The tumour suppressor and transcription factor p53 (TP53), a key regulator in preventing cancer formation,1,2 plays a vital role in cell cycling, growth, DNA repair, cell cycle arrest, and apoptosis.3 p53 mutations can be detected in many cancers and are associated with tumour progression, resistance to chemotherapy and poor prognosis.4 Recently, two related genes, p63 and p73, were identified, which share a high degree of sequence similarity in the DNA binding domain with p53 and transactivate p53-responsive genes causing cell cycle arrest and apoptosis.5,6 The present study demonstrates that p53, p63 and p73 have their own unique functions and each of them can express multiple mRNA variants due to multiple splicing and alternative promoters. To date, at least nine different p53 isoforms have been identified in humans, including p53, p53β, p53γ, Δ133p53, Δ133p53β, Δ133p53γ, Δ40p53, Δ40p53β, and Δ40p53γ. However, the function and expression of these isoforms are still not completely clear. In previous studies, these isoforms were differentially expressed in normal human tissue in a tissue-dependent manner.7 Furthermore, the expression of these isoforms in human cancers, such as breast cancer,8 neuroblastoma,9 acute myeloid leukaemia,10 and squamous cell carcinoma of the head and neck,11 suggests that they might be involved in tumour development or progression.
Renal cell carcinoma (RCC) is the most common malignancy in the adult kidney, with 30 000 new cases per year in US and 20 000 cases in the European Union.12 In China, the incidence of RCC is second only to bladder tumour in all malignant tumours in the urinary system. According to a new report, there are more than 23 000 new case of RCC per year in China, and the incidence is increasing rapidly due to the aging population and high smoking and obesity rates.13
p53 appears to be mutated in about 50% of many human cancers,14 while in RCC, there is a low incidence of p53 mutation.15-17 p53 mutation has been reported in 3%-33% of patients with RCC.18 Thus, a novel dominant mechanism of inactivation of p53 might be present in renal cells. In this study we analysed the expression patterns of p53 isoforms in RCC at the mRNA and protein levels and their associations with clinical and pathological factors to explore the mechanism of p53 isoforms in RCC.
Patients and specimens
Following approval by the Ethical Committee of Shandong Provincial Hospital, tumour samples were taken after informed consent from 41 patients with RCC, who underwent radical or partial nephrectomy from June 2007 to March 2008 in Shandong Provincial Hospital. The non-neoplastic renal tissue obtained from a region at least 2 cm distant from the tumour margin were also analysed for comparison. All tumour and normal tissues were confirmed by two pathologists. The samples were divided into three parts, 1/3 was fixed in formalin and embedded in paraffin, and the other 2/3 were frozen in liquid nitrogen and then stored at -80°C until used.
Forty-one patients, 29 men and 12 women, aged from 15 to 84 years old (mean 56 years) were all confirmed to be clear cell RCC by pathology according to the WHO (1998) classification and none of them underwent preoperative chemotherapy and/or radiation therapy. Patients were staged according to the 1997 TNM classification system and the nuclear grade of tumours was determined using the Fuhrman grading scheme.19
RNA and protein extraction
Total RNA was extracted from 1/3 of the tumour sample using TRIzol ® reagent (ABI, USA) according to the manufacturer’s protocol. RNA density and quality were measured with a NanoDrop-1000 spectrophotometer. The remaining 1/3 of the frozen tissues were homogenized and pulverized for protein extraction. The extraction was performed with the total protein extraction kit (Millipore, USA). Protein concentrations were measured with a BCA protein assay kit (Pierce, USA).
cDNA preparation and RT-PCR
Total RNA (1 μg) was used for preparation of first strand cDNA using reverse transcriptase. To measure the mRNA expression levels of p53 and its isoforms, primers were designed as described previously (Table 1).12 cDNA was amplified by nested PCR. PCR cycling conditions were 94°C for 1 minute, 58°C for 1 minute, and 72°C for 1 minute for 35 cycles using Taq DNA polymerase and the recommended protocol (Fermentas International Inc., USA). Expression intensity analysis was performed by 1D Image Analysis Software (Kodak, USA).
Immunohistochemical staining was performed in formalin fixed, paraffin embedded tissue sections. We used the avidin-biotin-peroxidase method with 1:100 diluted DO-1,20 1:40 diluted anti-p53 antibodies (Santa Cruz Biotechnology, Santa Cruz, USA), and 1:50 diluted DO-1221 antibody (Abcam, Cambridge, UK). As negative controls, normal sera were used in place of the primary antibodies. All samples were scored independently by two of the authors who were blinded to patient status. The percentage of the total number of tumour cells staining positively was categorized and given a score of 1 to 4 (1, 0-10% with weak intensity; 2, 11%—25% with weak or moderate intensity; 3, 26%-50% with weak or moderate intensity; and 4, more than 50% with strong intensity).
