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The Significance of Quantitative Evaluation of Telomerase Activity and hTERT mRNA Expression in Colorectal Cancers

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Introduction

Colorectal cancer is a common tumor in western countries. In the United States, during 2000, more than 130,000 new cases of colon cancer and rectal cancer were reported1 affecting about one person in 20 and representing 15% of cancers.2 Greater public awareness and acceptance of screening programs have contributed significantly to increasingly earlier detection of cancer and decreased mortality.3 Risk factors include familial polyposis syndromes, inflammatory bowel disease, previous malignant disease, and polyps, and it is now clearly demonstrated that colon cancer develops through an adenoma–carcinoma sequence.4 An estimated 90% of patients with colon cancer and 84% of patients with rectal cancer are treated surgically.5,6 Considerable advances in the molecular understanding of colorectal cancers have been made with the identification of mutations of adenomatous polyposis coli gene, 5q chromosomal mutations, ras oncogene expression, deletion of chromosome 18, and allelic loss of the DCC and p53 gene.7 In addition to these mutations, malignant cells must escape the control of cell senescence to reach immortality. Telomerase and telomere length have recently been shown to be involved in the control of cell proliferation, the regulation of cell senescence in most somatic cells, and the unlimited proliferation capacity of malignant cells.8

Telomerase and Colorectal Cancer

The presence of telomerase in colorectal cancer was initially reported by Chadeneau et al.,9 who detected telomerase activity in 14/15 (93%) adenocarcinomas but not in histologically normal colon mucosa and polyps. According to another report,10 telomerase was present in 81% of sporadic colon cancers and in 77% of hereditary nonpolyposis colorectal cancer (HNPCC). These findings were confirmed by Cheng et al.,11 who detected telomerase activity in 84% of non-HNPCC and in 96% of HNPCC samples. In this last study, the presence of telomerase in apparently normal mucosa was firstly reported in a consistent percentage of HNPCC patients (33%). Functionally, telomerase was found to correlate with the progression of adenomatous polyps in multistep colorectal carcinogenesis.12 In addition, a link between telomerase activity and tumor stage was found in colorectal cancer,13 suggesting that telomerase is upregulated as a function of tumor cell invasion, progression, and metastatic potential. Yoshida et al.,14 found that the weak telomerase activity in normal colonic mucosa is gradually increased during the course of colorectal carcinogenesis and that telomerase reactivation seems to occur later than k-ras mutation but earlier than p53 mutations.15 Telomerase was found to be significantly correlated with cell differentiation, proliferation, and lymph node metastasis,16 although other authors did not find any relationship with clinicopathological features.17,18 In the study from Abe et al.,18 telomerase was also detected in normal colorectal tissues as well as in adenoma, even if the enzyme activity was significantly lower than that of colon cancers. Finally, telomerase activity in colon cancers was not correlated to p53 mutations19,20 and microsatellite instability.21

The expression of the catalytic subunit of telomerase hTERT (telomerase reverse transcriptase) was demonstrated with immunohistochemical techniques both in human colorectal tumors and normal tissues.22 The same authors failed to demonstrate any relationship between hTERT expression and telomerase activity, as confirmed by Nakamura et al.,23 who found that telomerase activity in colon cancer is always lower than that expected from the level of expression of hTERT gene, suggesting posttranscriptional regulation of telomerase activity. However, more recent studies, performed with RT-PCR, demonstrated a qualitative24 and quantitative25 relation between hTERT mRNA expression and the activity of the enzyme. The presence of hTERT mRNA was also demonstrated in normal and adenoma colorectal tissues25 and in pre-neoplastic lesions.26

Quantification of Telomerase Activity in Cancer

Almost all data reported in the preceding section are based on the qualitative evaluation of telomerase activity (presence/absence) with little or no considerations on the possible significance of quantitative variations among patients or between neoplastic and nontumor tissues. Conventional TRAP (telomeric repeat amplification protocol assay),27 in spite of the high sensitivity of the assay, can provide only qualitative information. Several attempts were tried to overcome this limitation,28–30 and the application of quantitative protocols for the measurement of telomerase activity showed new potential applications of this marker.31,32

An indirect estimation of telomerase activity can also be obtained by the evaluation of mRNA expression of genes corresponding to telomerase subunits. According to this hypothesis quantitative RTPCR assays, based on realtime RT-PCR method were proposed.33

Here we present results from our study which evaluates simultaneously, using quantitative procedures, telomerase activity and hTERT mRNA expression in colorectal cancers and in corresponding nontumoral tissues.

