Analyses of p73 Protein Oligomerization and p73-MDM2 Interaction in Single Living Cells Using In Situ Single Molecule Spectroscopy.

Protein oligomerization and protein-protein interaction are crucial to regulate protein functions and biological processes. p73 protein is a very important transcriptional factor and can promote apoptosis and cell cycle arrest, and its transcriptional activity is regulated by p73 oligomerization and p73-MDM2 interaction. Although extracellular studies on p73 oligomerization and p73-MDM2 interaction have been carried out, it is unclear how p73 oligomerization and p73-MDM2 interaction occur in living cells. In our study, we described an in situ method for studying p73 oligomerization and p73-MDM2 interaction in living cells by combining fluorescence cross-correlation spectroscopy with a fluorescent protein labeling technique. Lentiviral transfection was used to transfect cells with a plasmid for either p73 or MDM2, each fused to a different fluorescent protein. p73 oligomerization was evaluated using brightness per particle, and the p73-MDM2 interaction was quantified using the cross-correlation value. We constructed a series of p73 mutants in three domains (transactivation domain, DNA binding domain, and oligomerization domain) and MDM2 mutants. We systematically studied p73 oligomerization and the effects of p73 oligomerization and the p73 and MDM2 structures on the p73-MDM2 interaction in single living cells. We have found that the p73 protein can form oligomers and that the p73 structure changes in the oligomerization domain significantly influence its oligomerization. p73 oligomerization and the structure changes significantly affect the p73-MDM2 interaction. Furthermore, the effects of inhibitors on p73 oligomerization and p73-MDM2 interaction were studied.

Immunohistochemical Profile of Tumor Suppressor Proteins RASSF1A and LATS1/2 in Relation to p73 and YAP Expression, of Human Inflammatory Bowel Disease and Normal Intestine.

The intestinal neoplastic transformation is a possible risk of chronic inflammatory bowel disease (IBD). Previous evidence in mice IBD provides a role for the RAS-association domain family tumor suppressor protein 1 A (RASSF1A), in the repairing process following mucosa epithelium damage, through cooperation with the HIPPO-signaling molecules p73, and YAP. HIPPO pathway which has been implicated in stem cell activity includes as key components for signal transduction the large tumor suppressor homology Ser/Thr kinases LATS1/2. The aim of this study was to assess immunohistochemically, using specific antibodies, the RASSF1A and LATS1/2 expression patterns in a cohort of patients with IBD including 52 ulcerative colitis (UC), 24 Crohn's disease (CD) and 24 IBD unclassified (IBD-U), compared with normal intestine from non-IBD patients (control group). The relationship between subtypes of IBD and RASSF1A and LATS1/2 expression, both individually and related to p73 and YAP/pYAP(Ser127) proteins was also investigated. Quantitative analyses of the immunohistochemical findings in mucosa cells revealed a significantly decreased expression in UC and IBD-U for RASSF1A expression and a significantly elevated expression in UC, IBD-U, and CD for LATS1/2 expression compared with normal mucosa (P < 0.05). However, ROC curve analysis showed that only LATS1/2 could differentiate IBD from control group. RASSF1A expression was significantly correlated with LATS1/2 in UC with dysplasia (P < 0.0001), and p73 in UC (P < 0.001), and IBD-U (P < 0.02). The expression of all proteins did not differ significantly between subtypes of IBD (P ≥ 0.05). RASSF1A-LATS1/2 co-expression was mainly observed in IBD samples. These findings suggest that tumor suppression proteins RASSF1A and LATS1/2 may be involved in the pathogenesis of human IBD and imply a potential cooperation of RASSF1A, and HIPPO signaling pathways in human bowel inflammation.

ΔNp73 status in peritoneal and ovarian dissemination of appendicular adenocarcinoids (goblet cells).

