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p53AIP1 Expression can be a Prognostic Marker in Non-small Cell Lung Cancer
Clinical Oncology (2008) 20: 148e151 doi:10.1016/j.clon.2007.08.006
Original Article
p53AIP1 Expression can be a Prognostic Marker in Non-small Cell Lung Cancer S.-i. Yamashita*y, Y. Masuda*, N. Yoshida*, H. Matsuzaki*, T. Kurizaki*, Y. Haga*, S. Ikei*, M. Miyawakiy, Y. Kawanoy, M. Chujyoy, K. Kawaharay *Department of Surgery, National Hospital Organization Kumamoto Medical Center, 1-5 Ninomaru, Kumamoto, 860-0008, Japan yDepartment of Surgery II, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama, Ufu, Oita, 879-5593, Japan
ABSTRACT: Aims: p53AIP1 is a potential mediator of p53-dependent apoptosis that is mutated in many kinds of carcinoma. To investigate the role of this gene for non-small cell lung cancer, we compared the relationship between p53AIP1 gene expression and clinicopathological status of lung cancer. Materials and methods: Seventy samples from non-small cell lung cancer patients were obtained between 1997 and 2003. For quantitative evaluation of RNA expression by polymerase chain reaction (PCR) we used the Taqman PCR methods. Exons 5e8 of the p53 gene were analysed using PCResingle-stranded conformation polymorphism and sequenced for mutation analysis. Results: p53AIP1 gene expression levels in the lymph node metastasis-positive group were significantly lower than in the negative group (positive 35.1 ± 83.9; negative 64.2 ± 113.4; P [ 0.0486). The overall survival of the p53AIP1 low expression group was significantly worse than that of the p53AIP1 high expression group (P [ 0.0206). In the multivariate Cox proportional hazard model, p53AIP1 (P [ 0.0489) was the independent predictor for overall survival. When we investigated mutation analyses of the p53 gene, we could find several point mutations in 15.7% of all samples. However, there was no relationship between p53AIP1 expression and p53 status. Conclusions: These data suggest that the p53AIP1 gene is important for non-small cell lung cancer progression and may be a possible prognostic marker. Yamashita, S.-i. et al. (2008). Clinical Oncology 20, 148e151 ª 2007 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved. Key words: Lung cancer, p53AIP1, prognostic marker
Introduction Many genes implicated in apoptosis play an important role in tumorigenesis [1]. Several abnormalities of genes were reported as prognostic markers of non-small cell lung cancer, such as p53 [2]. However, these processes are complex and still remain unclear. Abnormal expression of p53 is frequently reported in a variety of cancers [3]. p53 mutations are generally more common in smokers than in non-smokers and an excess of G to T transversions of p53 has been described as a molecular signature of tobacco smoke mutagens in smoking-associated lung cancers. There are also mutational hotspots (codons 157, 158, 245, 248 and 273) in the p53 gene in lung cancer [4]. Several reports have shown that p53 expression is a prognostic marker in non-small cell lung cancer [2]. p53 protein is a tumour suppressor gene and mediates cell cycle arrest or programmed cell death [5,6]. p53 mediated these events were triggered through transactivation of specific genes, including p21, GADD45, cyclin G1, Bax and fas [7,8]. 0936-6555/08/200148þ04 $35.00/0
Recently, p53AIP1, which is a new potential mediator of p53-dependent apoptosis, was reported [9]. p53AIP1 is not normally expressed in any tissues except thymus, but it was induced when Ser-46 of p53 was phosphorylated after severe DNA damage [9,10]. It is interesting to evaluate the expression of the p53AIP1 gene in primary non-small cell lung cancer. Here we show the expression of this gene in non-small cell lung cancer and normal lung tissue, and that p53AIP1 may be a possible prognostic marker.
Materials and Methods Patients and Samples The study was approved by the Institutional Review Board of the National Hospital Organization Kumamoto Medical Center (Kumamoto, Japan) and all patients completed informed consent forms. Seventy samples from non-small cell lung cancer patients were obtained at the National Hospital Organization Kumamoto Medical
ª 2007 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.
p53AIP1 EXPRESSION IN NON-SMALL CELL LUNG CANCER
Center (Kumamoto, Japan) between May 1997 and September 2003. The samples were histologically diagnosed for primary non-small cell lung cancer. None of all cases had received radiation therapy or chemotherapy before surgery. Adjacent normal lung tissue was also taken from all cases. Tissue specimens were frozen immediately with RNA later and stored at 80 C until RNA extraction. RNA from the tissue samples was prepared using TRIzol reagents (Invitrogen). To evaluate the consumption of cigarettes, a smoking index was used: cigarette consumption per day multiplied by smoking years. Referring to the index, smokers were divided into two groups: heavy smokers with indices R 400, and light ones !400.
