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Telomerase Activity and Bcl-2 Expression in Non-Small Cell Lung Cancer

Department of Surgery II, Okayama University School of Medicine, Okayama, 700-8558 Japan

	ABSTRACT

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Bcl-2 and p53 are the most relevant proteins in apoptosis and tumor development. Telomerase functions in the maintenance of telomeres and is indispensable for immortalization. Bcl-2 was reported as a direct modulator of telomerase activity, and a correlation between p53 and telomerase activity was reported. The aim of this study was to determine the relationships between Bcl-2, p53, and telomerase activity in non-small cell lung cancer.

Immunostaining for Bcl-2, p53, and Ki-67 was performed in 64 surgically resected non-small cell lung cancers, and a fluorescence-based telomeric repeat amplification protocol assay for semiquantitative analysis of telomerase activity was done. Twenty-eight (44%) and 33 (52%) cases showed positive staining for Bcl-2 and p53, respectively. Bcl-2 expression was associated with negative lymph node involvement (P = 0.0248). p53 expression was associated with tumor size (P = 0.0244), p stage (P = 0.0391), and proliferative activity (P = 0.0004). Telomerase activity was detected in 89.1% and was closely associated with aggressive clinicopathological features. Telomerase activity was higher in p53-positive tumors (P < 0.0001), but represented no correlation with Bcl-2 expression (P = 0.3239). Interestingly, when the cases were stratified by histological grade and the level of Ki-67 labeling index, Bcl-2 expression was more clearly associated with favorable clinicopathological features and lower telomerase activity only in low-grade tumors.

In conclusion, p53 is closely associated with telomerase activity. In low-grade tumors, Bcl-2 is inversely correlated to telomerase activity. Our results suggest that the biological role of the Bcl-2 protein alters according to tumor aggressiveness, thereby cofunctioning with telomerase against genetic instability.

	INTRODUCTION

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Malignant neoplasms generally arise from a multistep process characterized by various genetic alternations. Molecular genetic studies have revealed mutations in a number of oncogenes and tumor suppressor genes in malignant neoplasms. It has been demonstrated that tumor growth and aggressiveness may be determined by the proliferative rate, as well as by the rate of cell death, or apoptosis (1) . Apoptosis plays a key role in the development of malignant tumors (2) . The protein products of the bcl-2 and p53 genes are the most relevant proteins in apoptosis and tumor development.

The Bcl-2 oncoprotein is known to promote cell survival, even when the cell proliferation rate is not elevated, and it can act as a negative regulator at a certain point of the biological cascade, leading to physiological cell death, or apoptosis. This could provide a growth advantage eventually leading to neoplastic transformation (3) . Although expression of the Bcl-2 protein has been reported for a variety of human epithelial malignant tumors, including the lung, the precise biological role of Bcl-2 in the development of malignant tumors is still controversial. In NSCLC,² most reports found that Bcl-2 expression was associated with favorable clinicopathological characteristics and prognosis (4, 5, 6, 7, 8, 9) , although the antiapoptotic action of Bcl-2 is expected to confer a survival advantage to the cancer cell. However, there have been some reports describing no significant correlation between Bcl-2 expression and prognosis (10 , 11) .

The tumor suppressor gene p53 has been extensively studied in lung cancer tissues as well as in cultured cell lines, and it is the most frequently mutated gene. p53 status is thought to reflect the level of genomic stability, and it is also thought to be involved in the regulation of apoptosis. Alternations in the p53 tumor suppressor gene are believed to be crucial for the development and progression of most neoplasms (12) . However, the biological role of the p53 protein is also still controversial.

Telomerase is a specialized ribonucleoprotein polymerase that functions in the maintenance of telomeres and is considered to be necessary for the indefinite proliferation of human cancer cells. Progressive shortening of telomeres and activation of telomerase have been considered to be one of the key mechanisms in chromosome structural integrity, cellular immortalization, and tumor progression (13, 14, 15) . The activation of telomerase was reported in a number of human cancer tissues, using a sensitive PCR-based TRAP assay (16) . Recently, Mandal and Kumar (17) demonstrated that the forced expression of exogenous Bcl-2 is closely linked with increased levels of telomerase activity, as the overexpression of Bcl-2 was accompanied by increased telomerase activity: the modulation of telomerase activity by Bcl-2. In contrast, previous studies have demonstrated that telomerase activity is associated with aggressive tumor characteristics and poorer prognosis (18 , 19) , whereas Bcl-2 expression is linked with favorable tumor characteristics and better prognosis in NSCLCs (4, 5, 6, 7, 8, 9) . Thus, the estimation of the relationship between Bcl-2 expression and telomerase activity in clinical samples is of interest. Additionally, a positive correlation between p53 status and telomerase activity was reported (20) .

