Jia-Hua Zhou, Quan Chen, Dong-Dong Han, De-Tong Yang, Department of Biliary-pancreatic Surgery, Zhongda Hospital of Southeast University, Nanjing, Jiangsu Province, 210009, China
Hong-Mei Zhang, Li-Shan Zhang, Genetic Center, Southeast University, Nanjing, Jiangsu Province, 210009, China
Fei Pei, Department of General Surgery of Huangshi Central Hospital, Huangshi, Hubei Province, 435000, China
Supported by the Applied Basic Research Programs of Science and Technology Commission Foundation of Jiangsu Province, No BJ98080 (1998-2001)
Correspondence to: Jia-Hua Zhou, Department of Biliary-pancreatic Surgery, Zhongda Hospital Affiliated to Southeast University, 87 Dingjiaqiao, Nanjing 210009, Jiangsu Province, China.  zhoujiahua@hotmail.com
Telephone: +86-25-3249268    Fax: +86-25-3272356
Received: 2002-10-08    Accepted: 2003-02-09

Abstract
AIM: To directly investigate the relationship between telomerase activity and its subunit expression and the inhibitory effect of antisense hTR on pancreatic carcinogenesis.

METHODS: We examined the telomerase activity and its subunit expression by cell culture, polymerase chain reaction (PCR), PCR-silver staining, PCR-ELISA, DNA sequencing, MTT and flow cytometry methods.

RESULTS: PCR-silver staining and PCR-ELISA methods had the same specificity and sensitivity as the TRAP method. Telomerase activity was detected in the extract of the 10^th,20^thand 30^th passages of P3 cells,while it was absent in fibroblasts. Furthermore, after the 30th generation, the proliferation period of fibroblast cells was significantly prolonged. Telomerase activity and hTERTmRNA were detected in two pancreatic carcinoma cell lines, but were found to be negative in human fibroblast cells. Telomerase activity and hTERTmRNA were tested in pancreatic carcinoma specimens of 24 cases. The  telomerase activity was positive in 21 of the 24 cases (87.5 %), and the hTERTmRNA in 20 cases (83.3 %). In adjacent normal tissues positive rates were both 12.5 %. There was a significant difference between the two groups. This indicated a significant correlation between the expression level of telomerase activity and histologic differentiation, metastasis and advanced clinical stage of pancreatic carcinoma. Our findings showed that the expressions of hTR and TP1mRNA were not correlated with the activity of telomerase but the expression of hTERTmRNA was. After treatment with PS-ODNs, telomerase activity in P₃ cells weakened and the inhibiting effect became stronger with an increase in PS-ODNs concentration. There was a significant difference between different PS-ODN groups (P<0.05). Inhibition of telomerase activity occurred most significant with PS-ODN1.The results of the FCM test of pancreatic cancer P3 cells showed an increase in the apoptotic rate with increasing PS-ODN1 and PS-ODN2 concentrations.

CONCLUSION: The expression of telomerase activity has a significant relationship to carcinogenesis. A strong correlation exists between telomerase activity and hTERTmRNA expression. The up-regulation of hTERTmRNA expression may play a critical role in human carcinogenesis. The expression of telomerase activity and its subunit level in pancreatic carcinoma significantly correlate with the clinical stage of pancreatic carcinoma and hence, may be helpful in  its diagnosis and prognosis. The anti-hTR complementary to the template region of hTR is sufficient to inhibit P3 cell telomerase activity and cell proliferation in vitro, and can lead to a profound induction of programmed cell death.

Zhou JH, Zhang HM, Chen Q, Han DD, Pei F, Zhang LS, Yang DT. Relationship between telomerase activity and its subunit expression and inhibitory effect of antisense hTR on pancreatic carcinoma. World J Gastroenterol  2003; 9(8): 1808-1814
http://www.wjgnet.com/1007-9327/9/1808.asp

