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Esophageal tissues were collected from an esophageal carcinoma high-risk area of China and were used to detect the telomere length and the expression of human telomerase reverse transcriptase (hTERT) by immuhistochemistry and fluorescence
Esophageal carcinoma is one of the most common malignant gastrointestinal tumors with a high incidence in China and is characterized by a unique geographical distribution. Therefore, it is necessary to elucidate the underlying mechanisms and to search for chemopreventive tools or drugs to decrease the risk of carcinogenesis of esophageal epithelial cells.
Studies have indicated that maintenance of telomere length is important in preventing the consumption of telomere DNA during cell division. Studies found significant associations between short telomere length and increased esophageal cancer risk (
Telomerase is a ribonucleoprotein enzyme complex that adds 5′-TTAGGG-3′ repeats onto the ends of human chromosomes, providing a telomere maintenance mechanism for ~90% of cancers (
Studies have shown that inhibition of telomerase results in gradual erosion of telomeres followed by cessation of proliferation or apoptosis, and may thus be a promising target for cancer therapy. Pin2/TRF1 interacting protein X1 (PinX1) was previously found to be a tumor suppressor and telomerase inhibitor
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 (
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 (
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. OD490 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 = OD490 nm of the control group - OD490 nm of the treatment group/OD490 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,
The positive expression of hTERT protein was located in the nucleus of esophageal epithelial cells and cancerous cells by immunohistochemical staining (
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 (
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
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) (
There was a significant negative correlation between telomerase activity expression and PinX1 protein expression (rs=−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 (
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,
There was a significant positive correlation between telomerase activity expression and PI expression (rs=0.451, P=0.000).
Telomere shortening results in chromosomal instability which, in the absence of normal cellular senescence processes, can lead to cancer development (
Telomerase is a special reverse transcriptase that is composed of RNA and protein and regulates the length of telomere. hTERT is the key component in telomerase and plays an important role in genetic stability and maintenance of chromosomes. Several studies have indicated that telomerase is only slightly expressed in normal cells, but its expression and activity are enhanced in most immortalized tumor cells (
However, the mechanism of PinX1 functioning in tumor cells has yet to be fully elucidated. Some studies indicate that the PinX1 gene can inhibit telomerase activity and induce cell apoptosis and expression of PinX1 is negatively correlated with hTERT expression and telomerase activity in tumor cells. For example, Lai
Zhou and Lu (
In summary, our data demonstrate that the length shortening of telomere is an important characteristic in the carcinogenesis of esophageal epithelial cells, followed by increase of telomerase activity and downregulation of PinX1. Overexpression of PinX1 blocked Eca109 cell proliferation and induced cell apoptosis by downregulating the telomerase activity. However, further studies are required to examine the precise role of PinX1 in telomerase activity in the pathogenesis of esophageal cancer.
This study was supported by the Hebei Natural Science Foundation (H2012206107, 062611136D-5).
Upregulation of human telomerase reverse transcriptase (hTERT) protein in the carcinogenesis of esophageal epithelial cells. (A) Immunohistochemical staining for hTERT protein, positive expression located in the nuclei of epithelial cells. (B) Using FCM, the hTERT protein increased in the carcinoma group compared with the severe dysplasia and normal group.
Decrease of Pin2/TRF1 interacting protein X1 (PinX1) mRNA and protein in esophageal squamous cell carcinoma. (A) RT-PCR of PinX1 mRNA in esophageal tissue. *P<0.01 vs. normal group. (B) FCM of PinX1 protein, *P<0.05 vs. normal group; #P<0.05 vs. dysplasia group.
Overexpression of Pin2/TRF1 interacting protein X1 (PinX1) inhibits cell proliferation and induces cell apoptosis in Eca109 cells. (A) Detection of PinX1 protein by western blot analysis. (B) Cell growth by MTT. (C) Cell cycle distribution by FCM. (D) Cell apoptosis by FCM.
Length of telomere DNA in various lesions of esophageal epithelial cells by FCM (mean ± standard variation).
Telomere length, n (%) | |||||
---|---|---|---|---|---|
| |||||
Group | n | Q-FISH | Extend |
Shorten |
Normal |
Normal | 36 | 50.83±8.86 | |||
Mild dysplasia | 22 | 49.51±3.16 | 2 (9.09) | 13 (59.09) | 7 (31.82) |
Severe dysplasia | 28 | 36.96±8.02 |
2 (7.14) | 20 (71.43) |
6 (21.43) |
Carcinoma | 44 | 27.80±6.59 |
3 (6.82) | 35 (88.64) |
6 (13.64) |
Severe dysplasia group vs. mild dysplasia group, P<0.01
Carcinoma group vs. severe dysplasia group, P<0.01.
