Expression And Correlation Of △Np73 And P73 At Transcript Level In Esophageal Carcinoma

Objective: The p53 family member p73 displays significant structural and functional homology to p53,and it can induce growth arrest and apoptosis. But several pieces of evidence argue against p73 being a classical tumor suppressor. Overexpression of wild-type p73 has been reported in various tumor types compared with normal tissues. Despite initial reports suggesting tumor-associated deletion of p73,many subsequent studies failed to demonstrate mutational inactivation of TP73 gene in a wide variety of tumors. In contrast to mice lacking p53,p73-negative mice are not prone to tumor development. Together,these data raise the question about additional activities of p73 in cancer. It is reported that the human TP73 gene generates an NH2-terminally truncated isoform recently. ΔNp73 derives from an alternative promoter in intron 3 and lacks the transactivation domain of full-length TAp73. ΔNp73 is frequently overexpressed in variety of human cancers,but not in normal tissues. It acts as a potent transdominant inhibitor of wild-type p53 and transactivation-competent TAp73. Tumorigenisis progression of esophageal carcinoma is a complex and multi-step process involving polygenetic regulation. In this study,we analyzed the expression and correlation of p73mRNA andΔNp73mRNA in esophageal carcinoma to investigate the mechanism of oncogenesis and development of esophageal carcinoma initially. Methods: 34 fresh samples of esophageal carcinoma were analyzed and normal mucus that were resected furthest from the tumor specimens were as the control group. All subjects were the admitted patients for operative treatment in our department. Neither chemotherapy nor radiotherapy was carried out for these patients before esophagectomy. According to the classification of tumor stage suggested by UICC in 1997,of these subjects,17 were in stage Ⅰ,Ⅱ and 17 were in stage Ⅲ,Ⅳ. Tissue specimens were snap-frozen in liquid nitrogen and transferred to a –80℃ freezer for analysis. First,total RNA was prepared by using Trizol reagent. After being washed in ethanol and resuspension in DEPC-treated water,RNA yield and purity were measured by spctrophotometric determination at 260nm and 280nm. And it was visualized on formaldehyde denaturing agarose gel. Second,unique cDNA primers were designed for amplification ofΔNp73 and p73 from tissues by semiquantitative RT-PCR. They were as follows: ΔNp73, 5'- CGC CTA CCA TGC TGT ACG TC -3'(sence) and 5'- GTG CTG GAC TGC TGG AAA GT -3'(antisence); p73, 5'- CGA CGG CTG CAG AGC GAG -3' (sence) and 5'-TCG AAG GTG GAG CTG GGT TG -3' (antisence); andβ-actin,5'-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG-3' (sence) and 5'-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC -3'(antisence). RT-PCR products are 268bp,365bp and 838bp respectively. Third,products of RT-PCR were electrophoresed in 1.5% agarose. Then,ΔNp73mRNA and p73mRNA were evaluated by a Gel-analyzer software package respectively. The photon intensity as a ratio against that of corresponding β-actin mRNA was the expression parameter. Last, statistical analysis was performed. Differences were considered to be statistically significant when the probability value was less than 0.05. Results: 1. Expression of ΔNp73 at transcript level in esophageal carcinoma : 18 of 34 (53%)esophageal carcinomas were positive,and only 3 (9%)were positive in the corresponding normal mucous . The meanΔNp73mRNA expression level in esophageal carcinomas (1.01±0.24) was much higher than that in normal mucous (0.58±0.11). Both the ΔNp73mRNA positive rate and the expression quantity in esophageal carcinoma were remarkably higher than those in normal. They had obvious differences in statistics (P<0.01). 2. Relation between ΔNp73mRNA and characteristics of clinical pathology in esophageal carcinoma: the specific value of ΔNp73mRNA corrected in stage Ⅲ,Ⅳ (1.21±0.22) was outstandingly higher than that in stage Ⅰ,Ⅱ (0.86±0.13) ,they had significantly differences in statistics (P<0.01). The expression of...