Three different types of relationship were evaluated:

  1. telomerase activity and hTERT mRNA expression versus the clinicopathological characteristics of patients affected by colorectal cancer;
  2. the levels of telomerase activity and hTERT mRNA in colorectal cancers in comparison to the results obtained in corresponding non cancerous tissues;
  3. the levels of hTERT mRNA expression in comparison to telomerase activity in colorectal cancers and in non cancerous tissues.

Telomerase Activity and hTERT Expression in Colon Cancer

Telomerase activity was detected in 64/70 colon cancers and in 54/66 corresponding normal tissues, whereas hTERT mRNA was expressed in 98/100 colon cancers and in 90/92 normal tissues. However, telomerase activity was significantly higher in colon cancers (216.6±23.2 ng DNA/μg protein) than in control tissues (145.6±19.0, p<0.0001). Similarly the expression of hTERT mRNA was significantly higher in tumor sample (2762±661 fg RNA of HT1080/μg of total RNA) than in control sections (1178±290, p=0.002).

When results obtained in tumor samples were classified on the basis of the common clinical and pathological features of colon cancer, both telomerase activity and hTERT mRNA expression appeared tightly connected to tumor grade with a significant increase of both these markers in G3/G4 tumors. In addition, hTERT mRNA expression was significantly lower in right colon cancers in comparison to left and rectal carcinomas. However, we did not find any relationship with Dukes' stage, CEA levels, age, sex, pattern of growth, sex (see Table 1).

Table 1. Telomerase activity and hTERT mRNA expression (mean ± S.E.) in colon cancers.

Table 1

Telomerase activity and hTERT mRNA expression (mean ± S.E.) in colon cancers.

When telomerase activity and hTERT mRNA expression were compared in tumor/normal paired samples and classified according to the different clinicalpathological parameters of our patients, their levels were almost constantly and significantly higher in colon cancers than in normal corresponding control tissues (see Table 2).

Table 2. Telomerase activity and hTERT mRNA expression (mean ± S.E.) in colon cancers and in corresponding normal tissues.

Table 2

Telomerase activity and hTERT mRNA expression (mean ± S.E.) in colon cancers and in corresponding normal tissues.

The levels of telomerase activity in cancer samples were weakly correlated (p=0.03) to the expression of hTERT mRNA, obtained in the same samples (Fig. 1). Such correlation was totally absent in corresponding normal samples (data not shown). On the other hand, we found a surprisingly high relationship between the levels of telomerase activity found in colon cancer samples compared with the corresponding normal tissues (r=0.7972, p<0.0000) (Fig. 2). Similarly, we found for hTERT mRNA expression a significant correlation between colon cancer and related normal tissue (r=0.4218, p<0.0001) (Fig. 3).

Figure 1. Relationship between hTERT mRNA expression in colorectal cancers and the levels of telomerase activity measured in the same samples.

Figure 1

Relationship between hTERT mRNA expression in colorectal cancers and the levels of telomerase activity measured in the same samples.

Figure 2. Telomerase activity in colorectal cancers and in corresponding normal tissues, collected 10 cm away from the cancer.

Figure 2

Telomerase activity in colorectal cancers and in corresponding normal tissues, collected 10 cm away from the cancer.

Figure 3. Expression of hTERT mRNA in colorectal cancers and in corresponding normal tissues, collected 10 cm away from the cancer.

Figure 3

Expression of hTERT mRNA in colorectal cancers and in corresponding normal tissues, collected 10 cm away from the cancer.