INTRODUCTION: Goblet cell carcinoma (GCC) is an appendicular neoplasia representing less than 5% of all appendicular tumors, found in 0.3-0.9% of the appendectomies, 35-58% of all appendicular neoplasms, and less than 14% of malign appendix tumors. The most frequent clinical presentation is abdominal pain associated with a picture of acute appendicitis. MATERIALS AND METHODS: We present 3 clinical cases of appendix GCC, 2 subjected to cytoreductory surgery plus intraperitoneal hyperthermic chemotherapy and a third, who is currently receiving neoadjuvant treatment with a good response to chemotherapy and who will be offered the same treatment as the first two patients. Given the unpredictable behavior of these tumors, the use of molecular markers could help us to predict their behavior and prognosis. In this context, the TP73 gene would make an interesting putative marker. ∆Np73 has been described as overexpressed in a great variety of tumor types including colon cancer and this up-regulation is associated with a poor prognosis. To evidence its role in this malignancy, we evaluate here the status of ∆Np73 in the primary tumor and normal counterpart tissues, in the metastatic implants and in healthy areas of the peritoneum from the appendicular GCC patients. In addition, we checked the expression levels of this p73 variant in the tumor and normal tissue of 26 patients with colon cancer. RESULTS: Remarkably, 2 patients showed significant ∆Np73 down-regulation in both the primary tumor and the implants. Case 1 presented a fourfold decrease of levels in the primary tumor and 20-fold decrease in the implants. Case 2 showed a seven- and fourfold down-regulation in the primary tumor and implants, respectively. However, Case 3 showed an up-regulation of 53- and threefold in the primary tumor and implants, respectively. CONCLUSION: Goblet cell carcinoma of the appendix is very rate. It tends to seed throughout the peritoneum, making aggressive surgical cytoreduction and chemotherapy viable treatment options. Investigation into the molecular basis of these tumors may improve the diagnosis, prognosis and therapeutic decisions regarding these patients. ∆Np73 seems a good candidate for further analysis in longer series.

[Application of PLA Method for Detection of p53/p63/p73 Complexes in Situ in Tumour Cells and Tumour Tissue]. / Vyuzití metody PLA pro detekci komplexu p53/p63/p73 in situ v nádorových bunkách a nádorové tkáni.

BACKGROUND: PLA (proximity ligation assay) can be used for detection of protein-protein interactions in situ directly in cells and tissues. Due to its high sensitivity and specificity it is useful for detection, localization and quantification of protein complexes with single molecule resolution. One of the mechanisms of mutated p53 gain of function is formation of proten-protein complexes with other members of p53 family - p63 and p73. These interactions influences chemosensitivity and invasivity of cancer cells and this is why these complexes are potential targets of anti-cancer therapy. The aim of this work is to detect p53/p63/p73 interactions in situ in tumour cells and tumour tissue using PLA method. MATERIAL AND METHODS: Unique in-house antibodies for specific detection of p63 and p73 isoforms were developed and characterized. Potein complexes were detected using PLA in established cell lines SVK14, HCC1806 and FaDu and in paraffin sections of colorectal carcinoma tissue. Cell lines were also processed to paraffin blocks. RESULTS: p53/T-antigen and ΔNp63/T-antigen protein complexes were detected in SVK14 cells using PLA. Interactions of ΔNp63 and TAp73 isoforms were found in HCC1806 cell line with endogenous expression of these proteins. In FaDu cell line mut-p53/TAp73 complex was localized but not mut-p53/ΔNp63 complex. p53 tetramer was detected directly in colorectal cancer tissue. CONCLUSION: During development of PLA method for detection of protein complexes between p53 family members we detected interactions of p53 and p63 with T-antigen and mut-p53 and ΔNp63 with TAp73 tumour suppressor in tumour cell lines and p53 tetramers in paraffin sections of colorectal cancer tissue. PLA will be further used for detection of p53/p63, p53/p73 and p63/p73 interactions in tumour tissues and it could be also used for screening of compounds that can block formation of p53/p63/p73 protein complexes.Key words: p53 protein family - protein interaction mapping - immunofluorescence This work was supported by MEYS - NPS I - LO1413. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 13. 3. 2017Accepted: 26. 3. 2017.