TRIzol reagent and Lipofectamine^® 2000 were obtained from Invitrogen (Carlsbad, CA, USA). Horseradish peroxidase (HRP) AffiniPure goat anti-mouse/rabbit IgG(H+L) was purchased from Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China). FITC AffiniPure goat anti-mouse IgG was from Jackson ImmunoResearch. Telomere RNA probe kit was from (Dako, USA) and Telomerase activity kit from Jermaine gene Co., (USA). pCDNA3.1 vector and pCDNA3.1/PinX1 were obtained from Invitrogen. Geneticin^® (G418) was from Gibco (USA). The mouse anti-hTERT and mouse anti-PinX1 antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

One-thousand cases of esophageal tissues from patients without chemotherapy and radiotherapy were collected from an esophageal carcinoma high-risk area of China. All specimens were verified by pathologic diagnosis and we selected 44 cases of ESCC, 50 dysplasia of esophageal squamous epithelium (22 cases of mild dysplasia and 28 cases of severe dysplasia) and 36 normal esophageal mucosa to be used to detect the length of telomere and hTERT protein expression.

Moreover, esophageal carcinoma tissues, paired adjacent mucosa (2–5 cm from margin of esophageal carcinoma) and paired normal mucosa (at least 5 cm from margin of esophageal carcinoma) were obtained from resected surgical specimens of ESCC. All specimens were verified by pathologic diagnosis, 50 cases of ESCC, dysplasia of esophageal squamous epithelium and normal esophageal mucosa were selected from 130 specimens. Fifty ESCC tissues included 39 cases of well and moderately differentiated ESCC and 11 cases of poorly differentiated ESCC; fibrous membrane invasion (n=34), fibrous membrane untouched (n=16); lymph node metastasis positive (n=17), lymph node metastasis negative (n=33). Experimental protocols were approved by the Institutional Human Care and Use Committee of Hebei Medical University.

Human esophageal cancer cells (Eca109) were grown under humidified air with 5% CO₂ in an incubator at 37°C in RPMI-1640 supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin and 100 U/ml penicillin.

The single cell suspension was acquired as previously described and incubated with FITC-(CCCTAA)3PNA probe for 15 min at 87°C. Then, after overnight hybridization at room temperature, the cell suspensions were washed, followed by staining with propidium iodide (PI) solution for 2 h at 4°C. Subsequently, the cells were analyzed in an Epics-XL II Flow Cytometer (Beckman Coulter, Miami, FL, USA). Q-FISH was used to represent the telomere length. Q-FISH = fluorescence intensity in experimental group - fluorescence intensity in background group.

The tissues were fixed in 4% formaldehyde. Antigen recovery was performed using a microwave. The sections were incubated with primary antibodies against hTERT (1:100) overnight at 4°C. The following day, the sections were incubated with polyperoxidase-anti-mouse IgG at 37°C and finally stained with diaminobenzidine. The sections were imaged with an Olympus microscope. Positive staining of hTERT protein was located in the nuclei of epithelial cells. The result criteria: the number of positive cells was >10%.

The tissues in different groups were washed with phosphate buffered saline (PBS), fixed in 70% ethanol, and the single cell suspensions were then collected and washed with PBS, stained with mouse anti-hTERT or PinX1 antibodies at room temperature for 30 min. Then, the cells were washed 3 times with PBS and incubated with FITC-anti-mouse IgG at 37°C. The stained cells were analyzed in an Epics-XL II Flow Cytometer. Fluorescence index (FI) represents the relative protein expression content. FI = (fluorescence intensity in experimental group - fluorescence intensity in control group)/fluorescence intensity in normal control.

