Expression Of P53 Isoforms In Renal Cell Carcinoma And Its Significance

Background and objectsp53 gene is one of the most important human tumor suppressor and transcription factor genes to tumorigenesis.It is a key regulator in preventing cancer formation and plays a vital role in cell cycling,growth,DNA repair,cell cycle arrest,or apoptosis.Thus WTp53 has been classed as a "guardian of the genome" due to its ability to coordinate multiple and diverse signaling pathways involved in this response.Mutation of p53 is a common occurrence in many cancers and is associated with tumor progression,resistance to chemotherapy,and poor prognosis.Renal cell carcinoma(RCC) is the most common malignancy in adult kidney,with 30,000 new cases per year in the U.S.and 20,000 cases in the European Union.In China,the incidence of RCC is second only to bladder turnout 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,p53 appears mutation in about 50%of many human cancers,while in RCC,there is a pretty low incidence of p53 mutation.It has been reported in 3-33%of patients with renal-cell carcinoma.There may be a postulated the existence of a novel dominant mechanism of inactivation of p53 in renal cells carcinoma.Recently p53-related genes,p63,p73 and p53 splicing isoforms have been discovered,the role of the p53 regulatory mechanism was gradually in depth study.The high level of sequence similarity in the DNA-binding domain between p53 protein family members allows p63 and p73 to transactivate p53-responsive genes causing cell-cycle arrest and apoptosis.However, p53,p63 and p73 proteins are not entirely functionally redundant,each of them has specific biological functions and some of their isomers can be a negative regulator of p53 function.Alternative splicing is the RNA splicing variation mechanism in which the exons of the primary gene transcript,the pre-mRNA,are separated and reconnected so as to produce alternative ribonucleotide arrangements.It is an important mechanism for controlling gene expression which allows large proteomic complexity from a limited number of genes.Lots of research deemed that the variation of the alternative splicing have close relationship with cell physiology,development regulation,occurrence of disease,p53 gene family members can express multiple mRNA variants due to multiple splicing and alternative promoters.Hence, p53 gene family members express different forms of p53 protein containing different domain of the protein(isoforms).To date,at least nine different p53 isoforms have been identified in humans,including p53,p53β,p53γ,Δ133p53,Δ133p53β,Δ133p53γ,Δ40p53,Δ40p53βandΔ40p53γ.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.Furthermore,the expression of these isoforms in human cancers, such as breast cancer,neuroblastoma,acute myeloid leukemia,and squamous cell carcinoma of the head and neck,suggests that they may involve in tumor development or progression.The present study has confirmed that there are complex interactions mechanism between p53 splicing isoforms and WTp53.p53 splicing isoforms can negatively regulate the activity of WTp53.The expression of p53 splicing isoforms in many human cancers have been reported however,to our knowledge,no study has addressed the expression of p53 and its isoforms in renal cell carcinoma till now.Therefore,in this paper we aimed at analyzing the expression patterns of p53 isoforms in RCC at the mRNA and protein levels,and their associations with clinical and pathologic factors to explore the mechanism of p53 isoforms in RCC.Materials and Methods:MaterialsTumor samples were taken after informed consent from 47 patients with renal cell carcinoma (41patients were clear cell carcinoma,5 patients were chromophil cell carcinoma and 2 patients were chromophobe cell carcinoma),who underwent radical nephrectomy from June 2007 to March 2008 in Shandong Provincial Hospital.Patients were staged according to the 2002 staging system of the International Union Against Cancer(UICC) and the nuclear grade of tumors was determined by Fuhrman grading scheme.The nonneoplastic renal tissue resected from adjacent regions of each renal cell carcinoma were also analysed for comparison.Tissue samples were divided into three,1/3 were fixed in formalin and embedded in paraffin,the other 2/3 were frozen with liquid nitrogen after being taken from the patient quickly and then reserved in the