Telomerase, P53 and PCNA activity in osteosarcoma

EJSO 2003; 29: 564–567 doi:10.1016/S0748-7983(03)00117-3

Telomerase, P53 and PCNA activity in osteosarcoma H. Nakashima*, Y. Nishida*, H. Sugiura*, H. Katagiri*, M. Yonekawa*, Y. Yamada*, H. Iwata*, T. Nagasaka† and N. Ishiguro* Departments of *Orthopaedic Surgery, and †Pathology, Nagoya University School of Medicine, Nagoya, Japan

Aims: The aim of this study was to investigate telomerase activity and to assess the correlation between telomerase activity, tumor proliferative activity and p53 overexpression in osteosarcoma tumor samples. Methods: Using a telomerase polymerase chain reaction-enzyme-linked immunoassay based on the telomeric repeat amplification protocol method, we examined telomerase activity in 26 primary osteosarcoma specimens. P53 overexpression was identified using immunohistochemical staining, and tumor proliferative activity was assessed by immunohistochemical staining of PCNA. Results: Telomerase activity was detected at a relatively low level in five of 26 osteosarcoma tissue specimens. P53 was detected in eight of 21 cases. There was no significant correlation between telomerase activity and p53 overexpression ðp ¼ 0:325Þ: There was a significant correlation between PCNA staining and telomerase activity ðp ¼ 0:0075Þ: Conclusion: Difference between the telomerase activity and p53 overexpression in osteosarcoma suggests that p53 and telomerase may not cooperate in tumor proliferation. Correlation of telomerase activity to PCNA expression suggests that telomerase activity may also useful for evaluate proliferative activity in osteosarcoma. Q 2003 Elsevier Ltd. All rights reserved. Key words: telomerase activity; p53; PCNA; osteosarcoma.

INTRODUCTION Telomerase1 – 3 is a ribonucleoprotein that synthesizes telomeric DNA repeats at the end of chromosomes, using a complementary sequence of RNA as a template.4 Increased telomerase activity has been shown in human germ line cells, cancer cells, and immortal and tumorderived cell lines, but not in normal somatic cells. Escape from normal proliferative limitations of cellular senescence may contribute to tumor growth in vivo. Activation of telomerase may promote neoplasia.5 The p53 tumor suppressor gene may regulate cellular growth and proliferation and telomerase activation. P53 may also signal growth arrest in response to telomere shortening in aging cells.6 Several studies have investigated the association between telomerase activity and the status of cell cycle regulator such as p53 and Rb, in breast cancer,7,8 colorectal cancer9 and sarcoma cell lines.10 In this study, using telomerase repeat amplification protocol (TRAP), we analyzed telomerase activity in Correspondence to: Dr Hiroatsu Nakashima, Department of Orthopaedic Surgery, Nagoya Memorial Hospital, 4-035 Hirabari, Tenpaku-ku, Nagoya 468-8520, Japan. Tel.: þ81-52-804-1111; Fax: þ81-52-806-6257; E-mail: [email protected] 0748–7983/03/$30.00

osteosarcoma tissue, and investigated the possible correlation between telomerase activity and p53 accumulation, and proliferation, as assessed by immunostaining with the proliferating cell nuclear antigen (PCNA) antibody. The relationship between telomerase activity and clinical outcome, and chemosensitivity was also investigated.

MATERIALS AND METHODS Tumor specimens were obtained from 26 patients with primary osteosarcoma, seven patients before chemotherapy and 19 patients after chemotherapy who were treated between 1992 and 1997. Age range was 4 – 68, mean 24. Follow-up ranged from 3 months to 7 years. All tumor samples were taken immediately after resection or biopsy, then snap-frozen in liquid nitrogen and stored at 2 80 8C until telomerase activity analysis. Tissue from the core or middle of the tumor specimen was analyzed, to increase the probability that the tumor specimen contained neoplastic cells. Tumor specimens were formalin-fixed, paraffin-embedded and used for immunohistochemical staining. Q 2003 Elsevier Ltd. All rights reserved.

TELOMERASE ACTIVITY IN OSTEOSARCOMA

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Measurements of telomerase activity

Specimen evaluation

Telomerase activity was measured using the telomeric repeat amplification protocol (TRAP)-based telomerase polymerase chain reaction (PCR)-enzyme-linked immunoassay (ELISA) Kit (Roche-Boehringer-Mannheim, Mannheim, Germany). Cell protein (1.0 mg) was analyzed according to the manufactures instructions. Telomeric repeats were added to biotin-labeled P1-TS primer by telomerase present in cell extract during the first reaction for 30 min at 25 8C and telomerase was inactivated for 5 min at 94 8C. The elongation products were then amplified by PCR using the primer P1-TS and P2, with denaturation at 94 8C for 30 s, annealing at 50 8C for 30 s, and polymerization at 72 8C for 90 s. Thirty cycles were performed, and the last extension was performed at 72 8C for 10 min. Five microlitre of each PCR product was denatured, hybridized to a digoxigenin (Dig)-labeled, telomeric repeat-specific probe, and bound to a streptavidin-coated microtiter plate. Finally, the immobilized PCR product was detected with an antiDig-peroxidase antibody and visualized a color reaction product using the 3,30 5,50 -tetramethylbenzidine substrate. After the addition of a stop reagent, the absorbance of the samples was measured with ELISA Reader at the wavelength of 450 nm (reference wavelength, 690 nm). Immortalized human cells (293 cells) were used as the positive control. Inactivation of telomerase protein at 65 8C for 10 min was used as the negative control. The TRAP assay was considered valid when all of the following conditions were fulfilled: (1) the absorbance reading of positive control was higher than 2.0 A450 – 690 nm units after 20 min of substrate reaction using 0.5 mg cell equivalents in the assay; (2) the heat-treated negative control was , 0.2 A450 – 690 nm units. Samples were regarded as telomerase-positive if the difference in absorbance was higher than 0.2 A450 – 690 nm units, which was derived from the date that normal osseous tissues as negative control showed less than 0.2 A in telomerase activity.