Quantitative Polymerase Chain Reaction Analysis For quantitative evaluation of RNA expression by polymerase chain reaction (PCR) we used the Taqman PCR methods, as previously reported [11]. The p53AIP1 gene was amplified by the set of primers: reverse ggggacttctc aggtcgtgt, forward tggacttcttcatgccccga. The p53AIP1 gene internal probe is ttgcggtgcgagtcgtggaagtaa. The PCR amplification conditions were one cycle of 50 C, 2 min, and 95 C, 10 min followed by 50 cycles of 95 C, 15 sec and 60 C, 1 min. A measured value was calculated by comparative threshold cycle (Ct) methods [11] and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene amplification was used as the control. All reactions were duplicated. The amount of p53AIP1 mRNA was expressed as n-fold GAPDH mRNA and levels compared relative to adjacent normal lung tissues. A tumour/normal ratio of p53AIP1 mRNA expression greater than 1 was identified as high expression.
Polymerase Chain ReactioneSingle-stranded Conformation Polymorphism and Sequencing Exons 5e8 of the p53 gene were analysed using the PCResingle-stranded conformation polymorphism (SSCP) method [12]. Coding sequences including exoneintron boundaries were amplified with the primers and PCR conditions described previously [12]. After confirming PCR products as bands on 1e2% agarose gels, SSCP was carried out at 10 C, with GeneGel Excel 2.5/24 and analysed using a DNA silver staining kit (Amersham, Biosiences, USA) according to the instruction manual. When mutations were detected by comparison of fluorescence labelled SSCP findings for tumours and corresponding normal lung tissues, genomic DNAs were amplified with non-labelled primers under the same PCR conditions and products were electrophoresed in polyacrylamide gels (Gene Mutation Analysis gel, Elchrom Scientific, Switzerland). DNA bands with apparent mutation shifts were cored out under ultraviolet light and eluted DNAs were re-amplified with nonlabelled primer again and sequenced using a BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems, California, USA).
149
Fig. 1 e p53AIP1 gene expression by reverse transcriptionpolymerase chain reaction (PCR) revealed a 59 bp product. Realtime PCR products by a second PCR. M, size marker; lane 1, negative control; lanes 2e9, sample.
Statistical Analysis All statistical analyses were carried out using StatView J5.0 (SAS Institute). The different variables of the tumours and normal tissues were analysed with chi-squared tests or Fisher’s exact tests. Overall survival was analysed using the KaplaneMeier method and evaluated using the Log-rank test. Statistical significant differences were considered at P ! 0.05.
Table 1 e Correlation between clinicopathological status and p53AIP1 expression
Characteristics
n
p53AIP1 expression (mean SD)
Age !70 years R 70 years
34 36
98.2 242.1 51.7 92.6
0.8359
Gender Male Female
53 17
55.6 141.9 79.2 192.9
0.1952
Smoking index !400 R400
30 40
68.6 130.1 45.5 83.3
0.9102
Primary tumour T1 T2 T3
35 30 5
63.4 122.1 58.9 99.4 36.1 75.4
0.547
Lymph node metastasis N negative N positive
49 21
64.2 113.4 35.1 83.9
0.0486
Pathological stage IA IB IIA IIB IIIA
37 8 7 3 15
56.3 113.7 105.1 126.4 120.5 127.5 4.1 7.2 6.7 14.6
0.0248
Histological type Adenocarcinoma Squamous cell carcinoma Others
38 26 6
60.9 123.1 57.6 89.9 41.9 62.5
0.2848
P value
150
CLINICAL ONCOLOGY Table 3 e Correlation between smoking index and p53 mutation status, p53AIP1 expression n
Fig. 2 e Overall survival curves according to p53AIP1 gene expression. The differences are statistically significant (P ¼ 0.0206). Number of patients in each group: high 43; low 27.
Results p53AIP1 expression in primary non-small cell lung cancer was evaluated by real-time PCR. We designed a reverse transcription-PCR assay of the p53AIP1 gene in non-small cell lung cancer. Figure 1 shows the 59 bp amplification of the p53AIP1 gene and the GAPDH gene was co-amplified as the control. All 70 samples were studied with the paired histopathologically normal lung tissues, which were far from the tumour margin. Table 1 shows the correlation between clinicopathological status and p53AIP1 gene expression. Although it shows no relationship between p53AIP1 gene expression and age, tumour size, histological type, smoking index, p53AIP1 gene expression levels in the lymph node-positive group were significantly lower than in the negative group (positive 35.1 83.9; negative 64.2 113.4; P ¼ 0.0486). Furthermore, pathological stage was significantly related to p53AIP1 expression (P ¼ 0.0248). Figure 2 shows the overall survival curves by KaplaneMeier analysis for the patients with non-small cell lung cancer classified according to p53AIP1 expression (high tumour/ normal ratio R 1; low, !1). The patients in the high p53AIP1 expression group had better prognosis than patients in the low expression group (P ¼ 0.0206). The median follow-up period was 5.4 years (range 1.2e8.4 years). In the Table 2 e Multivariate Cox proportional hazard model Characteristics T; primary tumour (T1eT3) N; lymph node metastases (positive vs negative) Histological type (adenocarcinoma vs squamous cell carcinoma) p53AIP1 expression (low vs high) Chemotherapy (with vs without) CI, confidence interval.