INTRODUCTION
Telomerase is a DNA-dependent RNA polymerase^[1] carrying template features. It is different from reverse transcriptase, DNA polymerase of commonly pure proteins. The activated telomerase takes the 3 distal end of telomeres as the primer and its RNA component acts as the template. The protein component of telomerase regulates the catalytic activity in the synthesis of telomere repetitive sequence to maintain the telomere length. Human telomerase mainly consists of three subunits-human telomerase RNA (hTR), human telomerase-associated protein 1(TPl) and human telomerase reverse transcriptase (hTERT).Telomerase activity has been found in most human tumor cells^[2], while there is no evident expression in normal human tissues other than in germ cells, hemopoietic stem cells and cuticle basal cells^[3,4]. This suggests that telomerase is a broad spectrum tumor marker^[5-9]. It is recognized that activation of telomerase and stability of telomere length are necessary for tumor immortalization. However, it has been found that expression of hTERTmRNA is obviously related to telomerase activity. It is suggested that up-regulation of the expression level of hTERTmRNA is a key factor in the formation of tumor cells.
      Human pancreatic cancer is one of the most frequent tumors. The present clinical treatment however has a low curative effect. The mortality rate of human pancreatic cancer is very high. Post-operative metastases are common and less than 3 % of patients have a survival rate of 5-years. In recent years, a close relationship has been found between telomerase and pancreatic cancer. By cell culture, PCR-silver staining, PCR-ELISA, RT-PCR, DNA sequencing technology, MTT and Flow cytometry methods, we have investigated the expression of telomerase activity and its subunits in pancreatic cancer cell line, fibroblasts, and in pancreatic cancer samples of 24 cases and their adjacent normal tissues. The purpose of this research was to explore the relationship between the biological behavior and clinico-pathological characteristics of human pancreatic cancer, as well as the inhibitory effects of hTR antisense oligonucleotide on pancreatic cancer cells.

MATERIALS AND METHODS
Cell lines and tissuse samples
In this study, the pancreatic cancer P3 cells were provided by Peking Union Medical College Hospital, PaTu-8801 cells were provided by Shanghai Changhai Hospital, and the human skin fibroblasts were developed in the Zhongda Hospital. Cancer tissues and adjacent normal tissues from 24 patients with pancreatic cancer were provided and assayed by the Surgical Department of Zhongda Hospital Affiliated to Southeast University and Jiangsu Provincial People,s Hospital. These samples were frozen at -80 °C within 15 min after surgical removal and stored until use. Of the 24 patients, 13 were male and 12 female.Their age ranged between 55-74 years with an average of 63 years.

Cell culture
Culture of the pancreatic cancer P₃ cells and PaTu-8801 cells: P3 cells and human skin fibroblasts were cultured in 1640 culture medium and PaTu-8801 cells in DMEM culture medium (high glucose content) with 10 % inactivated calf serum and  all were put in 5 % CO₂ at 37 °C for generational culture at 100 % RH.

Telomerase assay
Telomerase extracts and assays of activity were done as TRAP method. Briefly frozen pancreatic tissue samples of approximately 200 mg were homogenized in 200 ml of lysis reagent containing 5 % CHPAS (3-(3-cholamidopropyl dimethyammonio)-1-propanesulfonate lysis buffer, Roche Co.). The cells obtained by cell culture were also treated with lysis reagent containing 5 % CHPAS (Roche Co.). After 30 min of incubation on ice,the lysates were centrifuged at 16 000×g (tissue) or 34 000×g (cell) for 20 min at 4 °C, and the supernatants were rapidly frozen in liquid nitrogen and stored at -80 °C until use. In the extracts from frozen tissues,the concentration of protein was measured using the Coomassie brilliant blue method. BCA. Extracts of 293 cell line with telomerase activity were used as the standard, while extracts obtained by inactivation for 10 min at 94 °C or treated with RNase for 10 min at 37 °C were used as the negative control.For cell samples, aliquots corresponding to extracts derived from approximately 10², 10³, 10⁴, 10⁵ cells were used for TRAP assay. Extracts of tissues containing 0.06, 0.6 and 6 mg of protein were used for TRAP assay. Each extract specimen was assayed in 25 ml reaction mixture (Roche Co assay kit containing dNTP, Taq enzyme, biotin tagged TS primer and CX primer) which was diluted to 50 ml with DEPC water solution. After 30 min incubation at 25 °C for telomerase-mediated extension of the TS primer, the reaction mixture was heated at 94 °C for 5 min and then subjected to 30 PCR cycles  at 94 °C for 30 s, at 50 °C for 30 s and at 72 °C for 50 s and then extended for 10 min at 72 °C. Firstly, the PCR product went through enzyme-linked immunosorbent assay (PCR-ELISA) containing DIG-labeled probes (Roche Co assay kit). After spectrophotometric determination, the absorbance value A(=A₄₅₀-A₆₉₀) was calculated. The result was positive if A >0.2. Secondly, the PCR product was electrophoresed on a 10 % polyacrylamide gel. The telomerase activity was positive if the specific band appeared. For each sample of pancreatic carcinoma tissue, the intensity of telomerase was graded according to the different contents of protein in the extract specimen. A sample intensity of telomerase was represented as (-) if the specimen containing 6 mg extracted protein was tested to be negative, or (+) if the specimen containing 6 mg extracted protein was tested positive. If both sample specimens containing 6 mg and 0.6 mg extracted protein were tested positive, while the specimen containing 0.06 mg extracted protein was tested to be negative, it was represented by (++),and (+++) if all three specimens containing 6 mg, 0.6 mg, and 0.06 mg extracted protein were tested positive^[10].