Immunostaing for p53 was evaluated at £ 200 magnification using a semiquantitative scale (0, , 10%, 10– 50%, or 50% , positive tumor cells). Overexpression of p53 was judged present when $ 10% of the tumor cells showed nuclear staining. Immunostaining for PCNA was graded as follow: grade 1: 0– 25% positive cells, grade 2: 26 –50% positive cells, grade 3: 51 – 75% positive cells and grade 4: 76– 100% positive cells. Breast (Hepatic) cancer tissues were used for positive control, showing more than 25% positive staining for PCNA and more than 10% positive staining for p53. Normal osseous tissues were used as a negative control. Positive staining of negative control was less than 10% in both PCNA and p53. PCNA staining was dichotomized as either low staining (0 –25%) or high staining (26 – 100%).

Statistical analysis The correlation between telomerase activity and the results of immunohistochemical staining was statistically analyzed by Fisher’s exact test. The probability of p , 0:05 was considered to be statistically significant.

RESULTS Telomerase activity Telomerase activity was found at a low level in five of 26 osteosarcoma patients, one patient before chemotherapy, and four patients after chemotherapy ranging from 0.23 to 0.69. There was no significant correlation between telomerase activity and staging, local recurrences, lung metastasis and survival rate (Fig. 1). Of seven biopsy cases, two specimens showed positive activity, whereas, of 19 resection cases, three specimens showed positive activity.

P53 and PCNA immnohistochemistry Twenty-one of the 26 osteosarcoma specimens (five telomerase positive, 16 telomerase negative) measured for telomerase activity were fixed in formalin, embedded in paraffin and stained by a standard protocol using streptavidinbiotin –peroxidase complex (histofine SABPO Kit, Nichirei, Tokyo, Japan), clone PC10, a mouse monoclonal antibody to the PCNA (DAKO), and clone DO-7, monoclonal mouse anti-Human p53 protein (DAKO). They were then treated with biotinylated rabbit anti-mouse antibody (Nichirei) and streptavidinHRP-conjugated (Nichirei). The bound peroxidase was visualized using 0.05% solution of DBA tetra-acetate. The sections were counterstained with hematoxylin.

Figure 1

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Table 1 Relationship between telomerase activity PCNA and p53 Telomerase activity

PCNA Negative Positive p53 Negative Positive

Negative

Positive

16 0

2* 3†

p ¼ 0:0075

11 5

2 3

p ¼ 0:325

prognosis in osteosarcoma is uncertain,13 as is the relationship with p53 expression.8,16,17 PCNA can be used to assess of cell proliferative activity in tumors.18 Telomerase activity may be relevance associated with KI-67 express in breast cancer,19 and lung cancer.20 Telomerase activity may a useful marker of cell proliferation in osteosarcoma.21 The prognosis of patients with osteosarcoma has been improved by chemotherapy. Tumor cells with high proliferative activity may be responsive to chemotherapy and telomerase activity might thus further investigated as a tool to predict chemosensitivity in osteosarcomas.

*Two cases were grade 3. †Two cases were grade 4 and one case was grade 5.

REFERENCES PCNA staining and telomerase activity PCNA staining, was grade 1 in 4 cases, grade 3 in 14 case, grade 4 in 2 cases, and grade 5 in 1 case. Telomerase activity was correlated with the PCNA staining (Table 1).

P53 Immunostaining and telomerase activity Expression of p53 protein by $ 10% of tumor cells present in eight cases. There was no correlation between telomerase activity and p53 expression (Table 1).

Necrotic rate and telomerase activity This was studied in 18 of 26 cases. The five cases with positive telomerase activity, and 8/13 with negative telomerase activity, displayed . 70% necrosis.

DISCUSSION Telomerase activity has been detected in a variety of human cancers and tumor-derived cell lines. It is rarely seen in normal somatic cells, include germ cells and hematopoietic stem cells.5 Aue et al.11 reported that eight of 17 skeletal sarcoma samples and three of the 10 osteosarcoma specimens showed low telomerase activity. Scheel et al.12 demonstrated telomerase activity in 15 of 29 osteosarcomas. Sangiorgi et al.13 investigated telomerase activity with telomerase-PCR-ELISA in seven osteosarcomas low telomerase activity was detected in all samples, morphology-preserving technique to detect telomerase expression and activity at a cellular level. Telomerase-negative tissues and tumours may posses telomerase activity inhibitors, or may have acquired an alternative mechanism of telomerase maintenance and elongation.14,15 The relationship between telomerase activity and

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TELOMERASE ACTIVITY IN OSTEOSARCOMA proliferation in lung adenocarcinomas. Mod Pathol 2000; 13(7): 723–9. 18. Bravo R, Frank R, Blundell PA, et al. Cyclin/PCNA is auxiliary protein of DNA polymerase delta. Nature 1987; 356: 515–7. 19. Carey LA, Kim NW, Goodman S, et al. Telomerase activity and prognosis in primary breast cancer. J Clin Oncol 1999; 17: 3075–81. 20. Albanell J, Lonardo F, Rusch V, et al. High telomerase activity in primary lung cancer: association with increased cell proliferation

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