Quantification and Localization of Protein-RNA Interactions in Patient-Derived Archival Tumor Tissue.

Abnormal post-transcriptional regulation induced by alterations of mRNA-protein interactions is critical during tumorigenesis and cancer progression and is a hallmark of cancer cells. A more thorough understanding is needed to develop treatments and foresee outcomes. Cellular and mouse tumor models are insufficient for vigorous investigation as they lack consistency and translatability to humans. Moreover, to date, studies in human tumor tissue are predominately limited to expression analysis of proteins and mRNA, which do not necessarily provide information about the frequency of mRNA-protein interactions. Here, we demonstrate novel optimization of a method that is based on FISH and proximity ligation techniques to quantify mRNA interactions with RNA-binding proteins relevant for tumorigenesis and cancer progression in archival patient-derived tumor tissue. This method was validated for multiple mRNA-protein pairs in several cellular models and in multiple types of archival human tumor samples. Furthermore, this approach allowed high-throughput analysis of mRNA-protein interactions across a wide range of tumor types and stages through tumor microarrays. This method is quantitative, specific, and sensitive for detecting interactions and their localization at both the individual cell and whole-tissue scales with single interaction sensitivity. This work presents an important tool in investigating post-transcriptional regulation in cancer on a high-throughput scale, with great potential for translatability into any applications where mRNA-protein interactions are of interest. SIGNIFICANCE: This work presents an approach to sensitively, specifically, and quantitatively detect and localize native mRNA and protein interactions for analysis of abnormal post-transcriptional regulation in patient-derived archival tumor samples.

A Modified Coupled Spectrophotometric Method to Detect 2-5 Oligoadenylate Synthetase Activity in Prostate Cell Lines.

BACKGROUND: 2'-5' oligoadenylate synthetases (OAS) are interferon inducible enzymes that polymerizes ATP to 2'-5'-linked oligomers of adenylate (2-5As). As part of the innate immune response, these enzymes are activated by viral double stranded RNA or mRNAs with significant double stranded structure. The 2-5As in turn activate RNaseL that degrade single stranded RNAs. Three distinct forms of OAS exist in human cells (OAS1, 2 and 3) with each form having multiple spliced variants. The OAS enzymes and their spliced variants have different enzyme activities. OAS enzymes also play a significant role in regulating multiple cellular processes such as proliferation and apoptosis. Moreover, Single nucleotide polymorphisms that alter OAS activity are also associated with viral infection, diabetes and cancer. Thus detection of OAS enzyme activity with a simple spectrophotometric method in cells will be important in clinical research. RESULTS: Here we propose a modified coupled spectrophotometric assay to detect 2-5 oligoadenylate synthetase (OAS) enzyme activity in prostate cell lines as a model system. The OAS enzyme from prostate cancer cell lysates was purified using Polyinosinic: polycytidylic acid (poly I:C) bound activated sepharose beads. The activated OAS enzyme eluted from Sepharose beads showed expression of p46 isoform of OAS1, generally considered the most abundant OAS isoform in elutes from DU14 cell line but not in other prostate cell line. In this assay the phosphates generated by the OAS enzymatic reaction is coupled with conversion of the substrate 2-amino-6-mercapto-7-methylpurine ribonucleoside (methylthioguanosine, a guanosine analogue; MESG) to a purine base product, 2-amino-6-mercapto-7-methylpurine and ribose1-phosphate via a catalyst purine nucleoside phosphorylase (phosphorylase) using a commercially available pyrophosphate kit. The absorbance of the purine base product is measured at 360 nm. The higher levels of phosphates detected in DU145 cell line indicates more activity of OAS in this prostate cancer cell line. CONCLUSION: The modified simple method detected OAS enzyme activity with sensitivity and specificity, which could help in detection of OAS enzymes avoiding the laborious and radioactive methods.

XMRV infection in patients with prostate cancer: novel serologic assay and correlation with PCR and FISH.