Content of hTERT protein in various lesions of esophageal epithelial cells by FCM (mean ± standard variation).
Group | n | hTERT FI | F | P-value |
---|---|---|---|---|
Normal | 36 | 0.87±0.18 | 73.52 | <0.01 |
Mild dysplasia | 22 | 1.13±0.19 | ||
Severe dysplasia | 28 | 1.39±0.24 |
||
Carcinoma | 44 | 1.84±0.21 |
Severe dysplasia group vs. mild dysplasia group, P<0.01.
Carcinoma group vs. severe dysplasia group, P<0.01. hTERT, human telomerase reverse transcriptase; FI, fluorescence index; F, F-value.
Detection of telomerase activity in various esophageal tissues.
Telomerase activity | ||||
---|---|---|---|---|
| ||||
Group | n | Positive no. | Rate | A-value |
Carcinoma group | 50 | 42 | 84 |
1.457±0.838 |
Dysplasia group | 50 | 31 | 62 |
0.429±0.346 |
Normal group | 50 | 3 | 6 | 0.073±0.039 |
P<0.05, compared with the normal group;
P<0.05, compared with the dysplasia group.
Relationship between telomerase activity and clinicopathological features in human esophageal squamous cell carcinomas.
Telomerase activity | |||||
---|---|---|---|---|---|
|
|||||
Clinicopathological feature | n | Positive no. | Rate (%) | A-value | P-value |
Gender | |||||
Male | 35 | 30 | 85.71 | 1.253±0.672 | >0.05 |
Female | 15 | 12 | 80 | 1.702±0.823 | |
Age (years) | |||||
≤60 | 36 | 30 | 83.33 | 1.465±0.672 | >0.05 |
>60 | 14 | 12 | 85.71 | 1.797±0.952 | |
Depth of invasion | |||||
Fibrous membrane | 16 | 13 | 81.25 | 1.467±0.923 | >0.05 |
Fibrous membrane | 34 | 29 | 85.29 | 1.534±0.782 | |
Untouched | |||||
Grade | |||||
Well/moderately differentiated | 39 | 32 | 82.05 | 1.163±0.438 | <0.05 |
Poorly differentiated | 11 | 10 | 90.91 | 2.235±0.814a | |
Lymph node metastasis | |||||
Positive | 17 | 16 | 94.12 | 1.917±0.814a | <0.05 |
Negative | 33 | 26 | 78.79 | 1.097±0.865 |
The relationship between PinX1 protein expression and clinicopathological features in ESCC.
Clinicopathological feature | n | PinX1 protein | P-value |
---|---|---|---|
Age (years) | |||
≤60 | 30 | 445.88±22.13 | |
>60 | 20 | 443.38±23.95 | 0.63 |
Gender | |||
Male | 35 | 442.70±22.57 | |
Female | 15 | 449.39±23.18 | 0.23 |
Differentiation grade | |||
Well/moderately | 39 | 441.04±22.02 | |
Poorly | 11 | 455.33±22.22 | 0.01 |
Depth of invasion | |||
Fibrous membrane | 34 | 448.80±22.76 | |
Fibrous membrane | 16 | 436.45±21.02 | 0.02a |
Untouched | |||
Lymph node metastasis | |||
Positive | 17 | 453.91±19.78 | |
Negative | 33 | 440.14±23.04 | 0.01 |
PinX1, Pin2/TRF1 interacting protein X1; ESCC, esophageal squamous cell carcinoma.
Changes of cell cycle distribution, PI and apoptotic rate in different groups.
Cell | G0/G1 | PI | Apoptotic rate |
---|---|---|---|
Eca109 | 53.14±4.83 | 33.17±0.47 | 0.27±0.18 |
Eca109/PCDNA3.1 | 47.27±3.73 | 35.64±0.65 | 3.40±1.09 |
Eca109/PinX1 | 70.58±5.26 |
13.58±0.59 |
23.28±5.73 |
P<0.05 vs. Eca109 cells and Eca109/PCDNA3.1 cells.
PI, proliferation index; PinX1, Pin2/TRF1 interacting protein X1.
Detection of telomerase activity in various cells.
Cell | A-value | F | P-value |
---|---|---|---|
Eca109 | 2.446±0.652 | ||
Eca109/PCDNA3.1 | 2.137±0.475 | ||
Eca109/PinX1 | 0.874±0.439 |
163.25 | 0.000 |
P<0.05 vs. Eca109 and Eca109/PCDNA3.1 cells.
PinX1, Pin2/TRF1 interacting protein X1; F, F-value.