Biopsy samples were stored in liquid nitrogen. The extraction of telomerase protein and evaluation of its activity were measured by the TRAP method using the TRAPeze-XL Telomerase Detection kit as previously described (12). Briefly, extracts from 100 mg of frozen esophageal tissues were homogenized in ~200 μl of lysis buffer. After 30 min of incubation on ice, the suspensions were centrifuged at 16,000 × g for 30 min at 4°C, after which the supernatant was frozen rapidly and stored at −70°C. The PCR conditions were: after 10 min for telomerase extension at 23°C, 95°C for 5 min to activate the Taq polymerase; 27 cycles at 94°C for 30 sec, 50°C for 30 sec, and 72°C for 90 sec; an extension at 72°C for 10 min; finally, a 4°C incubation. Equal amounts of amplified products were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gel was stained with 2% silver nitrate cream for 20 min after fixing with DNA fixative solution. After washing twice with d.d. water, the gel was placed in medium of 1.5% NaOH and 0.4% formaldehyde and waved lightly until clear DNA ladder appeared.

Total RNA was extracted from esophageal tissues or Eca109 cells with TRIzol reagent according to the manufacturer’s instructions. Total RNA (2 μg) was reverse transcribed into cDNA by AMV reverse transcriptase at 42°C for 1 h and then heated to 94°C for 5 min in a total reaction volume of 20 μl. PCR conditions used for PinX1 and internal reference GAPDH were: 94°C for 2 min followed by 30 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec, and 72°C for 5 min. The specific primers were: PinX1 forward, 5′-CCTCAGAACA CTGCCTGGAG-3′ and reverse, 5′-GTTCCACCTGCGTCT CAGAA-3′; GAPDH forward, 5′-CCTGAGGGTTCTTTGT GCTGA-3′ and reverse, 5′-AAAGGCTCAACCTTCCCCAT-3′. The expected PCR products were 577 bp and 122 bp for PinX1 and GAPDH, respectively. The amplicons were analyzed by electrophoresis, imaged using UVI gel imaging system and quantified using Gel-proAnalyzer3.1 software. Expression levels of PinX1 were normalized to internal reference GAPDH.

Stable transfections of Eca109 cells with pCDNA3.1 vector and pCDNA3.1/PinX1 were performed with Lipofectamine^® 2000, according to the manufacturer’s instructions. Subsequently, cells were cultured in selection medium containing 0.5 mg/ml Geneticin^® (G418) for 4 weeks before single clones were isolated. The clones were further expanded in selection medium containing Geneticin (0.5 mg/ml). Untransfected cells and cells transfected with pCDNA3.1 were used as controls. Cells were observed 24–48 h after transfection under a fluorescence microscope to examine transfection efficiency.

Total protein extraction from cultured Eca109 cells was performed as previously described (25). Protein was quantified with a Bio-Rad Protein Colorimetric Assay (Bio-Rad, Hercules, CA, USA). Equal amounts of protein extracts were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore Corp., Billerica, MA, USA) as previously described (26). The concentration of anti-PinX1 antibody and anti-β-actin antibody was 1:100 (41 kDa) and 1:1,000(42 kDa), respectively. PinX1 protein expression was quantified by comparison with β-actin.

Eca109 cells at the logarithmic phase were inoculated into 96-well plates with 1×10⁵ cells/well. Cell viability at 24 h was examined using the MTT method. OD_{490 nm} values were detected in 6 duplicate wells and their averages were used to plot growth curve and calculate the growth inhibition rate of each treatment using the following formula: Growth inhibition rate Ir = OD_{490 nm} of the control group - OD_{490 nm} of the treatment group/OD_{490 nm} of the control group × 100%.

Forty-eight hours after transfection, Eca109 cells were collected, washed with PBS, resuspended in PBS at 1×10⁶/ml, and stained with Annexin V and PI for 15 min in the dark. Apoptotic cells were then analyzed by flow cytometry and apoptotic index (AI) was calculated using AI = apoptotic cells/total cells × 100%. Cell cycle was determined after fixing with precooled 75% ethanol at 4°C and washing with PBS.

Data are expressed as mean ± standard variation and analyzed using SPSS 13.0 statistical software package. Differences between samples were tested using single factor analysis of variance and LSD method for multiple comparisons. A P-value <0.05 was considered to indicate a statistically significant difference. Prior to the comparison, data homogeneity of variance was first examined using F-test. In the case of heterogeneity of variance, the approximate variance F-test/Welch method was used.

