P53 Gene In DNA Research Articles

A multicancer-like syndrome in a dog characterized by p53 and cell cycle-checkpoint kinase 2 (CHK2) mutations and Sirtuin gene (SIRT1) down-regulation

IntroductionWe have investigated SIRT1, p53 and cell cycle-checkpoint kinase 2 (CHK2) gene dysfunction in a dog with a multicancer syndrome-like in order to evaluate their potential role in the determinism of the disease and to establish a possible correlation between SIRT1 transcript level and p53 expression status. Material and methodsBlood sample and tumour samples from a pure breed English Setter dog with different tumours were used for this study. Nucleotide sequence analysis was performed with a DNA autosequencer in order to examine p53 and CHK2 mutations. In addition, the expression level of SIRT1 was quantified by Southern Blot analysis of Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). ResultsCytological examination revealed five different tumours: a cutaneous sebaceous epithelioma, a cutaneous mast cell tumour, a testicular Sertoli cell tumour, an oral malignant melanoma, and a cutaneous squamous cell carcinoma. Sequencing analysis revealed the presence of a nucleotide substitution, (CGG>CAG) exon 7 of the p53 gene in DNA from peripheral blood mononuclear cells (PBMCs) as well as in the melanoma; whereas the other four cancers showed the loss of the wild-type allele. Furthermore, CHK2 mutation at codon 311 has been identified in the melanoma and sebaceous epithelioma. In addition, SIRT1 cDNA expression decreased in all tumour samples compared to cDNA SIRT1expression level in peripheral blood mononuclear cells (PBMCs) in the same dog. ConclusionsThese results suggest that the germ line mutation of the p53 gene at codon 248 might be, at least, one cause of the multicancer syndrome-like in our dog; furthermore, we show a possible correlation between SIRT1 transcript level and p53 mutations status. The regulatory role of SIRT1 in tumour suppressor pathways suggests that the net effect seen may represent both direct and indirect downstream regulation and it is likely to depend on the presence or absence of functional p53.

Oligonucleotide Probe Array for p53 Gene Alteration Analysis in DNA from Formalin‐Fixed Paraffin‐Embedded Breast Cancer Tissues

Mutations in the TP53 tumor-suppressor (p53) gene represent the most common molecular changes in various malignancies, including breast cancer (BC). We sequenced the p53 gene in DNA extracted from archival paraffin-embedded BC tissues and compared the results obtained from direct sequencing with those obtained by oligonucleotide probe array (OPA). DNA was extracted from 34 samples. OPA correctly detected 13 genetic alterations in 14 cases, with a mutation frequency of 41.2%. No changes were detected in exons 3, 4, 9, 10, and 11 and no polymorphisms were found. Direct manual sequencing in which DNA was amplified by PCR showed 21 genetic mutations in 19 (55.9%) cases. Eight mutations were identified by both OPA and PCR methods. Although OPA detected fewer gene alterations than direct sequencing, the difference was not significant (P= 0.11). In conclusion, the OPA may be safely used to identify individual genetic variations of human p53 gene in BC specimens.

Evaluation of oligonucleotide arrays for sequencing of the p53 gene in DNA from formalin-fixed, paraffin-embedded breast cancer specimens.

Routine tissue processing has generated banks of paraffin-embedded tissue that could be used in retrospective cohort studies to study the molecular changes that occur during cancer development. The purpose of this study was to determine whether a p53 microarray could be used to sequence the p53 gene in DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues. DNA was extracted from 70 FFPE breast cancer tissue specimens. p53 was sequenced with an oligonucleotide microarray (p53 GeneChip; Affymetrix), and the results were compared with the results obtained from direct sequencing. DNA was extracted from 62 of 70 cases. We identified 26 mutations in 24 of the 62 cases by the p53 GeneChip. No polymorphisms were detected, and exon 4 could not be evaluated in 20 cases. There were 43 genetic alterations detected by direct sequencing in 35 of the 62 cases. These consisted of 26 polymorphisms and 17 mutations in exons or splice sites. Fifteen mutations were identified by both methods. Direct sequencing detected significantly more gene alterations (43 of 54) in DNA extracted from FFPE tissue than the p53 GeneChip (26 of 54; P = 0.018). However, if the changes in exon 4 were eliminated from this comparison, the p53 GeneChip detected 26 of 27 mutations compared with direct sequencing, which identified 16 of 27 mutations. (P = 0.016). A combination of oligonucleotide microarray and direct sequencing may be necessary to accurately identify p53 gene alterations in FFPE breast cancer. The p53 GeneChip cannot be used to detect exon 4 polymorphisms (codon 72) in FFPE breast cancer tissue.

Loss of heterozygosity of p53 in oral cancers demonstrated by the polymerase chain reaction

Alterations in the tumor suppressor gene p53 are the most frequently detected genetic abnormalities in human cancers. Inactivated tumor suppressor genes, including p53, often are suggested by loss of heterozygosity (LOH) studies. p53 gene inactivation has been reported in esophageal cancers. Because the etiologic factors for esophageal and intraoral carcinomas often are the same, corresponding molecular events may occur in oral squamous cell carcinoma (SCC) development. The authors investigated LOH of the p53 gene in DNA from 27 primary oral cancers using a polymerase chain reaction (PCR)-based restriction fragment length polymorphism assay. DNA from fixed specimens of SCC and normal tissues was isolated and amplified at two p53 gene polymorphic restriction sites. In heterozygous individuals, 10 of 14 (71%) intraoral SCC demonstrated loss of p53 heterozygosity at one polymorphic restriction site. Two of five carcinomas showed LOH at a second site. These results suggest that inactivation of p53 is involved in the development or progression of SCC of the oral cavity.