Department of Surgery I, Gunma University Faculty of Medicine, Gunma, Japan
Received 28 March 2003; revised 22 October 2003; accepted 6 November 2003
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ABSTRACT |
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Cyclin B1 has an important role in the G2M phase transition of the cell cycle. Wee1 delays mitosis by suppressing the activity of the Cyclin B1/cdc2 complex. The objective of the present study was to elucidate the clinicopathological and prognostic significance of Cyclin B1 and Wee1 expression in non-small-cell lung cancer (NSCLC).
Patients and methods:
An immunohistochemical assessment of Cyclin B1 and Wee1 expression was performed in 79 patients with NSCLC.
Results:
The expression of Cyclin B1 was correlated with differentiation (P = 0.0423) and vascular invasion (P = 0.001). Patients with overexpression of Cyclin B1 had higher mean values for both the Ki-67 proliferative index (Ki-Index) (P <0.0001) and proliferating cell nuclear antigen labeling index (PCNA-LI) (P <0.0001), and a poorer prognosis (P = 0.0068). Patients lacking expression of Wee1 had a higher recurrence rate (P = 0.0084) and a poorer prognosis (P = 0.0457), and tended to have higher Ki-Index and PCNA-LI values. Multivariate analysis suggested that both Cyclin B1 (P = 0.0244) and Wee1 (P = 0.0444) expression were significant prognostic factors.
Conclusions:
These findings suggest that Cyclin B1 expression could be a significant prognostic parameter in NSCLC. The loss of Wee1 expression may have a potential role in promoting tumor progression and may be a significant prognostic indicator in NSCLC.
Key words: cell cycle, Cyclin B1, differential PCR, immunohistochemistry, non-small-cell lung cancer, Wee1
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Introduction |
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We carried out immunohistochemical analysis to assess the clinicopathological and prognostic significance, and the interrelationship, of Cyclin B1 and Wee1 expression. Furthermore, we examined by differential PCR analysis whether patients with non-small-cell lung cancer (NSCLC) have amplification or deletion of the Cyclin B1 and Wee1 genes.
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Patients and methods |
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Immunohistochemistry
Immunohistochemical staining was performed according to a previously described method [11]. The tissue sections were placed in 10 mM citrate buffer (pH 6.0 for Cyclin B1 and Ki-67, pH 7.0 for Wee1) and heated in an autoclave oven. The sections were incubated with either normal rabbit serum for the Cyclin B1, Ki-67 and proliferating cell nuclear antigen (PCNA) samples, or 4% Block Ace Solution (Dainippon Pharmaceutical Corp., Osaka, Japan) for the Wee1 samples. The sections were incubated with anti-Cyclin B1 monoclonal antibody (Novocastra Laboratories, Newcastle-upon-Tyne, UK) at a dilution of 1:200, and with anti-Wee1 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Ki-67 monoclonal antibody (DAKO, Glostrup, Denmark) and anti-PCNA monoclonal antibody (DAKO) all at a dilution of 1:100.
When >15% of tumor cells were immunoreactive [6], the sample was regarded as Cyclin B1-positive. The Wee1 labeling index defined the percentage of nuclear-stained tumor cells, and was classified as follows: 0 = <10%, 1+ = 1030%, 2+ = 3050% and 3+ = >50% of cell staining. Because this division was considered the most reliable prognostic indicator, we regarded a specimen with a score 0 or 1+ as negative. The Ki-67 proliferative index (Ki-Index) and PCNA labeling index (PCNA-LI) were defined as the percentages of nuclear-stained tumor cells.
Differential PCR analysis for the expression of Cyclin B1 and Wee1 genes
We studied 18 of the 79 patients whose specimens were subjected to immunohistochemical analysis. Total DNA was extracted from each fresh-frozen human lung tumor tissue and normal tissue using a DNeasy Tissue Kit (Qiagen, Valencia, CA, USA). For differential PCR analysis, a 150-bp fragment of the Cyclin B1 gene was co-amplified with an 82-bp fragment of the interferon- (IFNG) gene as an internal control [12]. Moreover, the same analysis was performed for a 440-bp fragment of the Wee1 gene and the IFNG gene. Specific primers used were as follows: 5'-CCTGCAAATGCCTGGTTTAT-3' and 5'-CATGCTTCGATGTGGCATAC-3' for Cyclin B1; 5'-ACTGATAGAATCCAGTTTGC-3' and 5'-ATGCAATGCCTACAAAGTGC-3' for Wee1 [13]; and 5'-GCAGAGCCAAATTGTCTCCT-3' and 5'-GGTCTCCACACTCTTTTGGA-3' for IFNG [12]. Differential PCR was performed by 25 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min.
The PCR products were electrophoresed in 3% agarose gel with ethidium bromide and autoradiographed by Fluor-S MAX2 MultiImager (Bio-Rad, Hercules, CA, USA). Analysis of gene density was performed using Quantity One v. 4.2.1 software (Bio-Rad). The ratio of the total target gene density relative to the total control gene density was calculated for each sample. Furthermore, using this result we analyzed the ratio of lung tumor tissue relative to normal tissue (T/N ratio) in each patient.
Statistical analysis
Statistical analysis was performed according to a previously described method [11], and Coxs proportional hazards model for risk ratio was used to assess the results of multivariate survival analysis. Differences were considered to be statistically significant at P <0.05.
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Results |
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Discussion |
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Similar to a previous study [16], our data also indicate that patients with NSCLC have no amplification or deletion of the Cyclin B1 gene. This result suggests that Cyclin B1 expression at the protein level may be regulated by other mechanisms, for example transcription, DNA methylation or translation, and not by gene expression at the DNA level. The activity of the Cyclin B1/cdc2 complex is negatively controlled by Wee1 kinase activity [8]. Thus, loss of Wee1 confers an advantage to neoplastic cells by allowing faster progression through the cell cycle. In this study, absence of Wee1 expression was observed immunohistochemically in about two-thirds of the NSCLC patients. The reduction in Wee1 expression was associated with a poorer prognosis and a higher recurrence rate. These tumors also tended to have higher Ki-Index and PCNA-LI values. This suggests that Wee1 may be only a weak suppressor of tumor progression in NSCLC. As for Cyclin B1, Wee1 expression at the protein level may be regulated by other mechanisms, and not by gene expression at the DNA level.
In conclusion, we suggest that assessment of Cyclin B1 expression could be useful for evaluating tumor progression and prognosis in NSCLC patients. Loss of Wee1 expression may play a role in promoting tumor aggressiveness, and may be a useful prognostic indicator.
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Acknowledgements |
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FOOTNOTES |
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