Japanese Journal of Clinical Oncology 31:532-535 (2001)
© 2001 Foundation for Promotion of Cancer Research
Expression of the Protein Gene Product 9.5, PGP9.5, is Correlated with T-status in Non-small Cell Lung Cancer
Department of Surgery II, Nagoya City University Medical School, Nagoya, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Background: In a tumor, increased deubiquitination of cyclins by a protein gene product gene, PGP9.5, could contribute to the uncontrolled growth of somatic cells that is a hallmark of cancer. We hypothesized that PGP9.5 may be expressed in tumors of patients with non-small cell lung cancer (NSCLC).
Methods: Expression of PGP9.5 messenger RNA was evaluated by reverse transcription polymerase chain reaction (RT-PCR) in 95 non-small cell lung carcinomas and adjacent histological normal lung samples. The data were analyzed with reference to clinicopathological factors.
Results: PGP9.5 transcripts were detected in 18 (12.8%) of the tumor samples, although some of paired normal lung samples showed very weak expression. There was no relationship between PGP9.5 gene expression and age, gender, N-status or pathological subtype. PGP9.5 gene was preferentially expressed in T3/T4 NSCLC (12/41, 29.3%) compared with T1/T2 NSCLC (6/54, 11.1%) (p = 0.0482).
Conclusions: Although the PGP9.5 gene was not expressed in a majority of NSCLC tumors, we suggest that PGP9.5 may correlate with tumor invasion or progression of NSCLC.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The high incidence and mortality of non-small lung cancer (NSCLC) makes lung cancer the second most common cancer and the leading cause of cancer death in men (1). The prognosis for patients with operable NSCLC remains gloomy in comparison with that observed for gastric, colon or breast cancer operable tumors (2). Over the past decade, there has been steadily accumulating evidence that numerous genetic markers influence the biological behavior of NSCLC (3)
Ubiquitination of cellular proteins and their targeting for subsequent degradation via ubiquitin-mediated proteolysis are an important mechanism that regulates cell cycle genes (4,5). Protein gene product 9.5, PGP9.5, is a ubiquitin hydrolase that is widely expressed in neuronal tissues at all stages of neuronal differentiation (6,7). In the tumor, increased deubiquitination of cyclins by PGP9.5 could contribute to the uncontrolled growth of somatic cells that is a hallmark of cancer (8). It has been reported that PGP9.5 gene was overexpressed in neuroblastoma (9).
Recently, using the serial analysis of gene expression (SAGE) method to analyze systematically transcripts present in NSCLC, several overexpressed clones were identified (10). One of these clones, PGP9.5, was overexpressed in 8/8 NSCLC cell lines but not in normal lung tissue (11). As available markers have proven inefficient for NSCLC, we investigated PGP9.5 transcript in patients with NSCLC by means of a reverse transcription polymerase chain reaction (RT-PCR) analysis. Also, we analyzed the data with reference to the clinicopathological factors.
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PATIENTS AND METHODS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Patients
The study groups included 95 NSCLC patients who had undergone surgery at the Department of Surgery II, Nagoya City University Medical School, between January 1997 and December 1999. The lung cancers were classified according to the general rules for clinical and pathological recording of lung cancer (12). All tumor and normal lung samples were collected at resection and immediately frozen in liquid nitrogen.
The clinical and pathological characteristics of the 95 NSCLC patients are shown in Table 1; they included 39 cases at stage I, 16 at stage II, 37 at stage III and three at stage IV. The mean age was 64.4 years (range, 42–88 years). Among the 95 NSCLC patients, 21 (22.1%) were women and 74 (77.9%) were men; 65 were lymph node metastasis negative and 30 were positive. Of 85 patients, 26 had squamous cell carcinoma and 59 adenocarcinoma (Table 1).
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Immunohistochemistry
Sections of 4 µm were made from paraffin tissue blocks from 61 NSCLC samples. The slides were treated with xylenes and then dehydrated in alcohol. Endogenous perioxidase was blocked with 0.3% H2O2. Microwave treatment was performed for 4 min in boiling citrate buffer (13). After blocking with Block Ace Solution (Dako Japan, Kyoto, Japan), the slides were incubated with monoclonal antiserum against PGP9.5 (Biogenesis, Kingston, NH, USA) at 1:500 dilution overnight at 4°C. Envision Kit and DAB substrate were used to visualize the antibody binding and the sections were counterstained with methyl green.