Expression of the subunit of telomerase
Analysis of the expression of each telomerase subunit was performed by RT-PCR amplification^[11-13]. hTERT mRAN was amplified by using the primer pair (145 bp): LT5 5'-CGGA AGAGTATCTGGAGCAA-3', LT6 5'-GGATGAAGCGGA GTCTGGA-3'. TP1 mRAN was amplified by using the primer pair (340 bp), TP1.1 5'-TCAAGCCAAACCTGAATCTGAG-3', TP1.2 5'-CCCCGAGTGAATCTTTCTACGC-3'. hTR was amplified by using the primer pair (134 bp): F3b 5'-TCTAACCCTAACTGAGAAGGGCGTAG-3', R3c 5'-GTTTGCTCTAGAATGAACGGTGGAAG-3'. The efficiency of cDNA synthesis from each sample was estimated by PCR with glyceraldehyde-3-phosphate-dehydrogenase (GAPDH)-specific primers of (450 bp): K136 5'-CTCAGACACCATG GGGAAGGTGA-3',K137 5'-ATGATCTTGAGGCTG TTGTCATA-3'. cDNA was synthesized in 20 ml of reaction mixture containing 5×RT Buffer 4 ml, RNasin 0.5 ml, total RNA 1 ml, and MLV 0.8 ml with 1 ml random primers. The reaction mixture was incubated at 94 °C for 5 min before it was heated at 95 °C for 5 min to inactivate MLV. To amplify the cDNA 2 ml aliquots of the reversely-transcribed cDNA was subjected to 35 cycles of PCR in 50 ml containing 10×buffer (10 mM Tris-HCl (pH8.3), 25 mM MgCl₂, 500 mM KCl) 10 mM dNTP 1 ml, 25 mM MgCl₂ 2-3 ml, Taq enzyme 4 unit and 10 pM of specific primers 22 m1. After heated at 94 °C for 5 min, each cycle consisted of de-naturation at 94 °C for 45 s, annealing at 56 °C for 45 s (hTERT) or at 61 °C for 45 s (hTR) or at 60 °C for 45 s (TP1) and extension at 72 °C for 90 s and then extended at 72 °C for 10 min. PCR products were electrophoresed on 3 % agarose gel with ladder marker to determine the concentrations and purity of PCR amplified products. hTERTmRNA PCR products were sequenced (sent to Shanghai Biolottering Co, Ltd.).