In this study, we examined the expression of Bcl-2 and p53 immunohistochemically in 64 surgically resected NSCLCs, as well as telomerase activity simultaneously with a fluorescence-based TRAP assay, to assess their correlation with clinicopathological features and to determine whether any correlation could be found between telomerase activity and each protein.

	MATERIALS AND METHODS

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Patients and Tissue Samples.
Tissue samples were obtained from 64 patients with NSCLC who underwent radical surgery for primary tumor. Their ages ranged from 24 to 82 years old (mean, 65.9 ± 11.6). Forty-four were male and 20 were female. The histological types were adenocarcinomas and squamous cell carcinomas: 45 and 19 cases, respectively. Patients received no other form of therapy before surgery. Surgically resected specimens were fixed in 10% formalin solution for histological examination. At the time of surgery, tissue samples were promptly dipped in liquid nitrogen and stored frozen at -80°C until use. All samples underwent histological examination by microscopy with H&E staining and were staged using the TNM classification according to the present International Union against Cancer (UICC) guideline (21 , 22) . For histological differentiation, well-, moderately, and poorly differentiated tumors were graded as grade 1, 2, and 3, respectively. The expression of both Bcl-2 and p53 proteins, and telomerase activity were compared with clinicopathological characteristics, including age, sex, histological type, histological grade, pT and pN status, and pTNM stage.

Immunostaining.
For immunostaining, we used a dextran polymer conjugate two-step visualization system (EnVision+) developed by Dako (Glostrup, Denmark; Refs. 23 and 24 ). Immunostaining was performed on 4-µm-thick cryostat sections and then fixed in 4% paraformaldehyde. Endogenous peroxidase activity was blocked with 0.3% H₂O₂ containing methanol (30 min; room temperature). After washing away excessive methanol in demineralized water, nonspecific bindings were blocked with blocking solution. For Bcl-2 oncoprotein staining, monoclonal mouse antihuman Bcl-2 oncoprotein was mounted on the tumor sections (clone bcl-2 124; dilution, 1:50; Dako), and for p53 immunostaining, monoclonal mouse antihuman p53 protein (clone DO-7; dilution, 1:50; Dako) was mounted on the tumor sections (20 min; room temperature). Excessive amounts of antibodies were washed off in 0.05 M Tris-buffered saline. EnVision+ polymer/horseradish peroxidase (Dako, Carpinteria) was mounted (40 min; room temperature) followed by rinsing with Tris-buffered saline. Staining was visualized using diaminobenzidine as a chromogen. Sections were counterstained with hematoxylin. We also examined the proliferative activity by detecting Ki-67 immunostaining in all specimens with rabbit antihuman Ki-67 antigen (dilution, 1:50; Dako), which has a reactivity similar to the one seen with the monoclonal anti-Ki-67, clone MIB 1. The EnVision+ method and frozen material were also used.

In most specimens, Bcl-2 expression was observed in the cytoplasm. We used a frozen section from a normal peribronchial lymph node removed during postsurgical sampling of a lung tumor as a positive control. At the same time, positive staining of small lymphocytes provided an internal control for Bcl-2 immunoreaction. Staining without the anti-Bcl-2 monoclonal antibody was performed as a negative control procedure. p53 expression was observed in the nuclei of tissue samples. Staining without the anti-p53 monoclonal antibody was performed as a negative control procedure, whereas p53-positive lung cancer tissue was used as a positive control.

Immunoreactivity was assessed by scoring with a minimum of five high-power fields (40x objective lens), and the mean number of positive cells was counted. The distribution of stained tumor cells across the sections was noted, and the percentage of positive cells was assessed. For the present study, immunohistochemically stained sections were judged positive for Bcl-2 and p53 expression when >20% of the cancer cells showed cytoplasmic and nucleolar staining, respectively. Ki-67 assessment was based on the percentage of stained nuclei; the Ki-67 labeling index. All slides were evaluated for immunostaining in a blinded fashion without any knowledge of the clinical outcome or other clinicopathological data.