Western blotting analysis
Protein samples (30 μg of total protein) were denatured in SDS-PAGE gel loading buffer supplemented with 5% mercaptoethanol for 5 minutes. After electrophoresis through 10% SDS-polyacrylamide gels, proteins were transferred to nitrocellulose membranes. The membranes were blocked with Tris-buffered saline with Tween 20 containing 5% skimmed milk powder for two hours at room temperature. The membranes were incubated with primary antibodies including DO-12 (directed against an epitope within the DNA-binding domain of p53, amino acids 256-270 and recognising all six isoforms; Abcam, Cambridge, UK), DO-1 (recognising an epitope located at amino acids 20-25 contained in p53, p53β and p53 only) and anti β-actin (Santa Cruz, USA) at 4°C overnight. Finally, the membranes were hybridized with a secondary antibody conjugated with peroxidase (Millipore, USA) and the chemoluminescent signal was detected using the enhanced chemiluminescence system (Pierce, Rockford, USA) with high-performance chemiluminescence film (Pierce).
Statistical analysis was performed using x2 test or Fisher’s exact test as appropriate. Correlations between variables were tested according to the Spearman correlation test. P values less than 0.05 were considered statistically significant. Two-sided tests were used throughout all the analyses. All calculations were performed using SPSS 11.0 statistical software package (SPSS Inc., Chicago, USA).
Expression of p53 isoform mRNA
The mRNA expressions of the p53 isoforms in RCC patients are summarized in Table 2. Using specific primers and nested RT-PCR, all six isoforms were detected in the tumour specimens, however only the p53β mRNA was significantly overexpressed compared with the adjacent non-neoplastic tissue (P <0.001). p53 mRNA was found in all the tumour and non-neoplastic tissues. Nevertheless, the expression of Δ133p53β mRNA was not detectable in adjacent non-neoplastic tissue, and even in the tumour tissues it was detected at very low levels (2/41). The other isoforms were expressed at different levels in both the tumour and non-neoplastic tissues but without statistical significance. Representative examples of the mRNA expression for p53 isoforms are shown in Figures 1 and 2.
Expression of p53 isoform proteins
Immunohistochemical analysis for p53 with three antibodies was performed in both tumour and non-neoplastic tissues. All specimens of non-neoplastic renal tissue lacked-immunoreactivity for the three antibodies. The immune-ohistochemistry results in tumour tissues are presented in Table 3. The expression was noted in 16, 10, and 11 specimens for the anti-p53 group, DO-1 group and DO-12 group respectively (P >0.05). All three groups showed that p53 was associated with the stage; however, only the anti-p53 antibody group showed that p53 was associated with both the stage and the grade (Table 3, Figure 3).
To identify which p53 isoforms were presented in the matched tissue samples, Western blotting analysis was also performed. Representative examples are shown in Figure 4. The DO-1 antibody recognises an epitope located at amino acids 20-25, and therefore it can recognise p53, p53β and p53γ. Western blotting analysis showed that DO-1 antibody could detect p53 at 53 kD, p53β and (or) p53γ at 46 kD, respectively (Figure 4A). The DO-12 antibody was directed against an epitope within the DNA-binding domain of p53, amino acids 256-270. Therefore, all six isoforms can be recognised by the DO-12 antibody. When the tumours and clinically normal tumour adjacent tissues were analysed with DO-12, we noted that some bands were located at the approximate sizes of 53, 46, 35, and 25 kD respectively which corresponded to the predicted sizes of these six isoforms (Figure 4B). The results were consistent with the immunohistochemistry result. In these immunoblots, β-actin was used as an internal control, whose corresponding bands located at an approximate sizes of 43 kD (Figure 4C).
Correlations between p53 isoforms and clinical variables
Due to the lack of specific antibodies for each p53 isoforms, we used the mRNA levels to correlate with the clinicopathological factors. The results are summarized in Table 4. Only p53β mRNA was significantly associated with tumour stage (P=0.009).
Although p53 variant isoforms have been identified in various tumours and are expressed in a tissue-dependent manner, a detailed analysis of the p53 isoforms in normal renal and renal cell carcinoma has not yet been performed. In this study we mapped the expression of p53 and its isoforms in renal cell carcinoma at the mRNA and protein level and correlated it with clinicopathological features. These findings not only showed the presence of the p53 isoforms, but also suggested that these isoforms might play an important role in tumour growth and differentiation.
