The transcription of this gene is regulated by a number of oncogene transcription factors including the E2F-1 transcription factor. Until quite recently the dominant view was that in normal cells, which are not subjected to various stresses, there is no or minimal activity of p The p53 gene is transcribed constitutively, and the mRNA is translated to p53 protein.
MDM2-p53 Cancer Pathway | BioOncology
However, the p53 protein is notoriously unstable, which is due to changeable degradation process that occurs in ubiquitin-dependent and ubiquitin-independent systems of 20S and 26S proteasomes. Degradation of p53 through the interaction with the ubiquitin ligase MDM-2 remains the most studied and, perhaps, the most important mechanism for regulation of its activity. The interaction of p53 with MDM-2 is subject to a fine regulation by a variety of mechanisms.
While some of the mechanisms are connected to regulation of MDM-2, others aim modifications in its target—the p53 itself. Transcription of the ARF gene is subject to positive and negative regulation by complexes that contain transcription factor E2F About 31 genes have been shown to be regulated by p53 and to have p53 Res responsive elements that bind the p53 protein. Several of these proteins mediate cell cycle arrest by p The p21 protein binds to Cyclin E—cdk2 and blocks it from phosphorylating the Rb protein which is required for entry into S phase.
The great majority of p53 responsive genes act in the proapoptotic pathway. Although the role of p53 in apoptosis is undisputed, the mechanism by which p53 induces apoptosis is not fully understood. Studies have suggested that pmediated apoptosis involves both transcription-dependent and -independent mechanisms. Cytochrome c release from mitochondria binds to apaf-1 resulting in apaf-1 cleavage and activation. This in turn cleaves caspase-9 which cleaves caspase-3 leading irreversibly to apoptosis. Thus, the p53 proapoptotic pathway acts upon many fronts that collectively execute programmed cell death.
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Another set of pregulated genes, for example, GADD45 and p53R2 a ribonucleotide reductase subunit , result in enhanced DNA repair in the cell or in the culture media. They are important for maintenance of the nucleotide pools during the DNA repair. Twitter Facebook.
Inquiry Basket. In the NUGC4 gastric cancer cell line p53 wild type , epigenetic treatment augmented apoptosis by chemotherapeutic drugs, partially through p53 transcription activity. On the other hand, in the KATO III cancer cell line p53 mutant , epigenetic treatment alone induced robust apoptosis, with no trans-activation of p In gastric cancer, p53 relevant and non-relevant pathways exist, and tumors with either pathway type exhibited unique clinical features.
Epigenetic treatments can induce apoptosis partially through p53 activation, however their apoptotic effects may be explained largely by mechanism other than through p53 pathways. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files. The funding agencies had no role in the design of the study, data collection, or analysis; in the interpretation of the results; in the preparation of the manuscript; or in the decision to submit the manuscript for publication. Competing interests: The authors have declared that no competing interests exist. DNA methylation plays a central role in gene silencing of tumor suppressor genes in human cancer.
A cancer-specific methylation gene is a rare entity, and frequent aberration of methylation in primary tumor tissues is even more rare [ 1 , 2 ]. We identified cancer-specific methylated genes in each organ using a pharmacological unmasking microarray PUM [ 1 , 2 ] and a modified PUM [ 3 — 5 ].
Moreover, we further compared the frequency of such aberrant methylation of candidate HRMGs with other reports of gastric cancer [ 16 ], and gene candidates were narrowed down to specific genes. Most importantly, these candidate tumor suppressor genes had been reported to be in the p53 tumor suppressor pathway Fig 1.
For example, PGP9. Hence, the p53 pathway is ablated in tumor tissues of primary cancers in an epigenetic manner together with wild type p53 , however there has been no report regarding the association of p53 mutation and epigenetic alterations in primary tumor tissues. In this study, we investigated the DNA methylation status of genes in the p53 pathway that are abnormally regulated in an epigenetic manner in primary gastric cancer, and compared their methylation pattern with the p53 mutation status in order to determine the clinical significance of p53 aberration phenotypes.
Neo-adjuvant therapy was not performed in this patient cohort.
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All tissue samples were collected at the Kitasato University Hospital, and written informed consent was obtained from all patients and healthy donors before sample collection. The present study was approved by the Ethics Committee of Kitasato University.
Mutations in exons 5, 6, 7 and 8 of the p53 gene were screened by non-radioactive single-strand conformation polymorphism SSCP analysis, which was performed using our previously established methods [ 23 ]. Electrophoresis was performed with 1. Bands were detected by staining gels using a silver stain plus kit Bio-Rad, Hercules, CA , followed by fixation, rinsing, development, and stopping of the reaction.
Bisulfite treatment results in the chemical modification of unmethylated, but not methylated, cytosines to uracils, allowing the distinction between methylated and unmethylated genomic DNA. PCR conditions and sequences are provided in S1 Table. Sections were counterstained with hematoxylin. After 24 hours of incubation, the reporter activity was measured using the Dual-luciferase Reporter Assay System Promega. The Kaplan-Meier method was used to estimate cumulative survival rates, and differences in survival rates were assessed using the log-rank test. Relapse free survival RFS and overall survival OS were measured from the date of operation to the date of recurrence and death, or the last follow up.