Odds ratio
95% CI
P
0.997 4.211
0.378e2.631 0.9957 1.115e12.325 0.0254
0.521
0.147e1.844
0.5045
2.523 0.673
1.004e6.339 0.267e1.698
0.0489 0.673
p53 mutation (%)
p53AIP1 expression (%)
Smoking index !400 30 R400 40
3 (10) 8 (20)
18 (60) 25 (62.5)
Total P
11 (15.7) 0.268
43 (61.4) 0.999
70
P
0.1337
multivariate Cox proportional hazard model analysis using tumour size, lymph node metastasis, histological type, with or without chemotherapy and p53AIP1 expression, both lymph node metastasis (P ¼ 0.0254) and p53AIP1 (P ¼ 0.0489) were independent predictors for overall survival (Table 2). When we examined p53 mutation status or p53AIP1 expression according to the smoking index, we could not find a significant relationship between the smoking index and p53 mutation (P ¼ 0.268) or p53AIP1 expression (P ¼ 0.999), even if the mutation rate in the heavy smokers was twice that of the light smokers (Table 3). The p53 mutation spectrum from heavy smokers was dominated by the presence of G to T transversions (4/8 samples) and from light smokers by a G to A transition (2/3 samples). Furthermore, the p53 mutation status was not related to p53AIP1 expression (P ¼ 0.1337).
Discussion The molecular mechanism of tumour progression and apoptosis is still unclear. A recent study reports that p53AIP1 is one of the candidates of p53-mediated apoptosis, and phosphorylation of Ser-46 in the p53 gene is a key role of apoptotic signalling under the p53-induced pathway [9]. From these results, we speculated that p53AIP1 in primary non-small cell lung cancer has the potential of tumour suppression. This gene is not normally expressed in any tissues except thymus, and there was no report about p53AIP1 expression in any type of cancer. Moreover, the p53AIP1 gene was induced by adenoviral p53 administration in a lung cancer cell line, followed by a rapid decrease and apoptosis [13]. In order to compare a very small amount of p53AIP1 expression in tumour or normal tissue, we used a real-time quantitative PCR method in this study. This gene is induced by DNA damaging agents, for example ultraviolet or gamma irradiation [9]. Although smoking is a possible DNA damaging agent and a cause of lung cancer, we could not find any relationship between p53AIP1 expression and the smoking index (Brinkman index). Our data showed that p53AIP1 gene expression was significantly lower in lymph node-positive patients than in negative patients. This result may suggest the possibility of p53AIP1 in a key role in lymph node metastasis and may lead to the close correlation between this gene and pathological stage, even if p53AIP1 gene expression is gradually decreased from T1 to T3, but not significant. We could not find histological type-specific expression of the p53AIP1 gene in this study. Further studies to clarify the tissue or
p53AIP1 EXPRESSION IN NON-SMALL CELL LUNG CANCER
histological type specificity should be carried out. p53AIP1 is a mediator of the normal apoptotic pathway related to the downstream of p53. In the present study, we examined the p53 status, especially exons 5e8, which are frequently reported as a mutational hotspot [14]. However, there was no relationship between p53 mutation and p53AIP1 expression. Although smoking status was not closely related to the p53 mutation rate in our study, the possibility of a detection bias for mutation might exist because the total mutation rate was relatively low compared with a previous report [4]. Our results suggest that p53AIP1 may be independent of p53 activity. As p53AIP1 is a direct mediator of apoptosis, p53AIP1 can induce apoptosis of various cancer cells regardless of their p53 status, i.e. p53AIP1 induced apoptosis effectively in both p53-sensitive and p53-resistant cancer cell lines [10]. p53AIP1 interacts with bcl-2 at mitochondria, which is an anti-apoptotic effector against a broad spectrum of apoptosis stimulators such as anticancer drugs. p53AIP1 induces the down-regulation of the mitochondrial membrane potential, which might be essential for p53- and p53AIP1-inducible apoptosis, and this regulation might be determined by the physical and functional interaction between bcl-2 and p53AIP1 [10]. If p53AIP1 increases in tumour cells with or without several stimulations, lead to overwhelm bcl-2 function to work as anti-apoptosis, the tumour cells apoptosis occur as result of p53AIP1 activation. Several predictors, such as nodal involvement, tumour stage and p53 were reported. However, the relationship between p53AIP1 and prognosis was unclear. Although the study population was heterogeneous with various disease stages and histology in the