The Immunocytochemical Detection of P53 Protein In Cytological Specimens: Technical Considerations

The p53 gene encodes a 393 amino acid nuclear phosphoprotein that appears to act as a cell cycle checkpoint, possibly by transactivating other target genes. Abnormalities of the p53 gene are common in a wide range of human tumours and are associated in many cases with immunologically detectable p53 protein. Detection of p53 immunoreactivity is uncommon in normal cells, but is frequently seen in neoplasia. Here we define the optimum conditions for the detection of p53 immunoreactivity in cytological material, including fixation and storage. Immersion in acetone-methanol for 10 min is optimal, and after air drying, smears or cytospin preparations can be stored at - 70°C for at least 6 months. We describe the range of controls necessary, including the use of positive control cell lines with known mutations of the p53 gene and defined abnormalities of p53 protein. Negative controls should include cell lines (or strains) with no p53 abnormality as well as the conventional negative immunological controls. It is only with these technical caveats and controls that p53 immunoreactivity can be performed reliably on cytological specimens. Le géne p53 code pour une phosphoprotéine nucléaire de 393 acides aminés qui semble jouer en rôle dans la régulation du cycle cellulaire, probablement par transactivation d'autres gènes cibles. Les anomalies du gène p53 sont présentes dans un large éventail de tumeurs humaines et sont associées a la présence d'une protéine p53 détectable immunologiquement. La détection d'une immunoréactivité anti p53 est rare dans les cellules normales alors qu'elle est fréquente dans les tumeurs. Nous avons défini dans ce travail les conditions optimales pour la détection de l'immunoréactivité anti p53 sur matériel cytologique, y compris les conditions de fixation et de conservation. L'immersion dans l'acétone-méthanol pendant 10 minutes est optimale. Aprés séchage à l'air, les frottis ou les préparations par cytocentrifugation peuvent être stockés à—70°C pendant au moins 6 mois. Nous décrivons aussi l'éventail des contrôles nécessaires incluant I'utilisation, comme contrôle positif, de lignées cellulaires avec des mutations connues du gène p53 et des anomalies définies de la protéine p53. Les contrôles négatifs doivent comporter des lignées cellulaires (ou des espèces) sans anomalie de p53 ainsi que les contrôles immunologiques négatifs convrentionnels. C'est seulement lorsque ces précautions techniques et ces contrôles sont respectés que l'immunoréactivité anti p53 peut être étudiée valablement sur les prélèvements cytologiques. Das p53 kodiert ein aus 393 Aminosären bestehendes Phosphoprotein, das offensichtlich den Zellzyklus blockiert, möglicherweise durch Aktivierung anderer Gene. Veränderungen des p53-Gens wurden in zahlreichen menschlischen Tumoren nachgewiesen, sodaß eine positive Reaktion in Neoplasien häufig, in Normalzellen jedoch ungewöhnlich ist. Die optimalen Bedingungen für den p53-Nachweis in cytologischem Material werden hinsichtlich Fixation und Lagerung untersucht. Eine 10minütige Aceton-Methanol-Fixierung mit anschlies-sender Lufttrocknung erlaubt die Lagerung von Ausstrichen und Cytozentrifugen-präparaten bei - 70°C für mindestens 6 Monate. Die erforderlichen Kontrollen einschließlich positiver Zellinien mit bekannten Mutationen des p53-Gens und definierten Anomalien des Proteins werden beschrieben. Negativkontrollen sollten Zellinien ohne p53-Anomalie ebenso umfassen wie die üblichen negativen immunologischen Kontrollen. Nur unter diesen Bedingungen ist ein zuverlässiger Nachweis von p53 mögloch.     

Investigation of role of aspartame on apoptosis process in HeLa cells -->

Aspartame is an artificial sweetener used as an alternate for sugar in several foods and beverages. The study reports that consumption of aspartame containing product could lead to cancer. However, the effect of aspartame on apoptosis process in cancer is not yet understood clearly. HeLa cells were exposed to different concentrations (0.01–0.05 mg/ml) of aspartame for 48 h. Cytotoxicity of aspartame on cancer cells was determined by SRB assay. The result indicates no significant changes on cell viability. Aspartame suppresses apoptosis process in cancer cells by down-regulation of mRNA expression of tumor suppressor gene p53, and pro-apoptotic gene bax. It up-regulates anti-apoptotic gene bcl-2 mRNA expression. In addition, Ki 67 and PCNA mRNA, and protein expressions were determined. Taking all these together, we conclude that aspartame may be a potent substance to slow-down the apoptosis process in HeLa cells. Further works are ongoing to understand the biochemical and molecular mechanism of aspartame in cancer cells.     

