analyses were performed using SPSS software version 13 (SPSS, Inc., Chicago, IL, USA). The variables were investigated using visual (histograms and probability plots) and analytical (Kolmogorov-Simirnov/Shapiro-Wilk's test) methods to determine whether or not they were normally distributed. The χ2 test was used to statistically analyze the cell cycle and apoptosis. The telomerase activity was evaluated using a Mann-Whitney U test. P<0.05 was considered to indicate a statistically significant difference.
The apoptotic ratios of the cells following exposure to irradiation were evaluated using the sub-G0 DNA content. Following treatment with 5 Gy γ-irradiation, the average apoptotic percentages of cell number significantly increased in a time-dependent manner in the p53+/+ and p53−/− cells (P<0.05), whereas there was no change in the apoptotic cell number in the non-irradiated control cells (
The G1, S and G2 phases of the cell cycle showed a normal distribution in the non-irradiated p53+/+ and p53−/− control cells (
The telomerase activity of the non-irradiated p53+/+ cells was nearly uniform at 0, 24 and 48 h. The telomerase activity of the non-irradiated p53−/− cells was marginally different to the p53+/+ cells at the designated time-points. In the irradiated p53+/+ cells, the telomerase activity was low at 0 h and continued to decrease at 24 and 48 h. In contrast to this, the telomerase activity was similar to the non-irradiated p53−/− cells at 0 h. However, at 24 h post-irradiation, the telomerase activity increased and remained at a higher level at 48 h (
γ-irradiation causes double or single strand breaks depending on the application dose, for example 5 or 7.5 Gy, in cells (
In the present study, the HCT116 cells expressing p53+/+ were arrested in the G1 phase of the cell cycle at 24 h and at 48 h, then escaped from G1 arrest and accumulated in the S phase, driving apoptosis at an additional 48 h after 5 Gy γ-irradiation. Attardi
While telomerase activity was decreased in the p53−/− cells of the present study, increased telomerase activity was identified in the p53+/+ cells following γ-irradiation. The activity of TERT, which is a catalytic subunit of telomerase, depends on whether p53 is expressed. Following irradiation, TERT activity is decreased depending upon the level of p53 expression. However, in p53−/− cells, TERT activity is increased due to the absence of p53 (
In summary, the exposure to 5 Gy γ-irradiation, telomerase activity and G1 cell cycle arrest were regulated depending on the p53 status in the HCT116 colon cancer cells. However, G2 arrest and the apoptotic response were promoted in a p53-independent pathway.
The p53+/+ and p53−/− HCT116 colon cancer cell lines were provided by Professor Bert Vogelstein.
(A) Average apoptotic percentages obtained from three independent experiments in irradiated and non-irradiated p53 wild-type (+/+) and deficient (−/−) HCT116 cells. (B) The average apoptotic cell numbers of irradiated and non-irradiated control cells. Values of <10% were considered to be the threshold in the irradiated control p53−/− and p53 +/+ cells. IR, irradiated.
Normal distribution of G1, S and G2 stages of the cell cycle in non-irradiated (A) p53 wild-type (+/+) and (B) p53 deficient (−/−) HCT116 cells at 0 and 24 h.
(A) G1 phase arrest at 24 h and accumulation in G2 phase arrest at 48 h in irradiated p53 deficient (−/−) HCT116 cells. (B) G1 and S phase arrest in irradiated p53 wild-type (+/+) HCT116 cells at 24 and 48 h. Blue areas of the histogram illustrate apoptotic cells associated with the sub-G0 DNA content.
Comparison of telomerase activity in p53 wild-type (+/+) and p53 deficient (−/−) cells in (A) irradiated and (B) non-irradiated cells. IR, irradiated.