Conclusions

To investigate the relationship between telomerase and clinico-pathological features in colorectal cancer, we quantified the hTERT mRNA levels by quantitative RT-PCR analysis in a large number of colon cancer samples and in corresponding noncancerous tissues. In most of the paired samples, the activity of telomerase was also assayed, using a modified telomeric repeat amplification protocol, which can provide a quantitative evaluation of the activity of this enzyme. Both telomerase activity and hTERT mRNA expression were significantly increased in cancer samples in comparison to corresponding tissues, as recently demonstrated.24,25 The difference of telomerase activity and hTERT expression in paired cancer/normal tissues remained almost constantly significant, even when patients were classified on the basis of their clinical parameters.

When the values of telomerase activity in cancer tissues were evaluated, we found a significant relationship with tumor grade but not with the other conventional clinicopathological features. In the case of hTERT, we still found that G0/G1 tumors had a significantly lower expression in comparison to G2/G3 cancers. Similarly, tumors of the right colon had a significantly lower expression of hTERT, in comparison to left colon or rectum cancer. Finally, we found a marked increase of hTERT expression in patients with less than 55 years, although this difference did not reach statistical significance (maybe due to the low number of cases).

Even if the determination of telomerase activity and/or of hTERT mRNA did not appear as relevant tools in diagnosis of colon cancer, an unsolved question remains on the real significance of their relatively high presence in corresponding normal colon mucosae that, on the basis of conventional morphological observations, were considered totally free from any neoplastic cells. The distance for the collection of normal tissues was at least 10 cm from primary lesion. It is important to remark that the progressive increase of sensitivity in methods for telomerase detection in human tissues is carrying to the mounting evidence that the expression of telomerase-related gene hTERT and the activation enzyme activity are not exclusive features of cancer cells. Several pieces of evidence seem to confirm the presence of one or both these markers in benign lesions and in normal tissues, collected apart from cancer district and without any evidence of cell abnormality or atypia (for reviews see refs. 39,40). The real significance of these presences into expected normal tissues is still to clarify. In particular it is not still fully demonstrated whether or not a generalized activation of telomerase in morphologically normal tissues might represent a favorable ground for the subsequent developing of a neoplasia. This hypothesis might be in agreement with an early activation of telomerase as precocious event in cell transformation. If this mechanism were true, a relationship between telomerase expression in normal and cancer tissues would be expected.

The results of our study demonstrated that the expression of hTERT mRNA in colon cancers was significantly (p < 0.00001) related to the values in corresponding normal tissues. The same relation appeared, even more evident, when we compared the level of telomerase activity in the paired cancer/normal samples. These data, taken together, seem to support the hypothesis of a precocious activation of both markers, which promote uncontrolled cellular proliferation and immortalization in the same tissue district from which, after additional mutational events, cancer will develop subsequently. Similar results, obtained in bladder cancer32 and in gastric cancer (unpublished data), seem to demonstrate that telomerase reactivation, in these cavitary tumors, covers an area of tissue that is significantly larger than that leading to tumor development.

The last aspect of our study was to demonstrate whether in colon cancer the levels of telomerase activity are related to the expression of its catalytic subunit hTERT. This relationship was demonstrated in hepatocellular carcinoma,41 in cervical cancer42 and, very recently, also in colon cancer.25 On the other hand, some reports, mainly based on qualitative data only, stated that telomerase activity and hTERT mRNA levels in cancer cells were not always in parallel, suggesting that telomerase activity is regulated in a posttranscriptional manner as well as a posttranslational manner in tumor cells. Interestingly, our results seem to indicate that hTERT expression and telomerase activity are weakly but significantly correlated in colon cancers, but not in corresponding normal tissues. This finding could be interpreted as a simultaneous and parallel increase of telomerase expression in tissues with rapid proliferation and transformation.

In conclusion, many aspects of telomerase function and regulation in cancer and normal tissues remain to be fully understood. Quantitative methods for the evaluation of telomerase activity as well as the expression of its catalytic subunit hTERT may open new insights in the study of these functional mechanisms.