multivariate analysis, including T factor, nodal metastasis, histological type, with or without chemotherapy and p53AIP1 gene expression, p53AIP1 was an independent prognostic factor for overall survival with the same potential as lymph node metastasis. p53AIP1 is induced after p53 Ser-46 phosphorylation. However, we did not examine the p53 Ser-46 phosphorylation levels in this study. Our results will require further investigation in the light of p53 Ser-46 phosphorylation levels. p53AIP1 expression in non-small cell lung cancer cells before chemotherapy can make a contribution as an independent predictor of the effect of chemotherapy. On the other hand, it was reported that p53AIP1 has three subtypes of transcript (i.e. a, b and g) [9]. We set the primers between exon 1 and 2 in order to amplify all three subtypes of p53AIP1. The function of these subtypes still remains unclear. In this study, although we detected all the subtypes amplified, we do not know which subtypes account for the PCR product. We tried quantitative analyses to elucidate the relationships between these isoforms and tumour grade, but any relationships were found in all isoforms (data not shown). Therefore, the important thing for tumour progression may not be so much the function of the isoforms but the total amount of p53AIP1. In conclusion, although the sample size was small, our study has shown p53AIP1 gene expression in non-small cell lung cancer as possibly an independent prognostic factor and the expression of p53AIP1 mRNA may predict a response
151
to chemotherapy in advanced non-small cell lung cancer. Further investigations of p53AIP1 may give new speculation to this possibility. Acknowledgements. This study was supported by a grant for National Hospital Clinical Research from the Ministry of Health, Labour and Welfare of Japan. We thank Dr Yuji Onodera, BML research company, for technical support and Ms Yoko Miyanari, Department of Surgery II, Oita University Faculty of Medicine. Author for correspondence: S.-i. Yamashita, Oita University, Faculty of Medicine, Department of Surgery II, 1-1 Idaigaoka, Hasama, Ufu, Oita 879-5593, Japan. Tel: þ81-97-586-5854; Fax: þ81-97-586-6449; E-mail: [email protected] Received 17 June 2006; received in revised form 14 May 2007; accepted 7 August 2007
References 1 Harris CC, Hollstein M. Clinical implications of the p53 tumorsuppressor gene. N Engl J Med 1993;329:1318e1327. 2 Mitsudomi T, Hamajima N, Ogawa M, Takahashi T. Prognostic significance of p53 alterations in patients with non-small cell lung cancer: a meta-analysis. Clin Cancer Res 2000;6:4055e4063. 3 Steele RJ, Thompson AM, Hall PA, Lane DP. The p53 tumour suppressor gene. Br J Surg 1998;85:1460e1467. 4 Pfeifer GP, Denissenko MF, Olivier M, Tretyakova N, Hecht SS, Hainaut P. Tobacco smoke carcinogens, DNA damage and p53 mutations in smoking-associated cancers. Oncogene 2002;21: 7435e7451. 5 Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature 2000;408:307e310. 6 Sionov RV, Haupt Y. The cellular response to p53: the decision between life and death. Oncogene 1999;18:6145e6157. 7 Asker C, Wiman KG, Selivanova G. p53-induced apoptosis as a safeguard against cancer. Biochem Biophys Res Commun 1999; 265:1e6. 8 Bates S, Vousden KH. Mechanisms of p53-mediated apoptosis. Cell Mol Life Sci 1999;55:28e37. 9 Oda K, Arakawa H, Tanaka T, et al. p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser-46-phosphorylated p53. Cell 2000;102:849e862. 10 Matsuda K, Yoshida K, Taya Y, Nakamura K, Nakamura Y, Arakawa H. p53AIP1 regulates the mitochondrial apoptotic pathway. Cancer Res 2002;62:2883e2889. 11 Aarskog NK, Vedeler CA. Real-time quantitative polymerase chain reaction. A new method that detects both the peripheral myelin protein 22 duplication in Charcot-Marie-Tooth type 1A disease and the peripheral myelin protein 22 deletion in hereditary neuropathy with liability to pressure palsies. Hum Genet 2000;107:494e498. 12 Matsuzoe D, Hideshima T, Ohshima K, Kawahara K, Shirakusa T, Kimura A. Discrimination of double primary lung cancer from intrapulmonary metastasis by p53 gene mutation. Br J Cancer 1999;79:1549e1552. 13 Ohtani S, Kagawa S, Tango Y, et al. Quantitative analysis of p53-targeted gene expression and visualization of p53 transcriptional activity following intratumoral administration of adenoviral p53 in vivo. Mol Cancer Ther 2004;3:93e100. 14 Gao WM, Mady HH, Yu GY, et al. Comparison of p53 mutations between adenocarcinoma and squamous cell carcinoma of the lung: unique spectra involving G to A transitions and G to T transversions in both histologic types. Lung Cancer 2003;40:141e150.