With regard to RFS or OS, patients who survived for more than 60 months 5-years were analyzed as survivors. We then analyzed the methylation profiles of PGP9. The TaqMeth value of all genes was higher in primary tumors of gastric cancer with wild type p53 than in those with mutant p53 gene methylation order: PGP9.
Importantly, a super-high methylation level of PGP9. Such super-high methylation of PGP9.
Human Oncoviruses and p53 Tumor Suppressor Pathway Deregulation at the Origin of Human Cancers
DAPK did not display this trend, because the DAPK methylation level was fairly high in the corresponding normal mucosa tissues of a considerable portion of the cases S2 Fig. A According to p53 gene mutation status and B according to the p53 aberration group. A Methylation of the indicated genes was analyzed using Q-MSP and TAqMeth values in tumors with p53 wild type were compared with those with p53 mutation. The threshold value for determination that a gene was methylated was determined as the maximum TaqMeth value of p53 wild type.
B Tumors with p53 wild type or p53 mutation were compared in terms of the presence of super-high methylation of the indicated genes.
Super-high methylation was defined that at least one gene showed higher TaqMeth value than each threshold values among three genes. Immunohistochemical staining for PGP9. A strong reduction in the expression of PGP9. On the other hand, no decrease in the protein expression of PGP9. Immunohistochemical staining of PGP9. We classified the gastric cancer patients into 3 categories based on the p53 mutation status as well as the DNA methylation status of PGP9. Although prognosis was not significantly different among the groups categorized according to GEC Fig 2B , more recurrences were found in the p53 aberration group as compared to the p53 non-aberration group Table 1 , no statistically significant difference.
This trend was preserved in terms of the recurrence pattern of each of the lymph nodes, peritoneum, and distant organs Table 2 , suggesting that the p53 aberration group is likely to exhibit a clinically unique phenotype even from a prognostic point of view. On the other hand, the diffuse type of gastric cancer showed significantly better prognosis than the intestinal type of gastric cancer in the p53 aberration group Fig 5A.
Since such patients tended to be distributed to a more advanced stage, these survival data suggested that S1 postoperative adjuvant chemotherapy is more effective for diffuse type gastric cancer than for intestinal type gastric cancer in cases with p53 pathway aberration Fig 5B. B Cancer stage distribution according to the p53 aberration group and histological findings. Interestingly, CDDP in combination with epigenetic treatments robustly increased the p53 protein level, which partially, but not totally, reflected p53 transcriptional activity of a luciferase reporter gene Fig 6A and 6B.
These findings suggested that p53 transcription activity can be reactivated by epigenetic treatments, however it also suggests that apoptosis is only partially induced through the p53 pathway. Subsequently the cells were analyzed for: A p53 protein expression by Western Blotting. B A dual reporter assay to confirm p53 transcription activity.
The dashed line indicates the optimal cut-off value 0. C Cell apoptosis was assayed using a Nexin Assay. A representative image is shown. Data are shown as the percentage of early and late apoptotic cells. CDDP in combination with epigenetic treatments did not increase the p53 protein level, which was reflected by the lack of p53 transcriptional activity of a luciferase reporter gene Fig 6A and 6B. These findings suggested that p53 transcription activity is not required for apoptosis by epigenetic treatments in KATOIII cells p53 null cells.
This consideration should be the first priority when considering further development of novel therapeutic strategies. Almost complete methylation of promoter DNA CpG islands is required for complete gene silencing, and even small proportions of unmethylated alleles can robustly induce gene expression [ 1 — 5 ]. In the primary tumor tissues, SHM must represent almost complete methylation in cancer cells, and SHM in the primary tumor tissues was indeed associated with reduced protein expression of such genes Fig 4.
This result suggested that SHM of genes in the p53 pathway could be functionally equivalent to p53 functional aberration that occurs as a result of p53 mutation, since these 3 molecules function in the same p53 pathway. Our current observations may be related to previous reports that demonstrated that p53 mutation is correlated with early stage of intestinal type gastric cancer, or with late stage of diffuse type gastric cancer [ 26 , 27 ], or elderly age [ 28 ], however, our clinicopathological analysis mainly included middle stage gastric cancer.
Interestingly, pathway aberration is unique and sustained even in recurrent patients Table 2. Positive prognostic relevance of p53 mutation in gastric cancer is controversial with both supporters [ 29 ] and dissenters [ 30 ] of this possibility. At first glance our data would appear to support the latter group, however, it should be taken into account that our survival data is likely to have been modified by post-operative adjuvant chemotherapy standard therapy in Japan. Whereas, in contrast to the present study, the diffuse type of gastric cancer showed poorer prognosis than the intestinal type of gastric cancer in our hospital decades ago [ 15 , 31 ], the latest updated survival data support the opposite results i.