It is widely acknowledged that P53 is an intra-nuclear phosphorylated protein, which half-life of wild-type P53 is only 20–30 minutes, and it exists in the nucleus for an extremely short time for hardly detected . Meanwhile mutated P53 is not digested quickly inside tumor cells that has prolonged half-life of 20–40 hours, and therefore accumulates inside tumor cells . In addition, as IHC has become an important indicator of various biomarker detection, P53 IHC is an effective substitute marker for TP53 mutation status . At present, P53 IHC is mainly applied for human tumor diseases, such as ovarian cancer, hepatocellular carcinoma, esophageal cancer, etc., and as an important measure to detect the stage and grade of cancers. But there are few studies in poultry tumor diseases.
In this study, immunohistochemical analysis revealed that P53 was detected clearly in the tumor tissues of liver, spleen and the bursa of fabricius infected with MDV. It is worth noticed that P53 are almost expressed in the cytoplasm, which may be due to the mutation of p53 leading to the loss of nuclear localization function, which eventually caused the mutant P53 overexpressed in the cytoplasm.
It has been known that p53 as the “hotspots”, in which mutations were frequently occurred, is shown in a variety of tumors . Of the 393 codons in the human p53, most mutations in the DBD occur at “mutation hotspots”, which had been found in R175, G245, R248, R249, R273, and R282. Previous studies have revealed that several point mutations were also found in neoplastic diseases of poultry such as MD, avian leukosis, but these mutations were not localized at the “hotspots” of the p53 . The locations and types of p53 gene mutation vary largely in different tumors. The DBD of the p53 is a “hotspot” for mutation, as the majority of tumor-associated mutations in p53 occur within this region. These mutations are mainly point mutations, which are frequently inactivated by base deletions and insertions; majority of p53 mutations are missense mutations . In the current study, p53 was detected to have multiple types of mutations dominated by point mutations in natural and experimental infections of MDV. Through mutational analyses, five base sites with the most frequent mutations were found, which were 651, 786, 828, 864, 879 respectively. And the altered codons compared with wild p53 were 217 (ACG-ACA), 262 (GCA-GCC), 276 (CGC-CGG), 288 (GCC-GCA), 293 (ACC-ACA), which encoded amino acid were synonymous mutations. It is generally believed that proteins are changed followed by gene mutation to participate in the regulation of tumorigenesis and development. However, in this study, p53 mutated at the frequent spots and amino acid occurred synonymy mutation. The amino acid of P53 was not changed.
At the same time, a short form of p53 transcript was detected in clinical cases infected with MDV. The deleted sequence was located at 631–677 bp in the open reading frame of the reference sequence, resulting in a frameshift with formation of missense mutations that would give rise to the ultimately termination of translation in amino acid at position 210. This was another short form of p53 transcript found after Qu’s research . In the present research, it is found that the p53 has high mutation frequency of 60% in poultry oncology, and the mutation region is mainly concentrated in the DBD. The DBD allows the specific recognition of target sequences, at the same time, the central part and the C-terminal base region comprise the nuclear localization signals, p53 oligomeric domain, and a domain that mediates non-sequence specific interaction of p53 with DNA and RNA . Once the p53 is deleted and point mutations in this region, it may affect the formation of tetramers, which in turn leads to the conversion of wild type p53 into a mutant type and loss of normal function.
The conformation of mutate P53 extends its half-life to several hours, thereby accumulating in the cells. The accumulated mutant P53 acts as a target antigen to elicit an autoimmune response of the body producing serum P53 antibodies . Several studies in human have shown that the production of serum P53 was closely related to the accumulation of P53 in the corresponding tumor. It had been found that the serum P53 concentration in cancer patients was significantly higher than that in healthy testers . But in this study, the serum P53 antibody concentrations of experimental infected group was significantly higher than the control group. Meanwhile, the concentration of P53 in clinical and experimental MDV-infected group was significantly lower than control group, which was different from cancer research in human. This might be that p53, as a tumor suppressor, was largely consumed in response to the occurrence of MD. And then, the tumor promoted mutation of p53 and further reduced P53 concentration.
This research indicates that the p53 in MDV infected chicken trend to mutate, which is consistent with cancer patients. However, P53 antibody concentration in MD-infected chicken’s serum did not show significant different from the control group. The concentration of P53 show a decrease in the MD-infected group, which may provide a new idea for the diagnosis and monitoring of MD.