RT-PCR Assays for PGP9.5 Gene
Total RNA was isolated from tumor and adjacent histological normal lung using an Isogen kit (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. RNA concentration was determined spectrophotometrically and adjusted to a concentration of 200 ng/ml. RNA (1 µg) was reverse transcribed by Superscript II enzyme (Gibco BRL, Gaithersburg, MD) with 0.5 mg of oligo(dT)12–16 (Amersham Pharmacia Biotech, Piscataway, NJ). The reaction mixture was incubated at 42°C for 50 min followed by incubation at 72°C for 15 min. To ensure the fidelity of reverse transcription, all samples were subjected to PCR amplification with oligonucleotide primers specific for the constitutively expressed gene glyceraldehyde-3-phosphate dehydrogenase. The primer sequences for PGP9.5 gene were as follows: forward primer 5-AGATCAACCCCGAGATGCTGAACAAAGTG-3 and reverse primer 5-ATTAGGCTGCCTTGCAGAGAGCCACGGCAGAGAA-3. The cycling conditions were as follows: initial denaturation at 94°C for 2 min, followed by eight cycles at 94°C for 40 s, 65°C for 30 s, 72°C for 60 s, eight cycles at 94°C for 40 s, 62°C for 30 s, 72°C for 60 s, eight cycles at 94°C for 40 s, 59°C for 30 s, 72°C for 60 s, 10 cycles at 94°C for 40 s, 56°C for 30 s, 72°C for 60 s and finally 72°C for 5 min (touchdown PCR). Amplified cDNAs were separated on 1% agarose gels and the bands were visualized with ethidium bromide and photographed under ultraviolet transillumination. The specificity of the PCR products of PGP9.5 was confirmed with a PCRII vector kit (Invitrogen, Carlsbad, CA) and Antosequencer. The intensities of the PGP9.5 and GAPDH bands were quantified using an NIH Imager and the ratio of the PGP9.5/GAPDH gene expressions from each samples was examined. Tumor/normal ratios > 2.0 were considered as a positive expression.
Statistical Methods
Statistical analysis was done using the Stat-View software package (Abacus Concepts, Berkeley, CA). Differences among the means of the age, gender, N- and T-status and pathological subtypes in the patients with NSCLC were examined using the 
2 method. The overall survival of NSCLC patients was examined by the Kaplan–Meier method and survival characteristics were compared using log rank tests. It was considered significant when the p-value was <0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PGP9.5 Protein Expression
An immunohistochemical approach was used to localize PGP9.5 protein expression. The majority of the normal lung epithelial cells were negative. On the other hand, some of the lung cancer tissue sections (21/61, 34.4%) stained positive for PGP9.5 (Fig. 1). Although the RNAs were isolated from different sections of frozen tissues, the PGP9.5 protein expression by immunohistochemistry was correlated with PGP9.5 mRNA expression (p < 0.0001) (Table 1).
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PGP9.5 mRNA Expression
Of 95 NSCLCs studied, PGP9.5 transcripts were detected in 18 (12.8%) of the tumor samples, although some of paired normal lung samples showed very weak expression. These18 tumor samples had overexpressed PGP9.5 transcript when compared with the normal lung tissues (Fig. 2). The relationship between the PGP9.5 expression in NSCLC and patients’ clinicopathological factors is shown in Table 1. There was no significant difference in the PGP9.5 overexpression between patient’s groups stratified according to age, gender or N-status. There was a significant difference in PGP9.5 expression between T-status. PGP9.5 gene was preferentially overexpressed in T3/T4 NSCLC (12/41, 29.3%) compared with T1/T2 NSCLC (6/54, 11.1%) (p = 0.0482). There was no difference among the histological subtypes (squamous cell carcinoma vs adenocarcinoma and squamous vs non-squamous cell carcinoma); 6/26 (23.1%) squamous cell carcinomas expressed PGP9.5 and 11/59 (18.6%) of adenocarcinoma express it (p = 0.8571) (Table 1). Compared with squamous cell carcinomas, 12/69 non-squamous cell carcinomas had PGP9.5 transcript (p = 0.7339).