Value of Q-FISH was analyzed by FCM assay in the normal esophageal epithelium group, the mild dysplasia group, the severe dysplasia group and the carcinoma group. Q-FISH in the carcinoma group was lower than in the severe dysplasia group (P<0.01), but it was lower in the severe dysplasia group than in the mild dysplasia group (P<0.01, Table I). There was a negative correlation between telomere length and cytologic grade in esophageal cancer (r=−0.79, P<0.01). As shown in Table I, the shorten rate of telomere length in the carcinoma group was higher compared with that in the mild dysplasia and severe dysplasia group.

The positive expression of hTERT protein was located in the nucleus of esophageal epithelial cells and cancerous cells by immunohistochemical staining (Fig. 1A). The positive rate of hTERT in incisal margin normal tissue was 11.1% (4/36) and those in para-tumorous dysplasia tissues and carcinoma were 44.0% (22/50) and 86.37% (38/44), the positive expression rate increased gradually with carcinogenesis of esophageal epithelial cells (P<0.01).

FI value of hTERT in the normal esophageal epithelium group, mild dysplasia group, severe dysplasia group and carcinoma group was 0.87±0.18, 1.13±0.19, 1.39±0.24 and 1.84±0.21 (Table II) (Fig. 1B). FI value of hTERT in the carcinoma group was higher than in the severe dysplasia group (P<0.01), and it was higher in the severe dysplasia group than in the mild dysplasia group (P<0.01). In addition, FI value of hTERT was positively related with cytologic grade (r=0.84, P<0.01).

There was a negative correlation between Q-FISH value of telomere and FI value of hTERT (r=−7.49, P<0.01).

The telomerase activity in different histological groups is summarized in Table III. The levels of telomerase activity were 0.073±0.039 in the normal group, 0.429±0.346 in the dysplasia group, 1.457±0.838 in the carcinoma group. Telomerase activity was higher in the carcinoma group compared to the control normal or dysplasia group (P=0.00). Moreover, the A-value of telomerase activity was related to the tumor tissue grade and lymph node metastasis (P<0.05), but not to other clinicopathological features in ESCCs (P>0.05, Table IV).

The results of RT-PCR showed that the relative expression of PinX1 mRNA in esophageal cancer tissues was significantly lower than that in normal tissues and dysplasia esophageal tissues (P<0.01) (Fig. 2A). As shown in Fig. 2B, the PinX1 protein level in esophageal cancer tissues was significantly lower than in the dysplasia and normal group, and there was a negative correlation with differentiation, invasive depth and lymph node metastasis (P<0.05, Table V) and no correlation with age and gender in ESCCs (P>0.05, Table V).

There was a significant negative correlation between telomerase activity expression and PinX1 protein expression (r_s=−0.883, P=0.000).

To investigate the effect of PinX1 on telomerase activity and cell growth, Eca109 cells were transfected with expression vector for PinX1 (pCR3.1/PinX1) or control vector (pCR3.1). The expression of PinX1 protein was 4.5-fold greater than that of the control group (Fig. 3A). We further examined the effect of PinX1 on the telomerase activity by TRAP-PCR, cell proliferation and apoptosis by FCM. In Eca109 cells, PinX1 overexpression decreased Eca109 cell growth (Fig. 3B) and blocked cells into the G0/G1 stage (Fig. 3C) (Table VI). The apoptosis rate was higher in PinX1-transfected Eca109 than in PinX1-untransfected cells or cells transfected with void vectors only by FCM (Fig. 3D) (Table VI).

The expression of telomerase activity was lower in PinX1-transfected Eca109 than in PinX1-untransfected cells or cells transfected with vectors only (P<0.05, Table VII).

There was a significant positive correlation between telomerase activity expression and PI expression (r_s=0.451, P=0.000).