Five colon cancer cell lines (LOVO, colo205, HCT15, HCT116 and HT29) and five gastric cancer cell lines (AGS, SGC7901, MKN-45, MKN-28 and SNU-1) were obtained from the China Center for Type Culture Collection (Wuhan, China). All the cells were cultured in RPMI-1640 medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (GE Healthcare Life Sciences HyClone Laboratories, Logan, UT, USA) at 37°C in a humidified atmosphere containing 5% CO₂.

Total RNA was isolated from cultured cells using the TRIzol® reagent (Invitrogen Life Technologies) and first-strand cDNA was synthesized using RevertAid first strand cDNA synthesis kit (MBI Fermentas, Vilnius, Lithuania) according to the manufacturer's instructions. To quantify full length POT1 mRNA levels, RT-qPCR was performed using 2 µl cDNA with SYBR-Green I (Takara Bio, Inc., Shiga, Japan) in a total volume of 50 µl with the primer 5′-CAGGAGCTG ACGTGGAAGAT-3′ (forward) and 5′-ATGTATTGTTCC TTGTATAAGAAATGGTGC-3′ (reverse). After enzyme activation for 10 min at 95°C, 40 three-step cycles were performed (30 sec at 94°C, 30 sec at 60°C and 20 sec at 72°C). RT-qPCR for POT1 v5 variant was performed as described above with primers 5′-CATCGGCTACAAAATCTG-3′ and 5′-ACCAT TTTCTCTTGGTCTCAG-3.′ β-actin expression was measured in all the samples as an endogenous control with primers. Threshold cycles (Ct) of β-actin were used to calculate the Ct values, which were corrected for input cDNA. The average ΔCt value was used to calculate the ΔΔCt values. Relative mRNA expression was calculated with the formula: 2 EXP (-ΔΔCt) × 100% and all the mRNA levels were indicated using the formula (target gene mRNA of sample/β-actin of sample) ×100. All the samples were measured in triplicate in two separate experiments.

PCR reactions were performed using a method by Cawthon (9) by aliquoting 15 µl of master mix into each reaction well of a 96-well plate compatible with the M×3000P qPCR system (Agilent Technologies, Santa Clara, CA, USA) containing ∼20 ng DNA diluted in pure water, for a final volume of 25 µl/reaction. Five concentrations of a reference DNA sample (the ‘Standard DNA’) were prepared by serial dilution and analyzed in duplicate in every 96-well plate, and these reactions provided the data for the generation of the standard curves used for relative quantitation. All the experimental DNA samples were assayed in triplicate. The final concentrations of reagents in the PCR reaction with SYBR-Green I (Takara Bio, Inc.) were 10 mmol/l Tris-HCl (pH 8.3), 50 mmol/l KCl 3 mmol/l MgCl₂, 0.2 mmol/l each deoxynucleotide, 1 mmol/l dithiothreitol and 1 M betaine. Each 25 µl reaction received 0.625 U AmpliTaq Gold DNA polymerase (Applied Biosystems, Inc., Foster City, CA, USA). For multiplex RT-qPCR, the telomere primer pair telg and telc (final concentration of 900 nM each), were combined either with the albumin primer pair albu and albd (final concentration of 900 nM each), or with the β-globin primer pair hbgu and hbgd (final concentration of 500 nM each) in the master mix. All the primer sequences and the rationale for their design are presented in the results section. The thermal cycling profile was stage 1: 15 min at 95°C; stage 2: 2 cycles of 15 sec at 94°C and 15 sec at 49°C; and stage 3: 32 cycles of 15 sec at 94°C, 10 sec at 62°C, 15 sec at 74°C with signal acquisition, 10 sec at 84°C and 15 sec at 88°C with signal acquisition. The 74°C reads provided the Ct values for the amplification of the telomere template, and the 88°C reads provided the Ct values for the amplification of the scg template. Following the completion of thermal cycling and raw data collection, two standard curves were generated for each plate; one for the telomere signal and one for the scg signal. The T/S ratio for an experimental DNA sample is T, which is the ‘Standard DNA’ that matches the experimental sample for copy number of the telomere template in nanograms, divided by S, which is the ‘Standard DNA’ that matches the experimental sample for copy number of the scg in nanograms. As each experimental sample was assayed in triplicate, three T/S results were obtained for each sample; therefore, the final reported result for a sample in a given run is the average of the three T/S values. Average T/S is expected to be proportional to the average telomere length/cell. Samples with a T/S >1.0 have an average telomere length greater than that of the ‘Standard DNA’ samples, and those with a T/S <1.0 have an average telomere length shorter than that of the ‘Standard DNA’.

Cells were trypsinized at 37°C for 5–10 min and pipetted eight times to keep the clumped cells as a single cell suspension. The single cell suspension was adjusted and seeded into 25 cm² flasks at various densities based on the results of the pre-experiments. Subsequently, the cells were left to settle overnight and were exposed to irradiation at room temperature, followed by immediate incubation at 37°C, 5% CO₂ for 14–20 days. Following fixation and staining with 1% w/v crystal violet (Sigma-Aldrich, St. Louis, MO, USA) in dehydrated alcohol, colonies of >50 cells were scored. Surviving fractions (SF2) were evaluated relative to 0 Gy radiation-treated controls.

The statistical analyses were performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) and assessed by the Mann-Whitney U test and Spearman's rank correlation test for equality of variances. P<0.05 was considered to indicate a statistically significant difference.

Transcript levels of full length POT1 and POT1 v5 mRNA were determined by RT-qPCR in all 10 cancerous cell samples. In all these cell lines, full length POT1 mRNA levels with a mean value of 198±54 ranged from 118 to 428, and had a significant difference when compared to each other. The expression difference of full length POT1 mRNA levels in different tissue-derived cancer cells is shown in Fig. 1A. In addition, the POT1 v5 mRNA levels with a mean value of 18±5 was in the range from 9 to 33 in all cancer cells. The expression of POT1 in different cancer cell types was relatively stable compared to the high variation of human telomerase reverse transcriptase. The expression difference of POT1 v5 mRNA levels in different tissue background cancer cells is shown in Fig. 1B.

There was an extremely weak correlation between the full length POT1 level and telomere length (R²=0.284, P<0.05). In particular, the linear correlation between them in colon cancer cells and gastric cancer cells was investigated. The mRNA levels of POT1 are positively correlated to telomere length in human gastric adenocarcinoma cell lines (Fig. 2A). However, a correlation in the colon cancer types was not found. The mRNA levels of POT1 are positively correlated to telomere length in human colon cell lines if HT29 is excluded from the group (P<0.05, Fig. 2B). No significant associations were observed between telomere length and POT1 v5 mRNA levels.

SF2 at 2 Gy was used as an index of clonogenic cellular radiosensitivity. The present results showed a correlation between radiosensitivity and POT1 mRNA levels in the 10 carcinoma cell lines in which linear regression analysis was used to establish a determination coefficient (r²) of 0.054 for the association between POT1 mRNA levels and radiosensitivity (SF2) (P>0.05). Evidently, different radiosensitivities of cancer cell lines did not depend on the levels of full length POT1 mRNA (Fig. 3A). Of note, there was a significant correlation between the POT1 v5 variant level and radiosensitivity in gastric cancer (Fig. 3B) and colon cancer cells (Fig. 3C). These results suggest that the POT1 v5 level is a critical factor in the regulation of radiosensitivity in colon and gastric adenocarcinoma cell lines. However, the correlation coefficient of only 0.633 (Fig. 3D) also suggests the presence of additional factors in the process of radiosensitivity regulation.