Telomerase Assay.
Extract of tissue specimens was done as described earlier (16) , and for semiquantitative analysis of telomerase activity, we used a nonradioisotopic fluorescence-based TRAP assay (25) , based on the TRAP-ese, Telomerase Detection Kit (Oncor, Gaithersburg, MD). The analysis of telomerase assay was conducted by the SRL Laboratory (Saitama, Japan). Briefly, frozen lung tissue samples of 10 mg were homogenized in 200 µl of CHAPS buffer (TRAP-ese). After 30 min of incubation on ice, the lysates were centrifuged at 12,000 x g for 20 min at 4°C. The resulting 160 µl of supernatant were recovered, and the concentration of protein was determined. An aliquot of the extract containing 1 µg of protein was used for each assay. TSR8 in the TRAP-ese Kit was used as a positive control. Aliquots of extracts were incubated with 0.1 ng of Cy-5-labeled TS primer (5'-AATCCGTCGAGCAGAGTT-3') in Master Mix (TRAP-ese). After incubation at 30°C for 30 min, PCR was performed at 94°C, 30 s; 60°C, 30 s; and 72°C, 45 s for 30 cycles. For analysis of amplified products, we used a 9% denaturing gel containing 6 M urea. The PCR products were diluted with an equal volume of formamide dye solution and heated at 94°C for 5 min, and 5 µl were applied to each lane of the gel fitted to an automated DNA sequencer (ALF red DNA Sequencer, Pharmacia Biotech). Electrophoresis was performed at 45 W, at a constant temperature of 45°C. The fluorescence data from the ALF red DNA Sequencer were collected and analyzed automatically by the Fragment Manager V1.1 (Pharmacia Biotech). Each fluorescent peak was quantitated in terms of size, peak height, and peak area. The telomerase quantitation results were expressed as TPG (see Fig. 1 for details).

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Fluorescence-based TRAP assay: Typical fluorescence curves representing telomerase activity detected by fluorescence-based TRAP assay. The first peak is the Cy-5-labeled TS primer, and the second peak is the internal control (36 bp; ITAS). The PCR product of telomerase extension yielded a six-nucleotide peak (50, 56, 62, 68 bp, and so forth) from the third peak (50 bp); the first amplifiable telomerase product. Lane 1, TSR8 from the TRAP-ese Kit was used as a positive control. Telomerase activity was detected in a waving-curve pattern with a periodicity of about six nucleotides, and all data were analyzed automatically by the Fragment Manager V1.1 (Pharmacia Biotech). Lane 2, a negative control, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid lysis buffer (no extract), was performed. The quantification of telomerase activity was determined as the TPG by following the formula: [Total of peak area originated from each sample/peak area of ITAS in each assay]/[Total of peak area originated from positive control/peak area of ITAS in positive control] x 100 = TPG (units). Lanes 3, 4, and 5, typical results of samples with high, medium, and low telomerase activity, respectively.

	RESULTS

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Bcl-2 and p53 Immunostaining.
The results of Bcl-2 and p53 immunostaining and their relationship with clinicopathological features are summarized in Table 1 . Of the 64 cases with NSCLC, 28 (44%) showed positive cytoplasmic staining for Bcl-2. A patchy and heterogeneous pattern of Bcl-2-positive cells was more prevalently recognized in adenocarcinomas than in squamous cell carcinomas (Fig. 2 a), whereas a diffuse and homogenous pattern was often found in squamous cell carcinomas (Fig. 2 b). The prevalence of Bcl-2 expression in primary tumors was significantly higher in patients who had no lymph node metastasis than in patients with positive nodes (P = 0.0248), and it was higher in pN₀ cases than in pN_1,2 cases (P = 0.0249). The cases of stage I, II expressed Bcl-2 immunostaining more frequently than the cases of stage III (P = 0.0329). There was no statistical significance in the frequency of Bcl-2 status with respect to other clinicopathological features, as well as Ki-67 labeling index. Stronger activation of telomerase was seen in Bcl-2-negative samples compared with positive ones, but without statistical significance (P = 0.3239).

View larger version (101K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Immunohistochemical detection of Bcl-2 and p53 expression: a, the distribution of Bcl-2-positive cells demonstrated a patchy and heterogeneous pattern in grade 2 adenocarcinoma. b and c, continuous sections of grade 3 squamous cell carcinoma were shown. Diffuse and homogenous Bcl-2-positive cells were seen (b), and p53 immunostaining (without counter-staining) also revealed a diffuse and intense pattern (c).