OBJECTIVES: To develop a serum-based assay to detect neutralizing antibodies to the xenotropic murine leukemia virus-related virus (XMRV) retrovirus and to use this assay with polymerase chain reaction and fluorescence in situ hybridization to identify patients with prostate cancer previously exposed to XMRV infection and those who carry XMRV viral sequences in their prostate. METHODS: Patients who had undergone radical prostatectomy were enrolled, and biologic specimens were obtained at surgery. The patients were genotyped for the R462Q RNASEL variant using a TaqMan genotyping assay on DNA from the peripheral blood. A serum assay that detects XMRV neutralizing antibodies was developed and used to determine which patients had serologic evidence of previous infection with XMRV virus. Some of these patients were also tested for the presence of XMRV nucleotide sequences in their prostate using polymerase chain reaction and fluorescence in situ hybridization analysis. RESULTS: At a serum dilution of 1:150, our assay detected 11 (27.5%) of 40 patients with XMRV neutralizing antibodies, including 8 (40%) of 20 with the RNASEL genotype QQ and 3 (15%) of 20 with either the RQ or RR genotype. These results were in complete concordance with 2 other assays (polymerase chain reaction and fluorescence in situ hybridization), which were designed to detect XMRV infection. CONCLUSIONS: XMRV infects some patients with prostate cancer. Neutralizing antibodies against XMRV correlated with 2 independent methods of detecting the virus in the prostate. The antibody response suggests that with clinical serologic assay development, it might be possible to screen patients for XMRV infection. The cases presented in the present report provided biologic samples that can be used for the development of a clinically relevant assay.

Apoptotic index as predictive marker for radiosensitivity of cervical carcinoma: evaluation of membrane fluidity, biochemical parameters and apoptosis after the first dose of fractionated radiotherapy to patients.

BACKGROUND: This study was aimed to develop possible predictive response of cervical carcinoma in stage IIIA and B patients by evaluating the changes in physical parameter, such as, membrane fluidity, biochemical parameters, such as, intracellular calcium, antioxidant enzymes [superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx)] and apoptotic cell death in cervical cancer cells from patients after treating with the first fractionated dose of 2 Gy in radiation therapy protocol. METHODS: Biopsies of cervical carcinoma patients were collected before and 24h after first fractionated radiation dose of 2 Gy. Cell suspensions and tissue of cervix cancer biopsies were used to measure various physical and biochemical parameters. RESULTS AND CONCLUSIONS: A negative correlation was found to exist between observed fluidity of membrane/SOD level with the degree of apoptosis in cervical cells. On the other hand, a positive correlation was observed between intracellular calcium level and percent cellular apoptosis. These results suggest that changes in membrane fluidity, SOD and calcium level were involved in the mechanism of radiation induced cervical apoptosis as measured by TUNEL assay. Moreover, apoptotic sensitivity of these cells after the first dose of radiation treatment showed a direct correlation with the radiation treatment outcome in patients after completion of radiotherapy protocol (70 Gy) in the clinic suggesting that apoptotic index may form a basis for prognosis in radiotherapy in stage III cervix cancer patients.

Enhancement of radiation-induced oxidative stress and cytotoxicity in tumor cells by ellagic acid.

BACKGROUND: Failure of treatment of cancer in clinic by radio/chemotherapy is generally attributed to tumor resistance. It is, therefore, important to develop strategies to increase the cytotoxicity of tumor cells by radiation in combination with new tumor selective cytotoxic agents. We describe the role of ellagic acid (EA) and gamma radiation on the oxidative stress and subsequent cytotoxicity of tumor cells in vitro as well as in vivo and their sparing effects on normal cells. METHODS: Ehrlich ascites carcinoma (EAC)-transplanted Swiss mice were intraperitoneally injected with EA followed by radiation treatment of 2 Gy for 4 alternate days. Hela cells were used for in vitro studies. Reactive oxygen species (ROS) level was measured by spectrofluorimetric method by using 2, 7-dichlorodihydrofluoresceindiacetate (DCHFDA) fluorescent probe. Cytotoxicity was measured by Trypan blue dye exclusion test and mitochondrial potential was measured using Rhodamine 123 as a probe. Antioxidant enzymes were measured by spectrophotometric methods. RESULTS: EA was found to generate ROS in tumor cells, which increased, by an order of magnitude when cells were treated with EA in combination with gamma radiation. The decrease in mitochondrial potential and the loss of cell viability were remarkably greater in tumor cells from mice treated with EA and radiation than alone treatment with either of them. Moreover, EA was found to protect against radiation-induced oxidative stress in splenic lymphocytes of tumor-transplanted mice. Measurement of antioxidant enzymes such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px) and glutathione reductase (GR) in tumor cells showed decrease after treatment with EA and radiation in vivo. Treatment of tumor bearing mice with EA and radiation showed significant decrease in animal's body weight suggesting reduced tumor burden. CONCLUSION: Combined treatment of tumor with EA and radiation enhances oxidative stress and cytotoxicity in tumor cells. EA protects normal cells against radiation damage. This may offer potential therapeutic benefit, which warrants clinical study for application in cancer radiotherapy.