Original Article
p53AIP1 Expression can be a Prognostic Marker in Non-small Cell Lung Cancer S.-i. Yamashita*y, Y. Masuda*, N. Yoshida*, H. Matsuzaki*, T. Kurizaki*, Y. Haga*, S. Ikei*, M. Miyawakiy, Y. Kawanoy, M. Chujyoy, K. Kawaharay *Department of Surgery, National Hospital Organization Kumamoto Medical Center, 1-5 Ninomaru, Kumamoto, 860-0008, Japan yDepartment of Surgery II, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama, Ufu, Oita, 879-5593, Japan
ABSTRACT: Aims: p53AIP1 is a potential mediator of p53-dependent apoptosis that is mutated in many kinds of carcinoma. To investigate the role of this gene for non-small cell lung cancer, we compared the relationship between p53AIP1 gene expression and clinicopathological status of lung cancer. Materials and methods: Seventy samples from non-small cell lung cancer patients were obtained between 1997 and 2003. For quantitative evaluation of RNA expression by polymerase chain reaction (PCR) we used the Taqman PCR methods. Exons 5e8 of the p53 gene were analysed using PCResingle-stranded conformation polymorphism and sequenced for mutation analysis. Results: p53AIP1 gene expression levels in the lymph node metastasis-positive group were significantly lower than in the negative group (positive 35.1 ± 83.9; negative 64.2 ± 113.4; P [ 0.0486). The overall survival of the p53AIP1 low expression group was significantly worse than that of the p53AIP1 high expression group (P [ 0.0206). In the multivariate Cox proportional hazard model, p53AIP1 (P [ 0.0489) was the independent predictor for overall survival. When we investigated mutation analyses of the p53 gene, we could find several point mutations in 15.7% of all samples. However, there was no relationship between p53AIP1 expression and p53 status. Conclusions: These data suggest that the p53AIP1 gene is important for non-small cell lung cancer progression and may be a possible prognostic marker. Yamashita, S.-i. et al. (2008). Clinical Oncology 20, 148e151 ª 2007 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved. Key words: Lung cancer, p53AIP1, prognostic marker
Introduction Many genes implicated in apoptosis play an important role in tumorigenesis [1]. Several abnormalities of genes were reported as prognostic markers of non-small cell lung cancer, such as p53 [2]. However, these processes are complex and still remain unclear. Abnormal expression of p53 is frequently reported in a variety of cancers [3]. p53 mutations are generally more common in smokers than in non-smokers and an excess of G to T transversions of p53 has been described as a molecular signature of tobacco smoke mutagens in smoking-associated lung cancers. There are also mutational hotspots (codons 157, 158, 245, 248 and 273) in the p53 gene in lung cancer [4]. Several reports have shown that p53 expression is a prognostic marker in non-small cell lung cancer [2]. p53 protein is a tumour suppressor gene and mediates cell cycle arrest or programmed cell death [5,6]. p53 mediated these events were triggered through transactivation of specific genes, including p21, GADD45, cyclin G1, Bax and fas [7,8]. 0936-6555/08/200148þ04 $35.00/0
Recently, p53AIP1, which is a new potential mediator of p53-dependent apoptosis, was reported [9]. p53AIP1 is not normally expressed in any tissues except thymus, but it was induced when Ser-46 of p53 was phosphorylated after severe DNA damage [9,10]. It is interesting to evaluate the expression of the p53AIP1 gene in primary non-small cell lung cancer. Here we show the expression of this gene in non-small cell lung cancer and normal lung tissue, and that p53AIP1 may be a possible prognostic marker.
Materials and Methods Patients and Samples The study was approved by the Institutional Review Board of the National Hospital Organization Kumamoto Medical Center (Kumamoto, Japan) and all patients completed informed consent forms. Seventy samples from non-small cell lung cancer patients were obtained at the National Hospital Organization Kumamoto Medical
ª 2007 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.
p53AIP1 EXPRESSION IN NON-SMALL CELL LUNG CANCER
Center (Kumamoto, Japan) between May 1997 and September 2003. The samples were histologically diagnosed for primary non-small cell lung cancer. None of all cases had received radiation therapy or chemotherapy before surgery. Adjacent normal lung tissue was also taken from all cases. Tissue specimens were frozen immediately with RNA later and stored at 80 C until RNA extraction. RNA from the tissue samples was prepared using TRIzol reagents (Invitrogen). To evaluate the consumption of cigarettes, a smoking index was used: cigarette consumption per day multiplied by smoking years. Referring to the index, smokers were divided into two groups: heavy smokers with indices R 400, and light ones !400.