Inhibitory effects of antisense hTR
Four nucleotides were synthesized as hTR template sequence (5'-CUAACCCUAAC-3') and modified by thiophosphoric acid. They were PS-ODN1: 5'-GTTAGG-3' (antisense), PS-ODN2: 5'-GTTAGGGTTAG-3' (antisense), PS-ODN3: 5'-CCTAAC-3' (pro-sense), and a PS-ODN4 with random sequence: 5'-AACTCGTAGTC-3'. After 5×10 P₃ cells and human fibroblasts were inoculated into culture bottles and replaced the old culture medium with a fresh one after 24-h-lasting cultivation, the cells stuck to the wall of bottle. The test group was arranged PS-ODN1, PS-ODN2, PS-ODN3, PS-ODN4 so that each had four concentrations, 3.16 mmol/L, 10 mmol/L, 31.6 mmol/L, and 100 mmo1/L. They were added into the culture bottles, respectively. Additional sets were the control groups which had no PS-ODN. The following tests were conducted when obvious change in cytomorphology was observed under the optical microscope. Firstly, P₃ and human fiber forming monolayer anchorage-dependent cells were digested. The suspended cells were cultured in RPMI 1640.After the suspended cell liquor was inoculated into a 96-cave culture board (100 m1 per cave), 20 m1 of freshly prepared 5 mg/ml MTT solution was added into each cave. This was incubated for 4 h at 37 °C, the culture medium in the caves was discarded, and then dimethyl sulfoxide (DMSO) 150 m1 was added into each cave to be shaken for 10 min to solve the crystal. The activity of succinic dehydrogenase (SDH) was determined according to the optical absorbance of the content in each cave at 540 mm in an enzyme-linked immunoassay analyzer. Secondly, after 2×10⁶ P₃ cells were treated with each of the different PS-ODNs, the activity of telomerase extracts was assayed according to the TRAP, PCR-ELISA and  polyacrylamidedel gel (10 %) electrophoresis (PAGE). The telomerase extract of 293 cell line was taken as the positive control. The extract that was subjected to inactivation for 10 min at 94 °C or RNase treatment for 10 min at 37 °C was taken as the negative control. Finally, the single cell suspension was prepared by digesting anchorage-dependent cells after treatment with PS-ODN1 and PS-ODN2, respectively. It was cool-washed three times, and then centrifuged for 10 min at 2 000 rpm. After the supernatant was discarded and dried, precooled 95 % alcohol was added and the sample was placed into a refrigerator at 4 °C for 1 h, and then incubated with 10 mg/ml RNase at 4 °C for 3 h. The apoptotic rate and the cell cycle distribution of pancreatic cancer cells were analyzed by a flow cytometer (DB Corp. United States) after 1 ml propidium iodide comprehensive staining solution was added in an ice-bath for 15 min. Data were collected by Cellquest software and analyzed by Mmodift Lt software.

Statistic analysis
The data were processed by t-test and variance analysis. The P value was determined according to the t value, F value and FI value.

RESULTS
Cell culture
There were morphological changes in the fibroblasts (HE stain) of the 32nd generation, such as karyopyknosis, cytoplasmic concentration, and cell body shrinkage. No changes were found in P₃ cells. P₃ cells, and fibroblasts of the 10^th, 20^th, and 30^th generations were used to detect the activity of telomerase in this test. All results were positive for P₃ cells, and negative for fibroblasts (Figure 1).

Figure 1(PDF) Telomerase activity of fibroblasts and P₃ cells of different generations (1, 3 and 5 were P₃ cells of the 10^th, 20^th, and 30^th generations; 2, 4, and 6 were fibroblasts of the 10^th, 20^th, and 30^th generations).
Figure 2(PDF) Specificity and sensitivity of telomerase activities in P₃ cells (1, 293 cells as the positive control; 2,104 P3 cells; 3,treated with RNase; 4, heat treated; 5, 6, 7, and 8 were 103, 102, 101, and 100 P₃ cells, respectively; 9, the lytic liquid).

Telomerase assay
The protein content was calculated from the sample extract, and standard curves were prepared by using the data. The protein contents in the sample extracts were determined by Coomassie brilliant blue method. PCR-silver staining method was used to detect the activities of telomerase in 10 cells or 1 mg protein extracted from a sample. PCR-ELISA method had a same sensitivity as PCR-silver staining (Figure 2).
     The results were positive for the activity of telomerase in pancreatic cancer cells, and negative for fibroblasts. There were 21 cases whose telomerase activity was detected in the 24 pancreatic cancer tissues by PCR-ELISA or PCR silver staining. Four cases of them were (+++), 11 cases (++), and 6 cases (+). The rate of positive findings was 87.5 %. However, there were only 3 cases with telomerase activity (+) was found in adjacent normal tissues, with a positive rate of 12.5 % (Figure 3).

Figure 3a(PDF) Telomerase activity of tumor and normal pancreas (T: tumor; N: normal).
Figure 3b(PDF) Semiquantitative telomerase activity of tumor and normal pancreas (1 2 3; 5 4 6; 9 10 11: T10, T15 T18 6 mg 0.6 mg 0.06 mg; 7: negative control, 8: positive control; T: tumor).

Detection of telomerase subunit
Pancreatic cancer cell P₃, and PaTu-8801 were taken as the positive control, while the fibroblast was taken as the negative control. There were 20 cases which expressed hTERTmRNA in cancer tissues of 24 cases, and 3 cases expressed hTERTmRNA in the adjacent normal tissues. 4 of 24 cases of cancer tissue did not express hTR and 3 cases of the adjacent normal tissues did not express hTR. Two cases tested were negative for TP1 (Figure 4).
    The cDNA sequencing of hTERTmRNA PCR products showed that there were 143 base groups that were significant sequences between the 51^st and the 193^rd base groups.The homology with hTERTcDNA reached 98.6 %, proving that the amplification products were the hTERTcDNA sequence amplified from hTERT.

