In GFP with high risk HPV-18E6 fusion protein expressed 293T and MCF-7 cells, the endogenous wild-type p53 could be transiently phosphorylated at multiple sites

<p>Abstract</p> <p>Background</p> <p>Infected cells recognize viral replication as a DNA damage stress and elicit the host surveillance mechanism to anti-virus infection. Modulation of the activity of tumor suppressor p53 is a key event in the replication of many viruses. They could manipulate p53 function through phosphorylation modification for their own purpose. But there is rarely research about p53 phosphorylation status in the context of HPV-E6. Therefore, we investigated whether p53 could be phosphorylated by HPV-E6.</p> <p>Methods</p> <p>We used a mammalian green fluorescence protein (GFP) expression system to express HPV-18E6 with GFP fusion proteins (GFP-18E6) in wild-type (wt) p53 cell lines, such as 293T and MCF-7 cells to trace the traffic and subcellular location of E6 protein. By immunofluorescence technique and immunoblotting, we determined the positive phosphorylated sites of p53 and observed the distribution of phosphorylated p53 in the context of GFP-18E6.</p> <p>Results</p> <p>GFP-18E6 was predominantly located in nuclei of wt p53 cell lines, and it could induce transient phosphorylation of p53 at multiple sites, such as Ser¹⁵, Ser²⁰, and Ser³⁹². All the three sites of phosphorylated p53s were localized in nuclei together with GFP-18E6.</p> <p>Conclusion</p> <p>In GFP with high risk HPV-18E6 fusion protein expressed 293T and MCF-7 cells, the endogenous wt p53 could be transiently phosphorylated at multiple sites.</p

MicroRNA profiling in human colon cancer cells during 5-fluorouracil-induced autophagy.

Autophagy modulation is now recognized as a potential therapeutic approach for cancer (including colorectal cancer), yet the molecular mechanisms regulating autophagy in response to cellular stress are still not well understood. MicroRNAs (miRNAs) have been found to play important roles in controlling many cellular functions, including growth, metabolism and stress response. The physiological importance of the miRNA-autophagy interconnection is only beginning to be elucidated. MiRNA microarray technology facilitates analysis of global miRNA expression in certain situations. In this study, we explored the expression profile of miRNAs during the response of human colon cancer cells (HT29s) to 5-FU treatment and nutrient starvation using miRNA microarray analysis. The alteration of miRNA expression showed the same pattern under both conditions was further testified by qRT-PCR in three human colon cancer cell lines. In addition, bioinformatic prediction of target genes, pathway analysis and gene network analysis were performed to better understand the roles of these miRNAs in the regulation of autophagy. We identified and selected four downregulated miRNAs including hsa-miR-302a-3p and 27 upregulated miRNAs under these two conditions as having the potential to target genes involved in the regulation of autophagy in human colon cancer cells. They have the potential to modulate autophagy in 5-FU-based chemotherapy in colorectal cancer

Identification of potential serum proteomic biomarkers for clear cell renal cell carcinoma.

OBJECTIVE: To investigate discriminating protein patterns and serum biomarkers between clear cell renal cell carcinoma (ccRCC) patients and healthy controls, as well as between paired pre- and post-operative ccRCC patients. METHODS: We used magnetic bead-based separation followed by matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) to identify patients with ccRCC. A total of 162 serum samples were analyzed in this study, among which there were 58 serum samples from ccRCC patients, 40 from additional paired pre- and post-operative ccRCC patients (n = 20), and 64 from healthy volunteers as healthy controls. ClinProTools software identified several distinct markers between ccRCC patients and healthy controls, as well as between pre- and post-operative patients. RESULTS: Patients with ccRCC could be identified with a mean sensitivity of 88.38% and a mean specificity of 91.67%. Of 67 m/z peaks that differed among the ccRCC, healthy controls, pre- and post-operative ccRCC patients, 24 were significantly different (P<0.05). Three candidate peaks, which were upregulated in ccRCC group and showed a tendency to return to healthy control values after surgery, were identified as peptide regions of RNA-binding protein 6 (RBP6), tubulin beta chain (TUBB), and zinc finger protein 3 (ZFP3) with the m/z values of 1466.98, 1618.22, and 5905.23, respectively. CONCLUSION: MB-MALDI-TOF-MS method could generate serum peptidome profiles of ccRCC, and provide a new approach to identify potential biomarkers for diagnosis as well as prognosis of this malignancy

Autophagy is activated by 5-FU treatment and starvation in HT29 cells.

<p>HT29 cells were treated with 5 µM of 5-FU or not. Activation of autophagy was observed by LC3 immunofluorescence (A). HT29 cells were starved in Krebs-Ringer buffer or not. Activation of autophagy was observed by LC3 immunofluorescence (B). DAPI staining was performed for identifying nucleus. LC3, p62 and mTOR immunoblotting was performed using the lysates of HT29 cells treated by 5 µM of 5-FU for 24 h or not, and starved for 7 h or not (C). Data are the representative of three independent experiments. Bar, 20 µm.</p