Quantitative Polymerase Chain Reaction Analysis For quantitative evaluation of RNA expression by polymerase chain reaction (PCR) we used the Taqman PCR methods, as previously reported [11]. The p53AIP1 gene was amplified by the set of primers: reverse ggggacttctc aggtcgtgt, forward tggacttcttcatgccccga. The p53AIP1 gene internal probe is ttgcggtgcgagtcgtggaagtaa. The PCR amplification conditions were one cycle of 50 C, 2 min, and 95 C, 10 min followed by 50 cycles of 95 C, 15 sec and 60 C, 1 min. A measured value was calculated by comparative threshold cycle (Ct) methods [11] and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene amplification was used as the control. All reactions were duplicated. The amount of p53AIP1 mRNA was expressed as n-fold GAPDH mRNA and levels compared relative to adjacent normal lung tissues. A tumour/normal ratio of p53AIP1 mRNA expression greater than 1 was identified as high expression.
Polymerase Chain ReactioneSingle-stranded Conformation Polymorphism and Sequencing Exons 5e8 of the p53 gene were analysed using the PCResingle-stranded conformation polymorphism (SSCP) method [12]. Coding sequences including exoneintron boundaries were amplified with the primers and PCR conditions described previously [12]. After confirming PCR products as bands on 1e2% agarose gels, SSCP was carried out at 10 C, with GeneGel Excel 2.5/24 and analysed using a DNA silver staining kit (Amersham, Biosiences, USA) according to the instruction manual. When mutations were detected by comparison of fluorescence labelled SSCP findings for tumours and corresponding normal lung tissues, genomic DNAs were amplified with non-labelled primers under the same PCR conditions and products were electrophoresed in polyacrylamide gels (Gene Mutation Analysis gel, Elchrom Scientific, Switzerland). DNA bands with apparent mutation shifts were cored out under ultraviolet light and eluted DNAs were re-amplified with nonlabelled primer again and sequenced using a BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems, California, USA).
149
Fig. 1 e p53AIP1 gene expression by reverse transcriptionpolymerase chain reaction (PCR) revealed a 59 bp product. Realtime PCR products by a second PCR. M, size marker; lane 1, negative control; lanes 2e9, sample.
Statistical Analysis All statistical analyses were carried out using StatView J5.0 (SAS Institute). The different variables of the tumours and normal tissues were analysed with chi-squared tests or Fisher’s exact tests. Overall survival was analysed using the KaplaneMeier method and evaluated using the Log-rank test. Statistical significant differences were considered at P ! 0.05.
Table 1 e Correlation between clinicopathological status and p53AIP1 expression
Characteristics
n
p53AIP1 expression (mean SD)
Age !70 years R 70 years
34 36
98.2 242.1 51.7 92.6
0.8359
Gender Male Female
53 17
55.6 141.9 79.2 192.9
0.1952
Smoking index !400 R400
30 40
68.6 130.1 45.5 83.3
0.9102
Primary tumour T1 T2 T3
35 30 5
63.4 122.1 58.9 99.4 36.1 75.4
0.547
Lymph node metastasis N negative N positive
49 21
64.2 113.4 35.1 83.9
0.0486
Pathological stage IA IB IIA IIB IIIA
37 8 7 3 15
56.3 113.7 105.1 126.4 120.5 127.5 4.1 7.2 6.7 14.6
0.0248
Histological type Adenocarcinoma Squamous cell carcinoma Others
38 26 6
60.9 123.1 57.6 89.9 41.9 62.5
0.2848
P value
150
CLINICAL ONCOLOGY Table 3 e Correlation between smoking index and p53 mutation status, p53AIP1 expression n
Fig. 2 e Overall survival curves according to p53AIP1 gene expression. The differences are statistically significant (P ¼ 0.0206). Number of patients in each group: high 43; low 27.
Results p53AIP1 expression in primary non-small cell lung cancer was evaluated by real-time PCR. We designed a reverse transcription-PCR assay of the p53AIP1 gene in non-small cell lung cancer. Figure 1 shows the 59 bp amplification of the p53AIP1 gene and the GAPDH gene was co-amplified as the control. All 70 samples were studied with the paired histopathologically normal lung tissues, which were far from the tumour margin. Table 1 shows the correlation between clinicopathological status and p53AIP1 gene expression. Although it shows no relationship between p53AIP1 gene expression and age, tumour size, histological type, smoking index, p53AIP1 gene expression levels in the lymph node-positive group were significantly lower than in the negative group (positive 35.1 83.9; negative 64.2 113.4; P ¼ 0.0486). Furthermore, pathological stage was significantly related to p53AIP1 expression (P ¼ 0.0248). Figure 2 shows the overall survival curves by KaplaneMeier analysis for the patients with non-small cell lung cancer classified according to p53AIP1 expression (high tumour/ normal ratio R 1; low, !1). The patients in the high p53AIP1 expression group had better prognosis than patients in the low expression group (P ¼ 0.0206). The median follow-up period was 5.4 years (range 1.2e8.4 years). In the Table 2 e Multivariate Cox proportional hazard model Characteristics T; primary tumour (T1eT3) N; lymph node metastases (positive vs negative) Histological type (adenocarcinoma vs squamous cell carcinoma) p53AIP1 expression (low vs high) Chemotherapy (with vs without) CI, confidence interval.