Experimental Procedures

Patients

For the study, 100 consecutive patients with CC (60 males and 38 females, with a range of age between 46 and 89 years), scheduled for elective resection, were considered for entry. For 92 patients, at least one sample of neoplastic and normal tissue (10 cm apart from the neoplasm) were obtained. In 70 patients a fragment of each sample was used for protein extraction and telomerase activity detection. Tumor histology and grade of differentiation were defined according to World Health Organization criteria.34 The pattern of cancer growth was assessed as expanding, when the tumor border was clearly demarcated, and as infiltrative, when cancer cells spread into the surrounding tissues without a distinct border.35 All cases were staged by the original Dukes system.36 For most patients, CEA (Carcinoembryonic Antigen) concentrations, measured before surgery, were also available.

Quantitative Assay for Telomerase Activity

We applied our recent modification of the classic TRAP assay, based on the detection of amplification products using the sensitive dsDNA intercalating, fluorescent dye Pico-Green.30

Protein was extracted from frozen tissues by homogenization in 200 ml of lysis buffer containing 10 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 1 mM EGTA, 0.1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 5 mM β-mercapto-ethanol, 0.5% of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and 10% of glycerol. The lysates were incubated for 30 min on ice and then centrifuged at 16,000 × g for 20 min at 4°C. The protein concentration was measured in an aliquot of each extract by the Bio-Rad Protein Assay. Samples were stored at −80°C.

For the telomerase activity assay, 1 μg of protein was used. For each sample an aliquot was treated with 0.5 μg of RNase A for 30 min at 37°C. Each extract was assayed in 47.2 μl of reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 4.5 mM MgCl2, 1 mM each dNTP, 20 pmol of forward TAG-U primer37 and 0.5 μM T4 gp 32. After a 60-min incubation at 30°C for telomerase-mediated extension of the forward primer and 3 min at 90°C to inactivate telomerase that has not reacted, 2.8 μl of the second reaction mixture containing 20 pmol of CTA-R reverse primer37 and 0.3 μl of 5 U/μl of Taq Gold (PE Biosystems) were added. For this assay, 60 PCR cycles are performed at 95°C for 30 s, 64°C for 30 s and 72°C for 30 s, followed by 10 min at 72°C. The detection of products of telomerase reaction and PCR amplification was by Pico-Green. This fluorescent dye, when excited at 480 nm, emits fluorescence at 520 nm that is proportional to the amount of dsDNA and thus to the telomerase activity of the initial sample. Ten μl of each amplification product was diluted with 490 μl of 10 mM Tris-HCl, 1 mM EDTA (pH 7.5) and 500 μl of Pico-Green. The concentration of each sample was measured with a spectrofluorophotometer (RF-540; Shimadzu) on a λ DNA standard curve from 0 to 100 ng. The final telomerase concentration, expressed in term of ng DNA/μg protein, was obtained by subtracting the DNA amount obtained in the same specimen after RNase treatment. In each assay positive and negative controls were also tested.

Quantitative Real-Time Assay for the Measurement of Telomerase Catalytic Subunit hTERT mRNA Expression by TaqMan Technology

Total RNA extraction was performed with RNeasy kit (Qiagen). Four hundred ng of total RNA were reverse transcribed in 80-ml final volume of a mixture containing 1× RT buffer, 5.5 mM MgCl2, 500 μM each dNTPs, 2.5 μM random hexamers, 0.4 U/μl RNAsin, 1.25 U/μl Reverse Transcriptase (MuLV). The reaction mixture was heated at 25°C for 10 min followed by 30 min at 48°C and 5 min at 95°C. Amplification and fluorescence detection were performed in the ABI PRISM 7700 Sequence Detector. Primers and probe sequences were as recently published.38 For the absolute quantification of hTERT mRNA, a standard curve was generated with 5-fold serial dilutions from 2.5 × 105 fg to 2.5 fg of HT1080 cDNA. Twenty-five ng of cDNA for each sample were assayed in triplicates. The reaction was performed in a final volume of 25 μl containing 12.5 μl of Universal Master Mix 1× (including AmpliTaq Gold DNA Polymerase, AmpErase UNG, dNTPs with dUTP, Passive Reference and optimized buffer components), each primer 300 nM, probe 200 nM. The thermal cycling condition included 15 s at 95°C and 1 min at 60°C for 40 cycles. The initial concentration of each sample, expressed in term of fg RNA of HT1080/μg of total RNA, was interpolated from the standard curve.

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