There was no correlation between Bcl-2 and p53 expression in immunochemistry (P = 0.8254; Table 2 ). Of note, among 28 Bcl-2-positive tumors, 12 of 14 (85.7%) Bcl-2+/p53+ phenotypes were grade 2 or 3 tumors (Fig. 2 , b and c). On the contrary, 9 of 14 (64.3%) Bcl-2+/p53- phenotypes were grade 1, whereas 5 cases (35.7%) were grade 2 or 3.

To estimate the correlation according to not only histological grade but also proliferative activity, we performed the same analysis using the Ki-67 labeling index (Table 4) . In NSCLCs with low proliferative rate (35 cases), Bcl-2 expression was significantly associated with small tumor size (P = 0.0411), negative lymph node involvement (P = 0.0354), and earlier pTNM stage (P = 0.0196). The pN₀ cases expressed Bcl-2 immunostaining more frequently than the pN_1,2 cases (P = 0.0303). Moreover, Bcl-2-negative tumors expressed significantly higher telomerase activity than did the Bcl-2-positive tumors (P = 0.0012). There was no significant correlation between Bcl-2 expression and clinicopathological features in tumors with high proliferative rate.

	DISCUSSION

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Although p53 alterations are the most common genetic lesions observed in lung cancers and have been extensively investigated to date, the association of p53 alteration with clinicopathological features and prognosis is also controversial in lung cancer. Some authors have reported that p53-positive immunoreactivity in tumor cells is a poor prognostic factor (28 , 29) , and others have reported that no correlation exists between p53 protein expression and prognosis (30 , 31) .

One of the reasons for these discrepancies in their results may be attributed to the differences in immunohistochemical methods: different antibodies, samples (paraffin-embedded or frozen samples), method, and criteria of positivity for Bcl-2 and p53 expression. In this study, cryostat sections were used because staining intensity and sensitivity are reported to decrease when tested on paraffin sections compared to frozen ones (32) . Additionally, a sensitive and simple procedure, the EnVision+ method (23 , 24) , was performed with the use of well-commercialized monoclonal antibodies. Immunopositivities of the Bcl-2 and p53 proteins were 44% and 52%, respectively. Bcl-2 expression was significantly associated with negative lymph node involvement and stage I, II tumors. Expression of p53 was associated with large tumors, advanced stage, and high proliferative activity. Such data are in agreement with results published previously. An inverse correlation between Bcl-2 and p53 expression was reported (5 , 33) , but our results did not find such a correlation. Notably, among Bcl-2-positive tumors, the distribution of histological grade was different between p53-positive and -negative tumors. In total, 85.7% of Bcl-2+/p53+ tumors were grade 2 or 3, whereas 64.3% of Bcl-2+/p53- tumors were grade 1. Therefore, we assessed the correlation between Bcl-2 expression and clinicopathological features, stratifying by histological grade. Subsequently, earlier pT status and smaller tumor size became statistically significant only in grade 1 tumors. This result suggests the possible alternation of the biological role of the Bcl-2 protein in accordance with histological grade, as well as the proliferative activity of the tumor. Interestingly, when the cases were subdivided by the level of the Ki-67 labeling index, favorable clinicopathological features in all parameters revealed a significant association with Bcl-2 positivity in tumors with low proliferative rate. On the other hand, there was no correlation in grade 2 or 3 tumors, or in tumors with high proliferative rate. There was only one report in which cases were stratified by histological grade; Ritter et al. (34) demonstrated statistically improved survival in Bcl-2-positive grade 1 tumors. Their results are supported by our findings.

A high frequency of telomerase activation in lung cancer has been reported (18, 19, 20 , 35) . In our study, 89.1% of NSCLCs expressed detectable telomerase activity, and the level of telomerase activation was well associated with the aggressive clinicopathological features. Also, a positive correlation between the level of telomerase activity and the Ki-67 labeling index was found as reported by Albanell et al. (18) . Recently, the relationship between telomerase activity and p53 was reported. Wu et al. (20) postulated that p53 status may be related to telomerase expression, although quantification of the level of telomerase activity was not performed. In our study, a semiquantitative TRAP assay was used, and a strong correlation between p53 and the level of telomerase activity was found (P < 0.0001). Mandal and Kumar (17) demonstrated that variable levels of the forced expression of exogenous Bcl-2 correlated with comparably enhanced levels of telomerase activity in clones, and reported that Bcl-2 directly modulates telomerase activity. But according to previous reports that investigated clinical samples, telomerase activity was reported to associate with aggressive characteristics, whereas overexpression of Bcl-2 with rather favorable tumor characteristics. Thus, we examined the relationship between Bcl-2 expression and telomerase activity simultaneously in lung cancer tissues. When considering all cases, there was no correlation between Bcl-2 expression and telomerase activity. But, when analyzing each histological grade, a significantly higher activation of telomerase was detected only in Bcl-2-negative grade 1 tumors. Moreover, in tumors with low proliferative rate, telomerase activity was also significantly higher in Bcl-2-negative than in Bcl-2-positive tumors.

