Hepatocyte dedifferentiation is a major source of hepatocellular carcinoma (HCC), but its mechanisms are unknown. We explored the p73 expression in HCC tumors and studied the effects of transcriptionally active p73β (TAp73β) in HCC cells. Expression profiles of p73 and patient clinical data were collected from the Genomic Data Commons (GDC) data portal and the TSVdb database, respectively. Global gene expression profiles were determined by pan-genomic 54K microarrays. The Gene Set Enrichment Analysis method was used to identify TAp73β-regulated gene sets. The effects of TAp73 isoforms were analyzed in monolayer cell culture, 3D-cell culture and xenograft models in zebrafish using western blot, flow cytometry, fluorescence imaging, real-time polymerase chain reaction (RT-PCR), immunohistochemistry and morphological examination. TAp73 isoforms were significantly upregulated in HCC, and high p73 expression correlated with poor patient survival. The induced expression of TAp73β caused landscape expression changes in genes involved in growth signaling, cell cycle, stress response, immunity, metabolism and development. Hep3B cells overexpressing TAp73β had lost hepatocyte lineage biomarkers including ALB, CYP3A4, AFP, HNF4α. In contrast, TAp73β upregulated genes promoting cholangiocyte lineage such as YAP, JAG1 and ZO-1, accompanied with an increase in metastatic ability. Our findings suggest that TAp73β may promote malignant dedifferentiation of HCC cells.

• Keywords
• TAp73, dedifferentiation, hepatocellular carcinoma, metastasis, yes-associated protein 1, zebrafish,
• Publication type
• Journal Article MeSH

The p73 protein is a member of the p53 family, and this protein is known to be essential for the maintenance of genomic stability, DNA repair, and apoptosis regulation. Transcription from two promoters leads to two main N-terminal isoforms: the TAp73 isoform is reported to have tumor suppressor function, whereas the ΔNp73 isoform likely has oncogenic potential. The present study is focused on the investigation of a possible role of both these p73 N-terminal isoforms in the process of centrosome amplification. HGG-02 and GM7 glioblastoma cell lines and the Daoy medulloblastoma cell line were used in this study. The cells were analyzed using indirect immunofluorescence to determine TAp73 and ΔNp73 expression patterns and possible co-localization with the BubR1 protein, as well as the number of centrosomes. A transiently transfected GM7 cell line was used to verify the results concerning the N-terminal isoforms in relation to centrosome amplification. We found that increased immunoreactivity for the ΔNp73 isoform is associated with the occurrence of an abnormal number of centrosomes in particular cells. Using the transiently transfected GM7 cell line, we confirmed that centrosome amplification is present in cells with overexpression of the ΔNp73 isoform. In contrast, the immunoreactivity for the TAp73 isoform was weak or medium in most of the cells with an aberrant number of centrosomes. To determine the putative counterpart of the p73 N-terminal isoforms among spindle assembly checkpoint (SAC) proteins, we also evaluated possible interactions between the N-terminal isoforms and BubR1 protein, but no co-localization of these proteins was observed.

• Keywords
• BubR1, Centrosome amplification, Glioblastoma multiforme, Medulloblastoma, TAp73, ΔNp73,
• MeSH
• Gene Amplification * MeSH
• Centrosome physiology   MeSH
• Chromosome Aberrations * MeSH
• DNA-Binding Proteins genetics   MeSH
• Fluorescent Antibody Technique, Indirect MeSH
• Nuclear Proteins genetics   MeSH
• Humans MeSH
• Tumor Cells, Cultured MeSH
• Tumor Suppressor Proteins genetics   MeSH
• Brain Neoplasms genetics   pathology   MeSH
• DNA Repair MeSH
• Promoter Regions, Genetic MeSH
• Protein Isoforms MeSH
• Tumor Protein p73 MeSH
• Protein Serine-Threonine Kinases genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• BUB1 protein, human MeSH Browser
• delta Np73 protein, human MeSH Browser
• DNA-Binding Proteins MeSH
• Nuclear Proteins MeSH
• Tumor Suppressor Proteins MeSH
• Protein Isoforms MeSH
• Tumor Protein p73 MeSH
• Protein Serine-Threonine Kinases MeSH
• TP73 protein, human MeSH Browser

Although methotrexate (MTX) is the most well-known antifolate included in many standard therapeutic regimens, substantial toxicity limits its wider use, particularly in pediatric oncology. Our study focused on a detailed analysis of MTX effects in cell lines derived from two types of pediatric solid tumors: medulloblastoma and osteosarcoma. The main aim of this study was to analyze the effects of treatment with MTX at concentrations comparable to MTX plasma levels in patients treated with high-dose or low-dose MTX. The results showed that treatment with MTX significantly decreased proliferation activity, inhibited the cell cycle at S-phase and induced apoptosis in Daoy and Saos-2 reference cell lines, which were found to be MTX-sensitive. Furthermore, no difference in these effects was observed following treatment with various doses of MTX ranging from 1 to 40 µM. These findings suggest the possibility of achieving the same outcome with the application of low-dose MTX, an extremely important result, particularly for clinical practice. Another important aspect of treatment with high-dose MTX in clinical practice is the administration of leucovorin (LV) as an antidote to reduce MTX toxicity in normal cells. For this reason, the combined application of MTX and LV was also included in our experiments; however, this application of MTX together with LV did not elicit any detectable effect. The expression analysis of genes involved in the mechanisms of resistance to MTX was a final component of our study, and the results helped us to elucidate the mechanisms of the various responses to MTX among the cell lines included in our study.

• MeSH
• Folic Acid Antagonists administration & dosage   MeSH
• Drug Resistance, Neoplasm genetics   MeSH
• Tetrahydrofolate Dehydrogenase metabolism   MeSH
• Humans MeSH
• Medulloblastoma * enzymology   genetics   MeSH
• Methotrexate administration & dosage   MeSH
• Cell Line, Tumor MeSH
• Bone Neoplasms * enzymology   genetics   MeSH
• Cerebellar Neoplasms * enzymology   genetics   MeSH
• Osteosarcoma * enzymology   genetics   MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Cell Proliferation drug effects   MeSH
• Flow Cytometry MeSH
• Dose-Response Relationship, Drug MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Folic Acid Antagonists MeSH
• Tetrahydrofolate Dehydrogenase MeSH
• Methotrexate MeSH