Relationship between the expression of telomerase activities and its subunit with human pancreatic cancer, its biological behavior and clinico-pathological characteristics
Table 1 shows the relationship between telomerase activity and tumor and adjacent normal tissue of pancreas.

Table 1  Telomerase activity of tumor and normal pancreas

^aP<0.01 (FI=28.82); ^bP<0.01 (FI=25.42).

     There were significant differences in the expressions of telomerase activity and hTERTmRNA between pancreatic cancer tissues and adjacent normal tissues
      Table 2 shows the relationship between telomerase activity and biological behavior of human pancreatic carcinoma.

Table 2  The relationship between telomerase activity and biological behavior of human pancreatic carcinoma

^aP<0.01 (F=26.06); ^bP<0.01 (t=3.22); ^cP<0.01 (F=9.31).

    A correlation existed between the expression level of telomerase activity in pancreatic cancer tissues with histologic differentiation, presence of lymph node metastasis, and clinical TNM stage of the tumor (no significant difference between stages III and IV). However, in pancreatic cancer patients no correlation was found between the expression level of telomerase activities and age or sex of patients, pathologic category or tumor infiltration.
      Table 3 shows the relationship between telomerase activity and telomerase subunit.
      There was also an obvious correlation between expression of hTERT and telomerase activities in pancreatic cancer tissues, but no correlation was found between expressions of hTR and TPlmRNA and telomerase activities.
      In 24 pancreatic cancer tissues and adjacent normal tissues, their hTR/GAPDH ratios were 0.592±0.056 and 0.510±0.059, respectively. No difference was found in the expression level of hTR.

Table 3  The relationship between telomerase activity and telomerase subunit

^aP>0.05 (c²=0.5); ^bP<0.01 (c²=13.76); ^cP<0.01(c²=15.70).

Inhibitory effects of antisense hTR
In the control groups, pancreatic cancer cells extended in polygon, showed large differences in their size and shape. The cells were more transparent, strong refractive, overlapped to grow after fully covering the bottom of the bottle and mitotic figures increased. After treatment with PS-ODN1 and PS-ODN2, morphologic changes of cells were obvious-refractiv, intercellular space became larger and the cells gradually became round, crenated, and fell off^[14]. Occasionally, ballooning could be seen. When P3 cells and human fibroblasts were treated with four PS-ODNs for reflecting the survival rate of cells, the results of SDH activity are shown in Table 4 and Table 5. There was a significant difference in the effects between the different PS-ODN groups (P<0.05). Along with an increase in concentrations of PS-ODN1 and PS-ODN2, the survival rate of cells significantly decreased. There was a significant difference between different concentrations in the same groups (P<0.05). No significant difference was found between PS-ODN1 and PS-ODN2. It was found that PS-ODN3 also might cause a decrease in SDH activity,but the decrease was less obvious than that of the former two. Inhibition of SDH activity occurred earliest with PS-ODN1. Comparison between different PS-ODNs and different concentrations of any PS-ODN (P<0.05) showed that the four PS-ODNs of any concentration had no significant effect on the survival rate of normal human fibroblasts.

Table 4  The viability of PS-ODNs treated P3 cells (%, )

PS-ODN	C1(3.16)V(%, )	C2(10)V(%, )	C3(31.6)V(%, )	C4(100)V(%, )
PS-ODN1	92.16±4.2	80.39±5.9	58.11±3.4	33.34±4.6
PS-ODN2	93.40±2.6	84.22±3.6	74.10±3.8	27.35±2.4
PS-ODN3	91.74±4.1	88.75±7.1	86.38±6.7	81.37±5.3
PS-ODN4	94.23±3.3	92.36±5.2	93.72±4.9	91.54±4.4

a.C is the concentration (unit). A is absorbance at 540 mm. V(%) is survival rate. V=_{ACONTRAL GROUP}*100 %.

Table 5  The viability of normal fibroblasts treated with PS-ODNs (%, )

PS-ODN	C1(3.16)V(%, )	C2(10)V(%, )	C3(31.6)V(%, )	C4(100)V(%, )
PS-ODN1	104.16±6.4	100.48±3.9	107.20±8.4