Odds ratio
95% CI
P
0.997 4.211
0.378e2.631 0.9957 1.115e12.325 0.0254
0.521
0.147e1.844
0.5045
2.523 0.673
1.004e6.339 0.267e1.698
0.0489 0.673
p53 mutation (%)
p53AIP1 expression (%)
Smoking index !400 30 R400 40
3 (10) 8 (20)
18 (60) 25 (62.5)
Total P
11 (15.7) 0.268
43 (61.4) 0.999
70
P
0.1337
multivariate Cox proportional hazard model analysis using tumour size, lymph node metastasis, histological type, with or without chemotherapy and p53AIP1 expression, both lymph node metastasis (P ¼ 0.0254) and p53AIP1 (P ¼ 0.0489) were independent predictors for overall survival (Table 2). When we examined p53 mutation status or p53AIP1 expression according to the smoking index, we could not find a significant relationship between the smoking index and p53 mutation (P ¼ 0.268) or p53AIP1 expression (P ¼ 0.999), even if the mutation rate in the heavy smokers was twice that of the light smokers (Table 3). The p53 mutation spectrum from heavy smokers was dominated by the presence of G to T transversions (4/8 samples) and from light smokers by a G to A transition (2/3 samples). Furthermore, the p53 mutation status was not related to p53AIP1 expression (P ¼ 0.1337).
Discussion The molecular mechanism of tumour progression and apoptosis is still unclear. A recent study reports that p53AIP1 is one of the candidates of p53-mediated apoptosis, and phosphorylation of Ser-46 in the p53 gene is a key role of apoptotic signalling under the p53-induced pathway [9]. From these results, we speculated that p53AIP1 in primary non-small cell lung cancer has the potential of tumour suppression. This gene is not normally expressed in any tissues except thymus, and there was no report about p53AIP1 expression in any type of cancer. Moreover, the p53AIP1 gene was induced by adenoviral p53 administration in a lung cancer cell line, followed by a rapid decrease and apoptosis [13]. In order to compare a very small amount of p53AIP1 expression in tumour or normal tissue, we used a real-time quantitative PCR method in this study. This gene is induced by DNA damaging agents, for example ultraviolet or gamma irradiation [9]. Although smoking is a possible DNA damaging agent and a cause of lung cancer, we could not find any relationship between p53AIP1 expression and the smoking index (Brinkman index). Our data showed that p53AIP1 gene expression was significantly lower in lymph node-positive patients than in negative patients. This result may suggest the possibility of p53AIP1 in a key role in lymph node metastasis and may lead to the close correlation between this gene and pathological stage, even if p53AIP1 gene expression is gradually decreased from T1 to T3, but not significant. We could not find histological type-specific expression of the p53AIP1 gene in this study. Further studies to clarify the tissue or
p53AIP1 EXPRESSION IN NON-SMALL CELL LUNG CANCER
histological type specificity should be carried out. p53AIP1 is a mediator of the normal apoptotic pathway related to the downstream of p53. In the present study, we examined the p53 status, especially exons 5e8, which are frequently reported as a mutational hotspot [14]. However, there was no relationship between p53 mutation and p53AIP1 expression. Although smoking status was not closely related to the p53 mutation rate in our study, the possibility of a detection bias for mutation might exist because the total mutation rate was relatively low compared with a previous report [4]. Our results suggest that p53AIP1 may be independent of p53 activity. As p53AIP1 is a direct mediator of apoptosis, p53AIP1 can induce apoptosis of various cancer cells regardless of their p53 status, i.e. p53AIP1 induced apoptosis effectively in both p53-sensitive and p53-resistant cancer cell lines [10]. p53AIP1 interacts with bcl-2 at mitochondria, which is an anti-apoptotic effector against a broad spectrum of apoptosis stimulators such as anticancer drugs. p53AIP1 induces the down-regulation of the mitochondrial membrane potential, which might be essential for p53- and p53AIP1-inducible apoptosis, and this regulation might be determined by the physical and functional interaction between bcl-2 and p53AIP1 [10]. If p53AIP1 increases in tumour cells with or without several stimulations, lead to overwhelm bcl-2 function to work as anti-apoptosis, the tumour cells apoptosis occur as result of p53AIP1 activation. Several predictors, such as nodal involvement, tumour stage and p53 were reported. However, the relationship between p53AIP1 and prognosis was unclear. Although the study population was heterogeneous with various disease stages and histology in the multivariate analysis, including