Background & Aims-Hepatic de-differentiation, liver development, and malignant transformation are processes in which the levels of hepatic S-adenosylmethionine (SAMe) are tightly regulated by two genes, MAT1A and MAT2A. MAT1A is expressed in the adult liver, whereas MAT2A expression is primarily extra-hepatic and is strongly associated with liver proliferation. The mechanisms that regulate these expression patterns are not completely understood. In silico analysis of the 3′ untranslated region of MAT1A and MAT2A revealed putative binding sites for the RNA-binding proteins AUF1 and HuR, respectively. We investigated the post-transcriptional regulation of MAT1A and MAT2A by AUF1, HuR and methyl-HuR in the aforementioned biological processes.
Lung cancer is the number one cancer killer in the United States. This disease is clinically divided into two sub-types, small cell lung cancer, (10-15% of lung cancer cases), and non-small cell lung cancer (NSCLC; 85-90% of cases). Early detection of NSCLC, which is the more common and less aggressive of the two sub-types, has the highest potential for saving lives. As yet, no routine screening method that enables early detection exists, and this is a key factor in the high mortality rate of this disease. Imaging and cytology-based screening strategies have been employed for early detection, and while some are sensitive, none have been demonstrated to reduce lung cancer mortality. However, mortality might be reduced by developing specific molecular markers that can complement imaging techniques. DNA methylation has emerged as a highly promising biomarker and is being actively studied in multiple cancers. The analysis of DNA methylation-based biomarkers is rapidly advancing, and a large number of potential biomarkers have been identified. Here we present a detailed review of the literature, focusing on DNA methylation-based markers developed using primary NSCLC tissue. Viable markers for clinical diagnosis must be detectable in 'remote media' such as blood, sputum, bronchoalveolar lavage, or even exhaled breath condensate. We discuss progress on their detection in such media and the sensitivity and specificity of the molecular marker panels identified to date. Lastly, we look to future advancements that will be made possible with the interrogation of the epigenome.
Background: Lung cancer is the leading cause of cancer death in men and women in the United States and Western Europe. Over 160,000 Americans die of this disease every year. The five-year survival rate is 15% -significantly lower than that of other major cancers. Early detection is a key factor in increasing lung cancer patient survival. DNA hypermethylation is recognized as an important mechanism for tumor suppressor gene inactivation in cancer and could yield powerful biomarkers for early detection of lung cancer. Here we focused on developing DNA methylation markers for squamous cell carcinoma of the lung. Using the sensitive, high-throughput DNA methylation analysis technique MethyLight, we examined the methylation profile of 42 loci in a collection of 45 squamous cell lung cancer samples and adjacent non-tumor lung tissues from the same patients.
Previous kinetic investigations of the N-terminal RNA Recognition Motif (RRM) domain of spliceosomal A protein of the U1 small nuclear ribonucleoprotein particle (U1A) interacting with its RNA target U1 hairpin II (U1hpII) provided experimental evidence for a ‘lure and lock’ model of binding. The final step of locking has been proposed to involve conformational changes in an α-helix immediately C-terminal to the RRM domain (helix C), which occludes the RNA binding surface in the unbound protein. Helix C must shift its position to accommodate RNA binding in the RNA–protein complex. This results in a new hydrophobic core, an intraprotein hydrogen bond and a quadruple stacking interaction between U1A and U1hpII. Here, we used a surface plasmon resonance-based biosensor to gain mechanistic insight into the role of helix C in mediating the interaction with U1hpII. Truncation, removal or disruption of the helix exposes the RNA-binding surface, resulting in an increase in the association rate, while simultaneously reducing the ability of the complex to lock, reflected in a loss of complex stability. Disruption of the quadruple stacking interaction has minor kinetic effects when compared with removal of the intraprotein hydrogen bonds. These data provide new insights into the mechanism whereby sequences C-terminal to an RRM can influence RNA binding.
Background: Lung cancer is the leading cause of cancer death worldwide. Over 1.3 million people succumbed to lung cancer in 2008 according to the World Health Organization (WHO). The predicted five year survival rate for non-small cell lung cancer (NSCLC) for the year 2010 is 16%. An early diagnosis would significantly increase survival of lung cancer patients. Low dose computed tomography (LDCT) detects tumors as small as T0. However, in a recent study by The National Lung Screening Trial Research Team, the false positive rate was 96%. DNA methylation, which has been shown to be associated with gene inactivation in various cancers, exhibits cancer-specific profiles. We hypothesize that DNA methylation in plasma/serum could be a powerful biomarker to complement LDCT when a lesion is detected. Results: Using candidate gene analysis in combination with epigenetic profiling we have identified 12 DNA methylation markers based on comparisons between archival lung cancer samples and matched adjacent non-tumor lung: 2C35, GDNF, HOXA1, HOXB4, MT1G, NEUROD1, NID2, OPCML/HNT, SFRP1, TNFRSF25, TRIM58, TWIST1 are all highly significantly and frequently hypermethylated in lung cancer tissues. Because detection of shed DNA in bodily fluids requires a very sensitive method, we have adapted Digital MethyLight to interrogate the 12 markers simultaneously on 384-well plates. Digital MethyLight is a sensitive real time-based limiting dilution method in which free floating DNA extracted from bodily fluids is diluted over a large number of wells (in our case 90) so that the number of detectable methylated DNA molecules can be counted. We used three different colors of probes to generate four sets of multiplexed probes, covering our 12-marker panel. Using this method, we examined the serum of 90 patients with lung cancer and 90 high-risk controls from New York City. We also examined serum from an additional 30 low risk, non-smoker controls. Many of our markers showed a statistically significant difference in methylation when comparing cases to non-smoker controls, but the difference with the high risk smokers was much less pronounced. Several subjects from the high-risk controls showed DNA methylation levels comparable to the cases, and several of these controls were ultimately diagnosed with cancer. Interestingly, we found no correlation between tumor stage and amount of DNA shed, and were able to detect methylated DNA in the serum of several stage 1A patients. Conclusion: Elevated DNA methylation is observed in lung cancer cases, as well as in a number of high risk control subjects. Serum DNA methylation levels are markedly lower in non-smoking controls. While sensitivity is low, methylated DNA can be detected in stage 1A patients, suggesting that even small tumors can shed sufficient DNA to allow detection. It is not clear why some patients do not show detectable DNA, even with progressed disease. This merits further investigation. Funding: R01 CA 119029, R01 CA120869, Canary Foundation and Thomas G. Labrecque Foundation.
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