In atypical adenomatous hyperplasia and dysplasia of the bronchial epithelium, which are possible precursor lesions for peripheral adenocarcinoma and squamous cell carcinoma, respectively, a high frequency of Bcl-2 expression was reported (36 , 37) . On the other hand, alveolar cells and areas of atypical adenomatous hyperplasia were reported to be telomerase-negative (38) . According to the speculation of previous reports, Bcl-2 deregulation may be a relatively early event and for some reason, lost in the later stage of carcinogenesis (6 , 39) . Similar opinions were reported in breast cancer (40) and colorectal cancer (41) , but the mechanism was not clarified. On the other hand, high prevalence of Bcl-2 oncoprotein expression was reported in SCLC: 55–93.7% (42, 43, 44, 45, 46) , with an aggressive biological behavior and correlated with extremely poor prognosis. Takayama et al. (44) reported that patients with Bcl-2-positive tumors have poor survival times compared with those with Bcl-2-negative tumors, although the response to chemotherapy was not significantly lower. Although the difference in the etiology of NSCLC and SCLC remains unclear, the combined histological type of SCLC, including components of squamous cell carcinoma and/or adenocarcinoma, is well known. Higashiyama et al. (47) investigated the distribution of Bcl-2 expression in combined histological type of SCLC, and demonstrated stronger and more frequent expression of the Bcl-2 protein in the portion of SCLC than in the portion of NSCLC. These results cause additional confusion in understanding the biological role of the Bcl-2 protein.

Overexpression of Bcl-2 was reported to occur as a reaction against a variety of cell stresses, including cytotoxic chemicals, growth factor depletion, heat shock, ionizing radiation, excess calcium influx, and a range of chemotherapeutic drugs (48, 49, 50, 51) . Therefore, the overexpressed status of the Bcl-2 protein in malignant tumors may present when cells cannot maintain genetic stability for various reasons. Thus, according to the results in the present study and previous reports, we established a hypothesis for the biological appearance of the Bcl-2 protein, as described below. First, the overexpression of Bcl-2 oncoprotein occurs as an early event in carcinogenesis to allow cells with DNA damage, such as gene mutations that causes genetic instability, to escape from the normal mechanisms of apoptotic cell death. In low-grade neoplasms, Bcl-2 overexpression disappears after activating enough level of telomerase to maintain genetic stability because telomerase has a function to heal the fragmented chromosome occurred by genetic mutations (52) . However, in cases that do not acquire enough telomerase activity, Bcl-2 overexpression remains strong. On the other hand, in high-grade neoplasms with severe genetic instability, Bcl-2 expression does not completely diminish and represents a "secondary" activation, and thus maintains a somewhat overexpressed status after activating telomerase and acts as a negative regulator of physiological cell death, while cofunctioning with telomerase. This hypothesis explains the high rate of Bcl-2 expression in SCLC. Although the prognostic evaluation of the Bcl-2 protein has not been clarified, the present results indicate the possibility that Bcl-2 expression could be a favorable prognostic indicator, whereas telomerase activity could be a negative indicator in low-grade NSCLCs. However, the number of analyzed samples was rather small, so a study with a large cohort and further laboratory exploration will be required to prove our hypothesis.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Department of Surgery II, Okayama University School of Medicine, 2-5-1, Shikata, Okayama, 700-8558 Japan. Phone: 81-86-235-7265; Fax: 81-86-235-7269; E-mail: surgery2{at}med.okayama-u.ac.jp

² The abbreviations used are: NSCLC, non-small cell lung cancer; TRAP, telomeric repeat amplification protocol; TPG, total product generated; SCLC, small cell lung cancer; ITAS, internal telomerase assay standard.

Received 2/17/00; accepted 4/27/00.

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