DDX21 is a newly discovered RNA G-quadruplex (rG4) binding protein with no known biological rG4 targets. In this study we used label-free proteomic MS/MS to identify 26 proteins that are expressed at significantly different levels in cells expressing an rG4-binding deficient DDX21 (M4). MS data are available via ProteomeXchange with identifier PXD013501. From this list we validate MAGED2 as a protein that is regulated by DDX21 through rG4 in its 5'-UTR. MAGED2 protein levels, but not mRNA levels, are reduced by half in cells expressing DDX21 M4. MAGED2 has a repressive effect on TRAIL-R2 expression that is relieved under these conditions, resulting in elevated TRAIL-R2 mRNA and protein in MCF-7 cells, rendering them sensitive to TRAIL-mediated apoptosis. Our work identifies the role of DDX21 in regulation at the translational level through biologically relevant rG4 and shows that MAGED2 protein levels are regulated, at least in part, by the potential to form rG4 in their 5'-UTRs.

The melanoma-associated antigen (MAGE) gene family consists of different expression patterns in various tumor types. They are considered tumor-specific antigens and are ideal targets for cancer immunotherapy. The purpose of this study is to identify the expression profiles of the MAGE family genes in Taiwanese colorectal cancer patients.

In this study, a well-constructed chip array platform was used to analyze the expression of the MAGE family genes of 100 colorectal cancer tissues. Statistical analysis of the experimental results and patients' clinical manifestations were also conducted.

The results showed MAGE-A2 (87%), -A7 (83%), -A8 (75%), -A12 (71%), -B2 (75%), -B3 (79%), -D2 (75%), -F1 (79%), and -H1 (70%) were significantly overexpressed genes in colorectal cancer tissues. MAGE-A2 was the most highly overexpressed gene among the MAGE family. MAGE-B3 gene expression is statistically correlated with tumor size, lymph node, and UICC stage. In addition, the overexpression of MAGE-D2 and -H1 genes are statistically correlated to the tumor size and depth, respectively (P < 0.05).

This is the first comprehensive report to clarify the differential expression profile of whole MAGE family in CRCs, and it might provide some crucial information about the carcinogenesis and progression in Taiwanese patients with CRC.

The current standard of treatment for patients with advanced ovarian cancer has been established in light of the results from various clinical trials. After debulking surgery, a combination of carboplatin and paclitaxel is considered to be the best treatment option in terms of survival and quality of life. However, since most patients on this chemotherapy modality will experience relapse, several studies have explored, and continue to do so, various modifications and alternatives to standard therapy in order to attain improved efficacy. Various modifications of dose, schedule, or route of standard regimens have shown no benefit, apart from intraperitoneal therapy, which has produced mixed results and would benefit from a definitive trial. Studies of maintenance/consolidation therapy have been mainly negative, although a small number of trials have produced enough positive data to prompt two new studies powered to detect survival benefits. Various phase II trials have investigated "targeted therapies," but until now no positive results have been recorded. Translational studies are needed to identify patients who will benefit from such specific treatment strategies. The current most evaluated modification of standard therapy is the addition of a third non-cross-resistant drug to carboplatin and paclitaxel. Data for the addition of anthracyclines have either been negative (epirubicin) or not yet analyzed (pegylated liposomal doxorubicin), while evaluable data are shortly expected for the addition of topotecan. Data on the addition of gemcitabine are eagerly awaited from two phase III trials.

Using cDNA microarray analysis, we identified a cDNA clone, DD74, from primary human bronchial epithelial cells, which exhibits increased expression in vitro after treatment with all-trans retinoic acid. This clone corresponded to MAGE D2 mRNA, a gene previously identified to be upregulated in several cancer tissues. Surprisingly, in situ hybridization of lung tissue demonstrated positive hybridization signals with sense, but not antisense, MAGE D2-specific cRNA probes. Examination of several cell lines by Northern blot hybridization confirmed significant expression of two RNA bands. With strand-specific riboprobes, we identified a 2.0kb RNA transcript with the antisense probe as expected and identified a 4.1kb transcript by the sense probe. Further sequence analysis of the 4.1kb transcript revealed at least a 509 nucleotide sequence exactly complementary to the 2.0kb MAGE D2 mRNA sequence. This MAGE D2i sequence contains unique structural features not shared with those of previously described antisense transcripts. Identification of this transcript potentially has important implications for future studies examining MAGE D2 expression patterns in cancer and normal tissues.