Alternative splicing is mechanism in which the exons of the primary gene transcript, the pre-mRNA, are separated and reconnected so as to produce alternative mRNA species.22 It is an important mechanism for controlling gene expression and allows large proteomic complexity from a limited number of genes. Many studies have shown that alternative splicing has a close relationship with cell physiology, developmental regulation, and occurrence of disease.23,24
The human p53 gene comprises 19 200 bp spanning 11 exons and 10 introns on chromosome 17p13.1. An initial study determined that the p53 gene structure was much simpler, which own only one promoter and three mRNA splice variants.3 However, recent research showed that the human p53 gene has a dual gene structure, similar to the p73 and p63 genes, and contains two alternative promoters.24 To our knowledge, the human p53 gene can encode at least nine different p53 protein isoforms. These include p53, p53β, p53γ, Δ133p53, Δ133p53β, and Δ133p53γ (due to alternative splicing of intron 9 and an alternative promoter in intron 4), and Δ40p53, Δ40p53β, and Δ40p53γ (due to alternative splicing of intron 2 or by alternative initiation of translation25,26).
Using nested RT-PCR technique described previously,11 we could specifically amplify six p53 isoforms. p53 mRNA was expressed in all samples. The Δ133p53 mRNA was expressed in tumour tissue higher than in normal tissue samples, but without statistical significance. Similar results were obtained for p53γ and Δ133p53γ. Δ133p53β, seldom expressed in tissues, is defective in inducing apoptosis and can inhibit the pro-apoptotic capabilities of full-length p53.7 In our study, Δ133p53β was not detected in the normal tissue samples, and at a low expression level in tumour tissues. This might be due to tissue specific expression. To confirm this result, further study is necessary using a specific antibody against Δ133p53β to determine its endogenous protein expression in normal tissues and tumours.
An important finding of this study was that the expression of p53β mRNA was significantly higher in tumours than in normal tissues. This result indicates that p53β might play a role in tumour differentiation in RCC. Though the impact of p53 expression on clinical severity and survival in RCC has been studied for many years, this subject is still a matter of some controversy between studies.27-29 The recent findings of the multiple isoforms encoded by the TP53 gene might partly explain the variable results. Bourdon et al7 found that co-transfection of p53 with p53β slightly increased p53-mediated apoptosis, while co-transfection of p53 with Δ133p53 strongly inhibited p53-mediated apoptosis. This suggests that the expression level of p53 isoforms may regulate cellular outcome in response to p53 activation.
p53β binds preferentially to the p53-responsive promoters p21 and Bax rather than Mdm2.7 Furthermore, p53β can form a protein complex with p53 and specifically enhance p53 transcriptional activity at the Bax promoter. However in 2006, Goldschneider et al30 found that the p53β isoform was the only p53 species to be endogenously expressed in the human neuroblastoma (NB) cell line SK-N-AS when they studied the expression of C-terminal deleted p53 isoforms in neuroblastoma. He suggested that the C-terminal truncated p53 isoforms might play an important role in NB tumour development. Avery-Kiejda et al31 studied the expression and subcellular localization of p53 and its isoforms in a panel of human melanoma cell lines using Western blotting, two-dimensional electrophoresis and RT-PCR. They reported that p53β was expressed in the majority of melanoma cell lines at the RNA level and protein level, but was absent or expressed at low levels in fibroblasts and melanocytes, suggesting that p53β might play a role in melanoma development.
The limitation of this study is the lack of a specific monoclonal antibody for each isoform, so we could not demonstrate the presence of bands at the predicted sizes of p53 isoforms by direct Western blotting of tissue. Consequently, the statistical analysis of p53 isoforms was performed mainly at the mRNA level. In the following work, raising a specific antibody which can recognize special p53 isoform is in badly need. Our data showed that the expression of p53β at the mRNA level was much higher in tumours. Therefore, we correlated its mRNA level with clinicopathological features to study the correlation between the p53β and the RCC. We found that the P53β mRNA was significantly associated with tumour stage, which supported our conjecture. We also hypothesized that deregulation of p53β isoforms expression might play a role early in tumor formation, as attenuation of the WTp53 response would render the cells more susceptible to further genetic damage and therefore to neoplastic transformation and tumor progression.
In conclusion, p53 isoforms are expressed in RCC and the normal tissue adjacent to the tumour at different levels. The expression of p53β in RCC is much higher than in normal tissues at the mRNA level. p53β might be not only the most easily identified isoform in RCC, but also play a vital role in tumour formation among these isoforms. The research about the relationship between p53 isoforms and the RCC is just beginning and its mechanism is not clear, so more research is still needed, such as the anti-isomform-specific antibodies, proteomics, microarray or tissue array. Because the isoforms are expressed differentially in individual RCCs, further studies are needed to determine the impact of their expression on p53 activities and the association between their expression patterns and clinical outcomes.
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