T factor, nodal metastasis, histological type, with or without chemotherapy and p53AIP1 gene expression, p53AIP1 was an independent prognostic factor for overall survival with the same potential as lymph node metastasis. p53AIP1 is induced after p53 Ser-46 phosphorylation. However, we did not examine the p53 Ser-46 phosphorylation levels in this study. Our results will require further investigation in the light of p53 Ser-46 phosphorylation levels. p53AIP1 expression in non-small cell lung cancer cells before chemotherapy can make a contribution as an independent predictor of the effect of chemotherapy. On the other hand, it was reported that p53AIP1 has three subtypes of transcript (i.e. a, b and g) [9]. We set the primers between exon 1 and 2 in order to amplify all three subtypes of p53AIP1. The function of these subtypes still remains unclear. In this study, although we detected all the subtypes amplified, we do not know which subtypes account for the PCR product. We tried quantitative analyses to elucidate the relationships between these isoforms and tumour grade, but any relationships were found in all isoforms (data not shown). Therefore, the important thing for tumour progression may not be so much the function of the isoforms but the total amount of p53AIP1. In conclusion, although the sample size was small, our study has shown p53AIP1 gene expression in non-small cell lung cancer as possibly an independent prognostic factor and the expression of p53AIP1 mRNA may predict a response
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to chemotherapy in advanced non-small cell lung cancer. Further investigations of p53AIP1 may give new speculation to this possibility. Acknowledgements. This study was supported by a grant for National Hospital Clinical Research from the Ministry of Health, Labour and Welfare of Japan. We thank Dr Yuji Onodera, BML research company, for technical support and Ms Yoko Miyanari, Department of Surgery II, Oita University Faculty of Medicine. Author for correspondence: S.-i. Yamashita, Oita University, Faculty of Medicine, Department of Surgery II, 1-1 Idaigaoka, Hasama, Ufu, Oita 879-5593, Japan. Tel: þ81-97-586-5854; Fax: þ81-97-586-6449; E-mail: [email protected] Received 17 June 2006; received in revised form 14 May 2007; accepted 7 August 2007
References 1 Harris CC, Hollstein M. Clinical implications of the p53 tumorsuppressor gene. N Engl J Med 1993;329:1318e1327. 2 Mitsudomi T, Hamajima N, Ogawa M, Takahashi T. Prognostic significance of p53 alterations in patients with non-small cell lung cancer: a meta-analysis. Clin Cancer Res 2000;6:4055e4063. 3 Steele RJ, Thompson AM, Hall PA, Lane DP. The p53 tumour suppressor gene. Br J Surg 1998;85:1460e1467. 4 Pfeifer GP, Denissenko MF, Olivier M, Tretyakova N, Hecht SS, Hainaut P. Tobacco smoke carcinogens, DNA damage and p53 mutations in smoking-associated cancers. Oncogene 2002;21: 7435e7451. 5 Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature 2000;408:307e310. 6 Sionov RV, Haupt Y. The cellular response to p53: the decision between life and death. Oncogene 1999;18:6145e6157. 7 Asker C, Wiman KG, Selivanova G. p53-induced apoptosis as a safeguard against cancer. Biochem Biophys Res Commun 1999; 265:1e6. 8 Bates S, Vousden KH. Mechanisms of p53-mediated apoptosis. Cell Mol Life Sci 1999;55:28e37. 9 Oda K, Arakawa H, Tanaka T, et al. p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser-46-phosphorylated p53. Cell 2000;102:849e862. 10 Matsuda K, Yoshida K, Taya Y, Nakamura K, Nakamura Y, Arakawa H. p53AIP1 regulates the mitochondrial apoptotic pathway. Cancer Res 2002;62:2883e2889. 11 Aarskog NK, Vedeler CA. Real-time quantitative polymerase chain reaction. A new method that detects both the peripheral myelin protein 22 duplication in Charcot-Marie-Tooth type 1A disease and the peripheral myelin protein 22 deletion in hereditary neuropathy with liability to pressure palsies. Hum Genet 2000;107:494e498. 12 Matsuzoe D, Hideshima T, Ohshima K, Kawahara K, Shirakusa T, Kimura A. Discrimination of double primary lung cancer from intrapulmonary metastasis by p53 gene mutation. Br J Cancer 1999;79:1549e1552. 13 Ohtani S, Kagawa S, Tango Y, et al. Quantitative analysis of p53-targeted gene expression and visualization of p53 transcriptional activity following intratumoral administration of adenoviral p53 in vivo. Mol Cancer Ther 2004;3:93e100. 14 Gao WM, Mady HH, Yu GY, et al. Comparison of p53 mutations between adenocarcinoma and squamous cell carcinoma of the lung: unique spectra involving G to A transitions and G to T transversions in both histologic types. Lung Cancer 2003;40:141e150.