Elevated PRC1 in gastric carcinoma exerts oncogenic function and is targeted by piperlongumine in a p53‐dependent manner

Gastric carcinoma is one of the most common malignancies worldwide and the second most frequent cause of cancer‐related death in China. Protein regulator of cytokinesis 1 (PRC1) is involved in cytokinesis and plays key roles in microtubule organization in eukaryotes. This study was aimed to analyse the expression and to investigate the functional role of PRC1 in gastric tumorigenesis. The expression of PRC1 was evaluated by qRT‐PCR, Western blot and immunohistochemistry. The biological function of PRC1 was determined by CCK‐8 proliferation assays, monolayer colony formation, xenografted nude mice and cell invasion assays by shRNA‐mediated knockdown in AGS and HGC27 cells. The regulation of PRC1 expression by piperlongumine was also investigated using dual‐luciferase reporter assay and ChIP‐qPCR analysis. PRC1 was up‐regulated in primary gastric cancers. Overexpression of PRC1 in gastric cancers was associated with poor disease‐specific survival and overall survival. PRC1 knockdown in AGS and HGC27 cell lines suppressed proliferation, reduced monolayer colony formation, inhibited cell invasion and migration ability and induced cell‐cycle arrest and apoptosis. Inhibition of PRC1 also suppressed tumour growth in vivo. We finally confirmed that PRC1 is a novel downstream target of piperlongumine in gastric cancer. Our findings supported the oncogenic role of PRC1 in gastric carcinogenesis. PRC1 might serve as a prognostic biomarker and potential therapeutic target for gastric carcinoma.     

The MDM2 RING Domain and Central Acidic Domain Play Distinct Roles in MDM2 Protein Homodimerization and MDM2-MDMX Protein Heterodimerization

The oncoprotein murine double minute 2 (MDM2) is an E3 ligase that plays a prominent role in p53 suppression by promoting its polyubiquitination and proteasomal degradation. In its active form, MDM2 forms homodimers as well as heterodimers with the homologous protein murine double minute 4 (MDMX), both of which are thought to occur through their respective C-terminal RING (really interesting new gene) domains. In this study, using multiple MDM2 mutants, we show evidence suggesting that MDM2 homo- and heterodimerization occur through distinct mechanisms because MDM2 RING domain mutations that inhibit MDM2 interaction with MDMX do not affect MDM2 interaction with WT MDM2. Intriguingly, deletion of a portion of the MDM2 central acidic domain selectively inhibits interaction with MDM2 while leaving intact the ability of MDM2 to interact with MDMX and to ubiquitinate p53. Further analysis of an MDM2 C-terminal deletion mutant reveals that the C-terminal residues of MDM2 are required for both MDM2 and MDMX interaction. Collectively, our results suggest a model in which MDM2-MDMX heterodimerization requires the extreme C terminus and proper RING domain structure of MDM2, whereas MDM2 homodimerization requires the extreme C terminus and the central acidic domain of MDM2, suggesting that MDM2 homo- and heterodimers utilize distinct MDM2 domains. Our study is the first to report mutations capable of separating MDM2 homo- and heterodimerization.     

The p53 isoforms are differentially modified by Mdm2

The discovery that the single p53 gene encodes several different p53 protein isoforms has initiated a flurry of research into the function and regulation of these novel p53 proteins. Full-length p53 protein level is primarily regulated by the E3-ligase Mdm2, which promotes p53 ubiquitination and degradation. Here, we report that all of the novel p53 isoforms are ubiquitinated and degraded to varying degrees in an Mdm2-dependent and -independent manner, and that high-risk human papillomavirus can degrade some but not all of the novel isoforms, demonstrating that full-length p53 and the p53 isoforms are differentially regulated. In addition, we provide the first evidence that Mdm2 promotes the NEDDylation of p53β. Altogether, our data indicates that Mdm2 can distinguish between the p53 isoforms and modify them differently.     

TP53 PIN3 and PEX4 polymorphisms and infertility associated with endometriosis or with post-in vitro fertilization implantation failure

p53 has a crucial role in human fertility by regulating the expression of leukemia inhibitory factor (LIF), a secreted cytokine critical for blastocyst implantation. To examine whether TP53 polymorphisms may be involved with in vitro fertilization (IVF) failure and endometriosis (END), we have assessed the associations between TP53 polymorphism in intron 2 (PIN2; G/C, intron 2), PIN3 (one (N, non-duplicated) or two (D, duplicated) repeats of a 16-bp motif, intron 3) and polymorphism in exon 4 (PEX4; C/G, p.P72R, exon 4) in 98 women with END and 115 women with post-IVF failure. In addition, 134 fertile women and 300 women unselected with respect to fertility-related features were assessed. TP53 polymorphisms and haplotypes were identified by amplification refractory mutation system polymerase chain reaction. TP53 PIN3 and PEX4 were associated with both END (P=0.042 and P=0.007, respectively) and IVF (P=0.004 and P=0.009, respectively) when compared with women both selected and unselected for fertility-related features. Haplotypes D-C and N-C were related to higher risk for END (P=0.002, P=0.001, respectively) and failure of IVF (P=0.018 and P=0.002, respectively) when compared with the Fertile group. These results support that specific TP53 haplotypes are associated with an increased risk of END-associated infertility and with post-IVF failure.     

新的抑癌基因家族--ASPP

P53是重要的抑癌基因之一的产物，其在抑制肿瘤功能的重要方面是诱导凋亡的能力。但多年来P53诱导凋亡的机制还不清楚，最近发现的ASPP蛋白较好的解释了这个问题，ASPP蛋白能和P53结合形成复合物，再作用于原凋亡基因的启动子区域，从而诱导细胞凋亡的发生，ASPP表达水平的改变能影响P53与DNA结合的能力，进而控制细胞是进入凋亡还是进入停滞的途径，影响P53诱导肿瘤抑制的效应。