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Relationship Between Clinical Course of Patients with NSCLC and PGP9.5 Expression
Of patients who did not have PGP9.5 transcript, 60/77 (77.9%) were alive. On the other hand, 17/18 (94.4%) patients who had PGP9.5 transcript were alive (Table 1). The NSCLC patients with PGP9.5 expression did not correlate with survival rate although the observation period was short (p = 0.4204, log rank test).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PGP9.5 mRNA expression was analyzed by RT-PCR assay in clinical NSCLC samples. It has been shown previously that the PGP9.5 is expressed during fetal and early postnatal development. Several human lung cancer cell lines also expressed PGP9.5 mRNA (11). In addition, PGP9.5 gene was cloned by the SAGE method from NSCLC (10). The increased expression of PGP9.5 was associated with lung cancer development (stage) by immunohistochemistry (11).
PGP9.5 was originally isolated as a cytoplasmic marker for neurons and neuroendocrine cells (14,15). Some 10–15% of NSCLCs demonstrate neuroendocrine features by electron microscopy or immunohistochemistry, despite the absence of neuroendocrine features by light microscopy. Therefore, PGP9.5 expression in cancers might be reflected in their endocrine status. However, Hibi et al. reported that PGP9.5 protein expression in lung cancers was independent of neural differentiation by immunohistochemistry (11). Functionally, PGP9.5 belongs to the ubiquitin C-terminal hydrolase (UCH) family (6). These enzymes are a part of the cellular proteolytic pathway that regulates many cellular processes, including cell cycle progression and cell death (8). Several UCH family members are overexpressed in different tumors and can be oncogenic. The tre-2 gene, which was identified as one of the UCH family proteins, was detectable in a variety of tumor cells but not in normal tissues (16). Unph was isolated as a human homologue of the mouse Unp proto-oncogene (17). Overexpression of mouse Unp leads to oncogenic transformation of NIH3T3 cells and the expression level of this gene is also elevated in lung tumors (18).
It has been reported that PGP9.5 protein expression was correlated with the histological type. PGP9.5 protein expression was correlated with squamous cell carcinoma (SCC) in lung cancer (11). However, our RT-PCR results showed no correlation with histological types. Also, there have been several reports about PGP9.5 overexpression in non-SCC tumors including pancreatic cancer (19) and lymphomas (20). Actually, these data showed that overexpression of PGP9.5 did not differ with histological type or pathological stage of pancreatic cancer (19) and lymphoma (20).
Although there was a correlation between the PGP9.5 protein expression by immunohistochemistry and PGP9.5 mRNA expression by RT-PCR, the positive rate was higher in immunohistochemistry. Hence immunohistochemistry seems to be more sensitive than our RT-PCR. However, our RT-PCR assay might be a more conventional method and has advantages with regard to time and cost. Biopsy specimens can be used in future studies. Also, detection of PGP9.5 gene expression will be a useful diagnostic tool in lung cancer using the microarray method.
Our results showed that expression of PGP9.5 were closely associated with advanced T-stages of NSCLC, but not with N-stages. Hence PGP9.5 expression might be more correlated with tumor invasion than with tumor metastasis. Documentation of mediastinal invasion (>T3) is important because it is a major prognostic variable (21). Although further study of the PGP9.5 gene will be needed to elucidate the mechanism of deubiquitination pathway downstream of the gene, it may provide unique insights into the molecular mechanism of PGP9.5 overexpression in lung cancer development and invasion.
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Acknowledgement |
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The authors thank Ms Miyazaki for excellent technical assistance.
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FOOTNOTES |
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+ For reprints and all correspondence: Hidefumi Sasaki, Department of Surgery II, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. E-mail: hisasaki@med.nagoya-cu.ac.jp
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Received May 9, 2001; accepted August 10, 2001.
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