Mammary ductal cells are the origin for 70-80% of breast cancers. Nipple aspirate fluid (NAF) contains proteins directly secreted by the ductal and lobular epithelium in non-lactating women. Proteomic approaches offer a largely unbiased way to evaluate NAF as a source of biomarkers and are sufficiently sensitive for analysis of small NAF volumes (10-50 microl). In this study, we initially evaluated a new process for obtaining NAF and discovered that this process resulted in a volume of NAF that was suitable for analysis in approximately 90% of subjects. Proteomic characterization of NAF identified 64 proteins. Although this list primarily includes abundant and moderately abundant NAF proteins, very few of these proteins have previously been reported in NAF. At least 15 of the NAF proteins identified have previously been reported to be altered in serum or tumor tissue from women with breast cancer, including cathepsin D and osteopontin. In summary, this study provides the first characterization of the NAF proteome and identifies several candidate proteins for future studies on breast cancer markers in NAF.

CD31, an adhesion molecule expressed by endothelial cells, leukocytes, and platelets, is used in surgical pathology as a marker of normal and neoplastic vascularization. During the assessment of angiogenesis in breast carcinomas, CD31 expression was observed in a single case of large (5.2 cm diameter) high nuclear grade ductal carcinoma in situ (HG-DCIS) associated with poorly differentiated invasive ductal carcinoma (G3-IDC). Expression was limited to the cell membrane. This study focused on 32 HG-DCIS> or = 2 cm, either pure or associated with IDC. Cancer cells wereCD31(+) in 11 cases. Double staining using anti-CD31 monoclonal antibody (MAb) and anti-CD44 MAb, the anti-hyaluronate receptor, showed that foci of CD31(+) and CD44(-) tumour cells could be traced throughout the glandular tree, marking the intraductal diffusion of tumour up to Paget's cells at the nipple. The associated G3-IDC and their lymph node metastases were instead CD31(+) and CD44(+). CD31(+) tumours were oestrogen receptor (ER)(-), frequently p53(+) and c-erb-B2(+), and infiltrated by CD4(+) T lymphocytes. Normal and hyperplastic epithelia were constantly CD31(-). Other endothelial markers (e.g Factor VIII-RA and CD34) were not expressed by carcinoma cells, as was CD38, the CD31 ligand. In conclusion, CD31 expression is a feature acquired by breast cancer cells in the DCIS model. CD31 expression mainly correlates with tumour cells spreading within the ductal system. Finally, the invasive phenotype requires the co-expression of CD31 and CD44.

The purpose of this study was to develop an imaging technique to measure and monitor tumor cells undergoing programmed death caused by radiation and chemotherapy using 99mTc-EC-annexin V. Annexin V has been used to measure programmed cell death both in vitro and in vivo. Assessment of apoptosis would be useful to evaluate the efficacy and mechanisms of therapy and disease progression or regression.

Ethylenedicysteine (EC) was conjugated to annexin V using sulfo-N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCl as coupling agents. The yield of EC-annexin V was 100%. In vitro cellular uptake, pre- and post-radiation (10-30 Gy) and paclitaxel treatment, was quantified using 99mTc-EC-annexin V. Tissue distribution and planar imaging of 99mTc-EC-annexin V were determined in breast tumor-bearing rats at 0.5, 2, and 4 hrs. To demonstrate in vivo cell apoptosis that occurred during chemotherapy, a group of rats was treated with paclitaxel and planar imaging studies were conducted at 0.5-4 hrs. Computer outlined region of interest (ROI) was used to quantify tumor uptake on day 3 and day 5 post-treatment.

In vitro cellular uptake showed that there was significantly increased uptake of 99mTc-EC-annexin V after irradiation (10-30 Gy) and paclitaxel treatment. In vivo biodistribution of 99mTc-EC-annexin in breast tumor-bearing rats showed increased tumor-to-blood, tumor-to-lung and tumor-to-muscle count density ratios as a function of time. Conversely, tumor-to-blood count density ratios showed a time-dependent decrease with 99mTc-EC in the same time period. Planar images confirmed that the tumors could be visualized clearly with 99mTc-EC-annexin. There was a significant difference of ROI ratios between pre- and post-paclitaxel treatment groups at 2 and 4 hrs post injection.

The results indicate that apoptosis can be quantified using 99mTc-EC-annexin and that it is feasible to use 99mTc-EC-annexin to image tumor apoptosis.