refrigerator(-80℃) until used.MethodsThis research was divided into two parts.Part 1.Dectet the mRNA expression of each pair of tumor specimens and the adjacent nonneoplastic tissue.Total RNA was extracted using Trizol reagent according to the manufacturer protocol,then reverse transcribed RNA into cDNA.At last cDNA was amplified by nested PCR using special primer.PCR cycling conditions for p53,p53βand p53γwere 94℃for 1min,60℃for 50sec,and 72℃for 1 min for 35 cycles using Taq DNA polymerase and the recommended protocol(Fermentas International Inc.,CA).PCR cycling conditions forΔ133p53,Δ133p53βandΔ133p53γwere 94℃for 1min,58℃for 50sec,and 72℃for 1 min for 35 cycles.Expression intensity analysis was performed by 1D Image Analysis Software (Kodak,USA).The expression of mRNA in RCC tissues and normal renal tissues were compared and the difference was compared by statistic analysis.Part 2.Dectet the isoform protein expression of each pair of tumor specimens and the adjacent nonneoplastic tissue.Immunohistochemical staining was performed in formalin fixed,paraffin embedded tissue sections.We used the avidin-biotin-peroxidase method with 1:100 diluted DO-1,1:40 diluted anti-p53 antibodies,1:50 diluted DO-12 antibody.As negative controls,normal serums 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 tumor 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 Blot analysis using DO-1 and DO-12 as primary antibodies.The membranes were hybridized with a secondary antibody conjugated with peroxidase and the chemoluminescent signal was detected using the enhanced chemiluminescence system with high-performance chemiluminescence film.Statistical analysis was performed usingχ~2 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 significant.Two-sided tests were used throughout all the analyses.All calculations were performed using SPSS 13.0 statistical software packageResultsⅠBy using specific primers and nested RT,all six isoforms were detected in the tumor specimens,however only the p53βmRNA was significantly overexpressed compared with the adjacent nonneoplastic tissue(P＜0.001),p53 mRNA were found in all the tumor and nonneoplastic tissues.Nevertheless,the expression ofΔ133p53βmRNA was not detectable in adjacent nonneoplastic tissue,and even in the tumor tissues it was detected at very low levels (2/47).The other isoforms expressed at different level in both the tumor and nonneoplastic tissues without statistical significance.ⅡThe immunohistochemistry for p53 with three antibodies were performed in both tumor and nonneoplastic tissue.In nonneoplastic renal tissue,all specimens lacked immunoreactivity for the three antibodies.The expression was noted in 18,11,13 specimens for anti- p53 group, DO-1 group and DO-12 group respectively,with no significant difference.Both anti- p53 group and DO-12 group showed that p53 was associated with the stage and the grade, however,only DO-1 antibody group showed that p53 was associated with both the stage. Western Blot analysis showed that DO-1 antibody could detect p53 at 53 kD,p53βand(or) p53γat 46 kD respectively.The DO-12 antibody could detect all these six isoforms.When the tumors and clinically normal tumor adjacent tissues were analysed with DO-12,we noted that some bands located at the approximate sizes of 53KD,46KD,35KD and 25KD respectively which correspondenced with the predicted sizes of these six isoforms.The results were consistent with the immunohistochemistry result.In these immunoBlots,β-actin was used as an internal control whose Corresponding bands located at the approximate sizes of 43KD clearly.As lack of specific antibodies for each p53 isoforms,we used the mRNA level to correlate with the clinicopathological factors.The results showed that only p53βmRNA was significant associated with tumor stage(P=0.009).ConclusionⅠp53 isoforms are expressed in RCC and the normal tissue adjacent to the tumour with different levels,p53βmRNA was significantly overexpressed compared with the adjacent nonneoplastic tissue.Δ133p53βmRNA might not be expressed in normal kidneys.ⅡThe expression of p53βmRNA was significant associated with tumor stage.Ⅲp53βmay not only be the most easily identified isoform in RCC,but also play a vital role in tumor formation among these isoforms.ⅣThe expression of p53 was associated with the stage of the RCC.Ⅴp53βmay be used as a new predictor and therapy target for RCC.